WO2015066439A2 - Methods of treating hematological malignancies - Google Patents

Methods of treating hematological malignancies Download PDF

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Publication number
WO2015066439A2
WO2015066439A2 PCT/US2014/063367 US2014063367W WO2015066439A2 WO 2015066439 A2 WO2015066439 A2 WO 2015066439A2 US 2014063367 W US2014063367 W US 2014063367W WO 2015066439 A2 WO2015066439 A2 WO 2015066439A2
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Prior art keywords
inhibitor
braf
nucleic acid
cll
alteration
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PCT/US2014/063367
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French (fr)
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WO2015066439A8 (en
WO2015066439A3 (en
Inventor
Matthew J. HAWRYLUK
Jie He
Vincent A. MILLER
Philip James STEPHENS
Omar Abdel-Wahab
Jeffrey R. GARDNER
Ross LEVINE
Marcel Van Den Brink
Andrew ZELENETZ
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Foundation Medicine, Inc.
Memorial Sloan-Kettering Cancer Center
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Publication of WO2015066439A8 publication Critical patent/WO2015066439A8/en
Publication of WO2015066439A3 publication Critical patent/WO2015066439A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the invention relates to methods and compositions for treating and detecting
  • Hematological malignancies are cancers that affect the blood, bone marrow, and lymph nodes. Hematological malignancies include myelomas, leukemias, and lymphomas. Myelomas are cancers of the plasma cells; lymphomas are cancers that start in the lymph system, mainly the lymph nodes; and leukemias are cancers of the bone marrow and blood and include both lymphocytic and myelogenous leukemias.
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • AML acute monocytic leukemia
  • Hodgkin's lymphomas Hodgkin's lymphomas
  • Non-hodgkin's lymphomas Non-hodgkin's lymphomas.
  • CLL Chronic lymphocytic leukemia
  • B cell-CLL is a common type of adulthood leukemia, and is characterized by the clonal proliferation and accumulation of mature B lymphocytes (Eichhorst B. et al. Ann Oncol (2011) 22 (6): vi50-vi54; Smolewski P. et al. ISRN Oncology Volume 2013 (2013), Article ID 740615). CLL progression is commonly
  • CLL characterized by staging systems including, for example, the Rai 4- stage system and the Binet classification system (National Cancer Institute. "Chronic Lymphocytic Leukemia (PDQ) Treatment: Stage Information". Archived from the original on 17 October 2007; Smolewski, supra). Treatment of CLL is typically stage dependent, with early stage CLL monitored without treatment. Once CLL progresses chemotherapeutic and biologic agents are commonly employed. Current treatment regimens include, for example, fludarabine, cyclochosphamide, and rituximab; chlorambucil; and cyclophosphamide, vincristine, doxorubicin, and prednisone.
  • Current treatment regimens include, for example, fludarabine, cyclochosphamide, and rituximab; chlorambucil; and cyclophosphamide, vincristine, doxorubicin, and prednisone.
  • Bone marrow or stem cell transplantation may also be used in younger patients with advanced or high risk CLL.
  • CLL relapsed or refractory CLL
  • the invention is based, at least in part, on the discovery of alterations, e.g., activating mutations, not previously identified in hematological malignancies, such as Chronic
  • the alteration includes an activating mutation in a MAP kinase (Mitogen-activated protein kinase or MAPK) pathway gene or gene product, e.g., an activating mutation in BRAF and/or KRAS (also referred to herein as "B-Raf ' and "K-Ras,” respectively).
  • MAPK Mitogen-activated protein kinase
  • BRAF and/or KRAS also referred to herein as "B-Raf ' and "K-Ras,” respectively.
  • one or more mutations resulting in constitutive MAPK signaling were identified at a high frequency in CLL (e.g., about 36% of the 59 CLL samples characterized, of which 10% were activating mutation in BRAF).
  • the presence of activating mutations in the MAPK pathway in CLL provides novel therapeutic approaches for CLL that include MAPK pathway inhibitors.
  • the invention provides, at least in part, methods for treating a hematological malignancy, e.g., CLL, as well as methods and reagents for identifying, assessing or detecting an alteration as described herein, in such malignancies.
  • a hematological malignancy e.g., CLL
  • the invention features, a method of treating a subject having a hematological malignancy.
  • a method of treating a subject having a CLL is disclosed. The method includes administering to the subject an effective amount of an agent (e.g., a therapeutic agent) that targets and/or inhibits a MAPK pathway gene or gene product (e.g., a MAPK pathway inhibitor as described herein), thereby treating the subject.
  • the method further includes acquiring knowledge of the presence (or absence) of an alteration in a MAPK pathway gene or gene product.
  • the method further includes identifying the subject, or a cancer sample (e.g., a CLL sample) from the subject, as having the presence (or absence) of an alteration in a MAPK pathway gene or gene product.
  • the alteration in the MAPK pathway gene or gene product is an alteration (e.g., one or more oncogenic alterations) of a RAF (e.g., one or more of A-Raf, B- Raf (BRAF) or C-Raf), a RAS (e.g., one or more of H-Ras, N-Ras or K-Ras (KRAS)), and/or MEK (MAP/ERK kinase) gene or gene product, or results in increased activity, e.g., constitutive action of the MAPK pathway gene or gene product.
  • a RAF e.g., one or more of A-Raf, B- Raf (BRAF) or C-Raf
  • RAS e.g., one or more
  • the alteration in the MAPK pathway gene or gene product is a mutation in a Raf gene or gene product, e.g., a mutation in one or more of the glycine-rich P loop of the N lobe, the activation segment and/or the flanking region(s) of BRAF.
  • a mutation in a Raf gene or gene product e.g., a mutation in one or more of the glycine-rich P loop of the N lobe, the activation segment and/or the flanking region(s) of BRAF.
  • the mutation is chosen from a mutation in codon 464, 465, 466, 468, 469, 580, 594, 595, 596, 597, 599, 600, 601 or 727, of BRAF.
  • the alteration in BRAF is not located at codon 600 (e.g., V600).
  • Exemplary alterations in the BRAF gene or gene product at a position other than BRAF at position 600 include but are not limited to, R461I, I462S, G463E, G463V, G464E, G464R, G464V, G465A, G465E, G465V, G466A, G466E, G466R, G466V, G468A, G468E, F468C, G469A, G469E, G469R, G469R, G469S, G469V, N580S, E585K, D593V, D594G, D594V, F594L, F595L, G595R, L596V, G596R, L597Q, L597R, L597S, L597V, T598I, T599I, V599D, V599E, V599K, V599R, K601E, K601N
  • the alteration in the MAPK pathway gene or gene product is a mutation in a Ras gene or gene product, e.g., a mutation in K-Ras, that includes, for example, a mutation in codon 12, 13 and/or 61, including but not limited to, G12A, G12N, G12R, G12C, G12S, G12V, G13N and Q61R.
  • Non-limiting examples of alterations in a KRAS gene include, but are not limited to, G12C, G12R, G12D, G12A, G12S, G12V, G13D, G13S, G13C, G13V, Q61H, Q61R, Q61P, Q61L, Q61K, Q61E, A59T and G12F.
  • the presence of the MAPK pathway alteration (e.g., the BRAF and/or KRAS alteration) in the subject is indicative that the subject is likely to respond to the agent, e.g., the therapeutic agent (e.g., the MAPK pathway inhibitor).
  • the agent, e.g., the therapeutic agent (e.g., the MAPK pathway inhibitor) is administered responsive to a determination of the presence of the MAPK pathway alteration (e.g., the BRAF and/or KRAS alteration) in the subject, or the cancer or tumor sample from the subject.
  • the cancer is a hematological malignancy.
  • the hematological malignancy is chosen from a cancer that affects one or more of the blood, the bone marrow, or the lymph nodes.
  • the hematological malignancy can be chosen from a myeloma, a leukemia, or a lymphoma.
  • the hematological malignancy is chosen from a chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (AML), Hodgkin' s lymphoma, or Non-hodgkin' s lymphoma.
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • AML acute monocytic leukemia
  • Hodgkin' s lymphoma or Non-hodgkin' s lymphoma.
  • the hematological malignancy is a CLL, e.g., a refractory CLL or a relapsed CLL.
  • the CLL can be at any stage or risk group of CLL defined according to suitable CLL classification systems known to those of skill in the art.
  • the CLL can be chosen from a stage 0, 1, II, III, or IV, wherein stage 0 CLL is characterized by an elevated level of lymphocytes in the blood, but there are no other detectable symptoms of leukemia; stage I CLL is characterized by an elevated level of lymphocytes in the blood and enlarged size of lymph nodes; stage II CLL is characterized by an elevated level of lymphocytes in the blood, the liver or spleen is larger than normal, and the lymph nodes may be larger than normal; stage III CLL is characterized by an elevated level of lymphocytes in the blood, reduced number of red blood cells, and the lymph nodes, liver, or spleen may be larger than normal, and stage IV CLL is characterized by an elevated level of lymphocytes in the blood, reduced platelets, the lymph nodes, liver, or spleen may be larger than normal, and there may be a reduced number of red blood cells.
  • the detected levels or tissue sizes can be compared to a reference value, e.g., a normal or a control value.
  • a reference value e.g., a normal or a control value.
  • Other classifications for CLL can be used, for example, the Rai Scale (Rai K et al. Blood Volume 46, Issue 2, 1975, Pages 219-234) and the Binet Scale as described in more detail below.
  • the CLL is a low risk disease, intermediate risk disease, or high risk disease on the modified Rai Scale, wherein low risk is characterized by lymphocytes less than 15 x lOg/1; intermediate risk is characterized by lymphocytes less than 15 x lOg/1 and hepato- or splenomegaly; and high risk is characterized as Anemia (Hb ⁇ 1 1 g/dL) or
  • thrombocytopenia platelets ⁇ 100 xl0 9 /L) (Hallek M et al. Blood June 15, 2008 vol. I l l no. 12 5446-5456).
  • the hematological malignancy e.g., CLL
  • the hematological malignancy comprises, or is identified or determined as having, an alteration in a MAPK pathway gene or gene product, e.g., an alteration in BRAF and/or KRAS alteration as described herein.
  • the alteration in a MAPK pathway gene or gene product results in increased activity of a BRAF and/or KRAS gene product (e.g., a BRAF and/or KRAS protein), compared to the wild-type activity protein.
  • the alteration can result in one or more of: increased kinase activity of a BRAF protein, constitutive activation of BRAF and/or KRAS, increased activity or phosphorylation of a downstream component of the MAPK pathway, including, but not limited to, MEK (MAP/ERK kinase) (e.g., MEKl and/or MEK2).
  • the alteration is, or comprises, a mutation (e.g., a somatic mutation), e.g., a substitution (e.g., a base substitution), a deletion, an insertion, or a missense mutation.
  • the alteration in the MAPK pathway gene or gene product is a mutation in a Raf gene or gene product, e.g., a mutation in one or more of the glycine-rich P loop of the N lobe, the activation segment and/or the flanking region(s) of BRAF.
  • a mutation in a Raf gene or gene product e.g., a mutation in one or more of the glycine-rich P loop of the N lobe, the activation segment and/or the flanking region(s) of BRAF.
  • the mutation is chosen from a mutation in codon 464, 465, 466, 468, 469, 580, 594, 595, 596, 597, 599, 600, 601 or 727, of BRAF.
  • the alteration in BRAF is not located at codon 600, e.g., V600.
  • Exemplary alterations in the BRAF gene or gene product at a position other than BRAF at position 600 include but are not limited to, R461I, I462S, G463E, G463V, G464E, G464R, G464V, G465A, G465E, G465V, G466A, G466E, G466R, G466V, G468A, G468E, F468C, G469A, G469E, G469R, G469R, G469S, G469V, N580S, E585K, D593V, D594G, D594V, F594L, F595L, G595R, L596V, G596R, L597Q, L597R, L597S, L597V, T598I, T599I, V599D, V599E, V599K, V599R, K601E, K601N
  • the alteration in the MAPK pathway gene or gene product is a mutation in a Ras gene or gene product a mutation in K-Ras, that includes, for example, a mutation in codon 12, 13 and/or 61, including but not limited to, G12A, G12N, G12R, G12C, G12S, G12V, G13N and Q61R.
  • Non-limiting examples of alterations in a KRAS gene include, but are not limited to, G12C, G12R, G12D, G12A, G12S, G12V, G13D, G13S, G13C, G13V, Q61H, Q61R, Q61P, Q61L, Q61K, Q61E, A59T and G12F.
  • the subject has an alteration in a MAPK pathway gene or gene product.
  • the subject has a hematological malignancy, e.g., CLL, that comprises an alteration described herein.
  • the subject is identified, or has been previously identified, as having a cancer (e.g., a hematological malignancy, e.g., CLL) comprising an alteration in a MAPK pathway gene or gene product as described herein.
  • the alteration in the MAPK pathway gene or gene product is an alteration (e.g., one or more oncogenic alterations) of a RAF (e.g., one or more of A-Raf, B-Raf (BRAF) or C-Raf), a RAS (e.g., one or more of H-Ras, N-Ras or K-Ras), and/or MEK (MAP/ERK kinase) gene or gene product, or results in increased activity, e.g., constitutive action of the MAPK pathway gene or gene product.
  • a RAF e.g., one or more of A-Raf, B-Raf (BRAF) or C-Raf
  • RAS e.g., one or more of H-Ras, N-Ras or K-Ras
  • MEK MAP/ERK kinase
  • the subject is a human.
  • the subject has, or is at risk of having a cancer (e.g., a hematological malignancy, e.g., CLL) as described herein at any stage of disease, e.g., any stage described herein, relapsed, recurrent, or refractory.
  • a cancer patient e.g., a patient having a hematological malignancy, e.g., CLL, as described herein.
  • the subject is undergoing or has undergone treatment with a different therapeutic agent or therapeutic modality (e.g., non-MAPK pathway inhibitor).
  • a different therapeutic agent or therapeutic modality e.g., non-MAPK pathway inhibitor.
  • the different therapeutic agent or therapeutic modality is a chemotherapy, an immunotherapy, or a surgical procedure.
  • the different therapeutic agent or therapeutic modality comprises one or more of: a chemotherapeutic agent (e.g., fludarabine, cyclophosphamide, doxorubicin, vincristine, chlorambucil, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, 5-azacytidine, pemetrexed disodium), an EGFR inhibitor (e.g., erlotinib), BTK inhibitor (e.g., PCI-32765), CD20 targeting agent (e.g., rituximab,
  • a chemotherapeutic agent e.g., fludarabine, cyclophosphamide, doxorubicin, vincristine, chlorambucil, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, 5-azacytidine,
  • CD52 targeting agent e.g., alemtuzumab
  • prednisolone darbepoetin alfa, lenalidomide
  • Bcl-2 inhibitor e.g., ABT-263
  • AT-101 immunotoxin
  • immunotoxin e.g., CAT- 8015, anti-Tac(Fv)-PE38 (LMB-2)
  • CD37 targeting agent e.g., TRU-016
  • radioimmunotherapy e.g., 131-tositumomab
  • hydroxychloroquine perifosine
  • SRC inhibitor e.g., dasatinib
  • thalidomide a steroid
  • prednisone chlorambucil
  • retinoid e.g., fenretinide
  • MDM2 antagonist e.g., RO5045337)
  • plerixafor Aurora kinase inhibitor
  • the different therapeutic agent or therapeutic modality is discontinued.
  • the subject has been identified as being likely or unlikely to respond to the different therapeutic agent or therapeutic modality.
  • the subject has participated previously in a clinical trial, e.g., a clinical trial for a different (e.g., non-MAPK pathway inhibitor) therapeutic agent or therapeutic modality.
  • the subject is a cancer patient who has participated in a clinical trial, e.g., a clinical trial for a different (e.g., non-MAPK pathway inhibitor) therapeutic agent or therapeutic modality.
  • the agent e.g., the therapeutic agent used in the methods described herein, targets and/or inhibits a MAPK pathway gene or gene product (e.g., is a MAPK pathway inhibitor as described herein).
  • the agent inhibits a RAF (e.g., one or more of A-Raf, B-Raf or C-Raf), a RAS (e.g., one or more of H-Ras, N-Ras or K-Ras), and/or MEK (MAP/ERK kinase) gene or gene product.
  • the agent inhibits BRAF and/or MEK.
  • the agent is a reversible or an irreversible inhibitor.
  • the agent is a kinase inhibitor.
  • the kinase inhibitor is chosen from: a multi- specific kinase inhibitor, a pan inhibitor, a serine/threonine kinase inhibitor, and/or an inhibitor that is selective for BRAF, KRAS or a MAPK downstream protein.
  • the kinase inhibitor is a small molecule inhibitor.
  • the agent is a BRAF inhibitor.
  • the BRAF inhibitor is chosen from: Vemurafenib (PLX4032, RG7204, R05185426), Sorafenib Tosylate (Bay 43-9006, Nexavar), PLX4720, GDC-0879, RAF265 (CHIR-265), MLN2480 (BIIB-024), PF-04880594, GW5074, CEP-32496, Dabrafenib (GSK2118436), AZ628, SB590885, Raf265 derivative, Regorafenib (BAY 73-4506, Fluoro-Sorafenib), DP-4978, DP-2514, DP-3346, ARQ736, XL281, RG7256, LGX818, PLX3603, trematinib, and/or ZM 336372.
  • Vemurafenib PLX4032, RG7204, R05185426)
  • Sorafenib Tosylate
  • the BRAF inhibitor is Vemurafenib (also known as PLX4032, RG7204, R05185426).
  • Vemurafenib has the chemical name: N-(3- ⁇ [5-(4- chlorophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]carbonyl ⁇ -2,4-difluorophenyl)propane- l- sulfonamide; and has the following structure:
  • the BRAF inhibitor is Sorafenib Tosylate (also known as Bay 43- 9006, Nexavar).
  • Sorafenib has the chemical name: 2-Pyridinecarboxamide, 4- [4- [ [[ [4-chloro-3- (trifluoromethyl)phenyl] amino] carbonyl] amino]phenoxy] -N-methyl- , 4- methylbenzenesulfonate (1 : 1); and has the following structure:
  • the BRAF inhibitor is PLX4720.
  • PLX4720 has the chemical name: N-(3-(5-chloro- lH-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4- difluorophenyl)propane- l-sulfonamide; and has the following structure:
  • the BRAF inhibitor is GDC-0879.
  • GDC-0879 has the chemical name: (E)-5-(l-(2-hydroxyethyl)-3-(pyridin-4-yl)-lH-pyrazol-4-yl)-2,3- dihydroinden-l-one oxime; and has the following structure:
  • the BRAF inhibitor is RAF265 (CHIR-265).
  • RAF265 has the chemical name: l-methyl-5-(2-(5-(trifluoromethyl)-lH-imidazol-2-yl)pyridin-4- yloxy)-N-(4-(trifluoromethyl)phenyl)-lH-benzo[d]imidazol-2-amine; and has the following structure:
  • the BRAF inhibitor is Raf265 derivative.
  • Raf265 derivative has the following structure:
  • the BRAF inhibitor is MLN2480 (BIIB-024).
  • MLN2480 is a pan-Raf inhibitor; has the chemical name: 4-Pyrimidinecarboxamide, 6-amino-5- chloro-N-[(lR)-l-[5-[[[5-chloro-4-(trifluoromethyl)-2-pyridinyl]arnino]carbonyl]-2- thiazolyl] ethyl]-; and has the following structure:
  • the BRAF inhibitor is PF-04880594.
  • PF- 04880594 has the chemical name: Propanenitrile, 3-[[4-[l-(2,2-difluoroethyl)-3-(lH-pyrrolo[ b]pyridin-5-yl)-lH-pyrazol-4-yl -2-pyrimidinyl]amino]-; and has the following structure:
  • the BRAF inhibitor is GW5074.
  • GW5074 has the chemical name: 2H-Indol-2-one, 3-[(3,5-dibromo-4-hydroxyphenyl)methylene]-l,3-dihydro- 5-iodo-; and has the following structure:
  • the BRAF inhibitor is CEP-32496.
  • CEP- 32496 has the chemical name: Urea, N-[3-[(6,7-dimethoxy-4-quinazolinyl)oxy]phenyl]-N-[5- (2,2,2-trifluoro-l,l-dimethylethyl)-3-isoxazolyl]-; and has the following structure:
  • the BRAF inhibitor is Dabrafenib (GSK2118436).
  • Dabrafenib has the chemical name: N-(3-(5-(2-aminopyrimidin-4-yl)-2-tert- butylthiazol-4-yl)-2-fluorophenyl)-2,6-difluorobenzenesulfonamide; and has the following structure:
  • the BRAF inhibitor is AZ628.
  • AZ628 has the chemical name: 3-(2-cyanopropan-2-yl)-N-(4-methyl-3-(3-methyl-4-oxo-3,4-dihydroquinazolin- 6-ylamino)phenyl)benzamide; and has the following structure:
  • the BRAF inhibitor is SB590885.
  • SB590885 has the chemical name: (E)-5-(2-(4-(2-(dimethylamino)ethoxy)phenyl)-4-(pyridin-4-yl)-lH- imidazol-5-yl)-2,3-dihydroinden-l-one oxime; and has the following structure:
  • the BRAF inhibitor is Regorafenib (also known as BAY 73-4506, Fluoro-Sorafenib).
  • Regorafenib has the chemical name: l-(4-chloro-3- (trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(methylcarbamoyl)pyridin-4-yloxy)phenyl)urea; and has the following structure:
  • the BRAF inhibitor is ZM 336372.
  • ZM 336372 has the chemical name: Benzamide, 3-(dimethylamino)-N-[3-[(4- hydroxybenzoyl)amino]-4-methylphenyl]-; and has the following structure:
  • the BRAF inhibitor is LGX818.
  • LGX818 has the following chemical name Methyl [(2S)- l- ⁇ [4-(3- ⁇ 5-chloro-2-fluoro-3- [(methylsulfonyl)amino]phenyl ⁇ -l-isopropyl- lH-pyrazol-4-yl)-2-pyrimidinyl]amino ⁇ -2- propanyl]carbamate; and the followin
  • the BRAF inhibitor is DP-4978.
  • the BRAF inhibitor is DP-2514.
  • the BRAF inhibitor is DP-3346.
  • the BRAF inhibitor is XL281.
  • the BRAF inhibitor is RG7256 (PLX3603).
  • the BRAF inhibitor is an antibody molecule, e.g., an anti-BRAF antibody molecule (e.g., a monoclonal or a bispecific antibody), or a conjugate thereof (e.g., an antibody to BRAF conjugated to a cytotoxic agent (e.g., mertansine DM1)).
  • an anti-BRAF antibody molecule e.g., a monoclonal or a bispecific antibody
  • a conjugate thereof e.g., an antibody to BRAF conjugated to a cytotoxic agent (e.g., mertansine DM1)).
  • the agent is chosen from a nucleic acid molecule (e.g., an antisense molecule, a ribozyme, a double stranded RNA, or a triple helix molecule) that hybridizes to and/or inhibits a BRAF nucleic acid, e.g., a BRAF nucleic acid encoding the alteration, or a transcription regulatory region that blocks or reduces mRNA expression of the alteration.
  • a nucleic acid molecule e.g., an antisense molecule, a ribozyme, a double stranded RNA, or a triple helix molecule
  • the agent used in the methods targets and/or inhibits MEK (mitogen activated protein kinase kinase or M AP/ERK Kinase) (e.g., a MEK1 and/or a MEK2 gene or gene product).
  • MEK mitogen activated protein kinase kinase or M AP/ERK Kinase
  • the agent binds to and/or inhibits MEK.
  • the agent is a reversible or an irreversible MEK inhibitor.
  • the agent is a MEK inhibitor.
  • a MEK inhibitor can include an agent that inhibits MEK1 and/or MEK2.
  • the MEK inhibitor is chosen from: ARRY-162 (MEK162), Trametinib (GSK1120212), Selumetinib (AZD6244, ARRY142886), XL518 (GDC-0973), CI-1040 (PDl 84352), PD035901, U0126-EtOH, PD198306, PD98059, BIX 02189, TAK-733, Honokiol, AZD8330 (ARRY-424704), PD318088, BIX 02188, AS703026 (Pimasertib), RG7167, E6201 ;
  • MSC2015103 MSC1936369, WX554 and/or SL327.
  • the inhibitor MEK is ARRY-162 (MEK 162).
  • ARRY- 162 has the chemical name: 5-((4-bromo-2-fluorophenyl)amino)-4- fluoro-N-(2-hydroxyethoxy)-l-methyl-lH-benzo[d]imidazole-6-carboxamide; and has the following structure:
  • the MEK inhibitor is Trametinib (GSK1120212).
  • Trametinib is a highly specific and potent MEKl/2 inhibitor with IC50 of 0.92 nM/1.8 Nm. Trametinib does not inhibit the kinase activities of c-Raf, B-Raf, ERKl/2.
  • Trametinib has the chemical name: N-(3- ⁇ 3-Cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7- tetrahydropyrido[4,3-d]pyrimidin- l(2H)-yl ⁇ phenyl)acetamide; and has the following structure:
  • the MEK inhibitor is Selumetinib (AZD6244, ARRY 142886).
  • Selumetinib is a potent, highly selective MEK1 inhibitor with IC50 of 14 nM, also inhibits ERKl/2 phosphorylation with IC50 of 10 nM.
  • Selumetinib has the chemical name: 6-(4-bromo- 2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide; and has the following structure:
  • the MEK inhibitor is XL518 (GDC-0973).
  • XL518 a potent, selective, orally bioavailable inhibitor of MEK1.
  • XL518 has the chemical name: [3,4-difluoro-2-[(2-iluoro-4- iodophenyl)amino]phenyl][3-hydroxy-3-[(2S)-2-piperidinyl]-l-azetidinyl]methanone; and has the following structure:
  • the MEK inhibitor is CI-1040 (PD 184352).
  • CI-1040 is an ATP noncompetitive MEKl/2 inhibitor with IC50 of about 17 nM, 100-fold more selective for MEKl/2 than MEK5.
  • CI-1040 has the chemical name: 2-(2-chloro-4-iodophenylamino)-N- (cyclopropylmethoxy)-3,4-difluorobenzamide; and has the following structure:
  • the MEK inhibitor is PD035901.
  • PD0325901 is selective and non ATP- competitive MEK inhibitor with IC50 of about 0.33 nM, roughly 500-fold more potent than CI-1040 on phosphorylation of ERKl and ERK2.
  • PD035901 has the chemical name: (R)-N-(2,3-dihydroxypropoxy)- 3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide; and has the following structure:
  • the MEK inhibitor is U0126-EtOH.
  • U0126-EtOH is a highly selective inhibitor of MEK1/2 with IC50 of about 0.07 ⁇ /0.06 ⁇ , 100-fold higher affinity for ⁇ 3- S218E/S222D MEK than PD098059.
  • PD098059 has the chemical name: (2Z,3Z)-2,3-bis(amino(2- aminophenylthio)methylene)succinonitrile,ethanol; and has the following structure:
  • the MEK inhibitor is PD198306.
  • PD198306 is a cell-permeable and highly selective MEK inhibitor with IC50 of 8 nM.
  • PD198306 has the chemical name: Benzamide, N- (cyclopropylmethoxy)-3,4,5-trifluoro-2-[(4-iodo-2-methylphenyl)amino]-; and has the following structure:
  • the MEK inhibitor is PD98059.
  • PD98059 is a non-ATP competitive MEK inhibitor with IC50 of 2 ⁇ , specifically inhibits MEK-1 -mediated activation of MAPK.
  • PD98059 does not directly inhibit ERKl or ERK2.
  • PD98059 has the chemical name: 2-(2-amino-3-methoxyphenyl)-4H- chromen-4-one; and has the following structure:
  • the MEK inhibitor is BIX 02189.
  • BIX 02189 is a selective inhibitor of MEK5 with IC50 of 1.5 nM, also inhibits ERK5 catalytic activity with IC50 of 810 nM.
  • BIX 02189 does not inhibit closely related kinases MEKl, MEK2, ERK2, and JNK2.
  • BIX 02189 has the chemical name: (Z)-3-((3-((dimethylamino)methyl)phenylamino) (phenyl)methylene)-N,N-dimethyl-2-oxoindoline-6- carboxamide; and has the following structure:
  • the MEK inhibitor is TAK-733.
  • TAK-733 is a potent and selective MEK allosteric site inhibitor for MEKl with IC50 of about 3.2 nM.
  • TAK-733 is inactive to Abll, AKT3, c- RAF, CamKl, CDK2, c-Met.
  • TAK-733 has the chemical name: (R)-3-(2,3-dihydroxypropyl)-6-fluoro-5- (2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione; and has the following structure:
  • the MEK inhibitor is Honokiol.
  • Honokiol is the active principle of magnolia extract that inhibits Akt-phosphorylation and promotes ERKl/2phosphorylation.
  • Honokiol has the chemical name: 2-(4-hydroxy-3-prop-2-enyl-phenyl)- 4-prop-2-enyl-phenol; and has the following structure:
  • the MEK inhibitor is AZD8330 (ARRY-424704).
  • AZD8330 is a novel, selective, non-ATP competitive MEK 1/2 inhibitor with IC50 of about 7 nM.
  • AZD8330 has the chemical name: 2-(2-fluoro-4-iodophenylamino)-N-(2-hydroxyethoxy)-l,5-dimethyl-6-oxo-l,6-dihydropyridine-3- carboxamide; and has the following structure:
  • the MEK inhibitor is PD318088.
  • PD318088 is a non-ATP competitive allosteric MEKl/2 inhibitor.
  • PD318088 has the chemical name: 5-bromo-N-(2,3- dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide; and has the following structure:
  • the MEK inhibitor is BIX 02188.
  • ⁇ 02188 is a selective inhibitor of MEK5 with IC50 of about 4.3 nM, also inhibits ERK5 catalytic activity with IC50 of 810 nM.
  • BIX 02188 does not significantly inhibit closely related kinases MEKl, MEK2, ERK2, and JNK2.
  • ⁇ 02188 has the chemical name: (Z)-3-((3-((dimethylamino)methyl)phenylamino) (phenyl)methylene)-2-oxoindoline-6-carboxamide; and has the following structure:
  • the MEK inhibitor is AS703026 (Pimasertib).
  • AS-703026 is a highly selective, potent, ATP non-competitive allosteric inhibitor of MEKl/2 with IC50 of about 0.005- 2 ⁇ in MM cell lines.
  • AS703026 has the chemical name: (S)-N-(2,3-dihydroxypropyl)-3-(2- fluoro-4-iodophenylamino)isonicotinamide; and has the following structure:
  • the MEK inhibitor is SL327.
  • SL327 is a selective inhibitor for MEK1/2 with IC50 of about 0.18 ⁇ / 0.22 ⁇ .
  • SL327 has no activity towards Erkl, MKK3, MKK4, c-JUN, PKC, PKA, or CamKII.
  • SL327 is capable of transport through the blood-brain barrier.
  • SL327 has the chemical name: (Z)-3-amino-3-(4-aminophenylthio)-2-(2- (trifluoromethyl)phenyl)acrylonitrile; and has the following structure:
  • the MEK inhibitor is RG7167.
  • RG7167 is a potent, orally
  • the MEK inhibitor is E6201.
  • E6201 is a synthetic, fungal metabolite analogue inhibitor of mitogen- activated protein kinase kinase 1 (MEK-1) and mitogen- activated protein kinase kinase kinase 1 (MEKK-1) with potential antipsoriatic and antineoplastic activities.
  • MEK-l/MEKK-1 inhibitor E6201 specifically binds to and inhibits the activities of MEK-1 and MEKK-1, which may result in the inhibition of tumor cell proliferation.
  • MEK-1 and MEKK-1 are key components in the RAS/RAF/MEK/MAPK signaling pathway, which regulates cell proliferation and is frequently activated in human cancers.
  • E6201 has the chemical name: ([(3S,4R,5Z,8S,9S,l lE)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro- lH-2-benzoxacyclotetradecine-l,7(8H)-dione]; and has the following structure:
  • the MEK inhibitor is MSC2015103.
  • MSC2015103 is an orally bio- available, selective, and highly potent small molecule inhibitor of MEK1/2.
  • the MEK inhibitor is WX-554.
  • WX-554 is an orally available small molecule mitogen- activated protein kinase kinase (MAP2K, MAPK/ERK kinase, or MEK) inhibitor, with potential antineoplastic activity.
  • MAP2K mitogen- activated protein kinase kinase
  • MEK MEK
  • the agent is an antibody molecule, e.g., an anti-MEK antibody molecule (e.g., a monoclonal or a bispecific antibody), or a conjugate thereof (e.g., an antibody to MEK conjugated to a cytotoxic agent (e.g., mertansine DM1)), and/or a MEK cellular immunotherapy.
  • the agent is a kinase inhibitor.
  • the MEK inhibitor is chosen from: a multi- specific kinase inhibitor, a tyrosine/threonine kinase inhibitor, and/or an inhibitor inhibitor that is selective for MEK.
  • the MEK inhibitor is a small molecule.
  • the agent is chosen from a nucleic acid molecule (e.g., an
  • antisense molecule a ribozyme, a double stranded RNA, or a triple helix molecule
  • a MEK nucleic acid hybridizes to and/or inhibits a MEK nucleic acid, e.g., a MEK nucleic acid encoding the
  • alteration or a transcription regulatory region that blocks or reduces mRNA expression of the alteration.
  • the agents can be administered in combination with a second therapeutic agent or a different therapeutic modality, e.g., anti-cancer agents, and/or in combination with surgical and/or radiation procedures.
  • a first therapeutic agent described herein is administered in combination with a second therapeutic agent, described herein.
  • the first therapeutic agent is a BRAF inhibitor described herein
  • the second therapeutic agent is a MEK inhibitor described herein.
  • the first therapeutic agent is a BRAF inhibitor and is chosen from: Vemurafenib (PLX4032, RG7204, R05185426), Sorafenib Tosylate (Bay 43- 9006, Nexavar), PLX4720, GDC-0879, RAF265 (CHIR-265), MLN2480 (BIIB-024), PF- 04880594, GW5074, CEP-32496, Dabrafenib (GSK2118436), AZ628, SB590885, Raf265 derivative, Regorafenib (BAY 73-4506, Fluoro-Sorafenib), and/or ZM 336372; and the second therapeutic agent is a MEK inhibitor and is chosen from ARRY- 162 (MEK162), Trametinib (GSK1120212), Selumetinib (AZD6244, ARRY142886), XL518 (GDC-0973), CI-1040
  • either the BRAF inhibitor or the MEK inhibitor, or both can be administered in combination with a therapeutic agent or therapeutic modality currently used in the treatment of a hematological malignancy, e.g., CLL.
  • a therapeutic agent or therapeutic modality currently used in the treatment of the hematological malignancy is a non-MAPK pathway inhibitor.
  • the non-MAPK pathway inhibitor therapeutic agent or therapeutic modality is a chemotherapy, an immunotherapy, or a surgical procedure.
  • the non-MAPK pathway inhibitor therapeutic agent or therapeutic modality comprises one or more of: a chemotherapeutic agent (e.g., fludarabine, cyclophosphamide, doxorubicin, vincristine, chlorambucil, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, 5-azacytidine, pemetrexed disodium), tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g., erlotinib), BTK inhibitor (e.g., PCI-32765), CD20 targeting agent (e.g., rituximab, ofatumumab, RO5072759, LFB-R603), CD52 targeting agent (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, Bcl-2 inhibitor
  • compositions e.g., pharmaceutical compositions, comprising one or more of the agents, e.g., the therapeutic agents described herein, for use, e.g., in treating a hematological
  • malignancy e.g., CLL
  • CLL malignancy
  • kits comprising the agents, e.g., the therapeutic agents (and compositions thereof), with instructions for use in treating a hematological malignancy, e.g., CLL, and/or determining the presence of an alteration described herein are also provided.
  • the invention features a kit comprising one or more detection reagents (e.g., probes, primers, antibodies), capable, e.g., of specific detection of a nucleic acid or protein comprising an alteration described herein.
  • detection reagents e.g., probes, primers, antibodies
  • the invention also provides methods of: identifying, assessing or detecting an alteration described herein, e.g., a BRAF mutation, a KRAS mutation, or a mutation identified in FIG. 1, FIG. 2 and/or Table 1, in a hematological malignancy, e.g., CLL, or in a sample derived from a patient diagnosed with or suspected of having a hematological malignancy, e.g., CLL.
  • the mutations include one or more of BRAF (e.g., a mutation as described herein), SPEN, FAT3 (e.g., a loss of function mutation in SPEN or FAT3), or a mutation described in FIG. 1, FIG. 2 and/or Table 1.
  • the mutation is associated with CLL disease progression, e.g., it is a mutation gained or lost at CLL progression as identified in FIG. 1, FIG. 2 and/or Table 1.
  • the mutation is associated with CLL progression and is chosen from one or more of: NOTCH1, KRAS, TP53, NRAS or BCOR.
  • the mutation is associated with clonal disease evolution include one or more of: DNMT3A, EED, IDH2, IRF4, VHL or RB I .
  • the mutation is retained at disease progression, if found in early disease states, for example, as chosen from a mutation in SF3B 1 or XPOl .
  • the invention features a method for evaluating progression of a hematological disease, e.g., CLL, in a subject (e.g., a subject described herein).
  • the method includes detecting (and/or acquiring information on the presence of) a mutation described herein, thereby evaluating progression of the disease.
  • the mutation is associated with CLL disease progression, e.g., it is a mutation gained or lost at CLL progression as identified in FIG. 1.
  • the mutation is associated with CLL progression and is chosen from one or more of: NOTCH 1, KRAS, TP53, NRAS or BCOR.
  • the mutation is associated with clonal disease evolution include one or more of: DNMT3A, EED, IDH2, IRF4, VHL or RB I .
  • the mutation is retained at disease progression, if found in early disease states, for example, as chosen from a mutation in SF3B 1 or XPOl .
  • nucleic acid molecules comprising the alterations, nucleic acid constructs, host cells containing the nucleic acid molecules; purified polypeptides comprising the alteration described herein and binding agents; detection reagents (e.g., probes, primers, antibodies, kits, capable, e.g., of specific detection of a nucleic acid or protein comprising an alteration described herein); screening assays for identifying molecules that interact with, e.g., inhibit the alterations, e.g., novel kinase inhibitors or binders of BRAF and/or KRAS.
  • the detection of the alteration comprises sequencing, e.g., nucleic acid sequencing or amino acid sequencing.
  • the invention features a method of determining the presence of an alteration described herein in a cancer, e.g., a hematological malignancy, e.g., CLL.
  • the method includes: acquiring knowledge (e.g., directly acquiring knowledge) that the alteration described herein is present in a subject, e.g., a sample (e.g., a cancer or tumor sample) from the subject.
  • the acquiring step comprises a determination of the presence of the alteration in a nucleic acid molecule from the subject, e.g., by performing a sequencing step.
  • the acquiring step comprises a determination of the presence of the alteration in a nucleic acid molecule from the subject, e.g., by performing a sequencing step.
  • the subject, or the sample comprises one or more cells or tissue from a hematological malignancy, e.g., CLL.
  • a hematological malignancy e.g., CLL.
  • the method further comprises administering an agent, e.g., a
  • the mutation is detected in a nucleic acid molecule or a polypeptide.
  • the method includes detecting whether a mutated nucleic acid molecule or polypeptide is present in a cell (e.g., a circulating cell), a tissue (e.g., a hematologic tissue, e.g., a CLL tissue), or a sample, e.g., a sample (e.g., a CLL sample), from a subject.
  • the sample is a nucleic acid sample.
  • the nucleic acid sample comprises DNA, e.g., genomic DNA or cDNA, or RNA, e.g., mRNA.
  • the sample is a protein sample.
  • the sample or tissue is, or has been, classified as non-malignant or malignant using other diagnostic techniques, e.g., immunohistochemistry.
  • the sample is acquired from a subject (e.g., a subject having or at risk of having a cancer, e.g., a patient), or alternatively, the method further includes acquiring a sample from the subject.
  • the sample is a blood sample, or a bone marrow biopsy or aspirate.
  • the sample is a blood sample, a whole blood sample, or a serum sample.
  • the sample can be chosen from one or more of: tissue, e.g., cancerous tissue (e.g., a tissue biopsy), whole blood, serum, plasma, circulating tumor cells, circulating nucleic acids, bone marrow, or any sample comprising hematologic or lymphocytic, e.g., CLL, cells.
  • tissue e.g., cancerous tissue (e.g., a tissue biopsy), whole blood, serum, plasma, circulating tumor cells, circulating nucleic acids, bone marrow, or any sample comprising hematologic or lymphocytic, e.g., CLL, cells.
  • the sample is a tissue (e.g., a bone marrow biopsy or aspirate), a circulating tumor cell or nucleic acid.
  • the cancer is a hematological malignancy, e.g., CLL.
  • the subject is at risk of having, or has CLL.
  • the mutation is detected in a nucleic acid molecule by a method chosen from one or more of: nucleic acid hybridization assay, amplification-based assays (e.g., polymerase chain reaction (PCR)), PCR-RFLP assay, real-time PCR, sequencing, screening analysis, SSP, HPLC or mass-spectrometric genotyping.
  • amplification-based assays e.g., polymerase chain reaction (PCR)
  • the method includes: contacting a nucleic acid sample, e.g., a genomic DNA sample (e.g., a chromosomal sample or a fractionated, enriched or otherwise pre- treated sample) or a gene product (mRNA, cDNA), obtained from the subject, with a nucleic acid fragment (e.g., a probe or primer as described herein (e.g., an exon-specific probe or primer) under conditions suitable for hybridization, and determining the presence or absence of the mutated nucleic acid molecule.
  • the method can, optionally, include enriching a sample for the gene or gene product.
  • the invention features a method for determining the presence of a mutated nucleic acid molecule, e.g., a mutated MAPK pathway gene or gene product.
  • the method includes: acquiring a sequence for a position in a nucleic acid molecule, e.g., by sequencing at least one nucleotide of the nucleic acid molecule (e.g., sequencing at least one nucleotide in the nucleic acid molecule that comprises the mutation), thereby determining that the mutation is present in the nucleic acid molecule.
  • the sequence acquired is compared to a reference sequence, or a wild type reference sequence. In one
  • the nucleic acid molecule is from a cell (e.g., a hematopoietic, lymphocytic, CLL cell, e.g., from a blood or bone marrow sample), a tissue, or any sample from a subject (e.g., blood or plasma sample).
  • the nucleic acid molecule from a tumor sample e.g., a tumor or cancer sample
  • the sequence is determined by a next generation sequencing method.
  • the method further can further include acquiring, e.g., directly or indirectly acquiring, a sample, e.g., a hematological malignancy, e.g., CLL.
  • the invention features a method of analyzing a malignancy or a circulating malignant cell.
  • the method includes acquiring a nucleic acid sample from the malignancy or the circulating cell; and sequencing, e.g., by a next generation sequencing method, a nucleic acid molecule, e.g., a nucleic acid molecule that includes an alteration as described herein, e.g., an alteration in a MAPK pathway gene or gene product.
  • a polypeptide comprising an alteration described herein is detected.
  • the method includes: contacting a protein sample with a reagent which specifically binds to a polypeptide comprising an alteration described herein; and detecting the formation of a complex of the polypeptide and the reagent.
  • the reagent is labeled with a detectable group to facilitate detection of the bound and unbound reagent.
  • the reagent is an antibody molecule, e.g., is selected from the group consisting of an antibody, and antibody derivative, and an antibody fragment.
  • the level (e.g., expression level) or activity the polypeptide comprising an alteration described herein is evaluated.
  • the level (e.g., expression level) or activity of the polypeptide e.g., mRNA or polypeptide
  • a pre-determined value e.g., a reference value (e.g., a control sample).
  • the alteration is detected prior to initiating, during, or after, a treatment in a subject having an alteration described herein.
  • the alteration is detected at the time of diagnosis with a cancer.
  • the alteration is detected at a pre-determined interval, e.g., a first point in time and at least at a subsequent point in time.
  • any of the methods described herein further include one or more of:
  • stratifying a patient or a patient population e.g., assigning a subject, e.g., a patient, to a group or class
  • MAPK pathway inhibitor e.g., BRAF and/or MEK inhibitor
  • a treatment option e.g., administering or not administering a preselected therapeutic agent, e.g., a MAPK pathway inhibitor (e.g., BRAF and/or MEK inhibitor) as described herein; or
  • prognosticating the time course of the disease in the subject e.g., evaluating the likelihood of increased or decreased patient survival, or the extent of disease progression.
  • the subject responsive to the determination of the presence of a mutation, is classified as a candidate to receive treatment with a therapy disclosed herein.
  • the subject e.g., a patient
  • the subject e.g., a patient, is assigned to a second class if the mutation is not present. For example, a patient who has a cancer that does not contain a mutation, may be determined as not being a candidate to receive a therapy disclosed herein.
  • the subject is identified as likely to respond to a treatment that comprises a therapy disclosed herein.
  • the method includes administering an agent, e.g., a therapeutic agent as described herein, e.g., a BRAF inhibitor and/or a MEK inhibitor, to the subject.
  • an agent e.g., a therapeutic agent as described herein, e.g., a BRAF inhibitor and/or a MEK inhibitor.
  • the invention features a method of evaluating a subject (e.g., a patient), e.g., for risk of having or developing a cancer, e.g., a hematological malignancy, e.g., CLL.
  • a subject e.g., a patient
  • a cancer e.g., a hematological malignancy, e.g., CLL.
  • the method includes: acquiring information or knowledge of the presence of a mutation as described herein in a subject (e.g., acquiring genotype information of the subject that identifies a mutation as being present in the subject); acquiring a sequence for a nucleic acid molecule identified herein (e.g., a nucleic acid molecule that includes a mutation sequence); or detecting the presence of a nucleic acid or polypeptide in the subject), wherein the presence of the mutation is positively correlated with increased risk for, or having, a cancer associated with such a mutation.
  • the method can further include acquiring, e.g., directly or indirectly, a sample from a patient and evaluating the sample for the present of an alteration as described herein.
  • the method can further include the step(s) of identifying (e.g., evaluating, diagnosing, screening, and/or selecting) the subject as being positively correlated with increased risk for, or having, a cancer associated with the alteration.
  • identifying e.g., evaluating, diagnosing, screening, and/or selecting
  • a subject identified as having the alteration is identified or selected as likely or unlikely to respond to a treatment, e.g., a therapy disclosed herein.
  • the method can further include treating the subject with a therapy disclosed herein.
  • a method of evaluating a patient or a patient population includes: identifying, selecting, or obtaining information or knowledge that the patient or patient population has participated in a clinical trial; acquiring information or knowledge of the presence of an alteration (e.g., an alteration as described herein) in the patient or patient population (e.g., acquiring genotype information of the subject that identifies an alteration as being present in the subject); acquiring a sequence for a nucleic acid molecule identified herein (e.g., a nucleic acid molecule that includes an alteration sequence); or detecting the presence of a mutated nucleic acid or polypeptide in the subject), wherein the presence of the alteration identifies the patient or patient population as being likely to respond to an agent as described herein (e.g., a MAPK pathway inhibitor (e.g., BRAF and/or MEK inhibitor)).
  • an agent e.g., a MAPK pathway inhibitor (e.g., BRAF and/or MEK inhibitor)
  • the method further includes treating the subject with an agent as described herein (e.g., a MAPK pathway inhibitor, e.g., a BRAF and/or a MEK inhibitor).
  • an agent as described herein e.g., a MAPK pathway inhibitor, e.g., a BRAF and/or a MEK inhibitor.
  • Reporting Methods described herein can include providing a report, such as, in electronic, web-based, or paper form, to the patient or to another person or entity, e.g., a caregiver, e.g., a physician, e.g., an oncologist, a hospital, clinic, third-party payor, insurance company or government office.
  • the report can include output from the method, e.g., the identification of nucleotide values, the indication of presence or absence of an alteration as described herein, or wildtype sequence.
  • a report is generated, such as in paper or electronic form, which identifies the presence or absence of an alteration described herein, and optionally includes an identifier for the patient from which the sequence was obtained.
  • the report can also include information on the role of a mutation as described herein, or wildtype sequence, in disease. Such information can include information on prognosis, resistance, or potential or suggested therapeutic options, e.g., an agent as described herein (e.g., a MAPK pathway inhibitor, e.g., a BRAF and/or a MEK inhibitor).
  • an agent as described herein e.g., a MAPK pathway inhibitor, e.g., a BRAF and/or a MEK inhibitor.
  • the report can include information on the likely effectiveness of a therapeutic option, the acceptability of a therapeutic option, or the advisability of applying the therapeutic option to a patient, e.g., a patient having a sequence, alteration or mutation identified in the test, and in embodiments, identified in the report.
  • the report can include information, or a recommendation on, the administration of a drug, e.g., the administration at a preselected dosage or in a preselected treatment regimen, e.g., in combination with other drugs, to the patient.
  • a drug e.g., the administration at a preselected dosage or in a preselected treatment regimen, e.g., in combination with other drugs.
  • not all mutations identified in the method are identified in the report.
  • the report can be limited to mutations in genes having a preselected level of correlation with the occurrence, prognosis, stage, or susceptibility of the cancer to treatment, e.g., with a preselected therapeutic option.
  • the report can be delivered, e.g., to an entity described herein, within 7, 14, or 21 days from receipt of the sample by the entity practicing the method.
  • the invention features a method for generating a report, e.g., a personalized cancer treatment report, by obtaining a sample, e.g., a tumor sample, from a subject, detecting a mutation as described herein in the sample, and selecting a treatment based on the mutation identified.
  • a report is generated that annotates the selected treatment, or that lists, e.g., in order of preference, two or more treatment options based on the mutation identified.
  • the subject e.g., a patient, is further administered the selected method of treatment.
  • all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
  • FIG. 1 depicts extensive deep targeted sequencing of logitudinal CLL samples which shows novel recurrent driver events and patterns of clonal evolution.
  • A Depicts sequencing of a panel of 435 genes at deep coverage in CLL which shows frequent mutations including novel recurrent mutations in BRAF, SPEN, FAT3, and TRAF3. Notably BRAF mutations in CLL are activating mutations predominately seen at codons other than V600.
  • B Depicts a tile plot comparison of paired sample sequencing which shows shifts in mutations at disease progression. Notably, leukemic cells sampled at disease progression during a time when chemotherpay had already commenced had much greater frequency of genetic alterations than those with untreated disease at the time of sample acquisition.
  • FIG. 2 depicts a tile plot showing the genomic alterations in each CLL case sample analyzed.
  • the analyzed genes are listed in the vertical column. The key distinguishes the genomic alterations of: substiutions/indels; truncations; and gene fusions.
  • Described at least in part herein is the identification of an alteration in a MAPK pathway gene or gene product, e.g., a BRAF and/or a MEK, in a series of human patients with chronic lymphocytic leukemia (CLL). Additionally described herein is a genomic analysis of a series of patients with CLL to characterize the genomic landscape of CLL. Also described herein is a genomic analysis of a series of patients with untreated CLL and previously treated CLL to characterize the genomic landscape of both untreated and treated CLL.
  • CLL chronic lymphocytic leukemia
  • an agent e.g., a therapeutic agent
  • targets and/or inhibits a MAPK pathway gene or gene product e.g., a BRAF and/or a MEK
  • methods and reagents for identifying, assessing and/or detecting an alteration as described herein, e.g., a BRAF and/or KRAS mutation, in CLL are disclosed herein.
  • CLL Chronic lymphocytic leukemia
  • B cell-CLL is a common type of adulthood leukemia, and is characterized by the clonal proliferation and accumulation of mature B lymphocytes (Eichhorst B et al. Ann Oncol (2011) 22 (suppl 6): vi50-vi54; Smolewski P et al. Oncology Volume 2013 (2013)).
  • CLL progression is characterized by stage.
  • Commonly used staging systemns include, for example, the Rai 4-stage system and the Binet classification system (National Cancer Institute. "Chronic Lymphocytic Leukemia (PDQ) Treatment: Stage
  • MAPK mitogen activated protein kinases
  • MAP kinases can be mediators of cancer-related disease mechanisms (Chen et al., Chem Rev (2001) 101:2449- 76; Pearson et al., Endocr Rev (2001) 22: 153-83; English et al., Trends Pharmacol Sci (2002) 23:40-45; Kohno et al., Prog Cell Cycle Res (2003) 5:219-24; and Sebolt-Leopold, Oncogene (2000) 19:6594-99).
  • a MAPK pathway comprises RAS, RAF, MEK, and ERK (MAPK) ⁇ e.g., Ras, Raf-1, A-Raf, B-Raf (BRAF)), MEK1 and/or MEK2.
  • MAPK pathway gene or gene product can also refer to either or both of the wild type or native gene, as well as or alternatively, certain mutations thereof ⁇ e.g., activating mutations thereof), and variants derived from any source (e.g., humans and other mammals), as described herein.
  • MAPK pathway gene product refers to mRNA, polypeptides and/or fragments thereof, of the encoding MAPK pathway gene.
  • V-Raf Murine Sarcoma Viral Oncogene Homolog Bl or “BRAF” (also known as BRAF1, proto-oncogene B-raf, RAFB1, NS7, P94, protein kinase 94, and serine/threonine protein kinase BRAF) refers to a BRAF gene or gene product (e.g., a nucleic acid or protein).
  • the BRAF protein refers to a protein, typically human BRAF that is encoded by the BRAF gene.
  • BRAF is a member of the raf/mil family of serine/threonine protein kinases, and plays a role in regulating the MAP kinase/ERKs signaling pathway, which affects, inter alia, cell division, differentiation, and secretion.
  • the BRAF amino and nucleotide sequences are known in the art.
  • An exemplary amino acid and nucleotide sequence for human BRAF is provided herein as SEQ ID NO: l and SEQ ID NO:2, respectively.
  • MEK refers to a gene or gene product having kinase activity on a MAP kinase.
  • This family of kinases is also known as MAP/ERK kinase (MEK) or Mitogen-activated protein kinase (MAPK) kinase, and includes at least MAPK kinase/MEK- 1 and -2.
  • MEK kinase 1 refers to a MEKl gene or gene product (e.g., a nucleic acid or protein).
  • the MEKl protein refers to a protein, typically human MEKl that is encoded by the MEKl gene.
  • MEKl is a member of the dual specificity protein kinase family, which acts as a mitogen-activated protein (MAP) kinase kinase.
  • MAP kinases also known as extracellular signal-regulated kinases (ERKs)
  • ERKs extracellular signal-regulated kinases
  • MEKl functions in the MAPK/ERK cascade, and catalyzes the concomitant phosphorylation of a threonine and a tyrosine residue in a Thr-Glu-Tyr sequence located in the extracellular signal-regulated kinases MAPK3/ERK1 and MAPK1/ERK2, leading to their activation and further transduction of the signal within the MAPK/ERK cascade.
  • MEKl amino and nucleotide sequences are known in the art.
  • An exemplary amino acid and nucleotide sequence for human MEKl is provided herein as SEQ ID NO:3 and SEQ ID NO:4, respectively.
  • Mitogen-activated protein kinase kinase 2 or “MEK2” (also known as PRKMK2, MKK2, MAPKK2, ERK activator kinase 2, MAP kinase kinase 2, and MAK/ERK kinase 2) refers to a MEK2 gene or gene product (e.g., a nucleic acid or protein).
  • the MEK2 protein refers to a protein, typically human MEK2 that is encoded by the MEK2 gene.
  • MEK2 is a member of the dual specificity protein kinase family, which acts as a mitogen-activated protein (MAP) kinase kinase.
  • MAP mitogen-activated protein
  • MAP kinases also known as extracellular signal-regulated kinases (ERKs) act as an integration point for multiple biochemical signals.
  • MEK2 functions in the MAPK/ERK cascade, and catalyzes the concomitant phosphorylation of a threonine and a tyrosine residue in a Thr-Glu-Tyr sequence located in the extracellular signal-regulated kinases MAPK3/ERK1 and MAPK1/ERK2, leading to their activation and further transduction of the signal within the MAPK/ERK cascade.
  • this pathway mediates diverse biological functions, including, inter alia, cell growth, adhesion, survival and
  • MEK2 amino and nucleotide sequences are known in the art.
  • An exemplary amino acid and nucleotide sequence for human MEK2 is provided herein as SEQ ID NO:5 and SEQ ID NO:6, respectively.
  • the articles “a” and “an” refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.
  • Directly acquiring means performing a process (e.g., performing a synthetic or analytical method) to obtain the physical entity or value.
  • Indirectly acquiring refers to receiving the physical entity or value from another party or source (e.g., a third party laboratory that directly acquired the physical entity or value).
  • Directly acquiring a physical entity includes performing a process that includes a physical change in a physical substance, e.g., a starting
  • Exemplary changes include making a physical entity from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, combining two or more separate entities into a mixture, performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond.
  • Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process which includes a physical change in a substance, e.g., a sample, analyte, or reagent (sometimes referred to herein as "physical analysis"), performing an analytical method, e.g., a method which includes one or more of the following: separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from another substance; combining an analyte, or fragment or other derivative thereof, with another substance, e.g., a buffer, solvent, or reactant; or changing the structure of an analyte, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the analyte; or by changing the structure of a reagent, or a fragment or other derivative
  • Acquiring a sequence refers to obtaining possession of a nucleotide sequence or amino acid sequence, by “directly acquiring” or “indirectly acquiring” the sequence.
  • Directly acquiring a sequence means performing a process (e.g., performing a synthetic or analytical method) to obtain the sequence, such as performing a sequencing method (e.g., a Next Generation Sequencing (NGS) method).
  • NGS Next Generation Sequencing
  • Indirectly acquiring a sequence refers to receiving information or knowledge of, or receiving, the sequence from another party or source (e.g., a third party laboratory that directly acquired the sequence).
  • the sequence acquired need not be a full sequence, e.g., sequencing of at least one nucleotide, or obtaining information or knowledge that identifies a mutation disclosed herein as being present in a subject constitutes acquiring a sequence.
  • Directly acquiring a sequence includes performing a process that includes a physical change in a physical substance, e.g., a starting material, such as a tissue sample, e.g., a biopsy, or an isolated nucleic acid (e.g., DNA or RNA) sample.
  • a starting material such as a tissue sample, e.g., a biopsy, or an isolated nucleic acid (e.g., DNA or RNA) sample.
  • Exemplary changes include making a physical entity from two or more starting materials, shearing or fragmenting a substance, such as a genomic DNA fragment; separating or purifying a substance (e.g., isolating a nucleic acid sample from a tissue); combining two or more separate entities into a mixture, performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond.
  • Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance as described above.
  • a sample refers to obtaining possession of a sample, e.g., a tissue sample or nucleic acid sample, by “directly acquiring” or “indirectly acquiring” the sample.
  • Directly acquiring a sample means performing a process (e.g., performing a physical method such as a surgery or extraction) to obtain the sample.
  • Indirectly acquiring a sample refers to receiving the sample from another party or source (e.g., a third party laboratory that directly acquired the sample).
  • Directly acquiring a sample includes performing a process that includes a physical change in a physical substance, e.g., a starting material, such as a tissue, e.g., a tissue in a human patient or a tissue that has was previously isolated from a patient.
  • a starting material such as a tissue
  • Exemplary changes include making a physical entity from a starting material, dissecting or scraping a tissue; separating or purifying a substance (e.g., a sample tissue or a nucleic acid sample); combining two or more separate entities into a mixture; performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond.
  • Directly acquiring a sample includes performing a process that includes a physical change in a sample or another substance, e.g., as described above.
  • alteration refers to the presence of a mutation or mutations within the gene or gene product, e.g., a mutation, which affects amount or activity of the gene or gene product, as compared to the normal or wild- type gene.
  • the alteration can be in amount, structure, and/or activity in a cancer tissue or cancer cell, as compared to its amount, structure, and/or activity, in a normal or healthy tissue or cell (e.g., a control), and is associated with a disease state, such as cancer.
  • a gene or gene product which is associated with cancer, or predictive of responsiveness to anti- cancer therapeutics can have an altered nucleotide sequence (e.g., a mutation), amino acid sequence, chromosomal translocation, intra-chromosomal inversion, copy number, expression level, protein level, protein activity, or methylation status, in a cancer tissue or cancer cell, as compared to a normal, healthy tissue or cell.
  • exemplary mutations include, but are not limited to, point mutations (e.g., silent, missense, or nonsense), deletions, insertions, inversions, linking mutations, duplications, translocations, inter- and intra-chromosomal rearrangements. Mutations can be present in the coding or non-coding region of the gene.
  • the alterations are associated (or not associated) with a phenotype, e.g., a cancerous phenotype (e.g., one or more of cancer risk, cancer progression, cancer treatment or resistance to cancer
  • Binding entity means any molecule to which molecular tags can be directly or indirectly attached that is capable of specifically binding to an analyte.
  • the binding entity can be an affinity tag on a nucleic acid sequence.
  • the binding entity allows for separation of the nucleic acid from a mixture, such as an avidin molecule, or an antibody that binds to the hapten or an antigen-binding fragment thereof.
  • Exemplary binding entities include, but are not limited to, a biotin molecule, a hapten, an antibody, an antibody binding fragment, a peptide, and a protein.
  • “Complementary” refers to sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine.
  • a first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
  • nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
  • cancer or “tumor” is used interchangeably herein. These terms refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features.
  • neoplasm or "neoplastic” cell refers to an abnormal proliferative stage, e.g., a hyperproliferative stage, in a cell or tissue that can include a benign, pre-malignant, malignant (cancer) or metastatic stage.
  • the cancer or neoplasm is a hematologic (or hematological) malignancy.
  • a hematological malignancy is a cancer that affects the blood, bone marrow, and lymph nodes. Hematological malignancies can include myelomas, leukemias, and lymphomas.
  • Cancer is "inhibited” if at least one symptom of the cancer is alleviated, terminated, slowed, or prevented. As used herein, cancer is also “inhibited” if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented.
  • “Chemo therapeutic agent” means a chemical substance, such as a cytotoxic or cytostatic agent that is used to treat a condition, particularly cancer.
  • cancer therapy and “cancer treatment” are synonymous terms.
  • chemotherapy and “chemotherapeutic” and “chemotherapeutic agent” are synonymous terms.
  • sequence similarity refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences, with identity being a more strict comparison.
  • percent identity or homology and “ident or homology” refer to the percentage of sequence similarity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences.
  • sequence similarity refers to the percent similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0-100% similar, or any integer value there between.
  • Identity or similarity can be determined by comparing a position in each sequence that can be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position.
  • a degree of similarity or identity between polynucleotide sequences is a function of the number of identical or matching nucleotides at positions shared by the polynucleotide sequences.
  • a degree of identity of polypeptide sequences is a function of the number of identical amino acids at positions shared by the polypeptide sequences.
  • a degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at positions shared by the polypeptide sequences.
  • substantially identical refers to an identity or homology of at least 75%, at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.
  • “Likely to” or “increased likelihood,” as used herein, refers to an increased probability that an item, object, thing or person will occur.
  • a subject that is likely to respond to treatment with a kinase inhibitor, alone or in combination has an increased
  • Unlikely to refers to a decreased probability that an event, item, object, thing or person will occur with respect to a reference.
  • a subject that is unlikely to respond to treatment with a kinase inhibitor, alone or in combination has a decreased probability of responding to treatment with a kinase inhibitor, alone or in combination, relative to a reference subject or group of subjects.
  • “Sequencing" a nucleic acid molecule requires determining the identity of at least 1 nucleotide in the molecule. In embodiments, the identity of less than all of the nucleotides in a molecule is determined. In other embodiments, the identity of a majority or all of the nucleotides in the molecule is determined.
  • next-generation sequencing or NGS or NG sequencing refers to any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules (e.g., in single molecule sequencing) or clonally expanded proxies for individual nucleic acid molecules in a highly parallel fashion (e.g., greater than 10 5 molecules are sequenced simultaneously).
  • the relative abundance of the nucleic acid species in the library can be estimated by counting the relative number of occurrences of their cognate sequences in the data generated by the sequencing experiment.
  • Next generation sequencing methods are known in the art, and are described, e.g., in Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46, incorporated herein by reference.
  • tissue sample tissue sample
  • patient sample patient cell or tissue sample
  • samples each refers to a collection of similar cells obtained from a tissue of a subject or patient.
  • the source of the tissue sample can be solid tissue as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; or cells from any time in gestation or
  • the sample is a blood sample, or a bone marrow biopsy or aspirate.
  • the sample comprises a hematologic cell (e.g., a blood cell, a bone marrow cell, and/or lymphatic cell).
  • the tissue sample can contain compounds that are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like.
  • the sample is preserved as a frozen sample or as formaldehyde- or paraformaldehyde-fixed paraffin-embedded (FFPE) tissue
  • the sample can be embedded in a matrix, e.g., an FFPE block or a frozen sample.
  • tumor nucleic acid sample refers to nucleic acid molecules from a tumor or cancer sample. Typically, it is DNA, e.g., genomic DNA, or cDNA derived from RNA, from a tumor or cancer sample. In certain embodiments, the tumor nucleic acid sample is purified or isolated (e.g., it is removed from its natural state).
  • the reference or control nucleic acid sample is a wild type or a non-mutated sequence.
  • the reference nucleic acid sample is purified or isolated (e.g., it is removed from its natural state).
  • the reference nucleic acid sample is from a non-tumor sample, e.g., a blood control, a normal adjacent tumor (NAT), or any other non-cancerous sample from the same or a different subject.
  • NAT normal adjacent tumor
  • adjacent to the interrogation position means that a site sufficiently close such that a detection reagent complementary with the site can be used to distinguish between a mutation, e.g., an alteration described herein, and a reference sequence, e.g., a non- mutant or wild- type sequence, in a target nucleic acid. Directly adjacent, as used herein, is where 2 nucleotides have no intervening nucleotides between them.
  • Associated mutation refers to a mutation within a preselected distance, in terms of nucleotide or primary amino acid sequence, from a definitional mutation, e.g., a mutant as described herein.
  • the associated mutation is within n, wherein n is 2, 5, 10, 20, 30, 50, 100, or 200 nucleotides from the definitional mutation (n does not include the nucleotides defining the associated and definitional mutations). In embodiments, the associated mutation is a translocation mutation.
  • Interrogation position comprises at least one nucleotide (or, in the case of polypeptides, an amino acid residue) which corresponds to a nucleotide (or amino acid residue) that is mutated in a mutation of interest, e.g., a mutation being identified, or in a nucleic acid (or protein) being analyzed, e.g., sequenced, or recovered.
  • a "reference sequence,” as used herein, e.g., as a comparator for a mutant sequence, is a sequence which has a different nucleotide or amino acid at an interrogation position than does the mutant(s) being analyzed.
  • the reference sequence is wild-type for at least the interrogation position.
  • the invention provides, at least in part, methods for treating a cancer, e.g., a cancer
  • the methods include treatment of a cancer, e.g., a hematological malignancy, e.g., a CLL harboring an alteration described herein (e.g., a BRAF and/or KRAS alteration described herein).
  • the methods include administering to the subject a therapeutic agent, e.g., an agent that antagonizes the function of a MAPK pathway gene or gene product (e.g., a MAPK pathway inhibitor as described herein).
  • the cancer is a hematological malignancy.
  • the hematological malignancy is chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (ALoL), a Hodgkin' s lymphoma (HL), e.g., nodular sclerosing HL, mixed cellularity HL, lymphocyte-rich HL (lymphocute predominance HL), lymphocyte depleted HL, unspecified HL) or a Non-hodgkin's lymphoma (NHL), e.g., Indolent (low grade) NHL, aggressive (high grade) NHL, a B-cell lymphoma, e.g., diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, mediastinal large B cell-ltmphoma
  • the cancer is a CLL. In certain embodiments the cancer is a refractory CLL. In certain embodiments the cancer is a relapsed CLL.
  • the hematological malignancy e.g., a CLL
  • the hematological malignancy is any stage or risk group defined according to any suitable classification system known to those of skill in the art.
  • the CLL is any stage or risk group of CLL defined according to any suitable CLL classification system known to those of skill in the art.
  • the CLL is a stage 0, 1, II, III, or IV, wherein stage 0 CLL is characterized by too many lymphocytes in the blood, e.g., compared to a reference value (e.g., a control), but there are no other symptoms of leukemia; stage I CLL is characterized by too many lymphocytes in the blood and the lymph nodes are larger than normal; stage II CLL is characterized by too many lymphocytes in the blood, the liver or spleen is larger than normal, and the lymph nodes may be larger than normal; stage III CLL is characterized by too many lymphocytes in the blood, there are too few red blood cells, and the lymph nodes, liver, or spleen may be larger than normal, and stage IV CLL is characterized by too many lymphocytes in the blood, too few platelets, the lymph nodes, liver, or spleen may be larger than normal, and there may be too few red blood cells.
  • stage I CLL is characterized by too many lymphocytes in the blood and the lymph nodes
  • the CLL is a stage 0, 1, II, III, or IV on the Rai Scale (Rai K et al. Blood Volume 46, Issue 2, 1975, Pages 219-234).
  • the CLL is a stage 0, 1, II, III, or IV on the Rai Scale wherein stage 0 CLL is characterized by bone marrow and blood lymphocytosis only; stage I CLL is characterized by lymphocytosis with enlarged nodes; stage II CLL is characterized by lymphocytosis with enlarged spleen or liver or both; stage III CLL is characterized by lymphocytosis with anemia; and stage IV CLL is characterized by lymphocytosis with thrombocytopenia (Rai, supra).
  • the CLL is a stage A, B, or C on the Binet Scale; wherein stage A is characterized by Hb 100 g/L (10 g/dL) or more and platelets 100 x 10 9 /L or more and up to 2 of the following areas are involved: 1. Head and neck, including the Waldeyer ring (this counts as one area, even if more than one group of nodes is enlarged); 2. Axillae (involvement of both axillae counts as one area); 3. Groins, including superficial femorals (involvement of both groins counts as one area); 4. Palpable spleen; and 5. Palpable liver (clinically enlarged); stage B is characterized as Hb 100 g/L (10 g/dL) or more and platelets 100 x 10 9 /L or more and
  • stage C organomegaly greater than that defined for stage A (ie, 3 or more areas of nodal or organ enlargement); and stage C is characterized as patients who have Hb less than 100 g/L (10 g/dL) and/or a platelet count less than 100 x 10 9 /L, irrespective of organomegaly (Hallek M et al. Blood June 15, 2008 vol. I l l no. 12 5446-5456).
  • the CLL is any stage of CLL defined according to any suitable CLL classification system known to those of skill in the art.
  • the CLL is a low risk disease, intermediate risk disease, or high risk diseaseon the modified Rai Scale, wherein low risk is characterized by lymphocytes less than 15 x lOg/1; intermediate risk is characterized by lymphocytes less than 15 x lOg/1 and hepato- or splenomegaly; and high risk is characterized as Anemia (Hb ⁇ 11 g/dL) or thrombocytopenia (platelets ⁇ 100 xl0 9 /L) (Hallek M et al. Blood June 15, 2008 vol. I l l no. 12 5446-5456).
  • Anemia Hb ⁇ 11 g/dL
  • thrombocytopenia platelets ⁇ 100 xl0 9 /L
  • the CLL is any risk group of CLL defined according to any suitable CLL classification system known to those of skill in the art.
  • the cancer e.g., the hematological malignancy, e.g., CLL
  • Treatment refers to the administration of an agent, e.g., a therapeutic agent, alone or in combination with a second agent in an amount effective to impede growth of a cancer, to cause a cancer to shrink by weight or volume, to extend the expected survival time of the subject and or time to progression of the tumor or the like.
  • treatment can include, but is not limited to, inhibiting tumor growth, reducing tumor mass, reducing size or number of metastatic lesions, inhibiting the development of new metastatic lesions, prolonged survival, prolonged progression-free survival, prolonged time to progression, and/or enhanced quality of life.
  • a cancer is "treated” if at least one symptom of the cancer is alleviated, terminated, slowed or prevented.
  • a cancer is also “treated” if recurrence or metastasis of the cancer is reduced, slowed, delayed or prevented.
  • the terms “prevent,” “preventing” and “prevention” contemplate an action that occurs before a subject begins to suffer from the re- growth of the cancer and/or which inhibits or reduces the severity of the cancer.
  • a “therapeutically effective amount” of an agent is an amount sufficient to provide a therapeutic benefit in the treatment or management of the cancer, or to delay or minimize one or more symptoms associated with the cancer.
  • a therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the cancer.
  • the term "therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the cancer, or enhances the therapeutic efficacy of another therapeutic agent.
  • a prophylactically effective amount of an agent is an amount sufficient to prevent re-growth of the cancer, or one or more symptoms associated with the cancer, or prevent its recurrence.
  • a prophylactically effective amount of an agent means an amount of the agent, alone or in combination with other therapeutic agents, which provides a prophylactic benefit in the prevention of the cancer.
  • the term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
  • patient or “subject” includes a human (e.g., a male or female of any age group, e.g., a pediatric patient (e.g., infant, child, adolescent) or adult patient (e.g., young adult, middle-aged adult or senior adult).
  • a human e.g., a male or female of any age group, e.g., a pediatric patient (e.g., infant, child, adolescent) or adult patient (e.g., young adult, middle-aged adult or senior adult).
  • pediatric patient e.g., infant, child, adolescent
  • adult patient e.g., young adult, middle-aged adult or senior adult.
  • these treatments can be provided to a patient having had an unsatisfactory response to a different (e.g., non-MAPK pathway inhibitor as described herein).
  • the subject is undergoing or has undergone treatment with a different therapeutic agent or therapeutic modality (e.g., non-MAPK pathway inhibitor).
  • the different therapeutic agent or therapeutic modality is a chemotherapy, an immunotherapy, or a surgical procedure.
  • the different therapeutic agent or therapeutic modality comprises one or more of: a chemotherapeutic agent (e.g., fludarabine, cyclophosphamide, doxorubicin, vincristine,
  • tyrosine kinase inhibitor e.g., EGFR inhibitor (e.g., erlotinib), BTK inhibitor (e.g., PCI-32765), CD20 targeting agent (e.g., rituximab, ofatumumab, RO5072759, LFB-R603), CD52 targeting agent (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, Bcl-2 inhibitor (e.g., ABT-263), AT-101, immunotoxin (e.g., CAT- 8015, anti-Tac(Fv)-PE38 (LMB-2)), CD37 targeting agent (e.g., TRU-016)
  • EGFR inhibitor e.g., erlotinib
  • BTK inhibitor e.g., PCI-32765
  • CD20 targeting agent e.g., rituximab, ofatum
  • the different therapeutic agent or therapeutic modality e.g., non-MAPK pathway inhibitor as described herein
  • the subject has been identified as being likely or unlikely to respond to the different therapeutic agent or therapeutic modality.
  • An agent, e.g., a therapeutic agent, described herein can be administered, alone or in combination, e.g., in combination with other chemotherapeutic agents or procedures, in an amount sufficient to reduce or inhibit the malignant cell growth, and/or treat or prevent the malignancy (e.g., CLL), in the subject.
  • the agent e.g., therapeutic agent
  • a nucleic acid e.g., a siRNA, an antisense or a micro RNA
  • exemplary agents and classes of agents are described herein.
  • the agent is a BRAF inhibitor.
  • the BRAF inhibitor is chosen from: Vemurafenib (PLX4032, RG7204, R05185426), Sorafenib Tosylate (Bay 43-9006, Nexavar), PLX4720, GDC-0879, RAF265 (CHIR-265), MLN2480 (BIIB-024), PF-04880594, GW5074, CEP-32496, Dabrafenib (GSK2118436), AZ628, SB590885, Raf265 derivative, Regorafenib (BAY 73-4506, Fluoro-Sorafenib), and/or ZM 336372.
  • the BRAF inhibitor is Vemurafenib (PLX4032, RG7204, R05185426).
  • Vemurafenib is a potent inhibitor of BRAF V600E with an IC50 of about 31 nM.
  • Vemurafenib inhibits B-RAF V600E , C-RAF, as well as wildtype B-RAF, with an IC50 of about 31 nM, 48 nM and 100 nM, respectively.
  • Vemurafenib also inhibits several non-RAF kinases, including ACK1, KHS 1, and SRMS, with IC50 of 18 nM to 51 nM.
  • Vemurafenib has the chemical name: N-(3- ⁇ [5-(4-chlorophenyl)- lH-pyrrolo[2,3-b]pyridin-3-yl]carbonyl ⁇ -2,4- difluorophenyl)propane- l-sulfonamide; and has the following structure:
  • the BRAF inhibitor is Sorafenib Tosylate (Bay 43-9006,
  • Sorafenib is a multikinase inhibitor of Raf-1, BRAF and VEGFR-2 with an IC50 about 6 nM, 22 nM and 90 nM, respectively.
  • Sorafenib has the chemical name: 2- Pyridinecarboxamide, 4-[4-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino] phenoxy] -N-methyl-, 4-methylbenzenesulfonate (1: 1); and has the following structure:
  • the BRAF inhibitor is PLX4720.
  • PLX4720 is a potent and selective inhibitor of BRAFV600E with IC50 of about 13 nM, modest potent to c-Raf- 1(Y340D and Y341D mutations), 10-fold selectivity for BRAFV600E than wild-type BRAF.
  • PLX4720 has the chemical name: N-(3-(5-chloro-lH-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4- difluorophenyl)propane-l-sulfonamide; and has the following structure:
  • the BRAF inhibitor is GDC-0879.
  • GDC-0879 is a potent, and selective BRAF inhibitor with an IC50 of about 0.13 nM with activity against c-Raf as well; no inhibition known to other protein kinases.
  • GDC-0879 has the chemical name: (E)-5-(l-(2- hydroxyethyl)-3-(pyridin-4-yl)-lH-pyrazol-4-yl)-2,3-dihydroinden-l-one oxime; and has the following structure:
  • the BRAF inhibitor is RAF265 (CHIR-265).
  • RAF265 is a highly selective BRAF and VEGFR2 inhibitor with an IC50 of about 3-60 nM and an EC50 of about 30 nM, including BRAF, C-Raf and mutant BRAF.
  • RAF265 has the chemical name: l-methyl-5-(2- (5-(trifluoromethyl)-lH-imidazol-2-yl)pyridin-4-yloxy)-N-(4-(trifluoromethyl)phenyl)-lH- benzo[d]imidazol-2- amine; and has the following structure:
  • the BRAF inhibitor is MLN2480 (BIIB-024).
  • MLN2480 is a pan- Raf inhibitor.
  • MLN2480 has the chemical name: 4-Pyrimidinecarboxamide, 6-amino-5-chloro- N-[(lR)-l-[5-[[[5-chloro-4-(trifluoromethyl)-2-pyridinyl]amino]carbonyl]-2-thiazolyl]ethyl]-; and has the following structure:
  • the BRAF inhibitor is PF-04880594.
  • PF-04880594 is a RAF inhibitor for BRAF/BRAFV599E and c-RAF with an IC50 of about 0.19 nM/0.13 nM and 0.39 nM, >100-fold selectivity over CSNKl, JNK2/3 and p38.
  • PF-04880594 has the chemical name: Propanenitrile, 3-[[4-[l-(2,2-difluoroethyl)-3-(lH-pyrrolo[2,3-b]pyridin-5-yl)-lH-pyrazol-4-yl]- 2-pyrimidinyl] amino]-; and has the following structure:
  • the BRAF inhibitor is GW5074.
  • GW5074 is a potent and selective c-Raf inhibitor with an IC50 of about 9 nM, no effect on the activities of JNK1/2/3, MEK1, MKK6/7, CDKl/2, c-Src, p38 MAP, VEGFR2 or c-Fms is noted.
  • GW5074 has the chemical name: 2H-Indol-2-one, 3-[(3,5-dibromo-4-hydroxyphenyl)methylene]-l,3-dihydro-5-iodo-; and has the following structure:
  • the BRAF inhibitor is CEP-32496.
  • CEP-32496 is a highly potent inhibitor of BRAF(V600E/WT) and c-Raf with K d of 14 nM/36 nM and 39 nM, modest potent to Abl-1, c-Kit, Ret, PDGFRp and VEGFR2, respectively; insignificant affinity for MEK-1, MEK- 2, ERK-1 and ERK-2.
  • CEP-32496 has the chemical name: Urea, N-[3-[(6,7-dimethoxy-4- quinazolinyl)oxy]phenyl]-N-[5-(2,2,2-trifluoro-l,l-dimethylethyl)-3-isoxazolyl]-; and has the following structure:
  • the BRAF inhibitor is Dabrafenib (GSK2118436).
  • Dabrafenib is a mutant BRAFV600 specific inhibitor with an IC50 of about 0.8 nM, with 4- and 6-fold less potency against BRAF(wt) and c-Raf, respectively.
  • Dabrafenib has the chemical name: N-(3-(5- (2-aminopyrimidin-4-yl)-2-tert-butylthiazol-4-yl)-2-fluorophenyl)-2,6- difluorobenzenesulfonamide; and has the following structure:
  • the BRAF inhibitor is AZ628.
  • AZ628 is a new pan-Raf inhibitor for BRAF, BRAFV600E, and c-Raf- 1 with an IC50 of about 105 nM, 34 nM and 29 nM, also inhibits VEGFR2, DDR2, Lyn, Fltl, FMS, etc.
  • AZ628 has the chemical name: 3-(2- cyanopropan-2-yl)-N-(4-methyl-3-(3-methyl-4-oxo-3,4-dihydroquinazolin-6- ylamino)phenyl)benzamide; and has the following structure:
  • the BRAF inhibitor is SB590885.
  • SB590885 is a potent BRAF inhibitor with a Kj of about 0.16 nM, 11-fold greater selectivity for BRAF over c-Raf, and shows no detectable inhibition of other human kinases.
  • SB590885 has the chemical name: (E)-5-(2-(4- (2-(dimethylamino)ethoxy)phenyl)-4-(pyridin-4-yl)-lH-imidazol-5-yl)-2,3-dihydroinden-l-one oxime; and has the following structure:
  • the BRAF inhibitor is Raf265 derivative.
  • Raf265 derivative is a derivative of Raf265.
  • Raf265 derivative has the following structure:
  • the BRAF inhibitor is Regorafenib (BAY 73-4506, Fluoro- Sorafenib).
  • Regorafenib is a multi-target inhibitor for VEGFRl, VEGFR2, VEGFR3, PDGFRp, Kit, RET and Raf-1 with an IC50 of about 13 nM/4.2 nM/46 nM, 22 nM, 7 nM, 1.5 nM and 2.5 nM, respectively.
  • Regorafenib has the chemical name: l-(4-chloro-3-(trifluoromethyl)phenyl)-3- (2-fluoro-4-(2-(methylcarbamoyl)pyridin-4-yloxy)phenyl)urea; and has the following structure:
  • the BRAF inhibitor is ZM 336372.
  • ZM 336372 is a potent and selective c-Raf inhibitor with an IC50 of about 70 nM, 10-fold selectivity over B-RAF.
  • ZM 336372 has the chemical name: Benzamide, 3-(dimethylamino)-N-[3-[(4- hydroxybenzoyl)amino]-4-methylphenyl]-; and has the following structure:
  • the agent used in the methods targets and/or inhibits MEK (mitogen activated protein kinase kinase) (e.g., MEKl and/or MEK2) (e.g., a MEKl and or MEK2 gene or gene product).
  • MEK mitogen activated protein kinase kinase
  • the agent binds and inhibits MEK.
  • the agent is a reversible or an irreversible MEK inhibitor.
  • the MEK inhibitor includes an agent that inhibits the mitogen- activated protein kinase kinase enzymes MEKl and/or MEK2.
  • the MEK inhibitor is chosen from: ARRY- 162 (MEK162), Trametinib (GSK1120212), Selumetinib (AZD6244, ARRY142886), XL518 (GDC-0973), CI-1040 (PD184352), PD035901, U0126- EtOH, PD198306, PD98059, BIX 02189, TAK-733, Honokiol, AZD8330 (ARRY-424704), PD318088, BIX 02188, AS703026 (Pimasertib) and/or SL327.
  • the MEK inhibitor is ARRY-162 (MEK 162).
  • ARRY-162 has the chemical name: 5-((4-bromo-2- fluorophenyl)amino)-4-fluoro-N-(2-hydroxyethoxy)-l-methyl-lH-benzo[d]imidazole-6- carboxamide; and has the following structure:
  • the MEK inhibitor is Trametinib (GSK1120212).
  • Trametinib is a highly specific and potent MEKl/2 inhibitor with an IC50 of about 0.92 nM/1.8 Nm. Trametinib does not inhibit the kinase activities of c-Raf, B-Raf, ERKl/2. Trametinib is currently FDA- approved for use in the treatment of BRAF-mutated melanoma. Trametinib has also been studied in combination with the BRAF inhibitor dabrafenib for use in the treatment of BRAF-mutated melanoma.
  • Trametinib has the chemical name: N-(3- ⁇ 3-Cyclopropyl-5-[(2-fluoro-4- iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-l(2H)- yl ⁇ phenyl) acetamide; and has the following structure:
  • the MEK inhibitor is Selumetinib (AZD6244, ARRY142886).
  • Selumetinib is a potent, highly selective MEK1 inhibitor with IC50 of 14 nM, also inhibits ERKl/2 phosphorylation with IC50 of 10 nM Selumetinib has been studied in phase 2 clinical trial for use in the treatment of non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • Selumetinib has the chemical name: 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3- methylbenzimidazole-5-carboxamide; and has the following structure:
  • the MEK inhibitor is XL518 (GDC-0973).
  • XL518 a potent, selective, orally bioavailable inhibitor of MEKl. It inhibits the proliferation and stimulates apoptosis in a variety of human tumor cell lines.
  • XL518 has the chemical name: [3,4-difluoro-2- [(2-fluoro-4-iodophenyl)amino]phenyl][3-hydroxy-3-[(2S)-2-piperidinyl]-l- azetidinyl]methanone; and has the following structure:
  • the MEK inhibitor is CI- 1040 (PD184352).
  • CI- 1040 is an ATP non-competitive MEKl/2 inhibitor with an IC50 of about 17 nM, 100-fold more selective for MEKl/2 than MEK5.
  • CI- 1040 has the chemical name: 2-(2-chloro-4-iodophenylamino)-N- (cyclopropylmethoxy)-3,4-difluorobenzamide; and has the following structure:
  • the MEK inhibitor is PD035901.
  • PD0325901 is selective and non ATP-competitive MEK inhibitor with an IC50 of about 0.33 nM, roughly 500-fold more potent than CI- 1040 on phosphorylation of ERKl and ERK2.
  • PD035901 has the chemical name: (R)-N- (2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide; and has the following structure:
  • the MEK inhibitor is U0126-EtOH.
  • U0126-EtOH is a highly selective inhibitor of MEKl/2 with an IC50 of about 0.07 ⁇ /0.06 ⁇ , 100-fold higher affinity for AN3-S218E/S222D MEK than PD098059.
  • U0126-EtOH has the chemical name: (2Z,3Z)- 2,3-bis(amino(2-aminophenylthio)methylene)succinonitrile,ethanol; and has the following structure:
  • the MEK inhibitor is PD198306.
  • PD198306 is a cell-permeable and highly selective MEK inhibitor with an IC50 of about 8 nM.
  • PD198306 has the chemical name: Benzamide, N-(cyclopropylmethoxy)-3,4,5-trifluoro-2-[(4-iodo-2-methylphenyl)amino]-; and has the following structure:
  • the MEK inhibitor is PD98059.
  • PD98059 is a non-ATP competitive MEK inhibitor with an IC50 of about 2 ⁇ , specifically inhibits MEK-1 -mediated activation of MAPK.
  • PD98059 does not directly inhibit ERK1 or ERK2.
  • PD98059 has the chemical name: 2-(2-amino-3-methoxyphenyl)-4H-chromen-4-one; and has the following structure:
  • the MEK inhibitor is BIX 02189.
  • BIX 02189 is a selective inhibitor of MEK5 with IC50 of 1.5 nM, also inhibits ERK5 catalytic activity with IC50 of 810 nM.
  • BIX 02189 does not inhibit closely related kinases MEKl, MEK2, ERK2, and JNK2.
  • BIX 02189 has the chemical name: (Z)-3-((3-((dimethylamino)methyl)phenylamino)(phenyl) methylene) -N,N-dimethyl-2-oxoindoline-6-carboxamide; and has the following structure:
  • the MEK inhibitor is TAK-733.
  • TAK-733 is a potent and selective MEK allosteric site inhibitor for MEKl with an IC50 of about 3.2 nM.
  • TAK-733 has the chemical name: (R)-3-(2,3-dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8- methylpyrido[2,3-d]pyrimidine-4,7( -dione; and has the following structure:
  • the MEK inhibitor is Honokiol.
  • Honokiol is the active principle of magnolia extract that inhibits Akt-phosphorylation and promotes ERK1 ⁇ phosphorylation.
  • Honokiol has the chemical name: 2-(4-hydroxy-3-prop-2-enyl-phenyl)- 4-prop-2-enyl-phenol; and has the following structure:
  • the MEK inhibitor is AZD8330 (ARRY-424704).
  • AZD8330 is a novel, selective, non-ATP competitive MEK 1/2 inhibitor with IC50 of 7 nM.
  • AZD8330 has the chemical name: 2-(2-fluoro-4-iodophenylamino)-N-(2-hydroxyethoxy)- 1 ,5-dimethyl-6-oxo- 1 ,6- dihydropyridine-3-carboxamide; and has the following structure:
  • the MEK inhibitor is PD318088.
  • PD318088 is a non-ATP competitive allosteric MEK1/2 inhibitor, binds simultaneously with ATP in a region of the MEKl active site that is adjacent to the ATP-binding site.
  • PD318088 has the chemical name: 5- bromo-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide; and has the following structure:
  • the MEK inhibitor is BIX 02188.
  • ⁇ 02188 is a selective inhibitor of MEK5 with an IC50 of about 4.3 nM, also inhibits ERK5 catalytic activity with an IC50 of about 810 nM.
  • BIX 02188 does not detectably inhibit closely related kinases MEKl, MEK2, ERK2, and JNK2.
  • ⁇ 02188 has the chemical name: (Z)-3-((3- ((dimethylamino)methyl) phenylamino) (phenyl)methylene) -2-oxoindoline-6-carboxamide; and has the following structure:
  • the MEK inhibitor is AS703026 (Pimasertib).
  • AS-703026 is a highly selective, potent, ATP non-competitive allosteric inhibitor of MEKl/2 with an IC50 of about 0.005-2 ⁇ in MM cell lines.
  • AS703026 has the chemical name: (S)-N-(2,3- dihydroxypropyl)-3-(2-fluoro-4-iodophenylamino)isonicotinamide; and has the following structure:
  • the MEK inhibitor is SL327.
  • SL327 is a selective inhibitor for MEKl/2 with an IC50 of about 0.18 ⁇ / 0.22 ⁇ .
  • SL327 has no detectable activity towards Erkl, MKK3, MKK4, c-JUN, PKC, PKA, or CamKII.
  • SL327 is capable of transport through the blood-brain barrier.
  • SL327 has the chemical name: (Z)-3-amino-3-(4-aminophenylthio)-2-(2- (trifluoromethyl)phenyl)acrylonitrile; and has the following structure:
  • agents e.g., the therapeutic agents described herein
  • a second therapeutic agent or a different therapeutic modality e.g., anti-cancer agents, and/or in combination with surgical and/or radiation procedures.
  • compositions can be administered concurrently with, prior to, or subsequent to, one or more other additional therapies or therapeutic agents.
  • each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • the additional therapeutic agent utilized in this combination can be administered together in a single composition or administered separately in different compositions.
  • the particular combination to employ in a regimen will take into account compatibility of the inventive pharmaceutical composition with the additional therapeutically active agent and/or the desired therapeutic effect to be achieved.
  • a first therapeutic agent described herein is administered in combination with a second therapeutic agent, described herein.
  • the first therapeutic agent is a BRAF inhibitor described herein
  • the second therapeutic agent is a MEK inhibitor described herein. Rationale for the combination of a BRAF inhibitor and a MEK inhibitor for therapeutic treatment is supported by, for example, Smalley K et al. Br J Cancer 2009 February 10; 100(3): 431-435; and Greger J et al. Mol Cancer Ther. 2012 Apr;l l(4):909- 20; the contents of which are incorporated herein by reference.
  • the first therapeutic agent is a BRAF inhibitor and is chosen from:
  • Vemurafenib (PLX4032, RG7204, R05185426), Sorafenib Tosylate (Bay 43-9006, Nexavar), PLX4720, GDC-0879, RAF265 (CHIR-265), MLN2480 (BIIB-024), PF-04880594, GW5074, CEP-32496, Dabrafenib (GSK2118436), AZ628, SB590885, Raf265 derivative, Regorafenib (BAY 73-4506, Fluoro-Sorafenib), and/or ZM 336372; and the second therapeutic agent is a MEK inhibitor and is chosen from ARRY- 162 (MEK162), Trametinib (GSK1120212),
  • either the BRAF inhibitor or the MEK inhibitor, or both can be administered in combination with a therapeutic agent or therapeutic modality currently used in the treatment of a hematological malignancy, e.g., CLL.
  • a therapeutic agent or therapeutic modality currently used in the treatment of the hematological malignancy is a non-MAPK pathway inhibitor.
  • the non-MAPK pathway inhibitor therapeutic agent or therapeutic modality is a chemotherapy, an immunotherapy, or a surgical procedure.
  • the non-MAPK pathway inhibitor therapeutic agent or therapeutic modality comprises one or more of: a chemotherapeutic agent (e.g., fludarabine, cyclophosphamide, doxorubicin, vincristine, chlorambucil, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, 5-azacytidine, pemetrexed disodium), tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g., erlotinib), BTK inhibitor (e.g., PCI-32765), multikinase inhibitor (e.g., MGCD265, RGB-286638), CD20 targeting agent (e.g., rituximab, ofatumumab, RO5072759, LFB-R603), CD52 targeting agent (e.g., alemtuzumab), prednisolone,
  • CD74 targeting agent e.g., milatuzumab
  • mTOR inhibitor e.g., everolimus
  • AT- 101 immunotoxin
  • immunotoxin e.g., CAT-8015, anti-Tac(Fv)-PE38 (LMB-2)
  • CD37 targeting agent e.g., TRU-016
  • radioimmunotherapy e.g., 131-tositumomab
  • hydroxychloroquine e.g., perifosine
  • SRC inhibitor e.g., dasatinib
  • thalidomide retinoid
  • retinoid e.g., fenretinide
  • MDM2 antagonist e.g., RO5045337
  • plerixafor, Aurora kinase inhibitor e.g., MLN8237, TAK-901
  • proteasome inhibitor e.g., bortezomib
  • the agent e.g., therapeutic agent, binds and inhibits a MAPK pathway protein, e.g., a BRAF or MEK protein.
  • the agent is an antibody molecule.
  • antibody and antibody molecule refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide featured in the invention.
  • a molecule which specifically binds to a given polypeptide featured in the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies.
  • the term "monoclonal antibody” or “monoclonal antibody composition,” as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope. Techniques for generating antibodies to a polypeptide target, e.g., BRAF (see e.g., WO 2012/092426, entitled "Optimization of Multigene Analysis of Tumor Samples,” incorporated herein by reference.
  • the agent is an antibody molecule, e.g., an anti- BRAF or anti-MEK antibody molecule (e.g., a monoclonal or a bispecific antibody), or a conjugate thereof (e.g., an antibody conjugated to a cytotoxic agent (e.g., mertansine DM1)).
  • an antibody molecule e.g., an anti- BRAF or anti-MEK antibody molecule (e.g., a monoclonal or a bispecific antibody), or a conjugate thereof (e.g., an antibody conjugated to a cytotoxic agent (e.g., mertansine DM1)).
  • a cytotoxic agent e.g., mertansine DM1
  • the agent e.g., the therapeutic agent, inhibits the expression of a nucleic acid encoding an alteration described herein.
  • agents include nucleic acid molecules, for example, antisense molecules, ribozymes, siRNA, triple helix molecules that hybridize to a nucleic acid encoding a mutation, or a transcription regulatory region, and blocks or reduces mRNA expression of the mutation.
  • the agent is chosen from a nucleic acid molecule (e.g., an antisense molecule, a ribozyme, a double stranded RNA, or a triple helix molecule) that hybridizes to and/or inhibits a MAPK kinase gene or gene product, e.g., a BRAF, KRAS or MEK nucleic acid, e.g., a nucleic acid encoding the
  • alteration or a transcription regulatory region that blocks or reduces mRNA expression of the alteration.
  • the nucleic acid antagonist is a siRNA that targets mRNA encoding a mutation.
  • Other types of antagonistic nucleic acids can also be used, e.g., a dsRNA, a ribozyme, a triple- helix former, or an antisense nucleic acid.
  • isolated nucleic acid molecules that are nucleic acid inhibitors, e.g., antisense, RNAi, to a mutation-encoding nucleic acid molecule are provided.
  • an “antisense” nucleic acid can include a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double- stranded cDNA molecule or complementary to an mRNA sequence.
  • the antisense nucleic acid can be complementary to an entire mutation coding strand, or to only a portion thereof.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding mutation (e.g., the 5' and 3' untranslated regions).
  • Anti-sense agents can include, for example, from about 8 to about 80 nucleobases (i.e., from about 8 to about 80 nucleotides), e.g., about 8 to about 50 nucleobases, or about 12 to about 30 nucleobases.
  • Anti-sense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.
  • Anti-sense compounds can include a stretch of at least eight consecutive nucleobases that are complementary to a sequence in the target gene. An oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable.
  • oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted.
  • Hybridization of antisense oligonucleotides with mRNA can interfere with one or more of the normal functions of mRNA.
  • the functions of mRNA to be interfered with include all key functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA. Binding of specific protein(s) to the RNA may also be interfered with by antisense oligonucleotide hybridization to the RNA.
  • Exemplary antisense compounds include DNA or RNA sequences that specifically hybridize to the target nucleic acid, e.g., the mRNA encoding a mutation described herein.
  • the complementary region can extend for between about 8 to about 80 nucleobases.
  • the compounds can include one or more modified nucleobases. Modified nucleobases are known in the art. Descriptions of modified nucleic acid agents are also available. See, e.g., U.S. Patent Nos. 4,987,071; 5,116,742; and 5,093,246; Woolf et al. (1992) Proc Natl Acad Sci USA; Antisense RNA and DNA, D.A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  • antisense nucleic acid molecules are typically administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a mutation to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • antisense nucleic acid molecules can be modified to target selected cells and then be administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
  • vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule is an cc-anomeric nucleic acid molecule.
  • An cc-anomeric nucleic acid molecule forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
  • siRNAs are small double stranded RNAs (dsRNAs) that optionally include overhangs.
  • the duplex region of an siRNA is about 18 to 25 nucleotides in length, e.g., about 19, 20, 21, 22, 23, or 24 nucleotides in length.
  • the siRNA sequences are exactly complementary to the target mRNA.
  • dsRNAs and siRNAs in particular can be used to silence gene expression in mammalian cells (e.g., human cells).
  • siRNAs also include short hairpin RNAs (shRNAs) with 29-base-pair stems and 2-nucleotide 3' overhangs. See, e.g., Clemens et al. (2000) Proc. Natl.
  • an antisense nucleic acid featured in the invention is a ribozyme.
  • a ribozyme having specificity for a mutation-encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of a mutation cDNA disclosed herein (i.e., SEQ ID NO:6), and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a mutation-encoding mRNA.
  • mutation mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261: 1411-1418.
  • Inhibition of a mutated gene can be accomplished by targeting nucleotide sequences
  • Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • the invention also provides detectably labeled oligonucleotide primer and probe molecules.
  • detectably labeled oligonucleotide primer and probe molecules are chemiluminescent, fluorescent, radioactive, or colorimetric.
  • a mutated nucleic acid molecule can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • synthetic oligonucleotides with modifications see Toulme (2001) Nature Biotech. 19: 17 and Faria et al. (2001) Nature Biotech. 19:40-44.
  • Such phosphoramidite oligonucleotides can be effective antisense agents.
  • the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4: 5- 23).
  • peptide nucleic acid or "PNA” refers to a nucleic acid mimic, e.g., a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • PNA oligomers can be synthesized using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra and Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.
  • PNAs of mutated nucleic acid molecules can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence- specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication.
  • PNAs of mutated nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup B. et al. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; W088/09810) or the blood-brain barrier (see, e.g., W0 89/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-6
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents (See, e.g., Zon (1988) Pharm. Res. 5:539-549).
  • the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).
  • a nucleic acid inhibitor described herein is provided for the inhibition of expression of a nucleic acid comprising the alteration in vitro.
  • Subjects can be evaluated for the presence of an alteration, e.g., an alteration as described herein.
  • a patient can be evaluated, for example, by determining the genomic sequence of the patient, e.g., by an NGS method.
  • evaluation of a patient can include directly assaying for the presence of a mutation in the patient, such as by an assay to detect a mutated nucleic acid (e.g., DNA or RNA), such as by, Southern blot, Northern blot, or RT-PCR, e.g., qRT-PCR.
  • a patient can be evaluated for the presence of a protein mutation, such as by immunohistochemistry, Western blot, immunoprecipitation, or
  • the results of a clinical trial can be repurposed to identify agents that target an alteration disclosed herein, e.g., a BRAF mutation.
  • a candidate agent used in a clinical trial can be reevaluated to determine if the agent in the trial targets a mutation, or is effective to treat a tumor containing a particular mutation.
  • subjects who participated in a clinical trial for an agent, such as a kinase inhibitor can be identified.
  • Reevaluation of patients can include, for example, determining the genomic sequence of the patients, or a subset of the clinical trial patients, e.g., by an NGS method.
  • reevaluation of the patients can include directly assaying for the presence of a mutation in the patient, such as by an assay to detect a mutated nucleic acid (e.g., RNA), such as by RT-PCR, e.g., qRT-PCR.
  • a patient can be evaluated for the presence of a protein mutation, such as by immunohistochemistry, Western blot, immunoprecipitation, or
  • the mutation is detected in a nucleic acid molecule by a method chosen from one or more of: nucleic acid hybridization assay, SSP, HPLC or mass- spectrometric genotyping.
  • Additional exemplary methods include traditional "direct probe” methods such as Southern blots and “comparative probe” methods such as comparative genomic hybridization (CGH), e.g., cDNA-based or oligonucleotide-based CGH, can be used.
  • CGH comparative genomic hybridization
  • the methods can be used in a wide variety of formats including, but not limited to, substrate (e.g., membrane or glass) bound methods or array-based approaches.
  • the evaluation methods include probes/primers against the alterations described herein.
  • probes/primers can be designed to detect a mutation or a reciprocal thereof. These probes/primers are suitable, e.g., for PCR amplification. Probes are used that contain DNA segments that are essentially complementary to DNA base sequences existing in different portions of chromosomes. Examples of probes useful according to the invention, and labeling and hybridization of probes to samples are described in two U.S. patents to Vysis, Inc. U.S. Patent Nos. 5,491,224 and 6,277,569 to Bittner, et al.
  • Chromosomal probes are typically about 50 to about 10 5 nucleotides in length. Longer probes typically comprise smaller fragments of about 100 to about 500 nucleotides in length. Probes that hybridize with centromeric DNA and locus-specific DNA are available
  • probes can be made non- commercially from chromosomal or genomic DNA through standard techniques.
  • sources of DNA include genomic DNA, cloned DNA sequences, somatic cell hybrids that contain one, or a part of one, chromosome (e.g., human chromosome) along with the normal chromosome complement of the host, and chromosomes purified by flow cytometry or microdissection.
  • the region of interest can be isolated through cloning, or by site-specific amplification via the polymerase chain reaction (PCR). See, for example, Nath and Johnson, Biotechnic Histochem., 1998, 73(l):6-22, Wheeless et al., Cytometry 1994, 17:319-326, and U.S. Patent No. 5,491,224.
  • PCR polymerase chain reaction
  • the probes to be used hybridize to a specific region of a chromosome to determine whether a cytogenetic abnormality is present in this region.
  • cytogenetic abnormality is a deletion. Although deletions can be of one or more entire chromosomes, deletions normally involve loss of part of one or more chromosomes. If the entire region of a chromosome that is contained in a probe is deleted from a cell, hybridization of that probe to the DNA from the cell will normally not occur and no signal will be present on that chromosome. If the region of a chromosome that is partially contained within a probe is deleted from a cell, hybridization of that probe to the DNA from the cell can still occur, but less of a signal can be present.
  • the loss of a signal is compared to probe hybridization to DNA from control cells that do not contain the genetic abnormalities which the probes are intended to detect.
  • at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more cells are enumerated for presence of the cytogenetic abnormality.
  • Cytogenetic abnormalities to be detected can include, but are not limited to, non- reciprocal translocations, balanced translocations, intra-chromosomal inversions, point mutations, deletions, gene copy number changes, gene expression level changes, and germ line mutations.
  • one type of cytogenetic abnormality is a duplication.
  • Duplications can be of entire chromosomes, or of regions smaller than an entire chromosome. If the region of a chromosome that is contained in a probe is duplicated in a cell, hybridization of that probe to the DNA from the cell will normally produce at least one additional signal as compared to the number of signals present in control cells with no abnormality of the chromosomal region contained in the probe.
  • Chromosomal probes are labeled so that the chromosomal region to which they hybridize can be detected.
  • Probes typically are directly labeled with a fluorophore, an organic molecule that fluoresces after absorbing light of lower wavelength/higher energy. The fluorophore allows the probe to be visualized without a secondary detection molecule.
  • the nucleotide can be directly incorporated into the probe with standard techniques such as nick translation, random priming, and PCR labeling.
  • deoxycytidine nucleotides within the probe can be transaminated with a linker. The fluorophore then is covalently attached to the transaminated deoxycytidine nucleotides. See, U.S. Patent No. 5,491,224.
  • U.S. Patent No. 5,491,224 describes probe labeling as a number of the cytosine residues having a fluorescent label covalently bonded thereto.
  • the number of fluorescently labeled cytosine bases is sufficient to generate a detectable fluorescent signal while the individual so labeled DNA segments essentially retain their specific complementary binding (hybridizing) properties with respect to the chromosome or chromosome region to be detected.
  • Such probes are made by taking the unlabeled DNA probe segment, transaminating with a linking group a number of deoxycytidine nucleotides in the segment, covalently bonding a fluorescent label to at least a portion of the transaminated deoxycytidine bases.
  • Probes can also be labeled by nick translation, random primer labeling or PCR labeling. Labeling is done using either fluorescent (direct)-or haptene (indirect)-labeled nucleotides.
  • labels include: AMCA-6-dUTP, CascadeBlue-4-dUTP, Fluorescein- 12-dUTP, Rhodamine-6-dUTP, TexasRed-6-dUTP, Cy3-6-dUTP, Cy5-dUTP, Biotin(BIO)-l l-dUTP, Digoxygenin(DIG)- l l-dUTP or Dinitrophenyl (DNP)- l l-dUTP.
  • Probes also can be indirectly labeled with biotin or digoxygenin, or labeled with
  • radioactive isotopes such as P and H
  • a probe labeled with biotin can be detected by avidin conjugated to a detectable marker.
  • avidin can be conjugated to an enzymatic marker such as alkaline phosphatase or horseradish peroxidase.
  • Enzymatic markers can be detected in standard colorimetric reactions using a substrate and/or a catalyst for the enzyme.
  • Catalysts for alkaline phosphatase include 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.
  • Diaminobenzoate can be used as a catalyst for horseradish
  • Probes can also be prepared such that a fluorescent or other label is not part of the DNA before or during the hybridization, and is added after hybridization to detect the probe hybridized to a chromosome.
  • probes can be used that have antigenic molecules incorporated into the DNA. After hybridization, these antigenic molecules are detected using specific antibodies reactive with the antigenic molecules. Such antibodies can themselves incorporate a fluorochrome, or can be detected using a second antibody with a bound fluorochrome.
  • the probe DNA is commonly purified in order to remove unreacted, residual products (e.g., fluorochrome molecules not incorporated into the DNA) before use in hybridization.
  • chromosomal probes Prior to hybridization, chromosomal probes are denatured according to methods well known in the art. Probes can be hybridized or annealed to the chromosomal DNA under hybridizing conditions. "Hybridizing conditions" are conditions that facilitate annealing between a probe and target chromosomal DNA. Since annealing of different probes will vary depending on probe length, base concentration and the like, annealing is facilitated by varying probe concentration, hybridization temperature, salt concentration and other factors well known in the art.
  • Hybridization conditions are facilitated by varying the concentrations, base compositions, complexities, and lengths of the probes, as well as salt concentrations, temperatures, and length of incubation.
  • in situ hybridizations are typically performed in hybridization buffer containing l-2x SSC, 50-65% formamide and blocking DNA to suppress non-specific hybridization.
  • hybridization conditions include temperatures of about 25° C to about 55° C, and incubation lengths of about 0.5 hours to about 96 hours.
  • Non-specific binding of chromosomal probes to DNA outside of the target region can be removed by a series of washes. Temperature and concentration of salt in each wash are varied to control stringency of the washes. For example, for high stringency conditions, washes can be carried out at about 65° C to about 80° C, using 0.2x to about 2x SSC, and about 0.1% to about 1% of a non-ionic detergent such as Nonidet P-40 (NP40). Stringency can be lowered by decreasing the temperature of the washes or by increasing the concentration of salt in the washes. In some applications it is necessary to block the hybridization capacity of repetitive sequences. Thus, in some embodiments, tRNA, human genomic DNA, or Cot-I DNA is used to block nonspecific hybridization. After washing, the slide is allowed to drain and air dry, then mounting medium, a counterstain such as DAPI, and a coverslip are applied to the slide. Slides can be viewed immediately or stored at -20° C. before examination.
  • a first collection of nucleic acids (e.g., from a sample, e.g., a possible tumor) is labeled with a first label
  • a second collection of nucleic acids e.g., a control, e.g., from a healthy cell/tissue
  • the ratio of hybridization of the nucleic acids is determined by the ratio of the two (first and second) labels binding to each fiber in the array. Where there are chromosomal deletions or multiplications, differences in the ratio of the signals from the two labels will be detected and the ratio will provide a measure of the copy number.
  • Array-based CGH can also be performed with single-color labeling (as opposed to labeling the control and the possible tumor sample with two different dyes and mixing them prior to hybridization, which will yield a ratio due to competitive hybridization of probes on the arrays).
  • single color CGH the control is labeled and hybridized to one array and absolute signals are read, and the possible tumor sample is labeled and hybridized to a second array (with identical content) and absolute signals are read. Copy number difference is calculated based on absolute signals from the two arrays.
  • Hybridization protocols suitable for use with the methods featured in the invention are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No. 430,402; Methods in Molecular Biology, Vol. 33: In situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, N.J. (1994), etc.
  • amplification-based assays can be used to measure presence/absence and copy number.
  • the nucleic acid sequences act as a template in an amplification reaction ⁇ e.g., Polymerase Chain Reaction (PCR).
  • PCR Polymerase Chain Reaction
  • the amount of amplification product will be proportional to the amount of template in the original sample.
  • Comparison to appropriate controls, e.g., healthy tissue, provides a measure of the copy number.
  • Quantitative amplification involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that can be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided in Innis, et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.).
  • LCR ligase chain reaction
  • tissue samples can be the source of the nucleic acid samples used in the present methods.
  • Genomic or subgenomic DNA fragments can be isolated from a subject's sample (e.g., a tumor sample, a normal adjacent tissue (NAT), a blood sample or any normal control)).
  • the tissue sample is preserved as a frozen sample or as formaldehyde- or
  • FFPE paraffin-embedded tissue preparation
  • the sample can be embedded in a matrix, e.g., an FFPE block or a frozen sample.
  • the isolating step can include flow-sorting of individual chromosomes; and/or micro-dissecting a subject's sample (e.g., a tumor sample, a NAT, a blood sample).
  • Protocols for DNA isolation, fragmentation and processing from a tissue sample are known in the art as described, e.g., in WO 2012/092426, entitled “Optimization of Multigene Analysis of Tumor Samples,” incorporated herein by reference in its entirety.
  • Additional methods to isolate nucleic acids (e.g., DNA) from formaldehyde- or paraformaldehyde-fixed, paraffin-embedded (FFPE) tissues are disclosed, e.g., in Cronin M. et al., (2004) Am J Pathol. 164(l):35-42; Masuda N. et al., (1999) Nucleic Acids Res. 27 (22): 4436-4443; Specht K. et al., (2001) Am J Pathol.
  • RecoverAllTM Total Nucleic Acid Isolation Kit uses xylene at elevated temperatures to solubilize paraffin-embedded samples and a glass-fiber filter to capture nucleic acids.
  • QIAamp® DNA FFPE Tissue Kit uses QIAamp® DNA Micro technology for purification of genomic and mitochondrial DNA.
  • a bait can be a nucleic acid molecule, e.g., a DNA or RNA molecule, which can hybridize to (e.g., be complementary to), and thereby allow capture of a target nucleic acid.
  • a bait is an RNA molecule.
  • a bait includes a binding entity, e.g., an affinity tag, that allows capture and separation, e.g., by binding to a binding entity, of a hybrid formed by a bait and a nucleic acid hybridized to the bait.
  • a bait is suitable for solution phase hybridization.
  • Baits can be produced and used by methods and hybridization conditions as described in US 2010/0029498 and Gnirke, A. et al. (2009) Nat Biotechnol. 27(2): 182-189, and WO 2012/092426, entitled "Optimization of Multigene Analysis of Tumor Samples, incorporated herein by reference.
  • the invention also includes methods of sequencing nucleic acids.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence at least a portion of a mutation.
  • the mutated sequence is compared to a
  • sequence of the nucleic acid molecule comprising an alteration described herein is determined by a method that includes one or more of: hybridizing an
  • oligonucleotide e.g., an allele specific oligonucleotide for one mutation described herein to said nucleic acid; hybridizing a primer, or a primer set (e.g., a primer pair), that amplifies a region comprising the mutation of the allele; amplifying, e.g., specifically amplifying, a region comprising the mutation of the allele; attaching an adapter oligonucleotide to one end of a nucleic acid that comprises the mutation of the allele; generating an optical, e.g., a colorimetric signal, specific to the presence of the one of the mutation; hybridizing a nucleic acid comprising the mutation to a second nucleic acid, e.g., a second nucleic acid attached to a substrate; generating a signal, e.g., an electrical or fluorescent signal, specific to the presence of the mutation; and incorporating a nucleotide into an oligonucleotide that is hybrid
  • the sequence is determined by a method that comprises one or more of: determining the nucleotide sequence from an individual nucleic acid molecule, e.g., where a signal corresponding to the sequence is derived from a single molecule as opposed, e.g., from a sum of signals from a plurality of clonally expanded molecules; determining the nucleotide sequence of clonally expanded proxies for individual nucleic acid molecules; massively parallel short-read sequencing; template-based sequencing; pyrosequencing; real-time sequencing comprising imaging the continuous incorporation of dye-labeling nucleotides during DNA synthesis; nanopore sequencing; sequencing by hybridization; nano-transistor array based sequencing; polony sequencing; scanning tunneling microscopy (STM) based sequencing; or nanowire-molecule sensor based sequencing.
  • a method comprises one or more of: determining the nucleotide sequence from an individual nucleic acid molecule, e.g., where a signal corresponding to the sequence is
  • sequencing reactions include those based on techniques developed by Maxam and Gilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger (Sanger et al. (1977) Proc. Nat. Acad. Sci 74:5463). Any of a variety of automated sequencing procedures can be utilized when performing the assays
  • Sequencing of nucleic acid molecules can also be carried out using next-generation sequencing (NGS).
  • Next-generation sequencing includes any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules or clonally expanded proxies for individual nucleic acid molecules in a highly parallel fashion (e.g., greater than 10 5 molecules are sequenced simultaneously).
  • the relative abundance of the nucleic acid species in the library can be estimated by counting the relative number of occurrences of their cognate sequences in the data generated by the sequencing experiment.
  • Next generation sequencing methods are known in the art, and are described, e.g., in Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46, incorporated herein by reference.
  • next-generation sequencing allows for the determination of the nucleotide sequence of an individual nucleic acid molecule (e.g., Helicos Biosciences' HeliScope Gene Sequencing system, and Pacific Biosciences' PacBio RS system).
  • an individual nucleic acid molecule e.g., Helicos Biosciences' HeliScope Gene Sequencing system, and Pacific Biosciences' PacBio RS system.
  • the sequencing method determines the nucleotide sequence of clonally expanded proxies for individual nucleic acid molecules (e.g., the Solexa sequencer, Alumina Inc., San Diego, Calif; 454 Life Sciences (Branford, Conn.), and Ion Torrent), e.g., massively parallel short-read sequencing (e.g., the Solexa sequencer, Illumina Inc., San Diego, Calif.), which generates more bases of sequence per sequencing unit than other sequencing methods that generate fewer but longer reads.
  • Other methods or machines for next-generation sequencing include, but are not limited to, the sequencers provided by 454 Life Sciences (Branford, Conn.), Applied Biosystems (Foster City, Calif.; SOLiD
  • NGS technologies can include one or more of steps, e.g., template preparation, sequencing and imaging, and data analysis as described in WO 2012/092426, entitled "Optimization of Multigene Analysis of Tumor Samples, incorporated herein by reference.
  • NGS reads After NGS reads have been generated, they can be aligned to a known reference sequence or assembled de novo. For example, identifying genetic variations such as single-nucleotide
  • polymorphism and structural variants in a sample can be accomplished by aligning NGS reads to a reference sequence (e.g., a wild- type sequence).
  • a reference sequence e.g., a wild- type sequence.
  • Methods of sequence alignment for NGS are described e.g., in Trapnell C. and Salzberg S.L. Nature Biotech., 2009, 27:455-457.
  • Examples of de novo assemblies are described, e.g., in Warren R. et al., Bioinformatics, 2007, 23:500-501; Butler J. et al., Genome Res., 2008, 18:810-820; and Zerbino D.R. and Birney E., Genome Res., 2008, 18:821-829.
  • Sequence alignment or assembly can be performed using read data from one or more NGS platforms, e.g., mixing Roche/454 and Illumina/Solexa read data. Algorithms and methods for data analysis are described in WO 2012/092426, entitled "Optimization of Multigene Analysis of Tumor Samples, incorporated herein by reference.
  • the activity or level of a mutated polypeptide can also be detected and/or quantified by detecting or quantifying the expressed polypeptide.
  • the mutated polypeptide can be detected and quantified by any of a number of means known to those of skill in the art.
  • analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), Immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, immunohistochemistry (IHC) and the like.
  • analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like
  • immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), Immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent
  • Another agent for detecting a mutated polypeptide is an antibody molecule capable of binding to a polypeptide corresponding to a polypeptide, e.g., an antibody with a detectable label.
  • Techniques for generating antibodies are described herein.
  • the term "labeled", with regard to the probe or antibody is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a
  • the antibody is labeled, e.g., a radio-labeled, chromophore- labeled, fluorophore-labeled, or enzyme-labeled antibody.
  • an antibody derivative e.g., an antibody conjugated with a substrate or with the protein or ligand of a protein-ligand pair ⁇ e.g., biotin-streptavidin ⁇
  • an antibody fragment e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.
  • Mutated polypeptides from cells can be isolated using techniques that are known to those of skill in the art.
  • the protein isolation methods employed can, for example, be such as those described in Harlow and Lane (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
  • Western blot (immunoblot) analysis is used to detect and quantify the presence of a polypeptide in the sample.
  • the polypeptide is detected using an immunoassay.
  • an immunoassay is an assay that utilizes an antibody to specifically bind to the analyte. The immunoassay is thus characterized by detection of specific binding of a polypeptide to an anti- antibody as opposed to the use of other physical or chemical properties to isolate, target, and quantify the analyte.
  • the mutated polypeptide is detected and/or quantified using any of a number of
  • the invention features, a kit, e.g., containing an oligonucleotide having an alteration described herein, e.g., a BRAF and/or KRAS mutation.
  • the kit can also contain an oligonucleotide that is the wildtype counterpart of the mutant oligonucleotide.
  • a kit can include a carrier, e.g., a means being compartmentalized to receive in close confinement one or more container means.
  • a carrier e.g., a means being compartmentalized to receive in close confinement one or more container means.
  • the container contains an
  • oligonucleotide e.g., a primer or probe as described above.
  • the components of the kit are useful, for example, to diagnose or identify a mutation in a tumor sample in a patient.
  • the probe or primer of the kit can be used in any sequencing or nucleotide detection assay known in the art, e.g., a sequencing assay, e.g., an NGS method, RT-PCR, or in situ hybridization.
  • the components of the kit are useful, for example, to diagnose or identify a mutation in a tumor sample in a patient, and to accordingly identify an appropriate therapeutic agent to treat the cancer.
  • a kit featured in the invention can include, e.g., assay positive and negative controls, nucleotides, enzymes (e.g., RNA or DNA polymerase or ligase), solvents or buffers, a stabilizer, a preservative, a secondary antibody, e.g., an anti-HRP antibody (IgG) and a detection reagent.
  • enzymes e.g., RNA or DNA polymerase or ligase
  • solvents or buffers e.g., a stabilizer, a preservative, a secondary antibody, e.g., an anti-HRP antibody (IgG) and a detection reagent.
  • secondary antibody e.g., an anti-HRP antibody (IgG) and a detection reagent.
  • An oligonucleotide can be provided in any form, e.g., liquid, dried, semi-dried, or lyophilized, or in a form for storage in a frozen condition.
  • an oligonucleotide, and other components in a kit are provided in a form that is sterile.
  • An oligonucleotide e.g., an oligonucleotide that contains a mutation, described herein, or an oligonucleotide complementary to an alteration described herein, is provided in a liquid solution, the liquid solution generally is an aqueous solution, e.g., a sterile aqueous solution.
  • a suitable solvent e.g., sterile buffer
  • the solvent e.g., sterile buffer, can optionally be provided in the kit.
  • the kit can include one or more containers for the composition containing an oligonucleotide in a concentration suitable for use in the assay or with instructions for dilution for use in the assay.
  • the kit contains separate containers, dividers or compartments for the
  • oligonucleotide and assay components and the informational material.
  • the informational material for example, the
  • oligonucleotides can be contained in a bottle or vial, and the informational material can be contained in a plastic sleeve or packet.
  • the separate elements of the kit are contained within a single, undivided container.
  • an oligonucleotide composition is contained in a bottle or vial that has attached thereto the informational material in the form of a label.
  • the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit forms (e.g., for use with one assay) of an oligonucleotide.
  • the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit forms (e.g., for use with one assay) of an oligonucleotide.
  • the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit forms (e.g., for use with one assay) of an
  • kits includes a plurality of ampoules, foil packets, or blister packs, each containing a single unit of oligonucleotide for use in sequencing or detecting a mutation in a tumor sample.
  • the containers of the kits can be air tight and/or waterproof.
  • the container can be labeled for use.
  • the kit can include: (1) a first antibody (e.g., attached to a solid support) which binds to a mutated polypeptide; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.
  • a first antibody e.g., attached to a solid support
  • a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.
  • the kit can include informational material for performing and
  • the kit can provide guidance as to where to report the results of the assay, e.g., to a treatment center or healthcare provider.
  • the kit can include forms for reporting the results of a sequencing or diagnostic assay described herein, and address and contact information regarding where to send such forms or other related information; or a URL (Uniform Resource Locator) address for reporting the results in an online database or an online application (e.g., an app).
  • the informational material can include guidance regarding whether a patient should receive treatment with a particular chemotherapeutic drug, depending on the results of the assay.
  • the informational material of the kits is not limited in its form. In many cases, the
  • informational material e.g., instructions
  • printed matter e.g., a printed text, drawings, and/or photographs, e.g., a label or printed sheet.
  • the informational material can also be provided in other formats, such as computer readable material, video recording, or audio recording.
  • the informational material of the kit is contact information, e.g., a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about the sequencing or diagnostic assay and/or its use in the methods described herein.
  • the informational material can also be provided in any combination of formats.
  • a biological sample is provided to an assay provider, e.g., a service provider (such as a third party facility) or a healthcare provider, who evaluates the sample in an assay and provides a read out.
  • an assay provider receives a biological sample from a subject, such as a blood or tissue sample, e.g., a biopsy sample, and evaluates the sample using an assay described herein, e.g., a sequencing assay or in situ
  • hybridization assay determines that the sample contains a mutation.
  • the assay provider e.g., a service provider or healthcare provider, can then conclude that the subject is, or is not, a candidate for a particular drug or a particular cancer treatment regimen.
  • the invention also features an isolated nucleic acid molecule, or an isolated preparation of nucleic acid molecules, that includes an alteration described herein.
  • nucleic acid molecules or preparations thereof can include an alteration described herein or can be used to detect, e.g., sequence, an alteration.
  • the invention also features a nucleic acid molecule, e.g., nucleic acid fragment, suitable as probe, primer, bait or library member that includes, flanks, hybridizes to, which are useful for identifying, or are otherwise based on, an alteration described herein.
  • the probe, primer or bait molecule is an oligonucleotide that allows capture, detection or isolation of a nucleic acid molecule containing an alteration described herein, e.g., an alteration in BRAF and/or KRAS.
  • the oligonucleotide can comprise a nucleotide sequence substantially complementary to nucleic acid molecules or fragments of nucleic acid molecules comprising an alteration described herein.
  • the sequence identity between the nucleic acid molecule, e.g., the oligonucleotide, and the target sequence need not be exact, so long as the sequences are sufficiently complementary to allow the capture, detection or isolation of the target sequence.
  • the nucleic acid fragment is a probe or primer that includes an oligonucleotide between about 5 and 25, e.g., between 10 and 20, or 10 and 15 nucleotides in length.
  • the nucleic acid fragment is a bait that includes an oligonucleotide between about 100 to 300 nucleotides, 130 and 230
  • nucleotides or 150 and 200 nucleotides, in length.
  • the nucleic acid fragment can be used to identify or capture, e.g., by hybridization, a nucleic acid molecules comprising an alteration described herein, e.g., an alteration in BRAF and/or KRAS.
  • the nucleic acid fragment can be a probe, a primer, or a bait, for use in identifying or capturing, e.g., by hybridization, an alteration described herein.
  • the probes or primers described herein can be used, for example, PCR amplification.
  • amplification of the mutation can be performed using a primer or a primer pair, e.g., for amplifying a sequence flanking an alteration described herein.
  • the nucleic acid fragment includes a bait that comprises a nucleotide sequence that hybridizes to a nucleic acid molecules comprising an alteration described herein, and thereby allows the capture or isolation said nucleic acid molecule.
  • a bait is suitable for solution phase hybridization.
  • a bait includes a binding entity, e.g., an affinity tag, that allows capture and separation, e.g., by binding to a binding entity, of a hybrid formed by a bait and a nucleic acid hybridized to the bait.
  • the nucleic acid fragment includes a library member comprising a nucleic acid molecule described herein.
  • the library member includes a mutation, e.g., a base substitution, that results in an alteration described herein.
  • the nucleic acid fragment can be detectably labeled with, e.g., a radiolabel, a fluorescent label, a bioluminescent label, a chemiluminescent label, an enzyme label, a binding pair label, or can include an affinity tag; a tag, or identifier (e.g., an adaptor, barcode or other sequence identifier).
  • a radiolabel e.g., a fluorescent label, a bioluminescent label, a chemiluminescent label, an enzyme label, a binding pair label, or can include an affinity tag; a tag, or identifier (e.g., an adaptor, barcode or other sequence identifier).
  • the disclosure features a polypeptide comprising an alteration described herein (e.g., a purified polypeptide comprising an alteration described herein), a biologically active or antigenic fragment thereof, as well as reagents (e.g., antibody molecules that bind to a polypeptide comprising an alteration described herein), methods for modulating the activity of a polypeptide comprising an alteration described herein and detection of a polypeptide comprising an alteration described herein.
  • reagents e.g., antibody molecules that bind to a polypeptide comprising an alteration described herein
  • the polypeptide or fragment is a peptide, e.g., an immunogenic peptide or protein that contains an alteration described herein.
  • immunogenic peptides or proteins can be used to raise antibodies specific to the polypeptide or protein comprising an alteration described herein.
  • immunogenic peptides or proteins can be used for vaccine preparation.
  • the vaccine preparation can include other components, e.g., an adjuvant.
  • the invention features antibody molecules that bind to a polypeptide comprising an alteration described herein or fragment described herein.
  • the antibody can distinguish wild type from the mutated polypeptide, e.g., the polypeptide comprising an alteration described herein.
  • Techniques for generating antibody molecules are known in the art, and are described, for example, in WO 2012/092426, entitled "Optimization of Multigene Analysis of Tumor Samples, incorporated herein by reference.
  • the invention features a detection reagent, e.g., a purified or an isolated preparation thereof.
  • Detection reagents can distinguish a nucleic acid, or protein sequence, having an alteration described herein, e.g., of a nucleic acid molecule comprising an alteration described herein, e.g., an alteration in BRAF and/or KRAS.
  • Detection reagents can be used to identify mutations in a target nucleic acid, e.g., DNA, e.g., genomic DNA or cDNA, or RNA, e.g., in a sample, e.g., a sample of nucleic acid derived from a CLL (e.g., a CLL cell).
  • Detection reagents e.g., antibody-based detection reagents, can be used to identify mutations in a target protein, e.g., in a sample, e.g., a sample of protein derived from, or produced by, CLL cell.
  • the detection reagent comprises a nucleic acid molecule, e.g., a DNA, RNA or mixed DNA/RNA molecule, comprising sequence which is complementary with a nucleic acid sequence on a target nucleic acid (the sequence on the target nucleic acid that is bound by the detection reagent is referred to herein as the "detection reagent binding site” and the portion of the detection reagent that corresponds to the detection reagent binding site is referred to as the "target binding site”).
  • a nucleic acid molecule e.g., a DNA, RNA or mixed DNA/RNA molecule
  • the detection reagent binding site is disposed in relationship to the interrogation position such that binding (or in embodiments, lack of binding) of the detection reagent to the detection reagent binding site allows differentiation of mutant and reference sequences for a mutant described herein (nucleic acid molecule comprising an alteration described herein, e.g., an alteration in BRAF and/or KRAS.
  • the detection reagent can be modified, e.g., with a label or other moiety, e.g., a moiety that allows capture.
  • the detection reagent comprises a nucleic acid molecule, e.g., a DNA, RNA or mixed DNA/RNA molecule, which, e.g., in its target binding site, includes the interrogation position and which can distinguish (e.g., by affinity of binding of the detection reagent to a target nucleic acid or the ability for a reaction, e.g., a ligation or extension reaction with the detection reagent) between a mutation, e.g., a translocation described herein, and a reference sequence.
  • a nucleic acid molecule e.g., a DNA, RNA or mixed DNA/RNA molecule
  • the interrogation position can correspond to a terminal, e.g., to a 3' or 5' terminal nucleotide, a nucleotide immediately adjacent to a 3' or 5' terminal nucleotide, or to another internal nucleotide, of the detection reagent or target binding site.
  • the difference in the affinity of the detection reagent for a target nucleic acid comprising the alteration described herein and that for a target nucleic acid comprising the reference sequence allows determination of the presence or absence of the mutation (or reference) sequence.
  • detection reagents, under assay conditions will exhibit substantially higher levels of binding only to the mutant or only to the reference sequence, e.g., will exhibit substantial levels of binding only to the mutation or only to the reference sequence.
  • binding allows (or inhibits) a subsequent reaction, e.g., a subsequent reaction involving the detection reagent or the target nucleic acid.
  • binding can allow ligation, or the addition of one or more nucleotides to a nucleic acid, e.g., the detection reagent, e.g., by DNA polymerase, which can be detected and used to distinguish mutant from reference.
  • the interrogation position is located at the terminus, or sufficiently close to the terminus, of the detection reagent or its target binding site, such that hybridization, or a chemical reaction, e.g., the addition of one or more nucleotides to the detection reagent, e.g., by DNA polymerase, only occurs, or occurs at a substantially higher rate, when there is a perfect match between the detection reagent and the target nucleic acid at the interrogation position or at a nucleotide position within 1, 2, or 3 nucleotides of the interrogation position.
  • hybridization or a chemical reaction, e.g., the addition of one or more nucleotides to the detection reagent, e.g., by DNA polymerase, only occurs, or occurs at a substantially higher rate, when there is a perfect match between the detection reagent and the target nucleic acid at the interrogation position or at a nucleotide position within 1, 2, or 3 nucleotides of the interrogation position
  • the detection reagent comprises a nucleic acid, e.g., a DNA, RNA or mixed DNA/RNA molecule wherein the molecule, or its target binding site, is adjacent (or flanks), e.g., directly adjacent, to the interrogation position, and which can distinguish between a mutation described herein, and a reference sequence, in a target nucleic acid.
  • a nucleic acid e.g., a DNA, RNA or mixed DNA/RNA molecule wherein the molecule, or its target binding site, is adjacent (or flanks), e.g., directly adjacent, to the interrogation position, and which can distinguish between a mutation described herein, and a reference sequence, in a target nucleic acid.
  • the detection reagent binding site is adjacent to the interrogation position, e.g., the 5' or 3 'terminal nucleotide of the detection reagent, or its target binding site, is adjacent, e.g., between 0 (directly adjacent) and 1,000, 500, 400, 200, 100, 50, 10, 5, 4, 3, 2, or 1 nucleotides from the interrogation position.
  • the outcome of a reaction will vary with the identity of the nucleotide at the interrogation position allowing one to distinguish between mutant and reference sequences. E.g., in the presence of a first nucleotide at the interrogation position a first reaction will be favored over a second reaction.
  • the product will differ, e.g., in charge, sequence, size, or susceptibility to a further reaction (e.g., restriction cleavage) depending on the identity of the nucleotide at the interrogation position.
  • the detection reagent comprises paired molecules (e.g., forward and reverse primers), allowing for amplification, e.g., by PCR amplification, of a duplex containing the interrogation position.
  • the presence of the mutation can be determined by a difference in the property of the amplification product, e.g., size, sequence, charge, or susceptibility to a reaction, resulting from a sequence comprising the interrogation position and a corresponding sequence having a reference nucleotide at the interrogation positions.
  • the presence or absence of a characteristic amplification product is indicative of the identity of the nucleotide at the interrogation site and thus allows detection of the mutation.
  • the detection reagent, or its target binding site is directly adjacent to the interrogation position, e.g., the 5' or 3'terminal nucleotide of the detection reagent is directly adjacent to the interrogation position.
  • the identity of the nucleotide at the interrogation position will determine the nature of a reaction, e.g., a reaction involving the detection reagent, e.g., the modification of one end of the detection reagent. E.g., in the presence of a first nucleotide at the interrogation position a first reaction will be favored over a second reaction.
  • the presence of a first nucleotide at the interrogation position can promote a first reaction, e.g., the addition of a complementary nucleotide to the detection reagent.
  • a first reaction e.g., the addition of a complementary nucleotide to the detection reagent.
  • the presence of an A at the interrogation position will cause the incorporation of a T, having, e.g., a first colorimetric label
  • the presence of a G and the interrogation position will cause the incorporation for a C, having, e.g., a second colorimetric label.
  • the presence of a first nucleotide at the nucleotide will result in ligation of the detection reagent to a second nucleic acid.
  • a third nucleic acid can be hybridized to the target nucleic acid sufficiently close to the interrogation site that if the third nucleic acid has an exact match at the interrogation site it will be ligated to the detection reagent. Detection of the ligation product, or its absence, is indicative of the identity of the nucleotide at the interrogation site and thus allows detection of the mutation.
  • binding of the detection reagent to the mutant or reference sequence can be followed by a moiety, e.g., a label, associated with the detection reagent, e.g., a radioactive or enzymatic label.
  • the label comprises a quenching agent and a signaling agent and hybridization results in altering the distance between those two elements, e.g., increasing the distance and un-quenching the signaling agent.
  • the detection reagent can include a moiety that allows separation from other components of a reaction mixture.
  • binding allows cleavage of the bound detection reagent, e.g., by an enzyme, e.g., by the nuclease activity of the DNA polymerase or by a restriction enzyme.
  • the cleavage can be detected by the appearance or disappearance of a nucleic acid or by the separation of a quenching agent and a signaling agent associated with the detection reagent.
  • binding protects, or renders the target susceptible, to further chemical reaction, e.g., labeling or degradation, e.g., by restriction enzymes.
  • binding with the detection reagent allows capture separation or physical manipulation of the target nucleic acid to thereby allow for identification.
  • binding can result in a detectable localization of the detection reagent or target, e.g., binding could capture the target nucleic acid or displace a third nucleic acid.
  • Binding can allow for the extension or other size change in a component, e.g., the detection reagent, allowing distinction between mutant and reference sequences. Binding can allow for the production, e.g., by PCR, of an amplicon that distinguishes mutant from reference sequence.
  • the detection reagent, or the target binding site is between 5 and 500, 5 and 300, 5 and 250, 5 and 200, 5 and 150, 5 and 100, 5 and 50, 5 and 25, 5 and 20, 5 and 15, or 5 and 10 nucleotides in length. In one embodiment the detection reagent, or the target binding site, is between 10 and 500, 10 and 300, 10 and 250, 10 and 200, 10 and 150, 10 and 100, 10 and 50, 10 and 25, 10 and 20, or 10 and 15, nucleotides in length. In one embodiment the detection reagent, or the target binding site, is between 20 and 500, 20 and 300, 20 and 250, 20 and 200, 20 and 150, 20 and 100, 20 and 50, or 20 and 25 nucleotides in length. In one embodiment the detection reagent, or the target binding site, is sufficiently long to distinguish between mutant and reference sequences and is less than 100, 200, 300, 400, or 500 nucleotides in length.
  • the invention features purified or isolated preparations of a neoplastic or tumor cell nucleic acid, e.g., DNA, e.g., genomic DNA or cDNA, or RNA, containing an interrogation position described herein, useful for determining if a mutation disclosed herein is present.
  • the nucleic acid includes the interrogation position, and typically additional sequence on one or both sides of the interrogation position.
  • the nucleic acid can contain heterologous sequences, e.g., adaptor or priming sequences, typically attached to one or both terminus of the nucleic acid.
  • the nucleic acid also includes a label or other moiety, e.g., a moiety that allows separation or localization.
  • the nucleic acid is between 20 and 1,000, 30 and 900, 40 and 800, 50 and 700, 60 and 600, 70 and 500, 80 and 400, 90 and 300, or 100 and 200 nucleotides in length (with or without heterologous sequences). In one embodiment, the nucleic acid is between 40 and 1,000, 50 and 900, 60 and 800, 70 and 700, 80 and 600, 90 and 500, 100 and 400, 110 and 300, or 120 and 200 nucleotides in length (with or without heterologous sequences).
  • the nucleic acid is between 50 and 1,000, 50 and 900, 50 and 800, 50 and 700, 50 and 600, 50 and 500, 50 and 400, 50 and 300, or 50 and 200 nucleotides in length (with or without heterologous sequences).
  • the nucleic acid is of sufficient length to allow sequencing (e.g., by chemical sequencing or by determining a difference in T m between mutant and reference preparations) but is optionally less than 100, 200, 300, 400, or 500 nucleotides in length (with or without heterologous sequences).
  • Such preparations can be used to sequence nucleic acid from a sample, e.g., a neoplastic or tumor sample.
  • a sample e.g., a neoplastic or tumor sample.
  • the purified preparation is provided by in situ
  • the purified preparation is spatially distinct from other nucleic acids, e.g., other amplified nucleic acids, on a substrate.
  • the purified or isolated preparation of nucleic acid is derived from a hematological malignancy, e.g., CLL. Such preparations can be used to determine if a sample comprises mutant sequence, e.g., an alteration described herein.
  • the invention features, a method of determining the sequence of an interrogation position for an alteration described herein, comprising:
  • nucleic acid e.g., DNA, e.g., genomic DNA or cDNA, or RNA, containing an interrogation position described herein,
  • sequencing by a method that breaks or forms a chemical bond, e.g., a covalent or non- covalent chemical bond, e.g., in a detection reagent or a target sequence, the nucleic acid so as to determine the identity of the nucleotide at an interrogation position.
  • a method that breaks or forms a chemical bond e.g., a covalent or non- covalent chemical bond, e.g., in a detection reagent or a target sequence
  • the method allows determining if an alteration described herein is present.
  • sequencing comprises contacting the nucleic acid comprising an alteration described herein with a detection reagent described herein.
  • sequencing comprises determining a physical property, e.g., stability of a duplex form of the nucleic acid comprising an alteration described herein, e.g., T m , that can distinguish mutant from reference sequence.
  • a physical property e.g., stability of a duplex form of the nucleic acid comprising an alteration described herein, e.g., T m , that can distinguish mutant from reference sequence.
  • the nucleic acid comprising an alteration described herein is derived from a hematological malignancy, e.g., CLL.
  • the invention features, purified or isolated preparations of a nucleic acid, e.g., DNA, e.g., genomic DNA or cDNA, or RNA, containing an interrogation position described herein, useful for determining if a mutation disclosed herein is present, disposed in sequencing device, or a sample holder for use in such a device.
  • a nucleic acid e.g., DNA, e.g., genomic DNA or cDNA, or RNA
  • the nucleic acid is derived from a hematological malignancy, e.g., CLL.
  • the invention features, purified or isolated preparations of a nucleic acid, e.g., DNA, e.g., genomic DNA or cDNA, or RNA, containing an interrogation position described herein, useful for determining if a mutation disclosed herein is present, disposed in a device for determining a physical or chemical property, e.g., stability of a duplex, e.g., T m or a sample holder for use in such a device.
  • the device is a calorimeter.
  • the nucleic acid comprising an alteration described herein is derived from a hematological malignancy, e.g., CLL.
  • the detection reagents described herein can be used to determine if an alteration described herein is present in a sample.
  • the sample comprises a nucleic acid that is derived from a hematological malignancy, e.g., CLL.
  • the cell can be from a neoplastic or a tumor sample, e.g., a biopsy taken from the neoplasm or the tumor; from circulating tumor cells, e.g., from peripheral blood; or from a blood or plasma sample.
  • the nucleic acid is derived from e.g., a hematological malignancy, e.g., CLL.
  • the invention features a method of making a reaction mixture, comprising: combining a detection reagent, or purified or isolated preparation thereof, described herein with a target nucleic acid derived from e.g., a hematological malignancy, e.g., CLL, which comprises a sequence having an interrogation position for an alteration described herein.
  • a detection reagent or purified or isolated preparation thereof, described herein with a target nucleic acid derived from e.g., a hematological malignancy, e.g., CLL, which comprises a sequence having an interrogation position for an alteration described herein.
  • CLL hematological malignancy
  • the invention features a reaction mixture, comprising:
  • a detection reagent or purified or isolated preparation thereof, described herein; and a target nucleic acid derived from e.g., a cell derived from a hematological malignancy, e.g., a CLL cell, which comprises a sequence having an interrogation position for an alteration described herein.
  • reaction mixture or the method of making the reaction mixture:
  • the detection reagent comprises a nucleic acid, e.g., a DNA, RNA or mixed DNA/RNA, molecule which is complementary with a nucleic acid sequence on a target nucleic acid (the detection reagent binding site) wherein the detection reagent binding site is disposed in relationship to the interrogation position such that binding of the detection reagent to the detection reagent binding site allows differentiation of mutant and reference sequences for a mutation sequence or event described herein.
  • a nucleic acid e.g., a DNA, RNA or mixed DNA/RNA
  • the target nucleic acid sequence is derived from a hematological malignancy, e.g., CLL, as described herein.
  • the mutation is an alteration described herein, including: a substitution, e.g., a substitution described herein.
  • An alteration described herein can be distinguished from a reference, e.g., a non-mutant or wildtype sequence, by reaction with an enzyme that reacts differentially with the mutation and the reference. E.g., they can be distinguished by cleavage with a restriction enzyme that has differing activity for the mutant and reference sequences.
  • the invention includes a method of contacting a nucleic acid comprising an alteration described herein with such an enzyme and determining if a product of that cleavage which can distinguish mutant form reference sequence is present.
  • the inventions provides a purified preparation of a restriction enzyme cleavage product which can distinguish between mutant and reference sequence, wherein one end of the cleavage product is defined by an enzyme that cleaves differentially between mutant and reference sequence.
  • the cleavage product includes the interrogation position.
  • a mutant protein described herein can be distinguished from a reference, e.g., a non- mutant or wild-type protein, by reaction with a reagent, e.g., a substrate, e.g, a substrate for catalytic activity or functional activity, or an antibody, that reacts differentially with the mutant and reference protein.
  • a reagent e.g., a substrate, e.g, a substrate for catalytic activity or functional activity, or an antibody, that reacts differentially with the mutant and reference protein.
  • the invention includes a method of contacting a sample comprising a mutant protein described herein with such reagent and determining if the mutant protein is present in the sample.
  • the invention features, an antibody that can distinguish a mutant protein described herein, or a fragment thereof, from a reference, e.g., a non-mutant or wild type protein.
  • the invention features a method of making a reaction mixture comprising:
  • a detection reagent or purified or isolated preparation thereof, e.g., a substrate, e.g., a substrate for phosphorylation or other activity, or an antibody, described herein with a target protein derived from e.g., a cell derived from a hematological malignancy, e.g., a CLL cell, which comprises a sequence having an interrogation position for an alteration described herein.
  • a reaction mixture comprising:
  • a detection reagent or purified or isolated preparation thereof, e.g., a substrate, e.g., a substrate for phosphorylation or other activity, or an antibody, described herein; and
  • a target protein derived from a hematological malignancy e.g., CLL cell
  • reaction mixture or the method of making the reaction
  • the detection reagent comprises an antibody specific for a mutant protein described herein.
  • reaction mixture or the method of making the reaction mixture that includes a cell derived from a hematological malignancy, e.g., a CLL cell.
  • reaction mixture or the method of making the reaction
  • the mutation is an alteration described herein (e.g., a BRAF and/or KRAS mutation described herein).
  • the invention features a method, or assay, for screening for agents that modulate, e.g., inhibit, the expression or activity of a nucleic acid or polypeptide or protein comprising a mutation as described herein.
  • the method includes contacting a nucleic acid or polypeptide or protein comprising an alteration described herein, or a cell expressing a nucleic acid or polypeptide or protein comprising an alteration described herein, with a candidate agent; and detecting a change in a parameter associated with a nucleic acid or polypeptide or protein comprising an alteration described herein, e.g., a change in the expression or an activity of the nucleic acid or polypeptide or protein comprising an alteration described herein.
  • the method can, optionally, include comparing the treated parameter to a reference value, e.g., a control sample (e.g., comparing a parameter obtained from a sample with the candidate agent to a parameter obtained from a sample without the candidate agent).
  • a reference value e.g., a control sample
  • the candidate agent if a decrease in expression or activity of the nucleic acid or polypeptide or protein comprising an alteration described herein is detected, the candidate agent is identified as an inhibitor. In another embodiment, if an increase in expression or activity of the nucleic acid or polypeptide or protein comprising an alteration described herein is detected, the candidate agent is identified as an activator.
  • the contacting step is effected in a cell-free system, e.g., a cell lysate or in a reconstituted system.
  • the contacting step is effected in a cell in culture, e.g., a cell expressing an alteration described herein (e.g., a mammalian cell, a tumor cell or cell line, a recombinant cell).
  • the contacting step is effected in a cell in vivo (a - expressing cell present in a subject, e.g., an animal subject (e.g., an in vivo animal model).
  • Exemplary parameters evaluated include one or more of:
  • a change in binding activity e.g., direct binding of the candidate agent to a polypeptide comprising an alteration described herein; a binding competition between a known ligand and the candidate agent to a polypeptide comprising an alteration described herein;
  • a change in kinase activity e.g., phosphorylation levels of a polypeptide comprising an alteration described herein (e.g., an increased or decreased autophosphorylation); or a change in a target of a polypeptide comprising an alteration described herein
  • a change in kinase activity is detected by any of Western blot (e.g., using an antibody which binds to a polypeptide comprising an alteration described herein, mass spectrometry, immunoprecipitation, immunohistochemistry, immunomagnetic beads, among others;
  • a change in an activity of a cell containing a tumor cell or a recombinant cell e.g., a change in proliferation, morphology or maligancy of the cell;
  • a change in the malignancy present in an animal subject e.g., number of lymphocytes, red cells, size of the liver, spleen; or
  • a change in a cell free assay in the presence of a candidate agent is evaluated. For example, an activity of a nucleic acid or polypeptide or protein comprising an alteration described herein, or interaction of a nucleic acid or polypeptide or protein comprising an alteration described herein with a downstream ligand can be detected.
  • the polypeptide or protein comprising an alteration described herein is contacted with a ligand, e.g., in solution, and a candidate agent is monitored for an ability to modulate, e.g., inhibit, an interaction, e.g., binding, between the nucleic acid or polypeptide or protein comprising an alteration described herein and the ligand.
  • a change in an activity of a cell is detected in a cell in culture, e.g., a cell expressing a mutation (e.g., a mammalian cell, a tumor cell or cell line, a recombinant cell).
  • the cell is a recombinant cell that is modified to express a nucleic acid comprising an alteration described herein, e.g., is a recombinant cell transfected with a nucleic acid comprising an alteration described herein.
  • the transfected cell can show a change in response to the expressed mutation, e.g., increased proliferation, changes in morphology, increased tumorigenicity, and/or acquired a transformed phenotype.
  • a change in any of the activities of the cell e.g., the
  • recombinant cell in the presence of the candidate agent can be detected.
  • a decrease in one or more of: proliferation, tumorigenicity, transformed morphology, in the presence of the candidate agent can be indicative of an inhibitor of a nucleic acid or polypeptide or protein comprising an alteration described herein.
  • a change in binding activity or phosphorylation as described herein is detected.
  • a change in a cancer present in an animal subject is detected.
  • the animal model contains the hematological malignancy animal or a xenograft comprising cells expressing a nucleic acid or polypeptide or protein comprising an alteration described herein.
  • the candidate agent can be administered to the animal subject and a change in the hematologic cells is detected.
  • the change in the hematologic cells includes one or more of: a change in the level of lymphocytes in the blood; a change in the size of lymph nodes; a change in the size of the liver or spleen; a change in the number of red blood cells, is evaluated.
  • a decrease in one or more of the above, or an increased survival is indicative that the candidate agent is an inhibitor.
  • a change in expression of a nucleic acid or polypeptide or protein comprising an alteration described herein can be monitored by detecting the nucleic acid or protein levels, e.g., using the methods described herein.
  • the screening methods described herein can be repeated and/or combined.
  • a candidate agent that is evaluated in a cell-free or cell-based described herein can be further tested in an animal subject.
  • the candidate agent is a small molecule compound, e.g., a kinase inhibitor, a nucleic acid (e.g., antisense, siRNA, aptamer, ribozymes, microRNA), an antibody molecule (e.g., a full antibody or antigen binding fragment thereof that binds to the mutation).
  • the candidate agent can be obtained from a library (e.g., a commercial library of kinase inhibitors) or rationally designed.
  • the method, or assay includes providing a step based on proximity- dependent signal generation, e.g., a two-hybrid assay that includes a first mutation protein (e.g., a mutated protein), and a second mutated protein (e.g., a ligand), contacting the two-hybrid assay with a test compound, under conditions wherein said two hybrid assay detects a change in the formation and/or stability of the complex, e.g., the formation of the complex initiates transcription activation of a reporter gene.
  • a two-hybrid assay that includes a first mutation protein (e.g., a mutated protein), and a second mutated protein (e.g., a ligand)
  • a test compound under conditions wherein said two hybrid assay detects a change in the formation and/or stability of the complex, e.g., the formation of the complex initiates transcription activation of a reporter gene.
  • the three-dimensional structure of the active site of a polypeptide or protein comprising an alteration described herein is determined by crystallizing the complex formed by the polypeptide or protein and a known inhibitor. Rational drug design is then used to identify new test agents by making alterations in the structure of a known inhibitor or by designing small molecule compounds that bind to the active site of the polypeptide or protein.
  • the candidate agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R.N. et al.
  • FET fluorescence energy transfer
  • determining the ability of the mutated protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705).
  • BiA Biomolecular Interaction Analysis
  • Example 1 Extensive high-depth sequencing of longitudinal CLL samples identifies frequent mutations in MAP kinase signaling and novel mutations activating Notch and Beta-Catenin.
  • Genomic DNA and total RNA was isolated from 59 CLL samples (including 29 CLL paired patients sampled at the time of initial presentation when no clinical indication for therapy was met and at a later time point of disease progression requiring therapy). The median time between samples was 2.07 years (range 0.08-4.95 years)).
  • Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 272 genes frequently rearranged for RNA-seq.
  • This targeted sequencing approach detected at least one clonal somatic mutation in 95% of CLL patients including 144 individual mutations, 3 functional rearrangements, and 1 homozygous deletion. Consistent with prior reports, mutations in NOTCH1 (25%), SF3B1 (19%), and TP53 (14%) were amongst the most commonly mutated genes in this cohort. Of note, activating mutations in KRAS (15%) and BRAF (10%) were also amongst the most commonly mutated genes targeted by somatic mutations in CLL (FIG. 1A). Taken together, mutations resulting in constitutive MAP kinase signaling occurred in 36% of all patients, second only to mutations activating Notch signaling which occurred in 40% of patients.
  • SPEN is a nuclear receptor transcription factor which blocks the differentiation of precursor B cells into marginal zone B cells through interactions factor which blocks the differentiation of precursor B cells into marginal zone B cells through interactions with RBP-J. Also identified as recurrent were mutations in FAT3, an inhibitor of ⁇ -catenin signaling. In addition, sequencing identified a novel fusion event in BIRC3 fusing BIRC3 to LRRC40 as well as a truncating mutation in one patient in BRCA2 at disease progression.

Abstract

Methods and compositions for treating and detecting hematological malignancies are disclosed.

Description

METHODS OF TREATING HEMATOLOGICAL MALIGNANCIES
FIELD OF THE INVENTION
The invention relates to methods and compositions for treating and detecting
hematological malignancies.
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of U.S. Provisional Application No.
61/898,818, filed November 1, 2013, the contents of which are hereby incorporated by reference in their entirety.
SEQUENCE LISTING
The present application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on October 27, 2014, is named "Sequence_Listing_ST25.txt" and is 24 KB in size.
BACKGROUND OF THE INVENTION
Hematological malignancies are cancers that affect the blood, bone marrow, and lymph nodes. Hematological malignancies include myelomas, leukemias, and lymphomas. Myelomas are cancers of the plasma cells; lymphomas are cancers that start in the lymph system, mainly the lymph nodes; and leukemias are cancers of the bone marrow and blood and include both lymphocytic and myelogenous leukemias. The most prevalent hematological malignancies include: chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (AML), Hodgkin's lymphomas, and Non-hodgkin's lymphomas.
Chronic lymphocytic leukemia (CLL), also known as B cell-CLL, is a common type of adulthood leukemia, and is characterized by the clonal proliferation and accumulation of mature B lymphocytes (Eichhorst B. et al. Ann Oncol (2011) 22 (6): vi50-vi54; Smolewski P. et al. ISRN Oncology Volume 2013 (2013), Article ID 740615). CLL progression is commonly
characterized by staging systems including, for example, the Rai 4- stage system and the Binet classification system (National Cancer Institute. "Chronic Lymphocytic Leukemia (PDQ) Treatment: Stage Information". Archived from the original on 17 October 2007; Smolewski, supra). Treatment of CLL is typically stage dependent, with early stage CLL monitored without treatment. Once CLL progresses chemotherapeutic and biologic agents are commonly employed. Current treatment regimens include, for example, fludarabine, cyclochosphamide, and rituximab; chlorambucil; and cyclophosphamide, vincristine, doxorubicin, and prednisone. Bone marrow or stem cell transplantation may also be used in younger patients with advanced or high risk CLL. Despite advances in treatment options a number of CLL patients suffer from relapsed or refractory CLL (Maddocks K et al. Journal of Hematology & Oncology 2009, 2:29). Therefore, the need exists for novel therapeutic approaches for treating hematological malignancies, including CLL.
SUMMARY OF THE INVENTION
The invention is based, at least in part, on the discovery of alterations, e.g., activating mutations, not previously identified in hematological malignancies, such as Chronic
Lymphocytic Leukemia (CLL). In certain embodiments, the alteration includes an activating mutation in a MAP kinase (Mitogen-activated protein kinase or MAPK) pathway gene or gene product, e.g., an activating mutation in BRAF and/or KRAS (also referred to herein as "B-Raf ' and "K-Ras," respectively). In one embodiment, one or more mutations resulting in constitutive MAPK signaling were identified at a high frequency in CLL (e.g., about 36% of the 59 CLL samples characterized, of which 10% were activating mutation in BRAF). The presence of activating mutations in the MAPK pathway in CLL provides novel therapeutic approaches for CLL that include MAPK pathway inhibitors. Therefore, the invention provides, at least in part, methods for treating a hematological malignancy, e.g., CLL, as well as methods and reagents for identifying, assessing or detecting an alteration as described herein, in such malignancies.
Accordingly, in one aspect, the invention features, a method of treating a subject having a hematological malignancy. In one embodiment, a method of treating a subject having a CLL is disclosed. The method includes administering to the subject an effective amount of an agent (e.g., a therapeutic agent) that targets and/or inhibits a MAPK pathway gene or gene product (e.g., a MAPK pathway inhibitor as described herein), thereby treating the subject. In one embodiment, the method further includes acquiring knowledge of the presence (or absence) of an alteration in a MAPK pathway gene or gene product. In another embodiment, the method further includes identifying the subject, or a cancer sample (e.g., a CLL sample) from the subject, as having the presence (or absence) of an alteration in a MAPK pathway gene or gene product. In certain embodiments, the alteration in the MAPK pathway gene or gene product is an alteration (e.g., one or more oncogenic alterations) of a RAF (e.g., one or more of A-Raf, B- Raf (BRAF) or C-Raf), a RAS (e.g., one or more of H-Ras, N-Ras or K-Ras (KRAS)), and/or MEK (MAP/ERK kinase) gene or gene product, or results in increased activity, e.g., constitutive action of the MAPK pathway gene or gene product.
In certain embodiments, the alteration in the MAPK pathway gene or gene product is a mutation in a Raf gene or gene product, e.g., a mutation in one or more of the glycine-rich P loop of the N lobe, the activation segment and/or the flanking region(s) of BRAF. In one
embodiment, the mutation is chosen from a mutation in codon 464, 465, 466, 468, 469, 580, 594, 595, 596, 597, 599, 600, 601 or 727, of BRAF. In certain embodiments, the alteration in BRAF is not located at codon 600 (e.g., V600). Exemplary alterations in the BRAF gene or gene product at a position other than BRAF at position 600, include but are not limited to, R461I, I462S, G463E, G463V, G464E, G464R, G464V, G465A, G465E, G465V, G466A, G466E, G466R, G466V, G468A, G468E, F468C, G469A, G469E, G469R, G469R, G469S, G469V, N580S, E585K, D593V, D594G, D594V, F594L, F595L, G595R, L596V, G596R, L597Q, L597R, L597S, L597V, T598I, T599I, V599D, V599E, V599K, V599R, K601E, K601N, and A727V. In other embodiments, the alteration in the BRAF gene or gene product is at a position 600 in BRAF, e.g., is a mutation chosen from V600E, V600K, V600L, or V600R.
In other embodiments, the alteration in the MAPK pathway gene or gene product is a mutation in a Ras gene or gene product, e.g., a mutation in K-Ras, that includes, for example, a mutation in codon 12, 13 and/or 61, including but not limited to, G12A, G12N, G12R, G12C, G12S, G12V, G13N and Q61R. Non-limiting examples of alterations in a KRAS gene include, but are not limited to, G12C, G12R, G12D, G12A, G12S, G12V, G13D, G13S, G13C, G13V, Q61H, Q61R, Q61P, Q61L, Q61K, Q61E, A59T and G12F.
In certain embodiments, the presence of the MAPK pathway alteration (e.g., the BRAF and/or KRAS alteration) in the subject is indicative that the subject is likely to respond to the agent, e.g., the therapeutic agent (e.g., the MAPK pathway inhibitor). In yet other embodiments, the agent, e.g., the therapeutic agent (e.g., the MAPK pathway inhibitor) is administered responsive to a determination of the presence of the MAPK pathway alteration (e.g., the BRAF and/or KRAS alteration) in the subject, or the cancer or tumor sample from the subject.
Cancers
In certain embodiments the cancer is a hematological malignancy. In one embodiment, the hematological malignancy is chosen from a cancer that affects one or more of the blood, the bone marrow, or the lymph nodes. The hematological malignancy can be chosen from a myeloma, a leukemia, or a lymphoma. In certain embodiments, the hematological malignancy is chosen from a chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (AML), Hodgkin' s lymphoma, or Non-hodgkin' s lymphoma.
In one embodiment, the hematological malignancy is a CLL, e.g., a refractory CLL or a relapsed CLL. The CLL can be at any stage or risk group of CLL defined according to suitable CLL classification systems known to those of skill in the art. For example, the CLL can be chosen from a stage 0, 1, II, III, or IV, wherein stage 0 CLL is characterized by an elevated level of lymphocytes in the blood, but there are no other detectable symptoms of leukemia; stage I CLL is characterized by an elevated level of lymphocytes in the blood and enlarged size of lymph nodes; stage II CLL is characterized by an elevated level of lymphocytes in the blood, the liver or spleen is larger than normal, and the lymph nodes may be larger than normal; stage III CLL is characterized by an elevated level of lymphocytes in the blood, reduced number of red blood cells, and the lymph nodes, liver, or spleen may be larger than normal, and stage IV CLL is characterized by an elevated level of lymphocytes in the blood, reduced platelets, the lymph nodes, liver, or spleen may be larger than normal, and there may be a reduced number of red blood cells. The detected levels or tissue sizes can be compared to a reference value, e.g., a normal or a control value. Other classifications for CLL can be used, for example, the Rai Scale (Rai K et al. Blood Volume 46, Issue 2, 1975, Pages 219-234) and the Binet Scale as described in more detail below.
In certain embodiments, the CLL is a low risk disease, intermediate risk disease, or high risk disease on the modified Rai Scale, wherein low risk is characterized by lymphocytes less than 15 x lOg/1; intermediate risk is characterized by lymphocytes less than 15 x lOg/1 and hepato- or splenomegaly; and high risk is characterized as Anemia (Hb < 1 1 g/dL) or
thrombocytopenia (platelets < 100 xl09/L) (Hallek M et al. Blood June 15, 2008 vol. I l l no. 12 5446-5456).
In other embodiments, the hematological malignancy, e.g., CLL, comprises, or is identified or determined as having, an alteration in a MAPK pathway gene or gene product, e.g., an alteration in BRAF and/or KRAS alteration as described herein.
In certain embodiments, the alteration in a MAPK pathway gene or gene product results in increased activity of a BRAF and/or KRAS gene product (e.g., a BRAF and/or KRAS protein), compared to the wild-type activity protein. For example, the alteration can result in one or more of: increased kinase activity of a BRAF protein, constitutive activation of BRAF and/or KRAS, increased activity or phosphorylation of a downstream component of the MAPK pathway, including, but not limited to, MEK (MAP/ERK kinase) (e.g., MEKl and/or MEK2). In one embodiment, the alteration is, or comprises, a mutation (e.g., a somatic mutation), e.g., a substitution (e.g., a base substitution), a deletion, an insertion, or a missense mutation.
In certain embodiments, the alteration in the MAPK pathway gene or gene product is a mutation in a Raf gene or gene product, e.g., a mutation in one or more of the glycine-rich P loop of the N lobe, the activation segment and/or the flanking region(s) of BRAF. In one
embodiment, the mutation is chosen from a mutation in codon 464, 465, 466, 468, 469, 580, 594, 595, 596, 597, 599, 600, 601 or 727, of BRAF. In certain embodiments, the alteration in BRAF is not located at codon 600, e.g., V600. Exemplary alterations in the BRAF gene or gene product at a position other than BRAF at position 600, include but are not limited to, R461I, I462S, G463E, G463V, G464E, G464R, G464V, G465A, G465E, G465V, G466A, G466E, G466R, G466V, G468A, G468E, F468C, G469A, G469E, G469R, G469R, G469S, G469V, N580S, E585K, D593V, D594G, D594V, F594L, F595L, G595R, L596V, G596R, L597Q, L597R, L597S, L597V, T598I, T599I, V599D, V599E, V599K, V599R, K601E, K601N, and A727V. In other embodiments, the alteration in the BRAF gene or gene product is at a position at position 600 in BRAF, e.g., is a mutation chosen from V600E, V600K, V600L, or V600R.
In other embodiments, the alteration in the MAPK pathway gene or gene product is a mutation in a Ras gene or gene product a mutation in K-Ras, that includes, for example, a mutation in codon 12, 13 and/or 61, including but not limited to, G12A, G12N, G12R, G12C, G12S, G12V, G13N and Q61R. Non-limiting examples of alterations in a KRAS gene include, but are not limited to, G12C, G12R, G12D, G12A, G12S, G12V, G13D, G13S, G13C, G13V, Q61H, Q61R, Q61P, Q61L, Q61K, Q61E, A59T and G12F.
Subjects
In certain embodiments, the subject has an alteration in a MAPK pathway gene or gene product. In one embodiment, the subject has a hematological malignancy, e.g., CLL, that comprises an alteration described herein. In other embodiments, the subject is identified, or has been previously identified, as having a cancer (e.g., a hematological malignancy, e.g., CLL) comprising an alteration in a MAPK pathway gene or gene product as described herein. In certain embodiments, the alteration in the MAPK pathway gene or gene product is an alteration (e.g., one or more oncogenic alterations) of a RAF (e.g., one or more of A-Raf, B-Raf (BRAF) or C-Raf), a RAS (e.g., one or more of H-Ras, N-Ras or K-Ras), and/or MEK (MAP/ERK kinase) gene or gene product, or results in increased activity, e.g., constitutive action of the MAPK pathway gene or gene product.
In one embodiment, the subject is a human. In one embodiment, the subject has, or is at risk of having a cancer (e.g., a hematological malignancy, e.g., CLL) as described herein at any stage of disease, e.g., any stage described herein, relapsed, recurrent, or refractory. In other embodiments, the subject is a cancer patient, e.g., a patient having a hematological malignancy, e.g., CLL, as described herein.
In one embodiment, the subject is undergoing or has undergone treatment with a different therapeutic agent or therapeutic modality (e.g., non-MAPK pathway inhibitor). In one embodiment, the different therapeutic agent or therapeutic modality is a chemotherapy, an immunotherapy, or a surgical procedure. In one embodiment, the different therapeutic agent or therapeutic modality comprises one or more of: a chemotherapeutic agent (e.g., fludarabine, cyclophosphamide, doxorubicin, vincristine, chlorambucil, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, 5-azacytidine, pemetrexed disodium), an EGFR inhibitor (e.g., erlotinib), BTK inhibitor (e.g., PCI-32765), CD20 targeting agent (e.g., rituximab,
ofatumumab, RO5072759, LFB-R603), CD52 targeting agent (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, Bcl-2 inhibitor (e.g., ABT-263), AT-101, immunotoxin (e.g., CAT- 8015, anti-Tac(Fv)-PE38 (LMB-2)), CD37 targeting agent (e.g., TRU-016), radioimmunotherapy (e.g., 131-tositumomab), hydroxychloroquine, perifosine, SRC inhibitor (e.g., dasatinib), thalidomide, a steroid, prednisone, chlorambucil, retinoid (e.g., fenretinide), MDM2 antagonist (e.g., RO5045337), plerixafor, Aurora kinase inhibitor (e.g., MLN8237, TAK-901), proteasome inhibitor (e.g., bortezomib), CD19 targeting agent (e.g., MEDI-551, MOR208), JAK-2 inhibitor (e.g., INCBO 18424), hypoxia-activated prodrug (e.g., TH-302), paclitaxel or a paclitaxel agent, AKT inhibitor (e.g., MK2206), HMG-CoA inhibitor (e.g., simvastatin), GNKG186, flebogamma, gamimune, gammagard S/D, gammaplex, panglobulin NF, polygamy S/D, privifen, sandoglobulin, radiation therapy, bone marrow transplantation, stem cell transplantation, immunotherapy, or a combination thereof.
In one embodiment, responsive to the determination of the presence of the alteration described herein, the different therapeutic agent or therapeutic modality is discontinued. In yet other embodiments, the subject has been identified as being likely or unlikely to respond to the different therapeutic agent or therapeutic modality.
In certain embodiments, the subject has participated previously in a clinical trial, e.g., a clinical trial for a different (e.g., non-MAPK pathway inhibitor) therapeutic agent or therapeutic modality. In other embodiments, the subject is a cancer patient who has participated in a clinical trial, e.g., a clinical trial for a different (e.g., non-MAPK pathway inhibitor) therapeutic agent or therapeutic modality.
Agents
In certain embodiments, the agent (e.g., the therapeutic agent) used in the methods described herein, targets and/or inhibits a MAPK pathway gene or gene product (e.g., is a MAPK pathway inhibitor as described herein). In one embodiment, the agent inhibits a RAF (e.g., one or more of A-Raf, B-Raf or C-Raf), a RAS (e.g., one or more of H-Ras, N-Ras or K-Ras), and/or MEK (MAP/ERK kinase) gene or gene product. In one embodiment, the agent inhibits BRAF and/or MEK. In one embodiment, the agent is a reversible or an irreversible inhibitor.
In one embodiment, the agent is a kinase inhibitor. In one embodiment, the kinase inhibitor is chosen from: a multi- specific kinase inhibitor, a pan inhibitor, a serine/threonine kinase inhibitor, and/or an inhibitor that is selective for BRAF, KRAS or a MAPK downstream protein. In one embodiment, the kinase inhibitor is a small molecule inhibitor. In one embodiment, the agent is a BRAF inhibitor. In certain embodiments, the BRAF inhibitor is chosen from: Vemurafenib (PLX4032, RG7204, R05185426), Sorafenib Tosylate (Bay 43-9006, Nexavar), PLX4720, GDC-0879, RAF265 (CHIR-265), MLN2480 (BIIB-024), PF-04880594, GW5074, CEP-32496, Dabrafenib (GSK2118436), AZ628, SB590885, Raf265 derivative, Regorafenib (BAY 73-4506, Fluoro-Sorafenib), DP-4978, DP-2514, DP-3346, ARQ736, XL281, RG7256, LGX818, PLX3603, trematinib, and/or ZM 336372.
In some embodiments, the BRAF inhibitor is Vemurafenib (also known as PLX4032, RG7204, R05185426). In one embodiment, Vemurafenib has the chemical name: N-(3-{ [5-(4- chlorophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]carbonyl}-2,4-difluorophenyl)propane- l- sulfonamide; and has the following structure:
Figure imgf000009_0001
In some embodiments, the BRAF inhibitor is Sorafenib Tosylate (also known as Bay 43- 9006, Nexavar). In one embodiment, Sorafenib has the chemical name: 2-Pyridinecarboxamide, 4- [4- [ [[ [4-chloro-3- (trifluoromethyl)phenyl] amino] carbonyl] amino]phenoxy] -N-methyl- , 4- methylbenzenesulfonate (1 : 1); and has the following structure:
Figure imgf000009_0002
G7H5Q3S
In some embodiments, the BRAF inhibitor is PLX4720. In one embodiment, PLX4720 has the chemical name: N-(3-(5-chloro- lH-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4- difluorophenyl)propane- l-sulfonamide; and has the following structure:
Figure imgf000010_0001
In some embodiments, the BRAF inhibitor is GDC-0879. In one embodiment, GDC-0879 has the chemical name: (E)-5-(l-(2-hydroxyethyl)-3-(pyridin-4-yl)-lH-pyrazol-4-yl)-2,3- dihydroinden-l-one oxime; and has the following structure:
Figure imgf000010_0002
In some embodiments, the BRAF inhibitor is RAF265 (CHIR-265). In one embodiment, RAF265 has the chemical name: l-methyl-5-(2-(5-(trifluoromethyl)-lH-imidazol-2-yl)pyridin-4- yloxy)-N-(4-(trifluoromethyl)phenyl)-lH-benzo[d]imidazol-2-amine; and has the following structure:
Figure imgf000010_0003
In some embodiments, the BRAF inhibitor is Raf265 derivative. In one embodiment, Raf265 derivative has the following structure:
Figure imgf000011_0001
In some embodiments, the BRAF inhibitor is MLN2480 (BIIB-024). In one embodiment, MLN2480 is a pan-Raf inhibitor; has the chemical name: 4-Pyrimidinecarboxamide, 6-amino-5- chloro-N-[(lR)-l-[5-[[[5-chloro-4-(trifluoromethyl)-2-pyridinyl]arnino]carbonyl]-2- thiazolyl] ethyl]-; and has the following structure:
Figure imgf000011_0002
In some embodiments, the BRAF inhibitor is PF-04880594. In one embodiment, PF- 04880594 has the chemical name: Propanenitrile, 3-[[4-[l-(2,2-difluoroethyl)-3-(lH-pyrrolo[ b]pyridin-5-yl)-lH-pyrazol-4-yl -2-pyrimidinyl]amino]-; and has the following structure:
Figure imgf000011_0003
In some embodiments, the BRAF inhibitor is GW5074. In one embodiment, GW5074 has the chemical name: 2H-Indol-2-one, 3-[(3,5-dibromo-4-hydroxyphenyl)methylene]-l,3-dihydro- 5-iodo-; and has the following structure:
Figure imgf000012_0001
In some embodiments, the BRAF inhibitor is CEP-32496. In one embodiment, CEP- 32496 has the chemical name: Urea, N-[3-[(6,7-dimethoxy-4-quinazolinyl)oxy]phenyl]-N-[5- (2,2,2-trifluoro-l,l-dimethylethyl)-3-isoxazolyl]-; and has the following structure:
Figure imgf000012_0002
In some embodiments, the BRAF inhibitor is Dabrafenib (GSK2118436). In one embodiment, Dabrafenib has the chemical name: N-(3-(5-(2-aminopyrimidin-4-yl)-2-tert- butylthiazol-4-yl)-2-fluorophenyl)-2,6-difluorobenzenesulfonamide; and has the following structure:
Figure imgf000012_0003
In some embodiments, the BRAF inhibitor is AZ628. In one embodiment, AZ628 has the chemical name: 3-(2-cyanopropan-2-yl)-N-(4-methyl-3-(3-methyl-4-oxo-3,4-dihydroquinazolin- 6-ylamino)phenyl)benzamide; and has the following structure:
Figure imgf000013_0001
In some embodiments, the BRAF inhibitor is SB590885. In one embodiment, SB590885 has the chemical name: (E)-5-(2-(4-(2-(dimethylamino)ethoxy)phenyl)-4-(pyridin-4-yl)-lH- imidazol-5-yl)-2,3-dihydroinden-l-one oxime; and has the following structure:
Figure imgf000013_0002
In some embodiments, the BRAF inhibitor is Regorafenib (also known as BAY 73-4506, Fluoro-Sorafenib). In one embodiment, Regorafenib has the chemical name: l-(4-chloro-3- (trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(methylcarbamoyl)pyridin-4-yloxy)phenyl)urea; and has the following structure:
Figure imgf000013_0003
In some embodiments, the BRAF inhibitor is ZM 336372. In one embodiment, ZM 336372 has the chemical name: Benzamide, 3-(dimethylamino)-N-[3-[(4- hydroxybenzoyl)amino]-4-methylphenyl]-; and has the following structure:
Figure imgf000014_0001
In some embodiments, the BRAF inhibitor is LGX818. In one embodiment, LGX818 has the following chemical name Methyl [(2S)- l-{ [4-(3-{ 5-chloro-2-fluoro-3- [(methylsulfonyl)amino]phenyl}-l-isopropyl- lH-pyrazol-4-yl)-2-pyrimidinyl]amino}-2- propanyl]carbamate; and the followin
Figure imgf000014_0002
In some embodiments, the BRAF inhibitor is DP-4978.
In some embodiments, the BRAF inhibitor is DP-2514.
In some embodiments, the BRAF inhibitor is DP-3346.
In some embodiments, the BRAF inhibitor is XL281.
In some embodiments, the BRAF inhibitor is RG7256 (PLX3603).
In other embodiments, the BRAF inhibitor is an antibody molecule, e.g., an anti-BRAF antibody molecule (e.g., a monoclonal or a bispecific antibody), or a conjugate thereof (e.g., an antibody to BRAF conjugated to a cytotoxic agent (e.g., mertansine DM1)).
In other embodiments, the agent is chosen from a nucleic acid molecule (e.g., an antisense molecule, a ribozyme, a double stranded RNA, or a triple helix molecule) that hybridizes to and/or inhibits a BRAF nucleic acid, e.g., a BRAF nucleic acid encoding the alteration, or a transcription regulatory region that blocks or reduces mRNA expression of the alteration.
In yet other embodiments, the agent (e.g., the therapeutic agent) used in the methods targets and/or inhibits MEK (mitogen activated protein kinase kinase or M AP/ERK Kinase) (e.g., a MEK1 and/or a MEK2 gene or gene product). In one embodiment, the agent binds to and/or inhibits MEK. In one embodiment, the agent is a reversible or an irreversible MEK inhibitor.
In some embodiments, the agent is a MEK inhibitor. A MEK inhibitor can include an agent that inhibits MEK1 and/or MEK2. In some embodiments, the MEK inhibitor is chosen from: ARRY-162 (MEK162), Trametinib (GSK1120212), Selumetinib (AZD6244, ARRY142886), XL518 (GDC-0973), CI-1040 (PDl 84352), PD035901, U0126-EtOH, PD198306, PD98059, BIX 02189, TAK-733, Honokiol, AZD8330 (ARRY-424704), PD318088, BIX 02188, AS703026 (Pimasertib), RG7167, E6201 ;
MSC2015103, MSC1936369, WX554 and/or SL327.
In one embodiment, the inhibitor MEK is ARRY-162 (MEK 162). ARRY- 162 is a potent, orally bioavailable and non-ATP competitive inhibitor of MEKl/2 (IC50 = 12 nM) and cellular pERK (IC50 = 11 nM). It shows ex vivo inhibition of cytokine production such as IL- 1, TNF and IL-6 in clinical trials. ARRY- 162 has the chemical name: 5-((4-bromo-2-fluorophenyl)amino)-4- fluoro-N-(2-hydroxyethoxy)-l-methyl-lH-benzo[d]imidazole-6-carboxamide; and has the following structure:
Figure imgf000015_0001
ARRY-162 Chemical Structure
Molecular Weight: 441.22681.
In one embodiment, the MEK inhibitor is Trametinib (GSK1120212). Trametinib is a highly specific and potent MEKl/2 inhibitor with IC50 of 0.92 nM/1.8 Nm. Trametinib does not inhibit the kinase activities of c-Raf, B-Raf, ERKl/2. Trametinib has the chemical name: N-(3- { 3-Cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7- tetrahydropyrido[4,3-d]pyrimidin- l(2H)-yl}phenyl)acetamide; and has the following structure:
Figure imgf000016_0001
Trametinib Chemical Structure
Molecular Weight: 615.39.
In one embodiment, the MEK inhibitor is Selumetinib (AZD6244, ARRY 142886). Selumetinib is a potent, highly selective MEK1 inhibitor with IC50 of 14 nM, also inhibits ERKl/2 phosphorylation with IC50 of 10 nM. Selumetinib has the chemical name: 6-(4-bromo- 2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide; and has the following structure:
Figure imgf000016_0002
Selumetinib Chemical Structure
Molecular Weight: 457.68.
In one embodiment, the MEK inhibitor is XL518 (GDC-0973). XL518 a potent, selective, orally bioavailable inhibitor of MEK1. XL518has the chemical name: [3,4-difluoro-2-[(2-iluoro-4- iodophenyl)amino]phenyl][3-hydroxy-3-[(2S)-2-piperidinyl]-l-azetidinyl]methanone; and has the following structure:
Figure imgf000017_0001
XL518 Chemical Structure
Molecular Weight: 531.31.
In one embodiment, the MEK inhibitor is CI-1040 (PD 184352). CI-1040 is an ATP noncompetitive MEKl/2 inhibitor with IC50 of about 17 nM, 100-fold more selective for MEKl/2 than MEK5. CI-1040 has the chemical name: 2-(2-chloro-4-iodophenylamino)-N- (cyclopropylmethoxy)-3,4-difluorobenzamide; and has the following structure:
Figure imgf000017_0002
CI-1040 Chemical Structure
Molecular Weight: 478.67.
In one embodiment, the MEK inhibitor is PD035901. PD0325901 is selective and non ATP- competitive MEK inhibitor with IC50 of about 0.33 nM, roughly 500-fold more potent than CI-1040 on phosphorylation of ERKl and ERK2. PD035901 has the chemical name: (R)-N-(2,3-dihydroxypropoxy)- 3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide; and has the following structure:
Figure imgf000017_0003
PD035901 Chemical Structure
Molecular Weight: 482.19. In one embodiment, the MEK inhibitor is U0126-EtOH. U0126-EtOH is a highly selective inhibitor of MEK1/2 with IC50 of about 0.07 μΜ/0.06 μΜ, 100-fold higher affinity for ΔΝ3- S218E/S222D MEK than PD098059. PD098059 has the chemical name: (2Z,3Z)-2,3-bis(amino(2- aminophenylthio)methylene)succinonitrile,ethanol; and has the following structure:
Figure imgf000018_0001
EtO H
U0126-EtOH Chemical Structure
Molecular Weight: 426.56.
In one embodiment, the MEK inhibitor is PD198306. PD198306 is a cell-permeable and highly selective MEK inhibitor with IC50 of 8 nM. PD198306 has the chemical name: Benzamide, N- (cyclopropylmethoxy)-3,4,5-trifluoro-2-[(4-iodo-2-methylphenyl)amino]-; and has the following structure:
Figure imgf000018_0002
PD198306 Chemical Structure
Molecular Weight: 476.23.
In one embodiment, the MEK inhibitor is PD98059. PD98059 is a non-ATP competitive MEK inhibitor with IC50 of 2 μΜ, specifically inhibits MEK-1 -mediated activation of MAPK. PD98059 does not directly inhibit ERKl or ERK2. PD98059 has the chemical name: 2-(2-amino-3-methoxyphenyl)-4H- chromen-4-one; and has the following structure:
Figure imgf000019_0001
PD98059 Chemical Structure
Molecular Weight: 267.28.
In one embodiment, the MEK inhibitor is BIX 02189. BIX 02189 is a selective inhibitor of MEK5 with IC50 of 1.5 nM, also inhibits ERK5 catalytic activity with IC50 of 810 nM. BIX 02189 does not inhibit closely related kinases MEKl, MEK2, ERK2, and JNK2. BIX 02189 has the chemical name: (Z)-3-((3-((dimethylamino)methyl)phenylamino) (phenyl)methylene)-N,N-dimethyl-2-oxoindoline-6- carboxamide; and has the following structure:
Figure imgf000019_0002
BIX 02189 Chemical Structure
Molecular Weight: 440.54.
In one embodiment, the MEK inhibitor is TAK-733. TAK-733 is a potent and selective MEK allosteric site inhibitor for MEKl with IC50 of about 3.2 nM. TAK-733 is inactive to Abll, AKT3, c- RAF, CamKl, CDK2, c-Met. TAK-733 has the chemical name: (R)-3-(2,3-dihydroxypropyl)-6-fluoro-5- (2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione; and has the following structure:
Figure imgf000019_0003
TAK-733 Chemical Structure
Molecular Weight: 267.28.
In one embodiment, the MEK inhibitor is Honokiol. Honokiol is the active principle of magnolia extract that inhibits Akt-phosphorylation and promotes ERKl/2phosphorylation. Honokiol has the chemical name: 2-(4-hydroxy-3-prop-2-enyl-phenyl)- 4-prop-2-enyl-phenol; and has the following structure:
Figure imgf000020_0001
Honokiol Chemical Structure
Molecular Weight: 266.334.
In one embodiment, the MEK inhibitor is AZD8330 (ARRY-424704). AZD8330 is a novel, selective, non-ATP competitive MEK 1/2 inhibitor with IC50 of about 7 nM. AZD8330 has the chemical name: 2-(2-fluoro-4-iodophenylamino)-N-(2-hydroxyethoxy)-l,5-dimethyl-6-oxo-l,6-dihydropyridine-3- carboxamide; and has the following structure:
Figure imgf000020_0002
AZD8330 Chemical Structure
Molecular Weight: 461.23.
In one embodiment, the MEK inhibitor is PD318088. PD318088 is a non-ATP competitive allosteric MEKl/2 inhibitor. PD318088 has the chemical name: 5-bromo-N-(2,3- dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide; and has the following structure:
Figure imgf000021_0001
PD318088 Chemical Structure
Molecular Weight: 561.09.
In some embodiments, the MEK inhibitor is BIX 02188. ΒΓΧ02188 is a selective inhibitor of MEK5 with IC50 of about 4.3 nM, also inhibits ERK5 catalytic activity with IC50 of 810 nM. BIX 02188 does not significantly inhibit closely related kinases MEKl, MEK2, ERK2, and JNK2. ΒΓΧ02188 has the chemical name: (Z)-3-((3-((dimethylamino)methyl)phenylamino) (phenyl)methylene)-2-oxoindoline-6-carboxamide; and has the following structure:
Figure imgf000021_0002
ΒΓΧ02188 Chemical Structure
Molecular Weight: 426.51.
In one embodiment, the MEK inhibitor is AS703026 (Pimasertib). AS-703026 is a highly selective, potent, ATP non-competitive allosteric inhibitor of MEKl/2 with IC50 of about 0.005- 2 μΜ in MM cell lines. AS703026 has the chemical name: (S)-N-(2,3-dihydroxypropyl)-3-(2- fluoro-4-iodophenylamino)isonicotinamide; and has the following structure:
Figure imgf000021_0003
AS703026 Chemical Structure
Molecular Weight: 431.20.
In one embodiment, the MEK inhibitor is SL327. SL327 is a selective inhibitor for MEK1/2 with IC50 of about 0.18 μΜ/ 0.22 μΜ. SL327 has no activity towards Erkl, MKK3, MKK4, c-JUN, PKC, PKA, or CamKII. SL327 is capable of transport through the blood-brain barrier. SL327 has the chemical name: (Z)-3-amino-3-(4-aminophenylthio)-2-(2- (trifluoromethyl)phenyl)acrylonitrile; and has the following structure:
Figure imgf000022_0001
SL327 Chemical Structure
Molecular Weight: 335.35.
In one embodiment, the MEK inhibitor is RG7167. RG7167 is a potent, orally
bioavailable, highly selective MEK inhibitor. It potently inhibits the MAPK signaling pathway activation and tumor cell growth.
In one embodiment, the MEK inhibitor is E6201. E6201 is a synthetic, fungal metabolite analogue inhibitor of mitogen- activated protein kinase kinase 1 (MEK-1) and mitogen- activated protein kinase kinase kinase 1 (MEKK-1) with potential antipsoriatic and antineoplastic activities. MEK-l/MEKK-1 inhibitor E6201 specifically binds to and inhibits the activities of MEK-1 and MEKK-1, which may result in the inhibition of tumor cell proliferation. MEK-1 and MEKK-1 are key components in the RAS/RAF/MEK/MAPK signaling pathway, which regulates cell proliferation and is frequently activated in human cancers. E6201 has the chemical name: ([(3S,4R,5Z,8S,9S,l lE)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro- lH-2-benzoxacyclotetradecine-l,7(8H)-dione]; and has the following structure:
Figure imgf000023_0001
E6201 Chemical Structure
Molecular Weight: 389.44.
In one embodiment, the MEK inhibitor is MSC2015103. MSC2015103 is an orally bio- available, selective, and highly potent small molecule inhibitor of MEK1/2.
In one embodiment, the MEK inhibitor is WX-554. WX-554 is an orally available small molecule mitogen- activated protein kinase kinase (MAP2K, MAPK/ERK kinase, or MEK) inhibitor, with potential antineoplastic activity.
In one embodiment, the agent is an antibody molecule, e.g., an anti-MEK antibody molecule (e.g., a monoclonal or a bispecific antibody), or a conjugate thereof (e.g., an antibody to MEK conjugated to a cytotoxic agent (e.g., mertansine DM1)), and/or a MEK cellular immunotherapy. In one embodiment, the agent is a kinase inhibitor. In one embodiment, the MEK inhibitor is chosen from: a multi- specific kinase inhibitor, a tyrosine/threonine kinase inhibitor, and/or an inhibitor inhibitor that is selective for MEK. In one embodiment, the MEK inhibitor is a small molecule.
In other embodiments, the agent is chosen from a nucleic acid molecule (e.g., an
antisense molecule, a ribozyme, a double stranded RNA, or a triple helix molecule) that
hybridizes to and/or inhibits a MEK nucleic acid, e.g., a MEK nucleic acid encoding the
alteration, or a transcription regulatory region that blocks or reduces mRNA expression of the alteration.
Combination therapies
The agents, e.g., the therapeutic agents described herein, can be administered in combination with a second therapeutic agent or a different therapeutic modality, e.g., anti-cancer agents, and/or in combination with surgical and/or radiation procedures. In certain embodiments, a first therapeutic agent described herein, is administered in combination with a second therapeutic agent, described herein. In certain embodiments, the first therapeutic agent is a BRAF inhibitor described herein, and the second therapeutic agent is a MEK inhibitor described herein. For example, the first therapeutic agent is a BRAF inhibitor and is chosen from: Vemurafenib (PLX4032, RG7204, R05185426), Sorafenib Tosylate (Bay 43- 9006, Nexavar), PLX4720, GDC-0879, RAF265 (CHIR-265), MLN2480 (BIIB-024), PF- 04880594, GW5074, CEP-32496, Dabrafenib (GSK2118436), AZ628, SB590885, Raf265 derivative, Regorafenib (BAY 73-4506, Fluoro-Sorafenib), and/or ZM 336372; and the second therapeutic agent is a MEK inhibitor and is chosen from ARRY- 162 (MEK162), Trametinib (GSK1120212), Selumetinib (AZD6244, ARRY142886), XL518 (GDC-0973), CI-1040 (PD184352), PD035901, U0126-EtOH, PD198306, PD98059, BIX 02189, TAK-733, Honokiol, AZD8330 (ARRY-424704), PD318088, BIX 02188, AS703026 (Pimasertib) and/or SL327.
In other embodiment, either the BRAF inhibitor or the MEK inhibitor, or both can be administered in combination with a therapeutic agent or therapeutic modality currently used in the treatment of a hematological malignancy, e.g., CLL. In one embodiment, the therapeutic agent or therapeutic modality currently used in the treatment of the hematological malignancy is a non-MAPK pathway inhibitor. In one embodiment, the non-MAPK pathway inhibitor therapeutic agent or therapeutic modality is a chemotherapy, an immunotherapy, or a surgical procedure. In one embodiment, the non-MAPK pathway inhibitor therapeutic agent or therapeutic modality comprises one or more of: a chemotherapeutic agent (e.g., fludarabine, cyclophosphamide, doxorubicin, vincristine, chlorambucil, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, 5-azacytidine, pemetrexed disodium), tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g., erlotinib), BTK inhibitor (e.g., PCI-32765), CD20 targeting agent (e.g., rituximab, ofatumumab, RO5072759, LFB-R603), CD52 targeting agent (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, Bcl-2 inhibitor (e.g., ABT-263), AT- 101, immunotoxin (e.g., CAT-8015, anti-Tac(Fv)-PE38 (LMB- 2)), CD37 targeting agent (e.g., TRU-016), radioimmunotherapy (e.g., 131-tositumomab), hydroxychloroquine, perifosine, SRC inhibitor (e.g., dasatinib), thalidomide, retinoid (e.g., fenretinide), MDM2 antagonist (e.g., RO5045337), plerixafor, Aurora kinase inhibitor (e.g., MLN8237, TAK-901), proteasome inhibitor (e.g., bortezomib), CD19 targeting agent (e.g., MEDI-551, MOR208), JAK-2 inhibitor (e.g., INCB018424), hypoxia-activated prodrug (e.g., TH-302), paclitaxel or a paclitaxel agent, AKT inhibitor (e.g., MK2206), HMG-CoA inhibitor (e.g., simvastatin), GNKG186, radiation therapy, bone marrow transplantation, stem cell transplantation, immunotherapy), or a combination thereof.
Compositions, e.g., pharmaceutical compositions, comprising one or more of the agents, e.g., the therapeutic agents described herein, for use, e.g., in treating a hematological
malignancy, e.g., CLL, as described herein are also disclosed.
Additionally, kits comprising the agents, e.g., the therapeutic agents (and compositions thereof), with instructions for use in treating a hematological malignancy, e.g., CLL, and/or determining the presence of an alteration described herein are also provided.
In another aspect, the invention features a kit comprising one or more detection reagents (e.g., probes, primers, antibodies), capable, e.g., of specific detection of a nucleic acid or protein comprising an alteration described herein.
Additional aspects or embodiments of the invention include one or more of the following.
The invention also provides methods of: identifying, assessing or detecting an alteration described herein, e.g., a BRAF mutation, a KRAS mutation, or a mutation identified in FIG. 1, FIG. 2 and/or Table 1, in a hematological malignancy, e.g., CLL, or in a sample derived from a patient diagnosed with or suspected of having a hematological malignancy, e.g., CLL. In certain embodiments, the mutations include one or more of BRAF (e.g., a mutation as described herein), SPEN, FAT3 (e.g., a loss of function mutation in SPEN or FAT3), or a mutation described in FIG. 1, FIG. 2 and/or Table 1. In other embodiments, the mutation is associated with CLL disease progression, e.g., it is a mutation gained or lost at CLL progression as identified in FIG. 1, FIG. 2 and/or Table 1. In one embodiment, the mutation is associated with CLL progression and is chosen from one or more of: NOTCH1, KRAS, TP53, NRAS or BCOR. In another embodiment, the mutation is associated with clonal disease evolution include one or more of: DNMT3A, EED, IDH2, IRF4, VHL or RB I . In yet other embodiments, the mutation is retained at disease progression, if found in early disease states, for example, as chosen from a mutation in SF3B 1 or XPOl .
In yet another aspect, the invention features a method for evaluating progression of a hematological disease, e.g., CLL, in a subject (e.g., a subject described herein). The method includes detecting (and/or acquiring information on the presence of) a mutation described herein, thereby evaluating progression of the disease. In certain embodiments, the mutation is associated with CLL disease progression, e.g., it is a mutation gained or lost at CLL progression as identified in FIG. 1. In one embodiment, the mutation is associated with CLL progression and is chosen from one or more of: NOTCH 1, KRAS, TP53, NRAS or BCOR. In another embodiment, the mutation is associated with clonal disease evolution include one or more of: DNMT3A, EED, IDH2, IRF4, VHL or RB I . In yet other embodiments, the mutation is retained at disease progression, if found in early disease states, for example, as chosen from a mutation in SF3B 1 or XPOl .
Included are isolated nucleic acid molecules comprising the alterations, nucleic acid constructs, host cells containing the nucleic acid molecules; purified polypeptides comprising the alteration described herein and binding agents; detection reagents (e.g., probes, primers, antibodies, kits, capable, e.g., of specific detection of a nucleic acid or protein comprising an alteration described herein); screening assays for identifying molecules that interact with, e.g., inhibit the alterations, e.g., novel kinase inhibitors or binders of BRAF and/or KRAS. In one embodiment, the detection of the alteration comprises sequencing, e.g., nucleic acid sequencing or amino acid sequencing.
Alternatively, or in combination with the methods described herein, the invention features a method of determining the presence of an alteration described herein in a cancer, e.g., a hematological malignancy, e.g., CLL. The method includes: acquiring knowledge (e.g., directly acquiring knowledge) that the alteration described herein is present in a subject, e.g., a sample (e.g., a cancer or tumor sample) from the subject. In one embodiment, the acquiring step comprises a determination of the presence of the alteration in a nucleic acid molecule from the subject, e.g., by performing a sequencing step. In other embodiments, the acquiring step
comprises a determination of the presence of a polypeptide or a protein comprising the alteration described herein in the sample from the subject.
In one embodiment, the subject, or the sample, comprises one or more cells or tissue from a hematological malignancy, e.g., CLL.
In one embodiment the method further comprises administering an agent, e.g., a
therapeutic agent that targets and/or inhibits a MAPK pathway inhibitor, e.g., an agent as described herein, to the subject responsive to the determination of the presence of the alteration in the sample (e.g., a CLL sample) from the subject. In one embodiment, the mutation is detected in a nucleic acid molecule or a polypeptide. The method includes detecting whether a mutated nucleic acid molecule or polypeptide is present in a cell (e.g., a circulating cell), a tissue (e.g., a hematologic tissue, e.g., a CLL tissue), or a sample, e.g., a sample (e.g., a CLL sample), from a subject. In one embodiment, the sample is a nucleic acid sample. In one embodiment, the nucleic acid sample comprises DNA, e.g., genomic DNA or cDNA, or RNA, e.g., mRNA. In other embodiments, the sample is a protein sample.
In one embodiment, the sample or tissue is, or has been, classified as non-malignant or malignant using other diagnostic techniques, e.g., immunohistochemistry.
In one embodiment, the sample is acquired from a subject (e.g., a subject having or at risk of having a cancer, e.g., a patient), or alternatively, the method further includes acquiring a sample from the subject. In certain embodiments, the sample is a blood sample, or a bone marrow biopsy or aspirate. In certain embodiments, the sample is a blood sample, a whole blood sample, or a serum sample. The sample can be chosen from one or more of: tissue, e.g., cancerous tissue (e.g., a tissue biopsy), whole blood, serum, plasma, circulating tumor cells, circulating nucleic acids, bone marrow, or any sample comprising hematologic or lymphocytic, e.g., CLL, cells. In certain embodiments, the sample is a tissue (e.g., a bone marrow biopsy or aspirate), a circulating tumor cell or nucleic acid.
In embodiments, the cancer is a hematological malignancy, e.g., CLL.
In one embodiment, the subject is at risk of having, or has CLL.
In other embodiments, the mutation is detected in a nucleic acid molecule by a method chosen from one or more of: nucleic acid hybridization assay, amplification-based assays (e.g., polymerase chain reaction (PCR)), PCR-RFLP assay, real-time PCR, sequencing, screening analysis, SSP, HPLC or mass-spectrometric genotyping.
In one embodiment, the method includes: contacting a nucleic acid sample, e.g., a genomic DNA sample (e.g., a chromosomal sample or a fractionated, enriched or otherwise pre- treated sample) or a gene product (mRNA, cDNA), obtained from the subject, with a nucleic acid fragment (e.g., a probe or primer as described herein (e.g., an exon-specific probe or primer) under conditions suitable for hybridization, and determining the presence or absence of the mutated nucleic acid molecule. The method can, optionally, include enriching a sample for the gene or gene product. Alternatively, or in combination with the methods described herein, the invention features a method for determining the presence of a mutated nucleic acid molecule, e.g., a mutated MAPK pathway gene or gene product. The method includes: acquiring a sequence for a position in a nucleic acid molecule, e.g., by sequencing at least one nucleotide of the nucleic acid molecule (e.g., sequencing at least one nucleotide in the nucleic acid molecule that comprises the mutation), thereby determining that the mutation is present in the nucleic acid molecule. Optionally, the sequence acquired is compared to a reference sequence, or a wild type reference sequence. In one
embodiment, the nucleic acid molecule is from a cell (e.g., a hematopoietic, lymphocytic, CLL cell, e.g., from a blood or bone marrow sample), a tissue, or any sample from a subject (e.g., blood or plasma sample). In other embodiments, the nucleic acid molecule from a tumor sample (e.g., a tumor or cancer sample) is sequenced. In one embodiment, the sequence is determined by a next generation sequencing method. The method further can further include acquiring, e.g., directly or indirectly acquiring, a sample, e.g., a hematological malignancy, e.g., CLL.
In another aspect, the invention features a method of analyzing a malignancy or a circulating malignant cell. The method includes acquiring a nucleic acid sample from the malignancy or the circulating cell; and sequencing, e.g., by a next generation sequencing method, a nucleic acid molecule, e.g., a nucleic acid molecule that includes an alteration as described herein, e.g., an alteration in a MAPK pathway gene or gene product.
In yet other embodiment, a polypeptide comprising an alteration described herein is detected. The method includes: contacting a protein sample with a reagent which specifically binds to a polypeptide comprising an alteration described herein; and detecting the formation of a complex of the polypeptide and the reagent. In one embodiment, the reagent is labeled with a detectable group to facilitate detection of the bound and unbound reagent. In one embodiment, the reagent is an antibody molecule, e.g., is selected from the group consisting of an antibody, and antibody derivative, and an antibody fragment.
In yet another embodiment, the level (e.g., expression level) or activity the polypeptide comprising an alteration described herein is evaluated. For example, the level (e.g., expression level) or activity of the polypeptide (e.g., mRNA or polypeptide) is detected and (optionally) compared to a pre-determined value, e.g., a reference value (e.g., a control sample).
In yet another embodiment, the alteration is detected prior to initiating, during, or after, a treatment in a subject having an alteration described herein. In one embodiment, the alteration is detected at the time of diagnosis with a cancer. In other embodiment, the alteration is detected at a pre-determined interval, e.g., a first point in time and at least at a subsequent point in time.
In certain embodiments, responsive to a determination of the presence of the alteration, any of the methods described herein further include one or more of:
(1) stratifying a patient or a patient population (e.g., assigning a subject, e.g., a patient, to a group or class);
(2) identifying or selecting the subject as being likely or unlikely to respond to a
treatment, e.g., a MAPK pathway inhibitor (e.g., BRAF and/or MEK inhibitor) treatment as described herein;
(3) selecting a treatment option, e.g., administering or not administering a preselected therapeutic agent, e.g., a MAPK pathway inhibitor (e.g., BRAF and/or MEK inhibitor) as described herein; or
(4) prognosticating the time course of the disease in the subject (e.g., evaluating the likelihood of increased or decreased patient survival, or the extent of disease progression).
In certain embodiments, responsive to the determination of the presence of a mutation, the subject is classified as a candidate to receive treatment with a therapy disclosed herein. In one embodiment, responsive to the determination of the presence of a mutation, the subject, e.g., a patient, can further be assigned to a particular class if a mutation is identified in a sample of the patient. For example, a patient identified as having a mutation can be classified as a candidate to receive treatment with a therapy disclosed herein. In one embodiment, the subject, e.g., a patient, is assigned to a second class if the mutation is not present. For example, a patient who has a cancer that does not contain a mutation, may be determined as not being a candidate to receive a therapy disclosed herein.
In another embodiment, responsive to the determination of the presence of the alteration, the subject is identified as likely to respond to a treatment that comprises a therapy disclosed herein.
In yet another embodiment, responsive to the determination of the presence of the alteration, the method includes administering an agent, e.g., a therapeutic agent as described herein, e.g., a BRAF inhibitor and/or a MEK inhibitor, to the subject. Method of Evaluating a Tumor or a Subject
In another aspect, the invention features a method of evaluating a subject (e.g., a patient), e.g., for risk of having or developing a cancer, e.g., a hematological malignancy, e.g., CLL. The method includes: acquiring information or knowledge of the presence of a mutation as described herein in a subject (e.g., acquiring genotype information of the subject that identifies a mutation as being present in the subject); acquiring a sequence for a nucleic acid molecule identified herein (e.g., a nucleic acid molecule that includes a mutation sequence); or detecting the presence of a nucleic acid or polypeptide in the subject), wherein the presence of the mutation is positively correlated with increased risk for, or having, a cancer associated with such a mutation.
The method can further include acquiring, e.g., directly or indirectly, a sample from a patient and evaluating the sample for the present of an alteration as described herein.
The method can further include the step(s) of identifying (e.g., evaluating, diagnosing, screening, and/or selecting) the subject as being positively correlated with increased risk for, or having, a cancer associated with the alteration.
In another embodiment, a subject identified as having the alteration is identified or selected as likely or unlikely to respond to a treatment, e.g., a therapy disclosed herein. The method can further include treating the subject with a therapy disclosed herein.
In a related aspect, a method of evaluating a patient or a patient population is provided. The method includes: identifying, selecting, or obtaining information or knowledge that the patient or patient population has participated in a clinical trial; acquiring information or knowledge of the presence of an alteration (e.g., an alteration as described herein) in the patient or patient population (e.g., acquiring genotype information of the subject that identifies an alteration as being present in the subject); acquiring a sequence for a nucleic acid molecule identified herein (e.g., a nucleic acid molecule that includes an alteration sequence); or detecting the presence of a mutated nucleic acid or polypeptide in the subject), wherein the presence of the alteration identifies the patient or patient population as being likely to respond to an agent as described herein (e.g., a MAPK pathway inhibitor (e.g., BRAF and/or MEK inhibitor)).
In some embodiments, the method further includes treating the subject with an agent as described herein (e.g., a MAPK pathway inhibitor, e.g., a BRAF and/or a MEK inhibitor).
Reporting Methods described herein can include providing a report, such as, in electronic, web-based, or paper form, to the patient or to another person or entity, e.g., a caregiver, e.g., a physician, e.g., an oncologist, a hospital, clinic, third-party payor, insurance company or government office. The report can include output from the method, e.g., the identification of nucleotide values, the indication of presence or absence of an alteration as described herein, or wildtype sequence. In one embodiment, a report is generated, such as in paper or electronic form, which identifies the presence or absence of an alteration described herein, and optionally includes an identifier for the patient from which the sequence was obtained.
The report can also include information on the role of a mutation as described herein, or wildtype sequence, in disease. Such information can include information on prognosis, resistance, or potential or suggested therapeutic options, e.g., an agent as described herein (e.g., a MAPK pathway inhibitor, e.g., a BRAF and/or a MEK inhibitor). The report can include information on the likely effectiveness of a therapeutic option, the acceptability of a therapeutic option, or the advisability of applying the therapeutic option to a patient, e.g., a patient having a sequence, alteration or mutation identified in the test, and in embodiments, identified in the report. For example, the report can include information, or a recommendation on, the administration of a drug, e.g., the administration at a preselected dosage or in a preselected treatment regimen, e.g., in combination with other drugs, to the patient. In one embodiment, not all mutations identified in the method are identified in the report. For example, the report can be limited to mutations in genes having a preselected level of correlation with the occurrence, prognosis, stage, or susceptibility of the cancer to treatment, e.g., with a preselected therapeutic option. The report can be delivered, e.g., to an entity described herein, within 7, 14, or 21 days from receipt of the sample by the entity practicing the method.
In another aspect, the invention features a method for generating a report, e.g., a personalized cancer treatment report, by obtaining a sample, e.g., a tumor sample, from a subject, detecting a mutation as described herein in the sample, and selecting a treatment based on the mutation identified. In one embodiment, a report is generated that annotates the selected treatment, or that lists, e.g., in order of preference, two or more treatment options based on the mutation identified. In another embodiment, the subject, e.g., a patient, is further administered the selected method of treatment. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and the example are illustrative only and not intended to be limiting.
The details of one or more embodiments featured in the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages featured in the invention will be apparent from the description and drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 depicts extensive deep targeted sequencing of logitudinal CLL samples which shows novel recurrent driver events and patterns of clonal evolution. (A) Depicts sequencing of a panel of 435 genes at deep coverage in CLL which shows frequent mutations including novel recurrent mutations in BRAF, SPEN, FAT3, and TRAF3. Notably BRAF mutations in CLL are activating mutations predominately seen at codons other than V600. (B) Depicts a tile plot comparison of paired sample sequencing which shows shifts in mutations at disease progression. Notably, leukemic cells sampled at disease progression during a time when chemotherpay had already commenced had much greater frequency of genetic alterations than those with untreated disease at the time of sample acquisition.
FIG. 2 depicts a tile plot showing the genomic alterations in each CLL case sample analyzed. The analyzed genes are listed in the vertical column. The key distinguishes the genomic alterations of: substiutions/indels; truncations; and gene fusions.
DETAILED DESCRIPTION
Described at least in part herein is the identification of an alteration in a MAPK pathway gene or gene product, e.g., a BRAF and/or a MEK, in a series of human patients with chronic lymphocytic leukemia (CLL). Additionally described herein is a genomic analysis of a series of patients with CLL to characterize the genomic landscape of CLL. Also described herein is a genomic analysis of a series of patients with untreated CLL and previously treated CLL to characterize the genomic landscape of both untreated and treated CLL. Accordingly, disclosed herein are methods for treating CLL, using an agent (e.g., a therapeutic agent) that targets and/or inhibits a MAPK pathway gene or gene product, e.g., a BRAF and/or a MEK, as well as methods and reagents for identifying, assessing and/or detecting an alteration as described herein, e.g., a BRAF and/or KRAS mutation, in CLL.
Chronic lymphocytic leukemia (CLL), also known as B cell-CLL, is a common type of adulthood leukemia, and is characterized by the clonal proliferation and accumulation of mature B lymphocytes (Eichhorst B et al. Ann Oncol (2011) 22 (suppl 6): vi50-vi54; Smolewski P et al. Oncology Volume 2013 (2013)). CLL progression is characterized by stage. Commonly used staging systemns include, for example, the Rai 4-stage system and the Binet classification system (National Cancer Institute. "Chronic Lymphocytic Leukemia (PDQ) Treatment: Stage
Information". Archived from the original on 17 October 2007; Smolewski, supra). Treatment of CLL is stage dependent, with early stage CLL monitored without treatment. Once CLL progresses chemotherapeutic and biologic agents are commonly employed. Current treatment regimens include, for example, fludarabine, cyclochosphamide, and ritiximab; chlorambucil; and cyclophosphamide, vincristine, doxorubicin, and prednisone. Bone marrow or stem cell transplantation may also be used in younger patients with advanced or high risk CLL. Despite advances in treatment options a number of CLL patients suffer from relapsed or refractory CLL (Maddocks K et al. Journal of Hematology & Oncology 2009, 2:29).
A "MAPK pathway gene or gene product" as used herein, refers to a gene or a gene product that is directly or indirectly involved in intracellular signaling via a mitogen activated protein kinases (MAPK). In some embodiments, this direct and/or indirect involvement can comprise a gene and/or a gene product upstream and/or downstream of MAPK. MAP kinases can be mediators of cancer-related disease mechanisms (Chen et al., Chem Rev (2001) 101:2449- 76; Pearson et al., Endocr Rev (2001) 22: 153-83; English et al., Trends Pharmacol Sci (2002) 23:40-45; Kohno et al., Prog Cell Cycle Res (2003) 5:219-24; and Sebolt-Leopold, Oncogene (2000) 19:6594-99). In one embodiment, a MAPK pathway comprises RAS, RAF, MEK, and ERK (MAPK) {e.g., Ras, Raf-1, A-Raf, B-Raf (BRAF)), MEK1 and/or MEK2. In some embodiments, MAPK pathway gene or gene product can also refer to either or both of the wild type or native gene, as well as or alternatively, certain mutations thereof {e.g., activating mutations thereof), and variants derived from any source (e.g., humans and other mammals), as described herein. In some embodiments, MAPK pathway gene product refers to mRNA, polypeptides and/or fragments thereof, of the encoding MAPK pathway gene.
"V-Raf Murine Sarcoma Viral Oncogene Homolog Bl" or "BRAF" (also known as BRAF1, proto-oncogene B-raf, RAFB1, NS7, P94, protein kinase 94, and serine/threonine protein kinase BRAF) refers to a BRAF gene or gene product (e.g., a nucleic acid or protein). The BRAF protein refers to a protein, typically human BRAF that is encoded by the BRAF gene. BRAF is a member of the raf/mil family of serine/threonine protein kinases, and plays a role in regulating the MAP kinase/ERKs signaling pathway, which affects, inter alia, cell division, differentiation, and secretion. The BRAF amino and nucleotide sequences are known in the art. An exemplary amino acid and nucleotide sequence for human BRAF is provided herein as SEQ ID NO: l and SEQ ID NO:2, respectively.
NCBI Reference Sequence: NP_004324
1 maalsggggg gaepgqalfn gdmepeagag agaaassaad paipeevwni kqmikltqeh 61 iealldkfgg ehnppsiyle ayeeytskld alqqreqqll eslgngtdfs vsssasmdtv 121 tsssssslsv lpsslsvfqn ptdvarsnpk spqkpivrvf lpnkqrtvvp arcgvtvrds 181 lkkalmmrgl ipeccavyri qdgekkpigw dtdiswltge elhvevlenv pltthnfvrk 241 tfftlafcdf crkllfqgfr cqtcgykfhq rcstevplmc vnydqldllf vskffehhpi 301 pqeeaslaet altsgsspsa pasdsigpqi ltspspsksi pipqpfrpad edhrnqfgqr 361 drsssapnvh intiepvnid dlirdqgfrg dggsttglsa tppaslpgsl tnvkalqksp 421 gpqrerksss ssedrnrmkt lgrrdssddw eipdgqitvg qrigsgsfgt vykgkwhgdv 481 avkmlnvtap tpqqlqafkn evgvlrktrh vnillfmgys tkpqlaivtq wcegsslyhh 541 lhiietkfem iklidiarqt aqgmdylhak siihrdlksn niflhedltv kigdfglatv 601 ksrwsgshqf eqlsgsilwm apevirmqdk npysfqsdvy afgivlyelm tgqlpysnin 661 nrdqiifmvg rgylspdlsk vrsncpkamk rlmaeclkkk rderplfpqi lasiellars 721 lpkihrsase pslnragfqt edfslyacas pktpiqaggy gafpvh
(SEQ ID NO: 1)
NCBI Reference Sequence: NM_004333
1 cgcctccctt ccccctcccc gcccgacagc ggccgctcgg gccccggctc tcggttataa 61 gatggcggcg ctgagcggtg gcggtggtgg cggcgcggag ccgggccagg ctctgttcaa 121 cggggacatg gagcccgagg ccggcgccgg cgccggcgcc gcggcctctt cggctgcgga 181 ccctgccatt ccggaggagg tgtggaatat caaacaaatg attaagttga cacaggaaca 241 tatagaggcc ctattggaca aatttggtgg ggagcataat ccaccatcaa tatatctgga 301 ggcctatgaa gaatacacca gcaagctaga tgcactccaa caaagagaac aacagttatt 361 ggaatctctg gggaacggaa ctgatttttc tgtttctagc tctgcatcaa tggataccgt 421 tacatcttct tcctcttcta gcctttcagt gctaccttca tctctttcag tttttcaaaa 481 tcccacagat gtggcacgga gcaaccccaa gtcaccacaa aaacctatcg ttagagtctt 541 cctgcccaac aaacagagga cagtggtacc tgcaaggtgt ggagttacag tccgagacag 601 tctaaagaaa gcactgatga tgagaggtct aatcccagag tgctgtgctg tttacagaat 661 tcaggatgga gagaagaaac caattggttg ggacactgat atttcctggc ttactggaga 721 agaattgcat gtggaagtgt tggagaatgt tccacttaca acacacaact ttgtacgaaa 781 aacgtttttc accttagcat tttgtgactt ttgtcgaaag ctgcttttcc agggtttccg
841 ctgtcaaaca tgtggttata aatttcacca gcgttgtagt acagaagttc cactgatgtg
901 tgttaattat gaccaacttg atttgctgtt tgtctccaag ttctttgaac accacccaat
961 accacaggaa gaggcgtcct tagcagagac tgccctaaca tctggatcat ccccttccgc
1021 acccgcctcg gactctattg ggccccaaat tctcaccagt ccgtctcctt caaaatccat
1081 tccaattcca cagcccttcc gaccagcaga tgaagatcat cgaaatcaat ttgggcaacg
1141 agaccgatcc tcatcagctc ccaatgtgca tataaacaca atagaacctg tcaatattga
1201 tgacttgatt agagaccaag gatttcgtgg tgatggagga tcaaccacag gtttgtctgc
1261 taccccccct gcctcattac ctggctcact aactaacgtg aaagccttac agaaatctcc
1321 aggacctcag cgagaaagga agtcatcttc atcctcagaa gacaggaatc gaatgaaaac
1381 acttggtaga cgggactcga gtgatgattg ggagattcct gatgggcaga ttacagtggg
1441 acaaagaatt ggatctggat catttggaac agtctacaag ggaaagtggc atggtgatgt
1501 ggcagtgaaa atgttgaatg tgacagcacc tacacctcag cagttacaag ccttcaaaaa
1561 tgaagtagga gtactcagga aaacacgaca tgtgaatatc ctactcttca tgggctattc
1621 cacaaagcca caactggcta ttgttaccca gtggtgtgag ggctccagct tgtatcacca
1681 tctccatatc attgagacca aatttgagat gatcaaactt atagatattg cacgacagac
1741 tgcacagggc atggattact tacacgccaa gtcaatcatc cacagagacc tcaagagtaa
1801 taatatattt cttcatgaag acctcacagt aaaaataggt gattttggtc tagctacagt
1861 gaaatctcga tggagtgggt cccatcagtt tgaacagttg tctggatcca ttttgtggat
1921 ggcaccagaa gtcatcagaa tgcaagataa aaatccatac agctttcagt cagatgtata
1981 tgcatttgga attgttctgt atgaattgat gactggacag ttaccttatt caaacatcaa
2041 caacagggac cagataattt ttatggtggg acgaggatac ctgtctccag atctcagtaa
2101 ggtacggagt aactgtccaa aagccatgaa gagattaatg gcagagtgcc tcaaaaagaa
2161 aagagatgag agaccactct ttccccaaat tctcgcctct attgagctgc tggcccgctc
2221 attgccaaaa attcaccgca gtgcatcaga accctccttg aatcgggctg gtttccaaac
2281 agaggatttt agtctatatg cttgtgcttc tccaaaaaca cccatccagg cagggggata
2341 tggtgcgttt cctgtccact gaaacaaatg agtgagagag ttcaggagag tagcaacaaa
2401 aggaaaataa atgaacatat gtttgcttat atgttaaatt gaataaaata ctctcttttt
2461 ttttaaggtg aaccaaagaa cacttgtgtg gttaaagact agatataatt tttccccaaa
2521 ctaaaattta tacttaacat tggattttta acatccaagg gttaaaatac atagacattg
2581 ctaaaaattg gcagagcctc ttctagaggc tttactttct gttccgggtt tgtatcattc
2641 acttggttat tttaagtagt aaacttcagt ttctcatgca acttttgttg ccagctatca
2701 catgtccact agggactcca gaagaagacc ctacctatgc ctgtgtttgc aggtgagaag
2761 ttggcagtcg gttagcctgg gttagataag gcaaactgaa cagatctaat ttaggaagtc
2821 agtagaattt aataattcta ttattattct taataatttt tctataacta tttcttttta
2881 taacaatttg gaaaatgtgg atgtctttta tttccttgaa gcaataaact aagtttcttt
2941 ttataaaaa
(SEQ ID NO:2)
The term "MEK" as used herein refers to a gene or gene product having kinase activity on a MAP kinase. This family of kinases is also known as MAP/ERK kinase (MEK) or Mitogen-activated protein kinase (MAPK) kinase, and includes at least MAPK kinase/MEK- 1 and -2.
"MAPK kinase 1" or "MEKl" (also known as PRKMK1, MKK1, MAPKK1, ERK activator kinase 1, MAP kinase kinase 1, and MAK/ERK kinase 1) refers to a MEKl gene or gene product (e.g., a nucleic acid or protein). The MEKl protein refers to a protein, typically human MEKl that is encoded by the MEKl gene. MEKl is a member of the dual specificity protein kinase family, which acts as a mitogen-activated protein (MAP) kinase kinase. MAP kinases, also known as extracellular signal-regulated kinases (ERKs), act as an integration point for multiple biochemical signals. MEKl functions in the MAPK/ERK cascade, and catalyzes the concomitant phosphorylation of a threonine and a tyrosine residue in a Thr-Glu-Tyr sequence located in the extracellular signal-regulated kinases MAPK3/ERK1 and MAPK1/ERK2, leading to their activation and further transduction of the signal within the MAPK/ERK cascade.
Depending on the cellular context, this pathway mediates diverse biological functions, including, inter alia, cell growth, adhesion, survival and differentiation, predominantly through the regulation of transcription, metabolism and cytoskeletal rearrangements. The MEKl amino and nucleotide sequences are known in the art. An exemplary amino acid and nucleotide sequence for human MEKl is provided herein as SEQ ID NO:3 and SEQ ID NO:4, respectively.
NCBI Reference Sequence: NP_002746.1
1 mpkkkptpiq lnpapdgsav ngtssaetnl ealqkkleel eldeqqrkrl eafltqkqkv 61 gelkdddfek iselgagngg vvfkvshkps glvmarklih leikpairnq iirelqvlhe 121 cnspyivgfy gafysdgeis icmehmdggs ldqvlkkagr ipeqilgkvs iavikgltyl 181 rekhkimhrd vkpsnilvns rgeiklcdfg vsgqlidsma nsfvgtrsym sperlqgthy 241 svqsdiwsmg lslvemavgr ypipppdake lelmfgcqve gdaaetpprp rtpgrplssy 301 gmdsrppmai felldyivne pppklpsgvf slefqdfvnk cliknpaera dlkqlmvhaf 361 ikrsdaeevd fagwlcstig lnqpstptha agv
(SEQ ID NO:3)
NCBI Reference Sequence: NM_002755.3
1 aggcgaggct tccccttccc cgcccctccc ccggcctcca gtccctccca gggccgcttc 61 gcagagcggc taggagcacg gcggcggcgg cactttcccc ggcaggagct ggagctgggc 121 tctggtgcgc gcgcggctgt gccgcccgag ccggagggac tggttggttg agagagagag 181 aggaagggaa tcccgggctg ccgaaccgca cgttcagccc gctccgctcc tgcagggcag 241 cctttcggct ctctgcgcgc gaagccgagt cccgggcggg tggggcgggg gtccactgag 301 accgctaccg gcccctcggc gctgacggga ccgcgcgggg cgcacccgct gaaggcagcc 361 ccggggcccg cggcccggac ttggtcctgc gcagcgggcg cggggcagcg cagcgggagg 421 aagcgagagg tgctgccctc cccccggagt tggaagcgcg ttacccgggt ccaaaatgcc 481 caagaagaag ccgacgccca tccagctgaa cccggccccc gacggctctg cagttaacgg 541 gaccagctct gcggagacca acttggaggc cttgcagaag aagctggagg agctagagct 601 tgatgagcag cagcgaaagc gccttgaggc ctttcttacc cagaagcaga aggtgggaga 661 actgaaggat gacgactttg agaagatcag tgagctgggg gctggcaatg gcggtgtggt 721 gttcaaggtc tcccacaagc cttctggcct ggtcatggcc agaaagctaa ttcatctgga 781 gatcaaaccc gcaatccgga accagatcat aagggagctg caggttctgc atgagtgcaa 841 ctctccgtac atcgtgggct tctatggtgc gttctacagc gatggcgaga tcagtatctg 901 catggagcac atggatggag gttctctgga tcaagtcctg aagaaagctg gaagaattcc 961 tgaacaaatt ttaggaaaag ttagcattgc tgtaataaaa ggcctgacat atctgaggga 1021 gaagcacaag atcatgcaca gagatgtcaa gccctccaac atcctagtca actcccgtgg 1081 ggagatcaag ctctgtgact ttggggtcag cgggcagctc atcgactcca tggccaactc 1141 cttcgtgggc acaaggtcct acatgtcgcc agaaagactc caggggactc attactctgt 1201 gcagtcagac atctggagca tgggactgtc tctggtagag atggcggttg ggaggtatcc 1261 catccctcct ccagatgcca aggagctgga gctgatgttt gggtgccagg tggaaggaga 1321 tgcggctgag accccaccca ggccaaggac ccccgggagg ccccttagct catacggaat 381 ggacagccga cctcccatgg caatttttga gttgttggat tacatagtca acgagcctcc
44 1 tccaaaactg cccagtggag tgttcagtct ggaatttcaa gattttgtga ataaatgctt
50 1 aataaaaaac cccgcagaga gagcagattt gaagcaactc atggttcatg cttttatcaa
56 1 gagatctgat gctgaggaag tggattttgc aggttggctc tgctccacca tcggccttaa
62 1 ccagcccagc acaccaaccc atgctgctgg cgtctaagtg tttgggaagc aacaaagagc
68 1 gagtcccctg cccggtggtt tgccatgtcg cttttgggcc tccttcccat gcctgtctct
74 1 gttcagatgt gcatttcacc tgtgacaaag gatgaagaac acagcatgtg ccaagattct
80 1 actcttgtca tttttaatat tactgtcttt attcttatta ctattattgt tcccctaagt
86 1 ggattggctt tgtgcttggg gctatttgtg tgtatgctga tgatcaaaac ctgtgccagg
92 1 ctgaattaca gtgaaatttt ggtgaatgtg ggtagtcatt cttacaattg cactgctgtt
98 1 cctgctccat gactggctgt ctgcctgtat tttcgggatt ctttgacatt tggtggtact
04 1 ttattcttgc tgggcatact ttctctctag gagggagcct tgtgagatcc ttcacaggca
10 1 gtgcatgtga agcatgcttt gctgctatga aaatgagcat cagagagtgt acatcatgtt
16 1 attttattat tattatttgc ttttcatgta gaactcagca gttgacatcc aaatctagcc
22 1 agagcccttc actgccatga tagctggggc ttcaccagtc tgtctactgt ggtgatctgt
28 1 agacttctgg ttgtatttct atatttattt tcagtatact gtgtgggata cttagtggta
34 1 tgtctcttta agttttgatt aatgtttctt aaatggaatt attttgaatg tcacaaattg
40 1 atcaagatat taaaatgtcg gatttatctt tccccatatc caagtaccaa tgctgttgta
46 1 aacaacgtgt atagtgccta aaattgtatg aaaatccttt taaccatttt aacctagatg
52 1 tttaacaaat ctaatctctt attctaataa atatactatg aaataaaaaa aaaaggatga
58 1 aagctaaaaa aaaaaaaaaa aaa
(SEQ ID NO:4)
"Mitogen-activated protein kinase kinase 2" or "MEK2" (also known as PRKMK2, MKK2, MAPKK2, ERK activator kinase 2, MAP kinase kinase 2, and MAK/ERK kinase 2) refers to a MEK2 gene or gene product (e.g., a nucleic acid or protein). The MEK2 protein refers to a protein, typically human MEK2 that is encoded by the MEK2 gene. MEK2 is a member of the dual specificity protein kinase family, which acts as a mitogen-activated protein (MAP) kinase kinase. MAP kinases, also known as extracellular signal-regulated kinases (ERKs), act as an integration point for multiple biochemical signals. MEK2 functions in the MAPK/ERK cascade, and catalyzes the concomitant phosphorylation of a threonine and a tyrosine residue in a Thr-Glu-Tyr sequence located in the extracellular signal-regulated kinases MAPK3/ERK1 and MAPK1/ERK2, leading to their activation and further transduction of the signal within the MAPK/ERK cascade. Depending on the cellular context, this pathway mediates diverse biological functions, including, inter alia, cell growth, adhesion, survival and
differentiation, predominantly through the regulation of transcription, metabolism and cytoskeletal rearrangements. The MEK2 amino and nucleotide sequences are known in the art. An exemplary amino acid and nucleotide sequence for human MEK2 is provided herein as SEQ ID NO:5 and SEQ ID NO:6, respectively.
NCBI Reference Sequence: NP_109587.1 1 mlarrkpvlp altinptiae gpsptsegas eanlvdlqkk leeleldeqq kkrleafltq 61 kakvgelkdd dferiselga gnggvvtkvq hrpsglimar klihleikpa irnqiirelq
121 vlhecnspyi vgfygafysd geisicmehm dggsldqvlk eakripeeil gkvsiavlrg
181 laylrekhqi mhrdvkpsni lvnsrgeikl cdfgvsgqli dsmansfvgt rsymaperlq
241 gthysvqsdi wsmglslvel avgrypippp dakeleaifg rpvvdgeege phsisprprp
301 pgrpvsghgm dsrpamaife lldyivnepp pklpngvftp dfqefvnkcl iknpaeradl
361 kmltnhtfik rseveevdfa gwlcktlrln qpgtptrtav
(SEQ ID NO:5)
NCBI Reference Sequence: NM_030662.3
1 cccctgcctc tcggactcgg gctgcggcgt cagccttctt cgggcctcgg cagcggtagc
61 ggctcgctcg cctcagcccc agcgcccctc ggctaccctc ggcccaggcc cgcagcgccg
121 cccgccctcg gccgccccga cgccggcctg ggccgcggcc gcagccccgg gctcgcgtag
181 gcgccgaccg ctcccggccc gccccctatg ggccccggct agaggcgccg ccgccgccgg
241 cccgcggagc cccgatgctg gcccggagga agccggtgct gccggcgctc accatcaacc
301 ctaccatcgc cgagggccca tcccctacca gcgagggcgc ctccgaggca aacctggtgg
361 acctgcagaa gaagctggag gagctggaac ttgacgagca gcagaagaag cggctggaag
421 cctttctcac ccagaaagcc aaggtcggcg aactcaaaga cgatgacttc gaaaggatct
481 cagagctggg cgcgggcaac ggcggggtgg tcaccaaagt ccagcacaga ccctcgggcc
541 tcatcatggc caggaagctg atccaccttg agatcaagcc ggccatccgg aaccagatca
601 tccgcgagct gcaggtcctg cacgaatgca actcgccgta catcgtgggc ttctacgggg
661 ccttctacag tgacggggag atcagcattt gcatggaaca catggacggc ggctccctgg
721 accaggtgct gaaagaggcc aagaggattc ccgaggagat cctggggaaa gtcagcatcg
781 cggttctccg gggcttggcg tacctccgag agaagcacca gatcatgcac cgagatgtga
841 agccctccaa catcctcgtg aactctagag gggagatcaa gctgtgtgac ttcggggtga
901 gcggccagct catcgactcc atggccaact ccttcgtggg cacgcgctcc tacatggctc
961 cggagcggtt gcagggcaca cattactcgg tgcagtcgga catctggagc atgggcctgt
1021 ccctggtgga gctggccgtc ggaaggtacc ccatcccccc gcccgacgcc aaagagctgg
1081 aggccatctt tggccggccc gtggtcgacg gggaagaagg agagcctcac agcatctcgc
1141 ctcggccgag gccccccggg cgccccgtca gcggtcacgg gatggatagc cggcctgcca
1201 tggccatctt tgaactcctg gactatattg tgaacgagcc acctcctaag ctgcccaacg
1261 gtgtgttcac ccccgacttc caggagtttg tcaataaatg cctcatcaag aacccagcgg
1321 agcgggcgga cctgaagatg ctcacaaacc acaccttcat caagcggtcc gaggtggaag
1381 aagtggattt tgccggctgg ttgtgtaaaa ccctgcggct gaaccagccc ggcacaccca
1441 cgcgcaccgc cgtgtgacag tggccgggct ccctgcgtcc cgctggtgac ctgcccaccg
1501 tccctgtcca tgccccgccc ttccagctga ggacaggctg gcgcctccac ccaccctcct
1561 gcctcacccc tgcggagagc accgtggcgg ggcgacagcg catgcaggaa cgggggtctc
1621 ctctcctgcc cgtcctggcc ggggtgcctc tggggacggg cgacgctgct gtgtgtggtc
1681 tcagaggctc tgcttcctta ggttacaaaa caaaacaggg agagaaaaag caaaaaaaaa
1741 aaaaaaaaaa aaaaaaaaa
(SEQ ID NO:6).
Additional terms are defined below and throughout the specification.
As used herein, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.
The term "or" is used herein to mean, and is used interchangeably with, the term "and/or", unless context clearly indicates otherwise. "About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.
"Acquire" or "acquiring" as the terms are used herein, refer to obtaining possession of a physical entity, or a value, e.g., a numerical value, by "directly acquiring" or "indirectly acquiring" the physical entity or value. "Directly acquiring" means performing a process (e.g., performing a synthetic or analytical method) to obtain the physical entity or value. "Indirectly acquiring" refers to receiving the physical entity or value from another party or source (e.g., a third party laboratory that directly acquired the physical entity or value). Directly acquiring a physical entity includes performing a process that includes a physical change in a physical substance, e.g., a starting
material. Exemplary changes include making a physical entity from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, combining two or more separate entities into a mixture, performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond. Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process which includes a physical change in a substance, e.g., a sample, analyte, or reagent (sometimes referred to herein as "physical analysis"), performing an analytical method, e.g., a method which includes one or more of the following: separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from another substance; combining an analyte, or fragment or other derivative thereof, with another substance, e.g., a buffer, solvent, or reactant; or changing the structure of an analyte, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the analyte; or by changing the structure of a reagent, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the reagent.
"Acquiring a sequence" as the term is used herein, refers to obtaining possession of a nucleotide sequence or amino acid sequence, by "directly acquiring" or "indirectly acquiring" the sequence. "Directly acquiring a sequence" means performing a process (e.g., performing a synthetic or analytical method) to obtain the sequence, such as performing a sequencing method (e.g., a Next Generation Sequencing (NGS) method). "Indirectly acquiring a sequence" refers to receiving information or knowledge of, or receiving, the sequence from another party or source (e.g., a third party laboratory that directly acquired the sequence). The sequence acquired need not be a full sequence, e.g., sequencing of at least one nucleotide, or obtaining information or knowledge that identifies a mutation disclosed herein as being present in a subject constitutes acquiring a sequence.
Directly acquiring a sequence includes performing a process that includes a physical change in a physical substance, e.g., a starting material, such as a tissue sample, e.g., a biopsy, or an isolated nucleic acid (e.g., DNA or RNA) sample. Exemplary changes include making a physical entity from two or more starting materials, shearing or fragmenting a substance, such as a genomic DNA fragment; separating or purifying a substance (e.g., isolating a nucleic acid sample from a tissue); combining two or more separate entities into a mixture, performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond. Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance as described above.
"Acquiring a sample" as the term is used herein, refers to obtaining possession of a sample, e.g., a tissue sample or nucleic acid sample, by "directly acquiring" or "indirectly acquiring" the sample. "Directly acquiring a sample" means performing a process (e.g., performing a physical method such as a surgery or extraction) to obtain the sample. "Indirectly acquiring a sample" refers to receiving the sample from another party or source (e.g., a third party laboratory that directly acquired the sample). Directly acquiring a sample includes performing a process that includes a physical change in a physical substance, e.g., a starting material, such as a tissue, e.g., a tissue in a human patient or a tissue that has was previously isolated from a patient. Exemplary changes include making a physical entity from a starting material, dissecting or scraping a tissue; separating or purifying a substance (e.g., a sample tissue or a nucleic acid sample); combining two or more separate entities into a mixture; performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond. Directly acquiring a sample includes performing a process that includes a physical change in a sample or another substance, e.g., as described above.
An "alteration" as used herein, of a gene or gene product (e.g., a BRAF gene or gene product) refers to the presence of a mutation or mutations within the gene or gene product, e.g., a mutation, which affects amount or activity of the gene or gene product, as compared to the normal or wild- type gene. The alteration can be in amount, structure, and/or activity in a cancer tissue or cancer cell, as compared to its amount, structure, and/or activity, in a normal or healthy tissue or cell (e.g., a control), and is associated with a disease state, such as cancer. For example, a gene or gene product which is associated with cancer, or predictive of responsiveness to anti- cancer therapeutics, can have an altered nucleotide sequence (e.g., a mutation), amino acid sequence, chromosomal translocation, intra-chromosomal inversion, copy number, expression level, protein level, protein activity, or methylation status, in a cancer tissue or cancer cell, as compared to a normal, healthy tissue or cell. Exemplary mutations include, but are not limited to, point mutations (e.g., silent, missense, or nonsense), deletions, insertions, inversions, linking mutations, duplications, translocations, inter- and intra-chromosomal rearrangements. Mutations can be present in the coding or non-coding region of the gene. In certain embodiments, the alterations are associated (or not associated) with a phenotype, e.g., a cancerous phenotype (e.g., one or more of cancer risk, cancer progression, cancer treatment or resistance to cancer
treatment).
"Binding entity" means any molecule to which molecular tags can be directly or indirectly attached that is capable of specifically binding to an analyte. The binding entity can be an affinity tag on a nucleic acid sequence. In certain embodiments, the binding entity allows for separation of the nucleic acid from a mixture, such as an avidin molecule, or an antibody that binds to the hapten or an antigen-binding fragment thereof. Exemplary binding entities include, but are not limited to, a biotin molecule, a hapten, an antibody, an antibody binding fragment, a peptide, and a protein.
"Complementary" refers to sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. In certain embodiments, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In other embodiments, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. The term "cancer" or "tumor" is used interchangeably herein. These terms refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features.
The term "neoplasm" or "neoplastic" cell refers to an abnormal proliferative stage, e.g., a hyperproliferative stage, in a cell or tissue that can include a benign, pre-malignant, malignant (cancer) or metastatic stage.
In certain embodiments, the cancer or neoplasm is a hematologic (or hematological) malignancy. A hematological malignancy is a cancer that affects the blood, bone marrow, and lymph nodes. Hematological malignancies can include myelomas, leukemias, and lymphomas.
Cancer is "inhibited" if at least one symptom of the cancer is alleviated, terminated, slowed, or prevented. As used herein, cancer is also "inhibited" if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented.
"Chemo therapeutic agent" means a chemical substance, such as a cytotoxic or cytostatic agent that is used to treat a condition, particularly cancer.
As used herein, "cancer therapy" and "cancer treatment" are synonymous terms.
As used herein, "chemotherapy" and "chemotherapeutic" and "chemotherapeutic agent" are synonymous terms.
The terms "homology" or "identity," as used interchangeably herein, refer to sequence similarity between two polynucleotide sequences or between two polypeptide sequences, with identity being a more strict comparison. The phrases "percent identity or homology" and " identity or homology" refer to the percentage of sequence similarity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences. "Sequence similarity" refers to the percent similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0-100% similar, or any integer value there between. Identity or similarity can be determined by comparing a position in each sequence that can be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of similarity or identity between polynucleotide sequences is a function of the number of identical or matching nucleotides at positions shared by the polynucleotide sequences. A degree of identity of polypeptide sequences is a function of the number of identical amino acids at positions shared by the polypeptide sequences. A degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at positions shared by the polypeptide sequences. The term "substantially identical," as used herein, refers to an identity or homology of at least 75%, at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.
"Likely to" or "increased likelihood," as used herein, refers to an increased probability that an item, object, thing or person will occur. Thus, in one example, a subject that is likely to respond to treatment with a kinase inhibitor, alone or in combination, has an increased
probability of responding to treatment with the inhibitor alone or in combination, relative to a reference subject or group of subjects.
"Unlikely to" refers to a decreased probability that an event, item, object, thing or person will occur with respect to a reference. Thus, a subject that is unlikely to respond to treatment with a kinase inhibitor, alone or in combination, has a decreased probability of responding to treatment with a kinase inhibitor, alone or in combination, relative to a reference subject or group of subjects.
"Sequencing" a nucleic acid molecule requires determining the identity of at least 1 nucleotide in the molecule. In embodiments, the identity of less than all of the nucleotides in a molecule is determined. In other embodiments, the identity of a majority or all of the nucleotides in the molecule is determined.
"Next-generation sequencing or NGS or NG sequencing" as used herein, refers to any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules (e.g., in single molecule sequencing) or clonally expanded proxies for individual nucleic acid molecules in a highly parallel fashion (e.g., greater than 105 molecules are sequenced simultaneously). In one embodiment, the relative abundance of the nucleic acid species in the library can be estimated by counting the relative number of occurrences of their cognate sequences in the data generated by the sequencing experiment. Next generation sequencing methods are known in the art, and are described, e.g., in Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46, incorporated herein by reference. Next generation sequencing can detect a variant present in less than 5% of the nucleic acids in a sample. "Sample," "tissue sample," "patient sample," "patient cell or tissue sample" or "specimen" each refers to a collection of similar cells obtained from a tissue of a subject or patient. The source of the tissue sample can be solid tissue as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; or cells from any time in gestation or
development of the subject. In one embodiment, the sample is a blood sample, or a bone marrow biopsy or aspirate. In certain embodiments, the sample comprises a hematologic cell (e.g., a blood cell, a bone marrow cell, and/or lymphatic cell). The tissue sample can contain compounds that are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like. In one embodiment, the sample is preserved as a frozen sample or as formaldehyde- or paraformaldehyde-fixed paraffin-embedded (FFPE) tissue
preparation. For example, the sample can be embedded in a matrix, e.g., an FFPE block or a frozen sample.
A "tumor" or "cancer" nucleic acid sample as used herein, refers to nucleic acid molecules from a tumor or cancer sample. Typically, it is DNA, e.g., genomic DNA, or cDNA derived from RNA, from a tumor or cancer sample. In certain embodiments, the tumor nucleic acid sample is purified or isolated (e.g., it is removed from its natural state).
A "control" or "reference" "nucleic acid sample" as used herein, refers to nucleic acid molecules from a control or reference sample. Typically, it is DNA, e.g., genomic DNA, or cDNA derived from RNA, not containing the alteration or variation in the gene or gene product, e.g., not containing a mutation. In certain embodiments, the reference or control nucleic acid sample is a wild type or a non-mutated sequence. In certain embodiments, the reference nucleic acid sample is purified or isolated (e.g., it is removed from its natural state). In other embodiments, the reference nucleic acid sample is from a non-tumor sample, e.g., a blood control, a normal adjacent tumor (NAT), or any other non-cancerous sample from the same or a different subject.
"Adjacent to the interrogation position," as used herein, means that a site sufficiently close such that a detection reagent complementary with the site can be used to distinguish between a mutation, e.g., an alteration described herein, and a reference sequence, e.g., a non- mutant or wild- type sequence, in a target nucleic acid. Directly adjacent, as used herein, is where 2 nucleotides have no intervening nucleotides between them. "Associated mutation," as used herein, refers to a mutation within a preselected distance, in terms of nucleotide or primary amino acid sequence, from a definitional mutation, e.g., a mutant as described herein. In embodiments, the associated mutation is within n, wherein n is 2, 5, 10, 20, 30, 50, 100, or 200 nucleotides from the definitional mutation (n does not include the nucleotides defining the associated and definitional mutations). In embodiments, the associated mutation is a translocation mutation.
"Interrogation position," as used herein, comprises at least one nucleotide (or, in the case of polypeptides, an amino acid residue) which corresponds to a nucleotide (or amino acid residue) that is mutated in a mutation of interest, e.g., a mutation being identified, or in a nucleic acid (or protein) being analyzed, e.g., sequenced, or recovered.
A "reference sequence," as used herein, e.g., as a comparator for a mutant sequence, is a sequence which has a different nucleotide or amino acid at an interrogation position than does the mutant(s) being analyzed. In one embodiment, the reference sequence is wild-type for at least the interrogation position.
Headings, e.g., (a), (b), (i) etc, are presented merely for ease of reading the specification and claims. The use of headings in the specification or claims does not require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.
Various aspects featured in the invention are described in further detail below. Additional definitions are set out throughout the specification.
Therapeutic Methods and Agents
The invention provides, at least in part, methods for treating a cancer, e.g., a
hematological malignancy, e.g., a CLL, in a subject. In certain embodiments, the methods include treatment of a cancer, e.g., a hematological malignancy, e.g., a CLL harboring an alteration described herein (e.g., a BRAF and/or KRAS alteration described herein). The methods include administering to the subject a therapeutic agent, e.g., an agent that antagonizes the function of a MAPK pathway gene or gene product (e.g., a MAPK pathway inhibitor as described herein).
In certain embodiments the cancer is a hematological malignancy. In certain
embodiments, the hematological malignancy is chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (ALoL), a Hodgkin' s lymphoma (HL), e.g., nodular sclerosing HL, mixed cellularity HL, lymphocyte-rich HL (lymphocute predominance HL), lymphocyte depleted HL, unspecified HL) or a Non-hodgkin's lymphoma (NHL), e.g., Indolent (low grade) NHL, aggressive (high grade) NHL, a B-cell lymphoma, e.g., diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, mediastinal large B cell-ltmphoma, splenic marginal zone B cell lymphoma, extranodal marginal zone B cell lymphoma of mucosa assoiated lymphoid tissue, nodal marginal zone B cell lymphoma, lymphoblastic lymphoma, primary effusion lymphoma, Burkitt lymphoma; a T cell lymphoma, e.g., anaplastic large cell lymphoma (primary cutaneous type), peripheral T cell lymphoma (NOS), angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, (systemic type), Precursor T-lymphoblastic lymphoma, Adult T-cell lymphoma, enteropathy- associated T-cell lymphoma, Gamma/delta hepatosplenic T-cell lymphoma, Subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides, a natural killer (NK )cell lymphoma, e.g., extranodal NK-cell lymphoma, a B cell leukemia, e.g., Burkitt cell leukemia, hairy cell leukemia. B-cell prolymphocyte leukemia; or a T cell leukemia, e.g., Adult T-cell leukemia, precursor T- lymphoblastic leukemia.
In certain embodiments, the cancer is a CLL. In certain embodiments the cancer is a refractory CLL. In certain embodiments the cancer is a relapsed CLL.
In some embodiments, the hematological malignancy, e.g., a CLL, is any stage or risk group defined according to any suitable classification system known to those of skill in the art.
In some embodiments, the CLL, is any stage or risk group of CLL defined according to any suitable CLL classification system known to those of skill in the art.
In certain embodiments, the CLL is a stage 0, 1, II, III, or IV, wherein stage 0 CLL is characterized by too many lymphocytes in the blood, e.g., compared to a reference value (e.g., a control), but there are no other symptoms of leukemia; stage I CLL is characterized by too many lymphocytes in the blood and the lymph nodes are larger than normal; stage II CLL is characterized by too many lymphocytes in the blood, the liver or spleen is larger than normal, and the lymph nodes may be larger than normal; stage III CLL is characterized by too many lymphocytes in the blood, there are too few red blood cells, and the lymph nodes, liver, or spleen may be larger than normal, and stage IV CLL is characterized by too many lymphocytes in the blood, too few platelets, the lymph nodes, liver, or spleen may be larger than normal, and there may be too few red blood cells.
In certain embodiments, the CLL is a stage 0, 1, II, III, or IV on the Rai Scale (Rai K et al. Blood Volume 46, Issue 2, 1975, Pages 219-234). In certain embodiments, the CLL is a stage 0, 1, II, III, or IV on the Rai Scale wherein stage 0 CLL is characterized by bone marrow and blood lymphocytosis only; stage I CLL is characterized by lymphocytosis with enlarged nodes; stage II CLL is characterized by lymphocytosis with enlarged spleen or liver or both; stage III CLL is characterized by lymphocytosis with anemia; and stage IV CLL is characterized by lymphocytosis with thrombocytopenia (Rai, supra).
In certain embodiments, the CLL is a stage A, B, or C on the Binet Scale; wherein stage A is characterized by Hb 100 g/L (10 g/dL) or more and platelets 100 x 109/L or more and up to 2 of the following areas are involved: 1. Head and neck, including the Waldeyer ring (this counts as one area, even if more than one group of nodes is enlarged); 2. Axillae (involvement of both axillae counts as one area); 3. Groins, including superficial femorals (involvement of both groins counts as one area); 4. Palpable spleen; and 5. Palpable liver (clinically enlarged); stage B is characterized as Hb 100 g/L (10 g/dL) or more and platelets 100 x 109/L or more and
organomegaly greater than that defined for stage A (ie, 3 or more areas of nodal or organ enlargement); and stage C is characterized as patients who have Hb less than 100 g/L (10 g/dL) and/or a platelet count less than 100 x 109/L, irrespective of organomegaly (Hallek M et al. Blood June 15, 2008 vol. I l l no. 12 5446-5456).
In some embodiments, the CLL, is any stage of CLL defined according to any suitable CLL classification system known to those of skill in the art.
In certain embodiments, the CLL is a low risk disease, intermediate risk disease, or high risk diseaseon the modified Rai Scale, wherein low risk is characterized by lymphocytes less than 15 x lOg/1; intermediate risk is characterized by lymphocytes less than 15 x lOg/1 and hepato- or splenomegaly; and high risk is characterized as Anemia (Hb < 11 g/dL) or thrombocytopenia (platelets < 100 xl09/L) (Hallek M et al. Blood June 15, 2008 vol. I l l no. 12 5446-5456).
In some embodiments, the CLL, is any risk group of CLL defined according to any suitable CLL classification system known to those of skill in the art. In other embodiment, the cancer, e.g., the hematological malignancy, e.g., CLL, comprises, or is identified or determined as having, an alteration in a MAPK pathway gene or gene product, e.g., an alteration in BRAF and/or KRAS as described herein.
"Treat," "treatment," and other forms of this word refer to the administration of an agent, e.g., a therapeutic agent, alone or in combination with a second agent in an amount effective to impede growth of a cancer, to cause a cancer to shrink by weight or volume, to extend the expected survival time of the subject and or time to progression of the tumor or the like. In those subjects, treatment can include, but is not limited to, inhibiting tumor growth, reducing tumor mass, reducing size or number of metastatic lesions, inhibiting the development of new metastatic lesions, prolonged survival, prolonged progression-free survival, prolonged time to progression, and/or enhanced quality of life. A cancer is "treated" if at least one symptom of the cancer is alleviated, terminated, slowed or prevented. A cancer is also "treated" if recurrence or metastasis of the cancer is reduced, slowed, delayed or prevented.
As used herein, unless otherwise specified, the terms "prevent," "preventing" and "prevention" contemplate an action that occurs before a subject begins to suffer from the re- growth of the cancer and/or which inhibits or reduces the severity of the cancer.
As used herein, and unless otherwise specified, a "therapeutically effective amount" of an agent is an amount sufficient to provide a therapeutic benefit in the treatment or management of the cancer, or to delay or minimize one or more symptoms associated with the cancer. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the cancer. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the cancer, or enhances the therapeutic efficacy of another therapeutic agent.
As used herein, and unless otherwise specified, a "prophylactically effective amount" of an agent is an amount sufficient to prevent re-growth of the cancer, or one or more symptoms associated with the cancer, or prevent its recurrence. A prophylactically effective amount of an agent means an amount of the agent, alone or in combination with other therapeutic agents, which provides a prophylactic benefit in the prevention of the cancer. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent. The term "patient" or "subject" includes a human (e.g., a male or female of any age group, e.g., a pediatric patient (e.g., infant, child, adolescent) or adult patient (e.g., young adult, middle-aged adult or senior adult). When the term is used in conjunction with administration of a compound or drug, then the patient has been the object of treatment, observation, and/or administration of the compound or drug.
These treatments can be provided to a patient having had an unsatisfactory response to a different (e.g., non-MAPK pathway inhibitor as described herein). In one embodiment, the subject is undergoing or has undergone treatment with a different therapeutic agent or therapeutic modality (e.g., non-MAPK pathway inhibitor). In one embodiment, the different therapeutic agent or therapeutic modality is a chemotherapy, an immunotherapy, or a surgical procedure. In one embodiment, the different therapeutic agent or therapeutic modality comprises one or more of: a chemotherapeutic agent (e.g., fludarabine, cyclophosphamide, doxorubicin, vincristine,
chlorambucil, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, 5-azacytidine, pemetrexed disodium), tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g., erlotinib), BTK inhibitor (e.g., PCI-32765), CD20 targeting agent (e.g., rituximab, ofatumumab, RO5072759, LFB-R603), CD52 targeting agent (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, Bcl-2 inhibitor (e.g., ABT-263), AT-101, immunotoxin (e.g., CAT- 8015, anti-Tac(Fv)-PE38 (LMB-2)), CD37 targeting agent (e.g., TRU-016), radioimmunotherapy (e.g., 131-tositumomab), hydroxychloroquine, perifosine, SRC inhibitor (e.g., dasatinib), thalidomide, retinoid (e.g., fenretinide), MDM2 antagonist (e.g., RO5045337), plerixafor, Aurora kinase inhibitor (e.g., MLN8237, TAK-901), proteasome inhibitor (e.g., bortezomib), CD19 targeting agent (e.g., MEDI-551, MOR208), JAK-2 inhibitor (e.g., INCB018424), hypoxia-activated prodrug (e.g., TH-302), paclitaxel or a paclitaxel agent, AKT inhibitor (e.g., MK2206), HMG-CoA inhibitor (e.g., simvastatin), GNKG186, radiation therapy, bone marrow transplantation, stem cell transplantation, immunotherapy, or a combination thereof.
In one embodiment, responsive to the determination of the presence of the alteration described herein, the different therapeutic agent or therapeutic modality (e.g., non-MAPK pathway inhibitor as described herein) is discontinued. In yet other embodiments, the subject has been identified as being likely or unlikely to respond to the different therapeutic agent or therapeutic modality. An agent, e.g., a therapeutic agent, described herein can be administered, alone or in combination, e.g., in combination with other chemotherapeutic agents or procedures, in an amount sufficient to reduce or inhibit the malignant cell growth, and/or treat or prevent the malignancy (e.g., CLL), in the subject.
The agent, e.g., therapeutic agent, can be a small molecule, a protein, a polypeptide, a peptide, an antibody molecule, a nucleic acid (e.g., a siRNA, an antisense or a micro RNA), a small molecule, or an immune cell therapy. Exemplary agents and classes of agents are described herein.
In one embodiment, the agent is a BRAF inhibitor. In one embodiment, the BRAF inhibitor is chosen from: Vemurafenib (PLX4032, RG7204, R05185426), Sorafenib Tosylate (Bay 43-9006, Nexavar), PLX4720, GDC-0879, RAF265 (CHIR-265), MLN2480 (BIIB-024), PF-04880594, GW5074, CEP-32496, Dabrafenib (GSK2118436), AZ628, SB590885, Raf265 derivative, Regorafenib (BAY 73-4506, Fluoro-Sorafenib), and/or ZM 336372.
In some embodiments, the BRAF inhibitor is Vemurafenib (PLX4032, RG7204, R05185426). Vemurafenib is a potent inhibitor of BRAFV600E with an IC50 of about 31 nM. Vemurafenib inhibits B-RAFV600E, C-RAF, as well as wildtype B-RAF, with an IC50 of about 31 nM, 48 nM and 100 nM, respectively. Vemurafenib also inhibits several non-RAF kinases, including ACK1, KHS 1, and SRMS, with IC50 of 18 nM to 51 nM. Vemurafenib has the chemical name: N-(3-{ [5-(4-chlorophenyl)- lH-pyrrolo[2,3-b]pyridin-3-yl]carbonyl}-2,4- difluorophenyl)propane- l-sulfonamide; and has the following structure:
Figure imgf000050_0001
Vemurafenib Chemical Structure
Molecular Weight: 489.92.
In some embodiments, the BRAF inhibitor is Sorafenib Tosylate (Bay 43-9006,
Nexavar). Sorafenib is a multikinase inhibitor of Raf-1, BRAF and VEGFR-2 with an IC50 about 6 nM, 22 nM and 90 nM, respectively. Sorafenib has the chemical name: 2- Pyridinecarboxamide, 4-[4-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino] phenoxy] -N-methyl-, 4-methylbenzenesulfonate (1: 1); and has the following structure:
Figure imgf000051_0001
C HROJS
Sorafenib Chemical Structure
Molecular Weight: 637.03.
In some embodiments, the BRAF inhibitor is PLX4720. PLX4720 is a potent and selective inhibitor of BRAFV600E with IC50 of about 13 nM, modest potent to c-Raf- 1(Y340D and Y341D mutations), 10-fold selectivity for BRAFV600E than wild-type BRAF. PLX4720 has the chemical name: N-(3-(5-chloro-lH-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4- difluorophenyl)propane-l-sulfonamide; and has the following structure:
Figure imgf000051_0002
PLX4720 Chemical Structure
Molecular Weight: 413.83.
In some embodiments, the BRAF inhibitor is GDC-0879. GDC-0879 is a potent, and selective BRAF inhibitor with an IC50 of about 0.13 nM with activity against c-Raf as well; no inhibition known to other protein kinases. GDC-0879 has the chemical name: (E)-5-(l-(2- hydroxyethyl)-3-(pyridin-4-yl)-lH-pyrazol-4-yl)-2,3-dihydroinden-l-one oxime; and has the following structure:
Figure imgf000052_0001
GDC-0879 Chemical Structure
Molecular Weight: 337.37.
In some embodiments, the BRAF inhibitor is RAF265 (CHIR-265). RAF265 is a highly selective BRAF and VEGFR2 inhibitor with an IC50 of about 3-60 nM and an EC50 of about 30 nM, including BRAF, C-Raf and mutant BRAF. RAF265 has the chemical name: l-methyl-5-(2- (5-(trifluoromethyl)-lH-imidazol-2-yl)pyridin-4-yloxy)-N-(4-(trifluoromethyl)phenyl)-lH- benzo[d]imidazol-2- amine; and has the following structure:
Figure imgf000052_0002
RAF265 Chemical Structure
Molecular Weight: 518.41.
In some embodiments, the BRAF inhibitor is MLN2480 (BIIB-024). MLN2480 is a pan- Raf inhibitor. MLN2480 has the chemical name: 4-Pyrimidinecarboxamide, 6-amino-5-chloro- N-[(lR)-l-[5-[[[5-chloro-4-(trifluoromethyl)-2-pyridinyl]amino]carbonyl]-2-thiazolyl]ethyl]-; and has the following structure:
Figure imgf000053_0001
MLN2480 Chemical Structure
Molecular Weight: 506.29.
In some embodiments, the BRAF inhibitor is PF-04880594. PF-04880594 is a RAF inhibitor for BRAF/BRAFV599E and c-RAF with an IC50 of about 0.19 nM/0.13 nM and 0.39 nM, >100-fold selectivity over CSNKl, JNK2/3 and p38. PF-04880594 has the chemical name: Propanenitrile, 3-[[4-[l-(2,2-difluoroethyl)-3-(lH-pyrrolo[2,3-b]pyridin-5-yl)-lH-pyrazol-4-yl]- 2-pyrimidinyl] amino]-; and has the following structure:
Figure imgf000053_0002
PF-04880594 Chemical Structure
Molecular Weight: 394.38.
In some embodiments, the BRAF inhibitor is GW5074. GW5074 is a potent and selective c-Raf inhibitor with an IC50 of about 9 nM, no effect on the activities of JNK1/2/3, MEK1, MKK6/7, CDKl/2, c-Src, p38 MAP, VEGFR2 or c-Fms is noted. GW5074 has the chemical name: 2H-Indol-2-one, 3-[(3,5-dibromo-4-hydroxyphenyl)methylene]-l,3-dihydro-5-iodo-; and has the following structure:
Figure imgf000054_0001
GW5074 Chemical Structure
Molecular Weight: 520.94.
In some embodiments, the BRAF inhibitor is CEP-32496. CEP-32496 is a highly potent inhibitor of BRAF(V600E/WT) and c-Raf with Kd of 14 nM/36 nM and 39 nM, modest potent to Abl-1, c-Kit, Ret, PDGFRp and VEGFR2, respectively; insignificant affinity for MEK-1, MEK- 2, ERK-1 and ERK-2. CEP-32496 has the chemical name: Urea, N-[3-[(6,7-dimethoxy-4- quinazolinyl)oxy]phenyl]-N-[5-(2,2,2-trifluoro-l,l-dimethylethyl)-3-isoxazolyl]-; and has the following structure:
Figure imgf000054_0002
CEP-32496 Chemical Structure
Molecular Weight: 517.46.
In some embodiments, the BRAF inhibitor is Dabrafenib (GSK2118436). Dabrafenib is a mutant BRAFV600 specific inhibitor with an IC50 of about 0.8 nM, with 4- and 6-fold less potency against BRAF(wt) and c-Raf, respectively. Dabrafenib has the chemical name: N-(3-(5- (2-aminopyrimidin-4-yl)-2-tert-butylthiazol-4-yl)-2-fluorophenyl)-2,6- difluorobenzenesulfonamide; and has the following structure:
Figure imgf000055_0001
Dabrafenib Chemical Structure
Molecular Weight: 519.56.
In some embodiments, the BRAF inhibitor is AZ628. AZ628 is a new pan-Raf inhibitor for BRAF, BRAFV600E, and c-Raf- 1 with an IC50 of about 105 nM, 34 nM and 29 nM, also inhibits VEGFR2, DDR2, Lyn, Fltl, FMS, etc. AZ628 has the chemical name: 3-(2- cyanopropan-2-yl)-N-(4-methyl-3-(3-methyl-4-oxo-3,4-dihydroquinazolin-6- ylamino)phenyl)benzamide; and has the following structure:
Figure imgf000055_0002
AZ628 Chemical Structure
Molecular Weight: 451.52.
In some embodiments, the BRAF inhibitor is SB590885. SB590885 is a potent BRAF inhibitor with a Kj of about 0.16 nM, 11-fold greater selectivity for BRAF over c-Raf, and shows no detectable inhibition of other human kinases. SB590885 has the chemical name: (E)-5-(2-(4- (2-(dimethylamino)ethoxy)phenyl)-4-(pyridin-4-yl)-lH-imidazol-5-yl)-2,3-dihydroinden-l-one oxime; and has the following structure:
Figure imgf000056_0001
SB590885 Chemical Structure
Molecular Weight: 453.54.
In some embodiments, the BRAF inhibitor is Raf265 derivative. Raf265 derivative is a derivative of Raf265. Raf265 derivative has the following structure:
Figure imgf000056_0002
Raf265 derivative Chemical Structure
Molecular Weight: 504.39.
In some embodiments, the BRAF inhibitor is Regorafenib (BAY 73-4506, Fluoro- Sorafenib). Regorafenib is a multi-target inhibitor for VEGFRl, VEGFR2, VEGFR3, PDGFRp, Kit, RET and Raf-1 with an IC50 of about 13 nM/4.2 nM/46 nM, 22 nM, 7 nM, 1.5 nM and 2.5 nM, respectively. Regorafenib has the chemical name: l-(4-chloro-3-(trifluoromethyl)phenyl)-3- (2-fluoro-4-(2-(methylcarbamoyl)pyridin-4-yloxy)phenyl)urea; and has the following structure:
Figure imgf000057_0001
Regorafenib Chemical Structure
Molecular Weight: 482.82.
In some embodiments, the BRAF inhibitor is ZM 336372. ZM 336372 is a potent and selective c-Raf inhibitor with an IC50 of about 70 nM, 10-fold selectivity over B-RAF. ZM 336372 has the chemical name: Benzamide, 3-(dimethylamino)-N-[3-[(4- hydroxybenzoyl)amino]-4-methylphenyl]-; and has the following structure:
Figure imgf000057_0002
ZM 336372 Chemical Structure
Molecular Weight: 389.45.
In certain embodiments, the agent (e.g., the therapeutic agent) used in the methods targets and/or inhibits MEK (mitogen activated protein kinase kinase) (e.g., MEKl and/or MEK2) (e.g., a MEKl and or MEK2 gene or gene product). In one embodiment, the agent binds and inhibits MEK. In one embodiment, the agent is a reversible or an irreversible MEK inhibitor.
In some embodiments, the MEK inhibitor includes an agent that inhibits the mitogen- activated protein kinase kinase enzymes MEKl and/or MEK2. In other embodiments, the MEK inhibitor is chosen from: ARRY- 162 (MEK162), Trametinib (GSK1120212), Selumetinib (AZD6244, ARRY142886), XL518 (GDC-0973), CI-1040 (PD184352), PD035901, U0126- EtOH, PD198306, PD98059, BIX 02189, TAK-733, Honokiol, AZD8330 (ARRY-424704), PD318088, BIX 02188, AS703026 (Pimasertib) and/or SL327.
In some embodiments, the MEK inhibitor is ARRY-162 (MEK 162). ARRY-162 is a potent, orally bioavailable and non-ATP competitive inhibitor of MEK1/2 (IC50 = 12 nM) and cellular pERK (IC50 = 11 nM). It shows ex vivo inhibition of cytokine production such as IL-1, TNF and IL-6 in clinical trials. ARRY-162 has the chemical name: 5-((4-bromo-2- fluorophenyl)amino)-4-fluoro-N-(2-hydroxyethoxy)-l-methyl-lH-benzo[d]imidazole-6- carboxamide; and has the following structure:
Figure imgf000059_0001
ARRY-162 Chemical Structure
Molecular Weight: 441.22681.
In some embodiments, the MEK inhibitor is Trametinib (GSK1120212). Trametinib is a highly specific and potent MEKl/2 inhibitor with an IC50 of about 0.92 nM/1.8 Nm. Trametinib does not inhibit the kinase activities of c-Raf, B-Raf, ERKl/2. Trametinib is currently FDA- approved for use in the treatment of BRAF-mutated melanoma. Trametinib has also been studied in combination with the BRAF inhibitor dabrafenib for use in the treatment of BRAF-mutated melanoma. Trametinib has the chemical name: N-(3-{ 3-Cyclopropyl-5-[(2-fluoro-4- iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-l(2H)- yl} phenyl) acetamide; and has the following structure:
Figure imgf000059_0002
Trametinib Chemical Structure
Molecular Weight: 615.39.
In some embodiments, the MEK inhibitor is Selumetinib (AZD6244, ARRY142886). Selumetinib is a potent, highly selective MEK1 inhibitor with IC50 of 14 nM, also inhibits ERKl/2 phosphorylation with IC50 of 10 nM Selumetinib has been studied in phase 2 clinical trial for use in the treatment of non-small cell lung cancer (NSCLC). Selumetinib has the chemical name: 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3- methylbenzimidazole-5-carboxamide; and has the following structure:
Figure imgf000060_0001
Selumetinib Chemical Structure
Molecular Weight: 457.68.
In some embodiments, the MEK inhibitor is XL518 (GDC-0973). XL518 a potent, selective, orally bioavailable inhibitor of MEKl. It inhibits the proliferation and stimulates apoptosis in a variety of human tumor cell lines. XL518 has the chemical name: [3,4-difluoro-2- [(2-fluoro-4-iodophenyl)amino]phenyl][3-hydroxy-3-[(2S)-2-piperidinyl]-l- azetidinyl]methanone; and has the following structure:
Figure imgf000060_0002
XL518 Chemical Structure
Molecular Weight: 531.31.
In some embodiments, the MEK inhibitor is CI- 1040 (PD184352). CI- 1040 is an ATP non-competitive MEKl/2 inhibitor with an IC50 of about 17 nM, 100-fold more selective for MEKl/2 than MEK5. CI- 1040 has the chemical name: 2-(2-chloro-4-iodophenylamino)-N- (cyclopropylmethoxy)-3,4-difluorobenzamide; and has the following structure:
Figure imgf000060_0003
CI- 1040 Chemical Structure
Molecular Weight: 478.67.
In some embodiments, the MEK inhibitor is PD035901. PD0325901 is selective and non ATP-competitive MEK inhibitor with an IC50 of about 0.33 nM, roughly 500-fold more potent than CI- 1040 on phosphorylation of ERKl and ERK2. PD035901 has the chemical name: (R)-N- (2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide; and has the following structure:
Figure imgf000061_0001
PD035901 Chemical Structure
Molecular Weight: 482.19.
In some embodiments, the MEK inhibitor is U0126-EtOH. U0126-EtOH is a highly selective inhibitor of MEKl/2 with an IC50 of about 0.07 μΜ/0.06 μΜ, 100-fold higher affinity for AN3-S218E/S222D MEK than PD098059. U0126-EtOH has the chemical name: (2Z,3Z)- 2,3-bis(amino(2-aminophenylthio)methylene)succinonitrile,ethanol; and has the following structure:
Figure imgf000061_0002
EtO H
U0126-EtOH Chemical Structure
Molecular Weight: 426.56.
In some embodiments, the MEK inhibitor is PD198306. PD198306 is a cell-permeable and highly selective MEK inhibitor with an IC50 of about 8 nM. PD198306 has the chemical name: Benzamide, N-(cyclopropylmethoxy)-3,4,5-trifluoro-2-[(4-iodo-2-methylphenyl)amino]-; and has the following structure:
Figure imgf000062_0001
PD 198306 Chemical Structure
Molecular Weight: 476.23.
In some embodiments, the MEK inhibitor is PD98059. PD98059 is a non-ATP competitive MEK inhibitor with an IC50 of about 2 μΜ, specifically inhibits MEK-1 -mediated activation of MAPK. PD98059 does not directly inhibit ERK1 or ERK2. PD98059 has the chemical name: 2-(2-amino-3-methoxyphenyl)-4H-chromen-4-one; and has the following structure:
Figure imgf000062_0002
PD98059 Chemical Structure
Molecular Weight: 267.28.
In some embodiments, the MEK inhibitor is BIX 02189. BIX 02189 is a selective inhibitor of MEK5 with IC50 of 1.5 nM, also inhibits ERK5 catalytic activity with IC50 of 810 nM. BIX 02189 does not inhibit closely related kinases MEKl, MEK2, ERK2, and JNK2. BIX 02189 has the chemical name: (Z)-3-((3-((dimethylamino)methyl)phenylamino)(phenyl) methylene) -N,N-dimethyl-2-oxoindoline-6-carboxamide; and has the following structure:
Figure imgf000063_0001
BIX 02189 Chemical Structure
Molecular Weight: 440.54.
In some embodiments, the MEK inhibitor is TAK-733. TAK-733 is a potent and selective MEK allosteric site inhibitor for MEKl with an IC50 of about 3.2 nM. TAK-733 has the chemical name: (R)-3-(2,3-dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8- methylpyrido[2,3-d]pyrimidine-4,7( -dione; and has the following structure:
Figure imgf000063_0002
TAK-733 Chemical Structure
Molecular Weight: 267.28.
In some embodiments, the MEK inhibitor is Honokiol. Honokiol is the active principle of magnolia extract that inhibits Akt-phosphorylation and promotes ERK1 ^phosphorylation.
Honokiol has the chemical name: 2-(4-hydroxy-3-prop-2-enyl-phenyl)- 4-prop-2-enyl-phenol; and has the following structure:
Figure imgf000063_0003
Honokiol Chemical Structure
Molecular Weight: 266.334. In some embodiments, the MEK inhibitor is AZD8330 (ARRY-424704). AZD8330 is a novel, selective, non-ATP competitive MEK 1/2 inhibitor with IC50 of 7 nM. AZD8330 has the chemical name: 2-(2-fluoro-4-iodophenylamino)-N-(2-hydroxyethoxy)- 1 ,5-dimethyl-6-oxo- 1 ,6- dihydropyridine-3-carboxamide; and has the following structure:
Figure imgf000064_0001
AZD8330 Chemical Structure
Molecular Weight: 461.23.
In some embodiments, the MEK inhibitor is PD318088. PD318088 is a non-ATP competitive allosteric MEK1/2 inhibitor, binds simultaneously with ATP in a region of the MEKl active site that is adjacent to the ATP-binding site. PD318088 has the chemical name: 5- bromo-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide; and has the following structure:
Figure imgf000064_0002
PD318088 Chemical Structure
Molecular Weight: 561.09.
In some embodiments, the MEK inhibitor is BIX 02188. ΒΓΧ02188 is a selective inhibitor of MEK5 with an IC50 of about 4.3 nM, also inhibits ERK5 catalytic activity with an IC50 of about 810 nM. BIX 02188 does not detectably inhibit closely related kinases MEKl, MEK2, ERK2, and JNK2. ΒΓΧ02188 has the chemical name: (Z)-3-((3- ((dimethylamino)methyl) phenylamino) (phenyl)methylene) -2-oxoindoline-6-carboxamide; and has the following structure:
Figure imgf000065_0001
ΒΓΧ02188 Chemical Structure
Molecular Weight: 426.51.
In some embodiments, the MEK inhibitor is AS703026 (Pimasertib). AS-703026 is a highly selective, potent, ATP non-competitive allosteric inhibitor of MEKl/2 with an IC50 of about 0.005-2 μΜ in MM cell lines. AS703026 has the chemical name: (S)-N-(2,3- dihydroxypropyl)-3-(2-fluoro-4-iodophenylamino)isonicotinamide; and has the following structure:
Figure imgf000065_0002
AS703026 Chemical Structure
Molecular Weight: 431.20.
In some embodiments, the MEK inhibitor is SL327. SL327 is a selective inhibitor for MEKl/2 with an IC50 of about 0.18 μΜ/ 0.22 μΜ. SL327 has no detectable activity towards Erkl, MKK3, MKK4, c-JUN, PKC, PKA, or CamKII. SL327 is capable of transport through the blood-brain barrier. SL327 has the chemical name: (Z)-3-amino-3-(4-aminophenylthio)-2-(2- (trifluoromethyl)phenyl)acrylonitrile; and has the following structure:
Figure imgf000066_0001
SL327 Chemical Structure
Molecular Weight: 335.35.
Combination Therapy
The agents, e.g., the therapeutic agents described herein, can be administered in combination with a second therapeutic agent or a different therapeutic modality, e.g., anti-cancer agents, and/or in combination with surgical and/or radiation procedures.
By "in combination with," it is not intended to imply that the therapy or the therapeutic agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the invention. The pharmaceutical
compositions can be administered concurrently with, prior to, or subsequent to, one or more other additional therapies or therapeutic agents. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In will further be appreciated that the additional therapeutic agent utilized in this combination can be administered together in a single composition or administered separately in different compositions. The particular combination to employ in a regimen will take into account compatibility of the inventive pharmaceutical composition with the additional therapeutically active agent and/or the desired therapeutic effect to be achieved.
In certain embodiments, a first therapeutic agent described herein, is administered in combination with a second therapeutic agent, described herein. In certain embodiments, the first therapeutic agent is a BRAF inhibitor described herein, and the second therapeutic agent is a MEK inhibitor described herein. Rationale for the combination of a BRAF inhibitor and a MEK inhibitor for therapeutic treatment is supported by, for example, Smalley K et al. Br J Cancer 2009 February 10; 100(3): 431-435; and Greger J et al. Mol Cancer Ther. 2012 Apr;l l(4):909- 20; the contents of which are incorporated herein by reference.
For example, the first therapeutic agent is a BRAF inhibitor and is chosen from:
Vemurafenib (PLX4032, RG7204, R05185426), Sorafenib Tosylate (Bay 43-9006, Nexavar), PLX4720, GDC-0879, RAF265 (CHIR-265), MLN2480 (BIIB-024), PF-04880594, GW5074, CEP-32496, Dabrafenib (GSK2118436), AZ628, SB590885, Raf265 derivative, Regorafenib (BAY 73-4506, Fluoro-Sorafenib), and/or ZM 336372; and the second therapeutic agent is a MEK inhibitor and is chosen from ARRY- 162 (MEK162), Trametinib (GSK1120212),
Selumetinib (AZD6244, ARRY142886), XL518 (GDC-0973), CI- 1040 (PD184352), PD035901, U0126-EtOH, PD198306, PD98059, BIX 02189, TAK-733, Honokiol, AZD8330 (ARRY- 424704), PD318088, BIX 02188, AS703026 (Pimasertib) and/or SL327.
In other embodiment, either the BRAF inhibitor or the MEK inhibitor, or both can be administered in combination with a therapeutic agent or therapeutic modality currently used in the treatment of a hematological malignancy, e.g., CLL. In one embodiment, the therapeutic agent or therapeutic modality currently used in the treatment of the hematological malignancy is a non-MAPK pathway inhibitor. In one embodiment, the non-MAPK pathway inhibitor therapeutic agent or therapeutic modality is a chemotherapy, an immunotherapy, or a surgical procedure. In one embodiment, the non-MAPK pathway inhibitor therapeutic agent or therapeutic modality comprises one or more of: a chemotherapeutic agent (e.g., fludarabine, cyclophosphamide, doxorubicin, vincristine, chlorambucil, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, 5-azacytidine, pemetrexed disodium), tyrosine kinase inhibitor (e.g., EGFR inhibitor (e.g., erlotinib), BTK inhibitor (e.g., PCI-32765), multikinase inhibitor (e.g., MGCD265, RGB-286638), CD20 targeting agent (e.g., rituximab, ofatumumab, RO5072759, LFB-R603), CD52 targeting agent (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, Bcl-2 inhibitor (e.g., ABT-263), HDAC inhibitor (e.g., vorinostat, valproic acid, LBH589, JNJ-26481585, AR-42), XIAP inhibitor (e.g.,
AEG35156), CD74 targeting agent (e.g., milatuzumab), mTOR inhibitor (e.g., everolimus), AT- 101, immunotoxin (e.g., CAT-8015, anti-Tac(Fv)-PE38 (LMB-2)), CD37 targeting agent (e.g., TRU-016), radioimmunotherapy (e.g., 131-tositumomab), hydroxychloroquine, perifosine, SRC inhibitor (e.g., dasatinib), thalidomide, retinoid (e.g., fenretinide), MDM2 antagonist (e.g., RO5045337), plerixafor, Aurora kinase inhibitor (e.g., MLN8237, TAK-901), proteasome inhibitor (e.g., bortezomib), CD19 targeting agent (e.g., MEDI-551, MOR208), JAK-2 inhibitor (e.g., INCBO 18424), hypoxia- activated prodrug (e.g., TH-302), paclitaxel or a paclitaxel agent, AKT inhibitor (e.g., MK2206), HMG-CoA inhibitor (e.g., simvastatin), GNKG186, radiation therapy, bone marrow transplantation, stem cell transplantation, immunotherapy (e.g., allogeneic CD4+ memory Thl-like T cells/microparticle -bound anti-CD3/anti-CD28, autologous cytokine induced killer cells (CIK)), or a combination thereof.
Antibody Molecules
In one embodiment, the agent, e.g., therapeutic agent, binds and inhibits a MAPK pathway protein, e.g., a BRAF or MEK protein. In one embodiment, the agent is an antibody molecule. The terms "antibody" and "antibody molecule" as used interchangeably herein refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide featured in the invention. A molecule which specifically binds to a given polypeptide featured in the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies. The term "monoclonal antibody" or "monoclonal antibody composition," as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope. Techniques for generating antibodies to a polypeptide target, e.g., BRAF (see e.g., WO 2012/092426, entitled "Optimization of Multigene Analysis of Tumor Samples," incorporated herein by reference.
In one embodiment, the agent is an antibody molecule, e.g., an anti- BRAF or anti-MEK antibody molecule (e.g., a monoclonal or a bispecific antibody), or a conjugate thereof (e.g., an antibody conjugated to a cytotoxic agent (e.g., mertansine DM1)).
Nucleic Acid Inhibitors
In yet another embodiment, the agent, e.g., the therapeutic agent, inhibits the expression of a nucleic acid encoding an alteration described herein. Examples of such agents include nucleic acid molecules, for example, antisense molecules, ribozymes, siRNA, triple helix molecules that hybridize to a nucleic acid encoding a mutation, or a transcription regulatory region, and blocks or reduces mRNA expression of the mutation. In other embodiments, the agent is chosen from a nucleic acid molecule (e.g., an antisense molecule, a ribozyme, a double stranded RNA, or a triple helix molecule) that hybridizes to and/or inhibits a MAPK kinase gene or gene product, e.g., a BRAF, KRAS or MEK nucleic acid, e.g., a nucleic acid encoding the
alteration, or a transcription regulatory region that blocks or reduces mRNA expression of the alteration.
In one embodiment, the nucleic acid antagonist is a siRNA that targets mRNA encoding a mutation. Other types of antagonistic nucleic acids can also be used, e.g., a dsRNA, a ribozyme, a triple- helix former, or an antisense nucleic acid. Accordingly, isolated nucleic acid molecules that are nucleic acid inhibitors, e.g., antisense, RNAi, to a mutation-encoding nucleic acid molecule are provided.
An "antisense" nucleic acid can include a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double- stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire mutation coding strand, or to only a portion thereof. In another
embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding mutation (e.g., the 5' and 3' untranslated regions). Anti-sense agents can include, for example, from about 8 to about 80 nucleobases (i.e., from about 8 to about 80 nucleotides), e.g., about 8 to about 50 nucleobases, or about 12 to about 30 nucleobases. Anti-sense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression. Anti-sense compounds can include a stretch of at least eight consecutive nucleobases that are complementary to a sequence in the target gene. An oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An
oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted.
Hybridization of antisense oligonucleotides with mRNA can interfere with one or more of the normal functions of mRNA. The functions of mRNA to be interfered with include all key functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA. Binding of specific protein(s) to the RNA may also be interfered with by antisense oligonucleotide hybridization to the RNA.
Exemplary antisense compounds include DNA or RNA sequences that specifically hybridize to the target nucleic acid, e.g., the mRNA encoding a mutation described herein. The complementary region can extend for between about 8 to about 80 nucleobases. The compounds can include one or more modified nucleobases. Modified nucleobases are known in the art. Descriptions of modified nucleic acid agents are also available. See, e.g., U.S. Patent Nos. 4,987,071; 5,116,742; and 5,093,246; Woolf et al. (1992) Proc Natl Acad Sci USA; Antisense RNA and DNA, D.A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988); 89:7305-9; Haselhoff and Gerlach (1988) Nature 334:585-59; Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14:807-15.
The antisense nucleic acid molecules are typically administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a mutation to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then be administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule is an cc-anomeric nucleic acid molecule. An cc-anomeric nucleic acid molecule forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
siRNAs are small double stranded RNAs (dsRNAs) that optionally include overhangs. For example, the duplex region of an siRNA is about 18 to 25 nucleotides in length, e.g., about 19, 20, 21, 22, 23, or 24 nucleotides in length. Typically, the siRNA sequences are exactly complementary to the target mRNA. dsRNAs and siRNAs in particular can be used to silence gene expression in mammalian cells (e.g., human cells). siRNAs also include short hairpin RNAs (shRNAs) with 29-base-pair stems and 2-nucleotide 3' overhangs. See, e.g., Clemens et al. (2000) Proc. Natl. Acad. Sci. USA 97:6499- 6503; Billy et al. (2001) Proc. Natl. Sci. USA 98: 14428-14433; Elbashir et al. (2001) Nature. 411:494-8; Yang et al. (2002) Proc. Natl. Acad. Sci. USA 99:9942-9947; Siolas et al. (2005), Nat. Biotechnol.
23(2):227-31; 20040086884; U.S. 20030166282; 20030143204; 20040038278; and 20030224432.
In still another embodiment, an antisense nucleic acid featured in the invention is a ribozyme. A ribozyme having specificity for a mutation-encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of a mutation cDNA disclosed herein (i.e., SEQ ID NO:6), and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a mutation-encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742. Alternatively, mutation mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261: 1411-1418.
Inhibition of a mutated gene can be accomplished by targeting nucleotide sequences
complementary to the regulatory region of the mutation to form triple helical structures that prevent transcription of the mutated gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene, C. i (1992) Am. NY. Acad. Sci. 660:27-36; and Maher, L.J. (1992) Bioassays 14:807- 15. The potential sequences that can be targeted for triple helix formation can be increased by creating a so-called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
The invention also provides detectably labeled oligonucleotide primer and probe molecules. Typically, such labels are chemiluminescent, fluorescent, radioactive, or colorimetric.
A mutated nucleic acid molecule can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For non-limiting examples of synthetic oligonucleotides with modifications see Toulme (2001) Nature Biotech. 19: 17 and Faria et al. (2001) Nature Biotech. 19:40-44. Such phosphoramidite oligonucleotides can be effective antisense agents.
For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4: 5- 23). As used herein, the terms "peptide nucleic acid" or "PNA" refers to a nucleic acid mimic, e.g., a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra and Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.
PNAs of mutated nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence- specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of mutated nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup B. et al. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; W088/09810) or the blood-brain barrier (see, e.g., W0 89/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents (See, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent). In some embodiments, a nucleic acid inhibitor described herein is provided for the inhibition of expression of a nucleic acid comprising the alteration in vitro.
Evaluation of Subjects
Subjects, e.g., patients, can be evaluated for the presence of an alteration, e.g., an alteration as described herein. A patient can be evaluated, for example, by determining the genomic sequence of the patient, e.g., by an NGS method. Alternatively, or in addition, evaluation of a patient can include directly assaying for the presence of a mutation in the patient, such as by an assay to detect a mutated nucleic acid (e.g., DNA or RNA), such as by, Southern blot, Northern blot, or RT-PCR, e.g., qRT-PCR. Alternatively, or in addition, a patient can be evaluated for the presence of a protein mutation, such as by immunohistochemistry, Western blot, immunoprecipitation, or
immunomagnetic bead assay.
In one aspect, the results of a clinical trial, e.g., a successful or unsuccessful clinical trial, can be repurposed to identify agents that target an alteration disclosed herein, e.g., a BRAF mutation. By one exemplary method, a candidate agent used in a clinical trial can be reevaluated to determine if the agent in the trial targets a mutation, or is effective to treat a tumor containing a particular mutation. For example, subjects who participated in a clinical trial for an agent, such as a kinase inhibitor, can be identified. Patients who experienced an improvement in symptoms, e.g., cancer (e.g., CLL) symptoms, such as decreased level of lymphocytes in the blood;
decreased size of lymph nodes; decreased size of the liver or spleen; increased number of red blood cells, can be evaluated for the presence of a mutation. Patients who did not experience an improvement in cancer symptoms can also be evaluated for the presence of a mutation. Where patients carrying a mutation are found to have been more likely to respond to the test agent than patients who did not carry such a mutation, then the agent is determined to be an appropriate treatment option for a patient carrying the mutation.
"Reevaluation" of patients can include, for example, determining the genomic sequence of the patients, or a subset of the clinical trial patients, e.g., by an NGS method. Alternatively, or in addition, reevaluation of the patients can include directly assaying for the presence of a mutation in the patient, such as by an assay to detect a mutated nucleic acid (e.g., RNA), such as by RT-PCR, e.g., qRT-PCR. Alternatively, or in addition, a patient can be evaluated for the presence of a protein mutation, such as by immunohistochemistry, Western blot, immunoprecipitation, or
immunomagnetic bead assay.
Methods for Detection of Nucleic Acids and Polypeptides
Methods for evaluating a mutated gene, mutations and/or gene products are known to those of skill in the art. In one embodiment, the mutation is detected in a nucleic acid molecule by a method chosen from one or more of: nucleic acid hybridization assay, SSP, HPLC or mass- spectrometric genotyping.
Additional exemplary methods include traditional "direct probe" methods such as Southern blots and "comparative probe" methods such as comparative genomic hybridization (CGH), e.g., cDNA-based or oligonucleotide-based CGH, can be used. The methods can be used in a wide variety of formats including, but not limited to, substrate (e.g., membrane or glass) bound methods or array-based approaches.
In certain embodiments, the evaluation methods include probes/primers against the alterations described herein.
In one embodiment, probes/primers can be designed to detect a mutation or a reciprocal thereof. These probes/primers are suitable, e.g., for PCR amplification. Probes are used that contain DNA segments that are essentially complementary to DNA base sequences existing in different portions of chromosomes. Examples of probes useful according to the invention, and labeling and hybridization of probes to samples are described in two U.S. patents to Vysis, Inc. U.S. Patent Nos. 5,491,224 and 6,277,569 to Bittner, et al.
Chromosomal probes are typically about 50 to about 105 nucleotides in length. Longer probes typically comprise smaller fragments of about 100 to about 500 nucleotides in length. Probes that hybridize with centromeric DNA and locus-specific DNA are available
commercially, for example, from Vysis, Inc. (Downers Grove, 111.), Molecular Probes, Inc. (Eugene, Oreg.) or from Cytocell (Oxfordshire, UK). Alternatively, probes can be made non- commercially from chromosomal or genomic DNA through standard techniques. For example, sources of DNA that can be used include genomic DNA, cloned DNA sequences, somatic cell hybrids that contain one, or a part of one, chromosome (e.g., human chromosome) along with the normal chromosome complement of the host, and chromosomes purified by flow cytometry or microdissection. The region of interest can be isolated through cloning, or by site-specific amplification via the polymerase chain reaction (PCR). See, for example, Nath and Johnson, Biotechnic Histochem., 1998, 73(l):6-22, Wheeless et al., Cytometry 1994, 17:319-326, and U.S. Patent No. 5,491,224.
The probes to be used hybridize to a specific region of a chromosome to determine whether a cytogenetic abnormality is present in this region. One type of cytogenetic abnormality is a deletion. Although deletions can be of one or more entire chromosomes, deletions normally involve loss of part of one or more chromosomes. If the entire region of a chromosome that is contained in a probe is deleted from a cell, hybridization of that probe to the DNA from the cell will normally not occur and no signal will be present on that chromosome. If the region of a chromosome that is partially contained within a probe is deleted from a cell, hybridization of that probe to the DNA from the cell can still occur, but less of a signal can be present. For example, the loss of a signal is compared to probe hybridization to DNA from control cells that do not contain the genetic abnormalities which the probes are intended to detect. In some embodiments, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more cells are enumerated for presence of the cytogenetic abnormality.
Cytogenetic abnormalities to be detected can include, but are not limited to, non- reciprocal translocations, balanced translocations, intra-chromosomal inversions, point mutations, deletions, gene copy number changes, gene expression level changes, and germ line mutations. In particular, one type of cytogenetic abnormality is a duplication. Duplications can be of entire chromosomes, or of regions smaller than an entire chromosome. If the region of a chromosome that is contained in a probe is duplicated in a cell, hybridization of that probe to the DNA from the cell will normally produce at least one additional signal as compared to the number of signals present in control cells with no abnormality of the chromosomal region contained in the probe.
Chromosomal probes are labeled so that the chromosomal region to which they hybridize can be detected. Probes typically are directly labeled with a fluorophore, an organic molecule that fluoresces after absorbing light of lower wavelength/higher energy. The fluorophore allows the probe to be visualized without a secondary detection molecule. After covalently attaching a fluorophore to a nucleotide, the nucleotide can be directly incorporated into the probe with standard techniques such as nick translation, random priming, and PCR labeling. Alternatively, deoxycytidine nucleotides within the probe can be transaminated with a linker. The fluorophore then is covalently attached to the transaminated deoxycytidine nucleotides. See, U.S. Patent No. 5,491,224.
U.S. Patent No. 5,491,224 describes probe labeling as a number of the cytosine residues having a fluorescent label covalently bonded thereto. The number of fluorescently labeled cytosine bases is sufficient to generate a detectable fluorescent signal while the individual so labeled DNA segments essentially retain their specific complementary binding (hybridizing) properties with respect to the chromosome or chromosome region to be detected. Such probes are made by taking the unlabeled DNA probe segment, transaminating with a linking group a number of deoxycytidine nucleotides in the segment, covalently bonding a fluorescent label to at least a portion of the transaminated deoxycytidine bases.
Probes can also be labeled by nick translation, random primer labeling or PCR labeling. Labeling is done using either fluorescent (direct)-or haptene (indirect)-labeled nucleotides.
Representative, non-limiting examples of labels include: AMCA-6-dUTP, CascadeBlue-4-dUTP, Fluorescein- 12-dUTP, Rhodamine-6-dUTP, TexasRed-6-dUTP, Cy3-6-dUTP, Cy5-dUTP, Biotin(BIO)-l l-dUTP, Digoxygenin(DIG)- l l-dUTP or Dinitrophenyl (DNP)- l l-dUTP.
Probes also can be indirectly labeled with biotin or digoxygenin, or labeled with
32 3
radioactive isotopes such as P and H, although secondary detection molecules or further processing then is required to visualize the probes. For example, a probe labeled with biotin can be detected by avidin conjugated to a detectable marker. For example, avidin can be conjugated to an enzymatic marker such as alkaline phosphatase or horseradish peroxidase. Enzymatic markers can be detected in standard colorimetric reactions using a substrate and/or a catalyst for the enzyme. Catalysts for alkaline phosphatase include 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium. Diaminobenzoate can be used as a catalyst for horseradish
peroxidase.
Probes can also be prepared such that a fluorescent or other label is not part of the DNA before or during the hybridization, and is added after hybridization to detect the probe hybridized to a chromosome. For example, probes can be used that have antigenic molecules incorporated into the DNA. After hybridization, these antigenic molecules are detected using specific antibodies reactive with the antigenic molecules. Such antibodies can themselves incorporate a fluorochrome, or can be detected using a second antibody with a bound fluorochrome.
However treated or modified, the probe DNA is commonly purified in order to remove unreacted, residual products (e.g., fluorochrome molecules not incorporated into the DNA) before use in hybridization.
Prior to hybridization, chromosomal probes are denatured according to methods well known in the art. Probes can be hybridized or annealed to the chromosomal DNA under hybridizing conditions. "Hybridizing conditions" are conditions that facilitate annealing between a probe and target chromosomal DNA. Since annealing of different probes will vary depending on probe length, base concentration and the like, annealing is facilitated by varying probe concentration, hybridization temperature, salt concentration and other factors well known in the art.
Hybridization conditions are facilitated by varying the concentrations, base compositions, complexities, and lengths of the probes, as well as salt concentrations, temperatures, and length of incubation. For example, in situ hybridizations are typically performed in hybridization buffer containing l-2x SSC, 50-65% formamide and blocking DNA to suppress non-specific hybridization. In general, hybridization conditions, as described above, include temperatures of about 25° C to about 55° C, and incubation lengths of about 0.5 hours to about 96 hours.
Non-specific binding of chromosomal probes to DNA outside of the target region can be removed by a series of washes. Temperature and concentration of salt in each wash are varied to control stringency of the washes. For example, for high stringency conditions, washes can be carried out at about 65° C to about 80° C, using 0.2x to about 2x SSC, and about 0.1% to about 1% of a non-ionic detergent such as Nonidet P-40 (NP40). Stringency can be lowered by decreasing the temperature of the washes or by increasing the concentration of salt in the washes. In some applications it is necessary to block the hybridization capacity of repetitive sequences. Thus, in some embodiments, tRNA, human genomic DNA, or Cot-I DNA is used to block nonspecific hybridization. After washing, the slide is allowed to drain and air dry, then mounting medium, a counterstain such as DAPI, and a coverslip are applied to the slide. Slides can be viewed immediately or stored at -20° C. before examination.
In CGH methods, a first collection of nucleic acids (e.g., from a sample, e.g., a possible tumor) is labeled with a first label, while a second collection of nucleic acids (e.g., a control, e.g., from a healthy cell/tissue) is labeled with a second label. The ratio of hybridization of the nucleic acids is determined by the ratio of the two (first and second) labels binding to each fiber in the array. Where there are chromosomal deletions or multiplications, differences in the ratio of the signals from the two labels will be detected and the ratio will provide a measure of the copy number. Array-based CGH can also be performed with single-color labeling (as opposed to labeling the control and the possible tumor sample with two different dyes and mixing them prior to hybridization, which will yield a ratio due to competitive hybridization of probes on the arrays). In single color CGH, the control is labeled and hybridized to one array and absolute signals are read, and the possible tumor sample is labeled and hybridized to a second array (with identical content) and absolute signals are read. Copy number difference is calculated based on absolute signals from the two arrays.
Hybridization protocols suitable for use with the methods featured in the invention are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No. 430,402; Methods in Molecular Biology, Vol. 33: In situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, N.J. (1994), etc. In one
embodiment, the hybridization protocol of Pinkel, et al. (1998) Nature Genetics 20: 207-211, or of Kallioniemi (1992) Proc. Natl Acad Sci USA 89:5321-5325 (1992) is used. Array-based CGH is described in U.S. Patent No. 6,455,258, the contents of each of which are incorporated herein by reference.
In still another embodiment, amplification-based assays can be used to measure presence/absence and copy number. In such amplification-based assays, the nucleic acid sequences act as a template in an amplification reaction {e.g., Polymerase Chain Reaction (PCR). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls, e.g., healthy tissue, provides a measure of the copy number.
Methods of "quantitative" amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that can be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided in Innis, et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.).
Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis is described in Ginzonger, et al. (2000) Cancer Research 60:5405-5409. The known nucleic acid sequence for the genes is sufficient to enable one of skill in the art to routinely select primers to amplify any portion of the gene. Fluorogenic quantitative PCR can also be used. In fluorogenic quantitative PCR, quantitation is based on amount of fluorescence signals, e.g., TaqMan and sybr green.
Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren, et al. (1988) Science
241: 1077, and Barringer et al. (1990) Gene 89: 117), transcription amplification (Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), self- sustained sequence replication (Guatelli, et al.
(1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc.
Nucleic Acid Samples
A variety of tissue samples can be the source of the nucleic acid samples used in the present methods. Genomic or subgenomic DNA fragments can be isolated from a subject's sample (e.g., a tumor sample, a normal adjacent tissue (NAT), a blood sample or any normal control)). In certain embodiments, the tissue sample is preserved as a frozen sample or as formaldehyde- or
paraformaldehyde-fixed paraffin-embedded (FFPE) tissue preparation. For example, the sample can be embedded in a matrix, e.g., an FFPE block or a frozen sample. The isolating step can include flow-sorting of individual chromosomes; and/or micro-dissecting a subject's sample (e.g., a tumor sample, a NAT, a blood sample).
Protocols for DNA isolation, fragmentation and processing from a tissue sample are known in the art as described, e.g., in WO 2012/092426, entitled "Optimization of Multigene Analysis of Tumor Samples," incorporated herein by reference in its entirety. Additional methods to isolate nucleic acids (e.g., DNA) from formaldehyde- or paraformaldehyde-fixed, paraffin-embedded (FFPE) tissues are disclosed, e.g., in Cronin M. et al., (2004) Am J Pathol. 164(l):35-42; Masuda N. et al., (1999) Nucleic Acids Res. 27 (22): 4436-4443; Specht K. et al., (2001) Am J Pathol.
158(2):419-429, Ambion RecoverAll™ Total Nucleic Acid Isolation Protocol (Ambion, Cat. No. AM1975, September 2008), and QIAamp® DNA FFPE Tissue Handbook (Qiagen, Cat. No. 37625, October 2007). RecoverAll™ Total Nucleic Acid Isolation Kit uses xylene at elevated temperatures to solubilize paraffin-embedded samples and a glass-fiber filter to capture nucleic acids. QIAamp® DNA FFPE Tissue Kit uses QIAamp® DNA Micro technology for purification of genomic and mitochondrial DNA.
Design of Baits
A bait can be a nucleic acid molecule, e.g., a DNA or RNA molecule, which can hybridize to (e.g., be complementary to), and thereby allow capture of a target nucleic acid. In one embodiment, a bait is an RNA molecule. In other embodiments, a bait includes a binding entity, e.g., an affinity tag, that allows capture and separation, e.g., by binding to a binding entity, of a hybrid formed by a bait and a nucleic acid hybridized to the bait. In one embodiment, a bait is suitable for solution phase hybridization.
Baits can be produced and used by methods and hybridization conditions as described in US 2010/0029498 and Gnirke, A. et al. (2009) Nat Biotechnol. 27(2): 182-189, and WO 2012/092426, entitled "Optimization of Multigene Analysis of Tumor Samples, incorporated herein by reference.
Sequencing
The invention also includes methods of sequencing nucleic acids. In one embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence at least a portion of a mutation. In one embodiment, the mutated sequence is compared to a
corresponding reference (control) sequence.
In one embodiment, the sequence of the nucleic acid molecule comprising an alteration described herein is determined by a method that includes one or more of: hybridizing an
oligonucleotide, e.g., an allele specific oligonucleotide for one mutation described herein to said nucleic acid; hybridizing a primer, or a primer set (e.g., a primer pair), that amplifies a region comprising the mutation of the allele; amplifying, e.g., specifically amplifying, a region comprising the mutation of the allele; attaching an adapter oligonucleotide to one end of a nucleic acid that comprises the mutation of the allele; generating an optical, e.g., a colorimetric signal, specific to the presence of the one of the mutation; hybridizing a nucleic acid comprising the mutation to a second nucleic acid, e.g., a second nucleic acid attached to a substrate; generating a signal, e.g., an electrical or fluorescent signal, specific to the presence of the mutation; and incorporating a nucleotide into an oligonucleotide that is hybridized to a nucleic acid that contains the mutation.
In another embodiment, the sequence is determined by a method that comprises one or more of: determining the nucleotide sequence from an individual nucleic acid molecule, e.g., where a signal corresponding to the sequence is derived from a single molecule as opposed, e.g., from a sum of signals from a plurality of clonally expanded molecules; determining the nucleotide sequence of clonally expanded proxies for individual nucleic acid molecules; massively parallel short-read sequencing; template-based sequencing; pyrosequencing; real-time sequencing comprising imaging the continuous incorporation of dye-labeling nucleotides during DNA synthesis; nanopore sequencing; sequencing by hybridization; nano-transistor array based sequencing; polony sequencing; scanning tunneling microscopy (STM) based sequencing; or nanowire-molecule sensor based sequencing.
Any method of sequencing known in the art can be used. Exemplary sequencing reactions include those based on techniques developed by Maxam and Gilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger (Sanger et al. (1977) Proc. Nat. Acad. Sci 74:5463). Any of a variety of automated sequencing procedures can be utilized when performing the assays
(Biotechniques (1995) 19:448), including sequencing by mass spectrometry (see, for example, U.S. Patent Number 5,547,835 and international patent application Publication Number WO
94/16101, entitled DNA Sequencing by Mass Spectrometry by H. Koster; U.S. Patent Number 5,547,835 and international patent application Publication Number WO 94/21822 entitled DNA Sequencing by Mass Spectrometry Via Exonuclease Degradation by H. Koster), and U.S. Patent Number 5,605,798 and International Patent Application No. PCT/US96/03651 entitled DNA Diagnostics Based on Mass Spectrometry by H. Koster; Cohen et al. (1996) Adv Chromatogr 36: 127-162; and Griffin et al. (1993) Appl Biochem Biotechnol 38: 147-159).
Sequencing of nucleic acid molecules can also be carried out using next-generation sequencing (NGS). Next-generation sequencing includes any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules or clonally expanded proxies for individual nucleic acid molecules in a highly parallel fashion (e.g., greater than 105 molecules are sequenced simultaneously). In one embodiment, the relative abundance of the nucleic acid species in the library can be estimated by counting the relative number of occurrences of their cognate sequences in the data generated by the sequencing experiment. Next generation sequencing methods are known in the art, and are described, e.g., in Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46, incorporated herein by reference.
In one embodiment, the next-generation sequencing allows for the determination of the nucleotide sequence of an individual nucleic acid molecule (e.g., Helicos Biosciences' HeliScope Gene Sequencing system, and Pacific Biosciences' PacBio RS system). In other embodiments, the sequencing method determines the nucleotide sequence of clonally expanded proxies for individual nucleic acid molecules (e.g., the Solexa sequencer, Alumina Inc., San Diego, Calif; 454 Life Sciences (Branford, Conn.), and Ion Torrent), e.g., massively parallel short-read sequencing (e.g., the Solexa sequencer, Illumina Inc., San Diego, Calif.), which generates more bases of sequence per sequencing unit than other sequencing methods that generate fewer but longer reads. Other methods or machines for next-generation sequencing include, but are not limited to, the sequencers provided by 454 Life Sciences (Branford, Conn.), Applied Biosystems (Foster City, Calif.; SOLiD
sequencer), and Helicos Biosciences Corporation (Cambridge, Mass.).
Platforms for next-generation sequencing include, but are not limited to, Roche/454' s Genome Sequencer (GS) FLX System, Illumina/Solexa's Genome Analyzer (GA), Life/APG's Support Oligonucleotide Ligation Detection (SOLiD) system, Polonator's G.007 system, Helicos Biosciences' HeliScope Gene Sequencing system, and Pacific Biosciences' PacBio RS system. NGS technologies can include one or more of steps, e.g., template preparation, sequencing and imaging, and data analysis as described in WO 2012/092426, entitled "Optimization of Multigene Analysis of Tumor Samples, incorporated herein by reference.
Data analysis
After NGS reads have been generated, they can be aligned to a known reference sequence or assembled de novo. For example, identifying genetic variations such as single-nucleotide
polymorphism and structural variants in a sample (e.g., a tumor sample) can be accomplished by aligning NGS reads to a reference sequence (e.g., a wild- type sequence). Methods of sequence alignment for NGS are described e.g., in Trapnell C. and Salzberg S.L. Nature Biotech., 2009, 27:455-457. Examples of de novo assemblies are described, e.g., in Warren R. et al., Bioinformatics, 2007, 23:500-501; Butler J. et al., Genome Res., 2008, 18:810-820; and Zerbino D.R. and Birney E., Genome Res., 2008, 18:821-829. Sequence alignment or assembly can be performed using read data from one or more NGS platforms, e.g., mixing Roche/454 and Illumina/Solexa read data. Algorithms and methods for data analysis are described in WO 2012/092426, entitled "Optimization of Multigene Analysis of Tumor Samples, incorporated herein by reference.
Detection of Mutated Polypeptide
The activity or level of a mutated polypeptide (e.g., a BRAF mutation) can also be detected and/or quantified by detecting or quantifying the expressed polypeptide. The mutated polypeptide can be detected and quantified by any of a number of means known to those of skill in the art. These can include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), Immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, immunohistochemistry (IHC) and the like. A skilled artisan can adapt known protein/antibody detection methods.
Another agent for detecting a mutated polypeptide is an antibody molecule capable of binding to a polypeptide corresponding to a polypeptide, e.g., an antibody with a detectable label. Techniques for generating antibodies are described herein. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a
fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
In another embodiment, the antibody is labeled, e.g., a radio-labeled, chromophore- labeled, fluorophore-labeled, or enzyme-labeled antibody. In another embodiment, an antibody derivative (e.g., an antibody conjugated with a substrate or with the protein or ligand of a protein-ligand pair { e.g., biotin-streptavidin}), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically with a mutated protein, is used.
Mutated polypeptides from cells can be isolated using techniques that are known to those of skill in the art. The protein isolation methods employed can, for example, be such as those described in Harlow and Lane (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
Means of detecting proteins using electrophoretic techniques are well known to those of skill in the art (see generally, R. Scopes (1982) Protein Purification, Springer- Verlag, N.Y.;
Deutscher, (1990) Methods in Enzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc., N.Y.).
In another embodiment, Western blot (immunoblot) analysis is used to detect and quantify the presence of a polypeptide in the sample.
In another embodiment, the polypeptide is detected using an immunoassay. As used herein, an immunoassay is an assay that utilizes an antibody to specifically bind to the analyte. The immunoassay is thus characterized by detection of specific binding of a polypeptide to an anti- antibody as opposed to the use of other physical or chemical properties to isolate, target, and quantify the analyte.
The mutated polypeptide is detected and/or quantified using any of a number of
immunological binding assays (see, e.g., U.S. Patent Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of the general immunoassays, see also Asai (1993) Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. New York; Stites & Terr (1991) Basic and Clinical Immunology 7th Edition.
Kits
In one aspect, the invention features, a kit, e.g., containing an oligonucleotide having an alteration described herein, e.g., a BRAF and/or KRAS mutation. Optionally, the kit can also contain an oligonucleotide that is the wildtype counterpart of the mutant oligonucleotide.
A kit can include a carrier, e.g., a means being compartmentalized to receive in close confinement one or more container means. In one embodiment the container contains an
oligonucleotide, e.g., a primer or probe as described above. The components of the kit are useful, for example, to diagnose or identify a mutation in a tumor sample in a patient. The probe or primer of the kit can be used in any sequencing or nucleotide detection assay known in the art, e.g., a sequencing assay, e.g., an NGS method, RT-PCR, or in situ hybridization.
In some embodiments, the components of the kit are useful, for example, to diagnose or identify a mutation in a tumor sample in a patient, and to accordingly identify an appropriate therapeutic agent to treat the cancer.
A kit featured in the invention can include, e.g., assay positive and negative controls, nucleotides, enzymes (e.g., RNA or DNA polymerase or ligase), solvents or buffers, a stabilizer, a preservative, a secondary antibody, e.g., an anti-HRP antibody (IgG) and a detection reagent.
An oligonucleotide can be provided in any form, e.g., liquid, dried, semi-dried, or lyophilized, or in a form for storage in a frozen condition.
Typically, an oligonucleotide, and other components in a kit are provided in a form that is sterile. An oligonucleotide, e.g., an oligonucleotide that contains a mutation, described herein, or an oligonucleotide complementary to an alteration described herein, is provided in a liquid solution, the liquid solution generally is an aqueous solution, e.g., a sterile aqueous solution. When the oligonucleotide is provided as a dried form, reconstitution generally is accomplished by the addition of a suitable solvent. The solvent, e.g., sterile buffer, can optionally be provided in the kit.
The kit can include one or more containers for the composition containing an oligonucleotide in a concentration suitable for use in the assay or with instructions for dilution for use in the assay. In some embodiments, the kit contains separate containers, dividers or compartments for the
oligonucleotide and assay components, and the informational material. For example, the
oligonucleotides can be contained in a bottle or vial, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, an oligonucleotide composition is contained in a bottle or vial that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit forms (e.g., for use with one assay) of an oligonucleotide. For example, the kit
includes a plurality of ampoules, foil packets, or blister packs, each containing a single unit of oligonucleotide for use in sequencing or detecting a mutation in a tumor sample. The containers of the kits can be air tight and/or waterproof. The container can be labeled for use.
For antibody-based kits, the kit can include: (1) a first antibody (e.g., attached to a solid support) which binds to a mutated polypeptide; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.
In one embodiment, the kit can include informational material for performing and
interpreting the sequencing or diagnostic. In another embodiment, the kit can provide guidance as to where to report the results of the assay, e.g., to a treatment center or healthcare provider. The kit can include forms for reporting the results of a sequencing or diagnostic assay described herein, and address and contact information regarding where to send such forms or other related information; or a URL (Uniform Resource Locator) address for reporting the results in an online database or an online application (e.g., an app). In another embodiment, the informational material can include guidance regarding whether a patient should receive treatment with a particular chemotherapeutic drug, depending on the results of the assay.
The informational material of the kits is not limited in its form. In many cases, the
informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawings, and/or photographs, e.g., a label or printed sheet. However, the informational material can also be provided in other formats, such as computer readable material, video recording, or audio recording. In another embodiment, the informational material of the kit is contact information, e.g., a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about the sequencing or diagnostic assay and/or its use in the methods described herein. The informational material can also be provided in any combination of formats.
In some embodiments, a biological sample is provided to an assay provider, e.g., a service provider (such as a third party facility) or a healthcare provider, who evaluates the sample in an assay and provides a read out. For example, in one embodiment, an assay provider receives a biological sample from a subject, such as a blood or tissue sample, e.g., a biopsy sample, and evaluates the sample using an assay described herein, e.g., a sequencing assay or in situ
hybridization assay, and determines that the sample contains a mutation. The assay provider, e.g., a service provider or healthcare provider, can then conclude that the subject is, or is not, a candidate for a particular drug or a particular cancer treatment regimen.
Other embodiments of the invention include the following.
Nucleic Acid Molecules, Detection and Capturing Reagents
The invention also features an isolated nucleic acid molecule, or an isolated preparation of nucleic acid molecules, that includes an alteration described herein. Such nucleic acid molecules or preparations thereof can include an alteration described herein or can be used to detect, e.g., sequence, an alteration.
The invention also features a nucleic acid molecule, e.g., nucleic acid fragment, suitable as probe, primer, bait or library member that includes, flanks, hybridizes to, which are useful for identifying, or are otherwise based on, an alteration described herein. In certain embodiments, the probe, primer or bait molecule is an oligonucleotide that allows capture, detection or isolation of a nucleic acid molecule containing an alteration described herein, e.g., an alteration in BRAF and/or KRAS.
The oligonucleotide can comprise a nucleotide sequence substantially complementary to nucleic acid molecules or fragments of nucleic acid molecules comprising an alteration described herein. The sequence identity between the nucleic acid molecule, e.g., the oligonucleotide, and the target sequence need not be exact, so long as the sequences are sufficiently complementary to allow the capture, detection or isolation of the target sequence. In one embodiment, the nucleic acid fragment is a probe or primer that includes an oligonucleotide between about 5 and 25, e.g., between 10 and 20, or 10 and 15 nucleotides in length. In other embodiments, the nucleic acid fragment is a bait that includes an oligonucleotide between about 100 to 300 nucleotides, 130 and 230
nucleotides, or 150 and 200 nucleotides, in length.
In one embodiment, the nucleic acid fragment can be used to identify or capture, e.g., by hybridization, a nucleic acid molecules comprising an alteration described herein, e.g., an alteration in BRAF and/or KRAS. For example, the nucleic acid fragment can be a probe, a primer, or a bait, for use in identifying or capturing, e.g., by hybridization, an alteration described herein.
The probes or primers described herein can be used, for example, PCR amplification. In one exemplary embodiment where detection is based on PCR, amplification of the mutation can be performed using a primer or a primer pair, e.g., for amplifying a sequence flanking an alteration described herein.
In other embodiments, the nucleic acid fragment includes a bait that comprises a nucleotide sequence that hybridizes to a nucleic acid molecules comprising an alteration described herein, and thereby allows the capture or isolation said nucleic acid molecule. In one embodiment, a bait is suitable for solution phase hybridization. In other embodiments, a bait includes a binding entity, e.g., an affinity tag, that allows capture and separation, e.g., by binding to a binding entity, of a hybrid formed by a bait and a nucleic acid hybridized to the bait.
In other embodiments, the nucleic acid fragment includes a library member comprising a nucleic acid molecule described herein. In one embodiment, the library member includes a mutation, e.g., a base substitution, that results in an alteration described herein.
The nucleic acid fragment can be detectably labeled with, e.g., a radiolabel, a fluorescent label, a bioluminescent label, a chemiluminescent label, an enzyme label, a binding pair label, or can include an affinity tag; a tag, or identifier (e.g., an adaptor, barcode or other sequence identifier).
Polypeptides
In another aspect, the disclosure features a polypeptide comprising an alteration described herein (e.g., a purified polypeptide comprising an alteration described herein), a biologically active or antigenic fragment thereof, as well as reagents (e.g., antibody molecules that bind to a polypeptide comprising an alteration described herein), methods for modulating the activity of a polypeptide comprising an alteration described herein and detection of a polypeptide comprising an alteration described herein.
In another embodiment, the polypeptide or fragment is a peptide, e.g., an immunogenic peptide or protein that contains an alteration described herein. Such immunogenic peptides or proteins can be used to raise antibodies specific to the polypeptide or protein comprising an alteration described herein. In other embodiments, such immunogenic peptides or proteins can be used for vaccine preparation. The vaccine preparation can include other components, e.g., an adjuvant.
In another aspect, the invention features antibody molecules that bind to a polypeptide comprising an alteration described herein or fragment described herein. In embodiments the antibody can distinguish wild type from the mutated polypeptide, e.g., the polypeptide comprising an alteration described herein. Techniques for generating antibody molecules are known in the art, and are described, for example, in WO 2012/092426, entitled "Optimization of Multigene Analysis of Tumor Samples, incorporated herein by reference.
Detection Reagents
In another aspect, the invention features a detection reagent, e.g., a purified or an isolated preparation thereof. Detection reagents can distinguish a nucleic acid, or protein sequence, having an alteration described herein, e.g., of a nucleic acid molecule comprising an alteration described herein, e.g., an alteration in BRAF and/or KRAS.
Detection reagents, e.g., nucleic acid-based detection reagents, can be used to identify mutations in a target nucleic acid, e.g., DNA, e.g., genomic DNA or cDNA, or RNA, e.g., in a sample, e.g., a sample of nucleic acid derived from a CLL (e.g., a CLL cell). Detection reagents, e.g., antibody-based detection reagents, can be used to identify mutations in a target protein, e.g., in a sample, e.g., a sample of protein derived from, or produced by, CLL cell.
Nucleic Acid-based Detection Reagents
In one embodiment, the detection reagent comprises a nucleic acid molecule, e.g., a DNA, RNA or mixed DNA/RNA molecule, comprising sequence which is complementary with a nucleic acid sequence on a target nucleic acid (the sequence on the target nucleic acid that is bound by the detection reagent is referred to herein as the "detection reagent binding site" and the portion of the detection reagent that corresponds to the detection reagent binding site is referred to as the "target binding site"). In one embodiment, the detection reagent binding site is disposed in relationship to the interrogation position such that binding (or in embodiments, lack of binding) of the detection reagent to the detection reagent binding site allows differentiation of mutant and reference sequences for a mutant described herein (nucleic acid molecule comprising an alteration described herein, e.g., an alteration in BRAF and/or KRAS. The detection reagent can be modified, e.g., with a label or other moiety, e.g., a moiety that allows capture.
In one embodiment, the detection reagent comprises a nucleic acid molecule, e.g., a DNA, RNA or mixed DNA/RNA molecule, which, e.g., in its target binding site, includes the interrogation position and which can distinguish (e.g., by affinity of binding of the detection reagent to a target nucleic acid or the ability for a reaction, e.g., a ligation or extension reaction with the detection reagent) between a mutation, e.g., a translocation described herein, and a reference sequence. In embodiments, the interrogation position can correspond to a terminal, e.g., to a 3' or 5' terminal nucleotide, a nucleotide immediately adjacent to a 3' or 5' terminal nucleotide, or to another internal nucleotide, of the detection reagent or target binding site.
In embodiments, the difference in the affinity of the detection reagent for a target nucleic acid comprising the alteration described herein and that for a target nucleic acid comprising the reference sequence allows determination of the presence or absence of the mutation (or reference) sequence. Typically, such detection reagents, under assay conditions, will exhibit substantially higher levels of binding only to the mutant or only to the reference sequence, e.g., will exhibit substantial levels of binding only to the mutation or only to the reference sequence.
In embodiments, binding allows (or inhibits) a subsequent reaction, e.g., a subsequent reaction involving the detection reagent or the target nucleic acid. E.g., binding can allow ligation, or the addition of one or more nucleotides to a nucleic acid, e.g., the detection reagent, e.g., by DNA polymerase, which can be detected and used to distinguish mutant from reference. In embodiments, the interrogation position is located at the terminus, or sufficiently close to the terminus, of the detection reagent or its target binding site, such that hybridization, or a chemical reaction, e.g., the addition of one or more nucleotides to the detection reagent, e.g., by DNA polymerase, only occurs, or occurs at a substantially higher rate, when there is a perfect match between the detection reagent and the target nucleic acid at the interrogation position or at a nucleotide position within 1, 2, or 3 nucleotides of the interrogation position. In one embodiment, the detection reagent comprises a nucleic acid, e.g., a DNA, RNA or mixed DNA/RNA molecule wherein the molecule, or its target binding site, is adjacent (or flanks), e.g., directly adjacent, to the interrogation position, and which can distinguish between a mutation described herein, and a reference sequence, in a target nucleic acid.
In embodiments, the detection reagent binding site is adjacent to the interrogation position, e.g., the 5' or 3 'terminal nucleotide of the detection reagent, or its target binding site, is adjacent, e.g., between 0 (directly adjacent) and 1,000, 500, 400, 200, 100, 50, 10, 5, 4, 3, 2, or 1 nucleotides from the interrogation position. In embodiments, the outcome of a reaction will vary with the identity of the nucleotide at the interrogation position allowing one to distinguish between mutant and reference sequences. E.g., in the presence of a first nucleotide at the interrogation position a first reaction will be favored over a second reaction. E.g., in a ligation or primer extension reaction, the product will differ, e.g., in charge, sequence, size, or susceptibility to a further reaction (e.g., restriction cleavage) depending on the identity of the nucleotide at the interrogation position. In embodiments the detection reagent comprises paired molecules (e.g., forward and reverse primers), allowing for amplification, e.g., by PCR amplification, of a duplex containing the interrogation position. In such embodiments, the presence of the mutation can be determined by a difference in the property of the amplification product, e.g., size, sequence, charge, or susceptibility to a reaction, resulting from a sequence comprising the interrogation position and a corresponding sequence having a reference nucleotide at the interrogation positions. In embodiments, the presence or absence of a characteristic amplification product is indicative of the identity of the nucleotide at the interrogation site and thus allows detection of the mutation.
In embodiments, the detection reagent, or its target binding site, is directly adjacent to the interrogation position, e.g., the 5' or 3'terminal nucleotide of the detection reagent is directly adjacent to the interrogation position. In embodiments, the identity of the nucleotide at the interrogation position will determine the nature of a reaction, e.g., a reaction involving the detection reagent, e.g., the modification of one end of the detection reagent. E.g., in the presence of a first nucleotide at the interrogation position a first reaction will be favored over a second reaction. By way of example, the presence of a first nucleotide at the interrogation position, e.g., a nucleotide associated with a mutation, can promote a first reaction, e.g., the addition of a complementary nucleotide to the detection reagent. By way of example, the presence of an A at the interrogation position will cause the incorporation of a T, having, e.g., a first colorimetric label, while the presence of a G and the interrogation position will cause the incorporation for a C, having, e.g., a second colorimetric label. In one embodiment, the presence of a first nucleotide at the nucleotide will result in ligation of the detection reagent to a second nucleic acid. E.g., a third nucleic acid can be hybridized to the target nucleic acid sufficiently close to the interrogation site that if the third nucleic acid has an exact match at the interrogation site it will be ligated to the detection reagent. Detection of the ligation product, or its absence, is indicative of the identity of the nucleotide at the interrogation site and thus allows detection of the mutation.
A variety of readouts can be employed. E.g., binding of the detection reagent to the mutant or reference sequence can be followed by a moiety, e.g., a label, associated with the detection reagent, e.g., a radioactive or enzymatic label. In embodiments the label comprises a quenching agent and a signaling agent and hybridization results in altering the distance between those two elements, e.g., increasing the distance and un-quenching the signaling agent. In embodiments, the detection reagent can include a moiety that allows separation from other components of a reaction mixture. In embodiments, binding allows cleavage of the bound detection reagent, e.g., by an enzyme, e.g., by the nuclease activity of the DNA polymerase or by a restriction enzyme. The cleavage can be detected by the appearance or disappearance of a nucleic acid or by the separation of a quenching agent and a signaling agent associated with the detection reagent. In embodiments, binding protects, or renders the target susceptible, to further chemical reaction, e.g., labeling or degradation, e.g., by restriction enzymes. In embodiments binding with the detection reagent allows capture separation or physical manipulation of the target nucleic acid to thereby allow for identification. In embodiments binding can result in a detectable localization of the detection reagent or target, e.g., binding could capture the target nucleic acid or displace a third nucleic acid. Binding can allow for the extension or other size change in a component, e.g., the detection reagent, allowing distinction between mutant and reference sequences. Binding can allow for the production, e.g., by PCR, of an amplicon that distinguishes mutant from reference sequence.
In one embodiment the detection reagent, or the target binding site, is between 5 and 500, 5 and 300, 5 and 250, 5 and 200, 5 and 150, 5 and 100, 5 and 50, 5 and 25, 5 and 20, 5 and 15, or 5 and 10 nucleotides in length. In one embodiment the detection reagent, or the target binding site, is between 10 and 500, 10 and 300, 10 and 250, 10 and 200, 10 and 150, 10 and 100, 10 and 50, 10 and 25, 10 and 20, or 10 and 15, nucleotides in length. In one embodiment the detection reagent, or the target binding site, is between 20 and 500, 20 and 300, 20 and 250, 20 and 200, 20 and 150, 20 and 100, 20 and 50, or 20 and 25 nucleotides in length. In one embodiment the detection reagent, or the target binding site, is sufficiently long to distinguish between mutant and reference sequences and is less than 100, 200, 300, 400, or 500 nucleotides in length.
Preparations of Nucleic Acids and Uses Thereof
In another aspect, the invention features purified or isolated preparations of a neoplastic or tumor cell nucleic acid, e.g., DNA, e.g., genomic DNA or cDNA, or RNA, containing an interrogation position described herein, useful for determining if a mutation disclosed herein is present. The nucleic acid includes the interrogation position, and typically additional sequence on one or both sides of the interrogation position. In addition the nucleic acid can contain heterologous sequences, e.g., adaptor or priming sequences, typically attached to one or both terminus of the nucleic acid. The nucleic acid also includes a label or other moiety, e.g., a moiety that allows separation or localization.
In embodiments, the nucleic acid is between 20 and 1,000, 30 and 900, 40 and 800, 50 and 700, 60 and 600, 70 and 500, 80 and 400, 90 and 300, or 100 and 200 nucleotides in length (with or without heterologous sequences). In one embodiment, the nucleic acid is between 40 and 1,000, 50 and 900, 60 and 800, 70 and 700, 80 and 600, 90 and 500, 100 and 400, 110 and 300, or 120 and 200 nucleotides in length (with or without heterologous sequences). In another embodiment, the nucleic acid is between 50 and 1,000, 50 and 900, 50 and 800, 50 and 700, 50 and 600, 50 and 500, 50 and 400, 50 and 300, or 50 and 200 nucleotides in length (with or without heterologous sequences). In embodiments, the nucleic acid is of sufficient length to allow sequencing (e.g., by chemical sequencing or by determining a difference in Tm between mutant and reference preparations) but is optionally less than 100, 200, 300, 400, or 500 nucleotides in length (with or without heterologous sequences).
Such preparations can be used to sequence nucleic acid from a sample, e.g., a neoplastic or tumor sample. In one embodiment the purified preparation is provided by in situ
amplification of a nucleic acid provided on a substrate. In embodiments the purified preparation is spatially distinct from other nucleic acids, e.g., other amplified nucleic acids, on a substrate. In one embodiment, the purified or isolated preparation of nucleic acid is derived from a hematological malignancy, e.g., CLL. Such preparations can be used to determine if a sample comprises mutant sequence, e.g., an alteration described herein.
In another aspect, the invention features, a method of determining the sequence of an interrogation position for an alteration described herein, comprising:
providing a purified or isolated preparations of nucleic acid, e.g., DNA, e.g., genomic DNA or cDNA, or RNA, containing an interrogation position described herein,
sequencing, by a method that breaks or forms a chemical bond, e.g., a covalent or non- covalent chemical bond, e.g., in a detection reagent or a target sequence, the nucleic acid so as to determine the identity of the nucleotide at an interrogation position. The method allows determining if an alteration described herein is present.
In one embodiment, sequencing comprises contacting the nucleic acid comprising an alteration described herein with a detection reagent described herein.
In one embodiment, sequencing comprises determining a physical property, e.g., stability of a duplex form of the nucleic acid comprising an alteration described herein, e.g., Tm, that can distinguish mutant from reference sequence.
In one embodiment, the nucleic acid comprising an alteration described herein is derived from a hematological malignancy, e.g., CLL.
Reaction Mixtures and Devices
In another aspect, the invention features, purified or isolated preparations of a nucleic acid, e.g., DNA, e.g., genomic DNA or cDNA, or RNA, containing an interrogation position described herein, useful for determining if a mutation disclosed herein is present, disposed in sequencing device, or a sample holder for use in such a device. In one embodiment, the nucleic acid is derived from a hematological malignancy, e.g., CLL.
In another aspect, the invention features, purified or isolated preparations of a nucleic acid, e.g., DNA, e.g., genomic DNA or cDNA, or RNA, containing an interrogation position described herein, useful for determining if a mutation disclosed herein is present, disposed in a device for determining a physical or chemical property, e.g., stability of a duplex, e.g., Tm or a sample holder for use in such a device. In one embodiment, the device is a calorimeter. In one embodiment the nucleic acid comprising an alteration described herein is derived from a hematological malignancy, e.g., CLL.
The detection reagents described herein can be used to determine if an alteration described herein is present in a sample. In embodiments, the sample comprises a nucleic acid that is derived from a hematological malignancy, e.g., CLL. The cell can be from a neoplastic or a tumor sample, e.g., a biopsy taken from the neoplasm or the tumor; from circulating tumor cells, e.g., from peripheral blood; or from a blood or plasma sample. In one embodiment, the nucleic acid is derived from e.g., a hematological malignancy, e.g., CLL.
Accordingly, in one aspect, the invention features a method of making a reaction mixture, comprising: combining a detection reagent, or purified or isolated preparation thereof, described herein with a target nucleic acid derived from e.g., a hematological malignancy, e.g., CLL, which comprises a sequence having an interrogation position for an alteration described herein.
In another aspect, the invention features a reaction mixture, comprising:
a detection reagent, or purified or isolated preparation thereof, described herein; and a target nucleic acid derived from e.g., a cell derived from a hematological malignancy, e.g., a CLL cell, which comprises a sequence having an interrogation position for an alteration described herein.
In one embodiment of the reaction mixture, or the method of making the reaction mixture:
the detection reagent comprises a nucleic acid, e.g., a DNA, RNA or mixed DNA/RNA, molecule which is complementary with a nucleic acid sequence on a target nucleic acid (the detection reagent binding site) wherein the detection reagent binding site is disposed in relationship to the interrogation position such that binding of the detection reagent to the detection reagent binding site allows differentiation of mutant and reference sequences for a mutation sequence or event described herein.
In one embodiment of the reaction mixture, or the method of making the reaction mixture: the target nucleic acid sequence is derived from a hematological malignancy, e.g., CLL, as described herein. In one embodiment of the reaction mixture, or the method of making the reaction mixture: the mutation is an alteration described herein, including: a substitution, e.g., a substitution described herein. An alteration described herein, can be distinguished from a reference, e.g., a non-mutant or wildtype sequence, by reaction with an enzyme that reacts differentially with the mutation and the reference. E.g., they can be distinguished by cleavage with a restriction enzyme that has differing activity for the mutant and reference sequences. E.g., the invention includes a method of contacting a nucleic acid comprising an alteration described herein with such an enzyme and determining if a product of that cleavage which can distinguish mutant form reference sequence is present.
In one aspect, the inventions provides a purified preparation of a restriction enzyme cleavage product which can distinguish between mutant and reference sequence, wherein one end of the cleavage product is defined by an enzyme that cleaves differentially between mutant and reference sequence. In one embodiment, the cleavage product includes the interrogation position.
Protein-based Detection Reagents, Methods, Reaction Mixtures and Devices
A mutant protein described herein can be distinguished from a reference, e.g., a non- mutant or wild-type protein, by reaction with a reagent, e.g., a substrate, e.g, a substrate for catalytic activity or functional activity, or an antibody, that reacts differentially with the mutant and reference protein. In one aspect, the invention includes a method of contacting a sample comprising a mutant protein described herein with such reagent and determining if the mutant protein is present in the sample.
In another embodiment, the invention features, an antibody that can distinguish a mutant protein described herein, or a fragment thereof, from a reference, e.g., a non-mutant or wild type protein.
Accordingly, in one aspect, the invention features a method of making a reaction mixture comprising:
combining a detection reagent, or purified or isolated preparation thereof, e.g., a substrate, e.g., a substrate for phosphorylation or other activity, or an antibody, described herein with a target protein derived from e.g., a cell derived from a hematological malignancy, e.g., a CLL cell, which comprises a sequence having an interrogation position for an alteration described herein. In another aspect, the invention features, a reaction mixture comprising:
a detection reagent, or purified or isolated preparation thereof, e.g., a substrate, e.g., a substrate for phosphorylation or other activity, or an antibody, described herein; and
a target protein derived from a hematological malignancy, e.g., CLL cell, which
comprises a sequence having an interrogation position for an alteration described herein.
In one embodiment of the reaction mixture, or the method of making the reaction
mixture:
the detection reagent comprises an antibody specific for a mutant protein described herein.
In one embodiment of the reaction mixture, or the method of making the reaction mixture that includes a cell derived from a hematological malignancy, e.g., a CLL cell.
In one embodiment of the reaction mixture, or the method of making the reaction
mixture: the mutation is an alteration described herein (e.g., a BRAF and/or KRAS mutation described herein).
Screening Methods
In another aspect, the invention features a method, or assay, for screening for agents that modulate, e.g., inhibit, the expression or activity of a nucleic acid or polypeptide or protein comprising a mutation as described herein. The method includes contacting a nucleic acid or polypeptide or protein comprising an alteration described herein, or a cell expressing a nucleic acid or polypeptide or protein comprising an alteration described herein, with a candidate agent; and detecting a change in a parameter associated with a nucleic acid or polypeptide or protein comprising an alteration described herein, e.g., a change in the expression or an activity of the nucleic acid or polypeptide or protein comprising an alteration described herein. The method can, optionally, include comparing the treated parameter to a reference value, e.g., a control sample (e.g., comparing a parameter obtained from a sample with the candidate agent to a parameter obtained from a sample without the candidate agent). In one embodiment, if a decrease in expression or activity of the nucleic acid or polypeptide or protein comprising an alteration described herein is detected, the candidate agent is identified as an inhibitor. In another embodiment, if an increase in expression or activity of the nucleic acid or polypeptide or protein comprising an alteration described herein is detected, the candidate agent is identified as an activator. In one embodiment, the contacting step is effected in a cell-free system, e.g., a cell lysate or in a reconstituted system. In other embodiments, the contacting step is effected in a cell in culture, e.g., a cell expressing an alteration described herein (e.g., a mammalian cell, a tumor cell or cell line, a recombinant cell). In yet other embodiments, the contacting step is effected in a cell in vivo (a - expressing cell present in a subject, e.g., an animal subject (e.g., an in vivo animal model).
Exemplary parameters evaluated include one or more of:
(i) a change in binding activity, e.g., direct binding of the candidate agent to a polypeptide comprising an alteration described herein; a binding competition between a known ligand and the candidate agent to a polypeptide comprising an alteration described herein;
(ii) a change in kinase activity, e.g., phosphorylation levels of a polypeptide comprising an alteration described herein (e.g., an increased or decreased autophosphorylation); or a change in a target of a polypeptide comprising an alteration described herein, In certain embodiments, a change in kinase activity, e.g., phosphorylation, is detected by any of Western blot (e.g., using an antibody which binds to a polypeptide comprising an alteration described herein, mass spectrometry, immunoprecipitation, immunohistochemistry, immunomagnetic beads, among others;
(iii) a change in an activity of a cell containing a tumor cell or a recombinant cell, e.g., a change in proliferation, morphology or maligancy of the cell;
(iv) a change in the malignancy present in an animal subject, e.g., number of lymphocytes, red cells, size of the liver, spleen; or
(v) a change in the level, e.g., expression level, of a nucleic acid or polypeptide or protein comprising an alteration described herein.
In one embodiment, a change in a cell free assay in the presence of a candidate agent is evaluated. For example, an activity of a nucleic acid or polypeptide or protein comprising an alteration described herein, or interaction of a nucleic acid or polypeptide or protein comprising an alteration described herein with a downstream ligand can be detected. In one embodiment, the polypeptide or protein comprising an alteration described herein is contacted with a ligand, e.g., in solution, and a candidate agent is monitored for an ability to modulate, e.g., inhibit, an interaction, e.g., binding, between the nucleic acid or polypeptide or protein comprising an alteration described herein and the ligand.
In other embodiments, a change in an activity of a cell is detected in a cell in culture, e.g., a cell expressing a mutation (e.g., a mammalian cell, a tumor cell or cell line, a recombinant cell). In one embodiment, the cell is a recombinant cell that is modified to express a nucleic acid comprising an alteration described herein, e.g., is a recombinant cell transfected with a nucleic acid comprising an alteration described herein. The transfected cell can show a change in response to the expressed mutation, e.g., increased proliferation, changes in morphology, increased tumorigenicity, and/or acquired a transformed phenotype. A change in any of the activities of the cell, e.g., the
recombinant cell, in the presence of the candidate agent can be detected. For example, a decrease in one or more of: proliferation, tumorigenicity, transformed morphology, in the presence of the candidate agent can be indicative of an inhibitor of a nucleic acid or polypeptide or protein comprising an alteration described herein. In other embodiments, a change in binding activity or phosphorylation as described herein is detected.
In yet other embodiment, a change in a cancer present in an animal subject (e.g., an in vivo animal model) is detected. In one embodiment, the animal model contains the hematological malignancy animal or a xenograft comprising cells expressing a nucleic acid or polypeptide or protein comprising an alteration described herein. The candidate agent can be administered to the animal subject and a change in the hematologic cells is detected. In one embodiment, the change in the hematologic cells includes one or more of: a change in the level of lymphocytes in the blood; a change in the size of lymph nodes; a change in the size of the liver or spleen; a change in the number of red blood cells, is evaluated. A decrease in one or more of the above, or an increased survival is indicative that the candidate agent is an inhibitor.
In other embodiments, a change in expression of a nucleic acid or polypeptide or protein comprising an alteration described herein can be monitored by detecting the nucleic acid or protein levels, e.g., using the methods described herein.
In certain embodiments, the screening methods described herein can be repeated and/or combined. In one embodiment, a candidate agent that is evaluated in a cell-free or cell-based described herein can be further tested in an animal subject.
In one embodiment, the candidate agent is a small molecule compound, e.g., a kinase inhibitor, a nucleic acid (e.g., antisense, siRNA, aptamer, ribozymes, microRNA), an antibody molecule (e.g., a full antibody or antigen binding fragment thereof that binds to the mutation). The candidate agent can be obtained from a library (e.g., a commercial library of kinase inhibitors) or rationally designed. In other embodiments, the method, or assay, includes providing a step based on proximity- dependent signal generation, e.g., a two-hybrid assay that includes a first mutation protein (e.g., a mutated protein), and a second mutated protein (e.g., a ligand), contacting the two-hybrid assay with a test compound, under conditions wherein said two hybrid assay detects a change in the formation and/or stability of the complex, e.g., the formation of the complex initiates transcription activation of a reporter gene.
In one non-limiting example, the three-dimensional structure of the active site of a polypeptide or protein comprising an alteration described herein is determined by crystallizing the complex formed by the polypeptide or protein and a known inhibitor. Rational drug design is then used to identify new test agents by making alterations in the structure of a known inhibitor or by designing small molecule compounds that bind to the active site of the polypeptide or protein.
The candidate agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R.N. et al.
(1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12: 145).
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261: 1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37: 1233.
Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner U.S. Patent No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89: 1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378- 6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).
The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FET) (see, for example, Lakowicz et al., U.S. Patent No. 5,631,169; Stavrianopoulos, et al., U.S. Patent No. 4,868,103). An FET binding event can be conveniently measured through standard fluorometric detection means known in the art (e.g., using a fluorimeter).
In another embodiment, determining the ability of the mutated protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). "Surface plasmon resonance" or "BIA" detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.
EXAMPLES
Example 1. Extensive high-depth sequencing of longitudinal CLL samples identifies frequent mutations in MAP kinase signaling and novel mutations activating Notch and Beta-Catenin.
Background:
Whole-genome sequencing, whole-exome sequencing, and copy number analysis studies in small sets of patients with chronic lymphocytic leukemia (CLL) at presentation versus
progression have demonstrated that clonal evolution has clinical and biologic relevance in CLL.
Although the time and cost required to perform whole genome and exome sequencing are
improving, challenges still exist in implementing these genomic strategies in real-time clinical practice. Here we performed DNA and RNA sequencing of an extensive panel of all genes
known to be recurrently mutated in lymphoid, myeloid, and solid tumor malignancies, at high sequencing depth in patients with CLL at clinical presentation and progression. This enabled the acquisition of the integrated mutation, copy number, and gene fusion events in CLL and to use this data to obtain new insights into clonal evolution of CLL. Methods:
DNA and RNA sequencing of an extensive panel of all genes known to be recurrently mutated in lymphoid, myeloid, and solid tumor malignancies, at high sequencing depth in patients with CLL at clinical presentation and progression were performed. Genomic DNA and total RNA was isolated from 59 CLL samples (including 29 CLL paired patients sampled at the time of initial presentation when no clinical indication for therapy was met and at a later time point of disease progression requiring therapy). The median time between samples was 2.07 years (range 0.08-4.95 years)). Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 272 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >590X for DNA and >20, 000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. All clinical data as well as biomarkers previously validated for clinical use in CLL (IGHV mutational status, CD38 expression, metaphase cytogenetics and FISH) were also obtained for all samples at each time point.
Results:
This targeted sequencing approach detected at least one clonal somatic mutation in 95% of CLL patients including 144 individual mutations, 3 functional rearrangements, and 1 homozygous deletion. Consistent with prior reports, mutations in NOTCH1 (25%), SF3B1 (19%), and TP53 (14%) were amongst the most commonly mutated genes in this cohort. Of note, activating mutations in KRAS (15%) and BRAF (10%) were also amongst the most commonly mutated genes targeted by somatic mutations in CLL (FIG. 1A). Taken together, mutations resulting in constitutive MAP kinase signaling occurred in 36% of all patients, second only to mutations activating Notch signaling which occurred in 40% of patients. The two most commonly mutated genes in the Notch signaling pathway were mutations in NOTCH1 followed by loss of function mutations in SPEN (7%). SPEN is a nuclear receptor transcription factor which blocks the differentiation of precursor B cells into marginal zone B cells through interactions factor which blocks the differentiation of precursor B cells into marginal zone B cells through interactions with RBP-J. Also identified as recurrent were mutations in FAT3, an inhibitor of β-catenin signaling. In addition, sequencing identified a novel fusion event in BIRC3 fusing BIRC3 to LRRC40 as well as a truncating mutation in one patient in BRCA2 at disease progression.
Paired mutational analysis of samples at initial sample acquisition and at disease progression requiring therapy was performed (FIG. IB). Mutational analysis at the time of clonal evolution identified mutations in DNMT3A, EED, IDH2, IRF4, VHL, and RBI only at the time of disease evolution. Mutations in NOTCH1, KRAS, TP53, NRAS, and BCOR were more common at disease progression than initial presentation. In contrast, mutations in SF3B1 and XPOl were always retained at disease progression if found at earlier disease states.
Conclusions:
These data demonstrate the utility of clinical grade, high throughput targeted sequencing in CLL to identify targetable genomic alterations, discover novel mutations not previously reported, and identify important markers of disease evolution which may represent incipient biomarkers for clinical disease progression. This includes a much more frequent occurrence of targetable mutations in the MAP kinase pathway than previously described in CLL and recurrent loss of function mutations in SPEN and FAT3 in CLL.
Table 1 : Exemplary Genomic Alterations in CLL
KRAS_c.437C>T_p.A146V BCOR_c.2967_2971delTCAGC_p.L991fs*25,
NOTCHl_c.7353_7353delG_p.A2452fs*25,
TRAF3_c.856_857delAA_p.K286fs*l l
KRAS_c.437C>T_p.A146V NOTCHl_c.7353_7353delG_p.A2452fs*25,
TRAF3_c.856_857delAA_p.K286fs*l l
TP53_c.830G>A_p.C277Y, NOTCHl_c.7541_7542delCT_p.P2514fs*4
XP01_c.l711G>A_p.E571K
FAT3_c.8773G>A_p.A2925T, NOTCHl_c.7541_7542delCT_p.P2514fs*4
SF3B l_c.1874G>A_p.R625H,
TP53_c.818G>C_p.R273P
FAT3_c.8773G>A_p.A2925T, NOTCHl_c.7541_7542delCT_p.P2514fs*4
SF3B l_c.1874G>A_p.R625H,
TP53_c.818G>C_p.R273P
FAT3_c.8773G>A_p.A2925T, NOTCHl_c.7541_7542delCT_p.P2514fs*4
SF3B l_c.1874G>A_p.R625H,
TP53_c.818G>C_p.R273P,
TP53_c.695T>C_p.I232T,
TP53_c.524G>A_p.R175H
KRAS_c.351A>C_p.K117N ATM_c.8672-lG>T_p.splice,
ATM_c.364_370delAATTATA_p.N122fs*5
KRAS_c.351A>C_p.K117N ATM_c.6019G>T_p.E2007*,
ATM_c.8672-lG>T_p.splice,
ATM_c.364_370delAATTATA_p.N122fs*5,
Figure imgf000103_0001
(0.48,249), ZNF703_c.l205_1205delA_p.H402fs*8
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS We claim:
1. A method of treating a subject having a chronic lymphocytic leukemia (CLL), comprising administering to the subject an effective amount of a MAPK pathway inhibitor chosen from a BRAF inhibitor, a MEK inhibitor, or a combination thereof, thereby treating the CLL.
2. The method of claim 1, further comprising acquiring knowledge of the presence or the absence of an alteration in B-Raf, K-Ras, or both, in the subject having the CLL.
3. The method of claim 1, wherein the MAPK pathway inhibitor is administered to the subject responsive to a determination of the presence of the alteration in B-Raf, K-Ras, or both, in the subject, or a sample from the subject.
4. The method of claim 2, wherein the alteration is chosen from one or more of:
(i) a mutation in codon 464, 465, 466, 468, 469, 580, 594, 595, 596, 597, 599, 600, 601 or 727, of BRAF;
(ii) a mutation in BRAF other than at codon 600;
(iii) a mutation in BRAF at a position chosen from one or more of R461I, I462S, G463E, G463V, G464E, G464R, G464V, G465A, G465E, G465V, G466A, G466E, G466R, G466V, G468A, G468E, F468C, G469A, G469E, G469R, G469R, G469S, G469V, N580S, E585K, D593V, D594G, D594V, F594L, F595L, G595R, L596V, G596R, L597Q, L597R, L597S, L597V, T598I, T599I, V599D, V599E, V599K, V599R, K601E, K601N, or A727V;
(iv) a mutation in BRAF at codon 600;
(v) a mutation in BRAF chosen from V600E, V600K, V600L, or V600R;
(vi) a mutation in K-Ras in codon 12, 13 or 61; or
(vii) a mutation in K-Ras at a position chosen from one or more of G12C, G12R, G12D, G12A, G12S, G12V, G13D, G13N, G13S, G13C, G13V, Q61H, Q61R, Q61P, Q61L, Q61K, Q61E, A59T or G12F.
5. The method of claim 1, wherein the CLL is a refractory or a relapsed CLL.
6. The method of claim 1, wherein the CLL is at a stage 0, 1, II, III, or IV, wherein stage 0 CLL is characterized by an elevated level of lymphocytes in the blood compared to a reference value, without other detectable symptoms of leukemia; stage I CLL is characterized by an elevated level of lymphocytes in the blood and enlarged lymph nodes; stage II CLL is
characterized by an elevated level of lymphocytes in the blood, the liver or spleen is larger than normal, and the lymph nodes may be larger than normal; stage III CLL is characterized by an elevated level of lymphocytes in the blood, decreased red blood cells, and the lymph nodes, liver, or spleen may be larger than normal; and stage IV CLL is characterized by an elevated level of lymphocytes in the blood reduced platelets, the lymph nodes, liver, or spleen may be larger than normal, and there are reduced red blood cells.
7. The method of claim 1, wherein the subject is a human having, or is at risk of having,
CLL.
8. The method of claim 1, wherein the subject is identified, or has been previously identified, as having CLL.
9. The method of any of the preceding claims, wherein the subject is undergoing or has undergone treatment with a non-MAPK pathway therapeutic agent or therapeutic modality.
10. The method of claim 9, wherein the non- MAPK pathway therapeutic agent or therapeutic modality comprises one or more of: ritixumab (Rituxan), ofatumumab (Arzerra), alamtuzumab (Campath), flebogamma, gamimune, gammagard S/D, gammaplex, panglobulin NF, polygamy S/D, privifen, sandoglobulin, treanda, fludarabine, cyclophosphamide, bendamustine, pentostatin, cladribine, doxorubicin, vincristine, prednisone, chlorambucil, a steroid, radiotherapy, or a bone marrow transplant.
11. The method of claim 9, wherein, responsive to the determination of the presence of the alteration in B-Raf, K-Ras, or both, the non- MAPK pathway therapeutic agent or therapeutic modality is discontinued.
12. The method of claim 9, wherein the agent is administered after cessation of the non- MAPK pathway therapeutic agent or therapeutic modality.
13. The method of any claims 1-8, wherein the MAPK pathway inhibitor is an inhibitor of a BRAF gene or gene product.
14. The method of of claim 13, wherein the BRAF inhibitor is chosen from: a kinase inhibitor, a multi- specific kinase inhibitor; a pan inhibitor, or an inhibitor that is selective for BRAF.
15. The method of claim 13, wherein the BRAF inhibitor is chosen from one or more of: Vemurafenib, Sorafenib Tosylate, PLX4720, GDC-0879, RAF265, GW5074, CEP-32496, Dabrafenib, AZ628, SB590885, Raf265 derivative, Regorafenib or ZM 336372.
16. The method of claim 13, wherein the BRAF inhibitor is chosen from an antisense molecule, a ribozyme, a double stranded RNA, or a triple helix molecule that hybridizes to and/or inhibits a BRAF nucleic acid encoding the alteration, or a transcription regulatory region that blocks or reduces mRNA expression of the alteration.
17. The method of any of claims 1-8, wherein the MAPK pathway inhibitor is an inhibitor of a MEK gene or gene product.
18. The method of of claim 17, wherein the MEK inhibitor is chosen from: a kinase inhibitor, a multi- specific kinase inhibitor; a pan inhibitor, or an inhibitor that is selective for MEK.
19. The method of claim 17, wherein the MEK inhibitor is chosen from one or more of: ARRY-162, Trametinib, Selumetinib, XL518, CI-1040, PD035901, U0126-EtOH, PD198306, PD98059, BIX 02189, TAK-733, Honokiol, AZD8330, PD318088, BIX 02188, AS703026
(Pimasertib) or SL327.
20. The method of claim 17, wherein the MEK inhibitor is chosen from an antisense molecule, a ribozyme, a double stranded RNA, or a triple helix molecule that hybridizes to and/or inhibits a MEK nucleic acid encoding the alteration, or a transcription regulatory region that blocks or reduces mRNA expression of the alteration.
21. The method of claims 14-16, wherein the BRAF inhibitor is administered in
combination with the MEK inhibitor chosen from one or more of: ARRY-162, Trametinib,
Selumetinib, XL518, CI-1040, PD035901, U0126-EtOH, PD198306, PD98059, BIX 02189, TAK-733, Honokiol, AZD8330, PD318088, BIX 02188, AS703026 (Pimasertib) or SL327.
22. The method of claim 2, wherein the determination of the presence of the alteration in BRAF, KRAS, or both, comprises nucleic acid sequencing.
23. A method of determining the presence of a BRAF alteration, a KRAS alteration, or both, in a subject with CLL, comprising directly acquiring knowledge that a nucleic acid molecule comprising the BRAF alteration is present in the subject, thereby determining the presence of the BRAF alteration, a KRAS alteration, or both, in the subject.
24. The method of claim 23, further comprises one or more of:
(1) stratifying the subject;
(2) identifying or selecting the subject as being likely or unlikely to respond to a MAPK pathway inhibitor treatment;
(3) selecting a treatment option comprising an inhibitor of BRAF, MEK, or both;
(4) administering a BRAF inhibitor, a MEK inhibitor, or both to the subject; or
(5) prognosticating the time course of the disease in the subject.
25. The method of claims 23-24, wherein the acquiring step comprising sequencing an alteration chosen from:
(i) a mutation in codon 464, 465, 466, 468, 469, 580, 594, 595, 596, 597, 599, 600, 601 or 727, of BRAF;
(ii) a mutation in BRAF other than at codon 600;
(iii) a mutation in BRAF at a position chosen from one or more of R461I, I462S, G463E, G463V, G464E, G464R, G464V, G465A, G465E, G465V, G466A, G466E, G466R, G466V, G468A, G468E, F468C, G469A, G469E, G469R, G469R, G469S, G469V, N580S, E585K, D593V, D594G, D594V, F594L, F595L, G595R, L596V, G596R, L597Q, L597R, L597S, L597V, T598I, T599I, V599D, V599E, V599K, V599R, K601E, K601N, or A727V;
(iv) a mutation in BRAF at codon 600;
(v) a mutation in BRAF chosen from V600E, V600K, V600L, or V600R;
(vi) a mutation in K-Ras in codon 12, 13 or 61; or
(vii) a mutation in K-Ras at a position chosen from one or more of G12C, G12R, G12D, G12A, G12S, G12V, G13D, G13N, G13S, G13C, G13V, Q61H, Q61R, Q61P, Q61L, Q61K, Q61E, A59T and G12F.
26. A composition, comprising a MAPK pathway inhibitor chosen from a BRAF inhibitor, a MEK inhibitor, or a combination thereof, for use in treating a CLL.
27. A kit comprising a MAPK pathway inhibitor chosen from a BRAF inhibitor, a MEK inhibitor, or a combination thereof, with instructions for use in treating a CLL, and/or
determining the presence of an alteration described herein.
28. A kit comprising one or more detection reagents, capable of specific detection of a nucleic acid or protein comprising an alteration described herein in a CLL sample.
29. A purified or an isolated preparation of a nucleic acid derived from a CLL, containing an interrogation position described herein, useful for determining if a mutation disclosed herein is present, disposed in sequencing device, or a sample holder for use in such a device.
30. A reaction mixture, comprising:
a detection reagent, or purified or isolated preparation thereof, described herein; and a target nucleic acid derived from a CLL sample, which comprises a sequence having an interrogation position for an alteration described herein.
31. A method of making a reaction mixture, comprising:
combining a detection reagent, or purified or isolated preparation thereof, described herein with a target nucleic acid derived from a CLL, which comprises a sequence having an interrogation position for an alteration described herein.
32. The reaction mixture of claim 30, or the method of making the reaction mixture of claim 31, wherein:
the detection reagent comprises a nucleic acidmolecule which is complementary with a nucleic acid sequence on a target nucleic acid (the detection reagent binding site) wherein the detection reagent binding site is disposed in relationship to the interrogation position such that binding of the detection reagent to the detection reagent binding site allows differentiation of mutant and reference sequences for a mutation sequence or event described herein.
33. The composition of claim 26, wherein the composition is a pharmaceutical composition.
34. The kit of claim 28, wherein the one or more detection reagents comprise one or more of: probes, primers, or antibodies.
35. The purified or isolated preparation of the nucleic acid of claim 29, wherein the nucleic acid comprises one or more of: genomic DNA, cDNA, or RNA.
36. The reaction mixture of claim 32, wherein the nucleic acid molecule is chosen from: a DNA, RNA or mixed DNA/RNA molecule.
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