WO2015092614A1 - Altérations d'activation de notch dans le cancer du sein - Google Patents

Altérations d'activation de notch dans le cancer du sein Download PDF

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WO2015092614A1
WO2015092614A1 PCT/IB2014/066728 IB2014066728W WO2015092614A1 WO 2015092614 A1 WO2015092614 A1 WO 2015092614A1 IB 2014066728 W IB2014066728 W IB 2014066728W WO 2015092614 A1 WO2015092614 A1 WO 2015092614A1
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notch
cancer
patient
pest domain
signaling pathway
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PCT/IB2014/066728
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Keith Anthony CHING
Peter A. OLSON
Kai Wang
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Pfizer Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention is directed to methods of identifying and treating patients having activating Notch PEST domain alterations who will benefit from treatment with a Notch signaling pathway inhibitor, in particular a ⁇ -secretase inhibitor (GSI).
  • a Notch signaling pathway inhibitor in particular a ⁇ -secretase inhibitor (GSI).
  • Notch signaling pathway is a highly conserved developmental pathway responsible for a variety of cell fate decisions. Andersson et al. Development (201 1 ), 138(17):3593-3612. Notch is cleaved at the S1 site within its extracellular domain (ECD), forming a bipartite receptor held together by non-covalent interactions within the heterodimerization (HD) domain. Notch signaling is activated by ligand binding to the ECD, followed by conformational change of the negative regulatory region (NRR), which permits cleavage at the S2 site by ADAM/Tumor necrosis factor-a-converting enzyme (TACE) metalloprotease.
  • ECD extracellular domain
  • HD heterodimerization
  • Notch signaling is activated by ligand binding to the ECD, followed by conformational change of the negative regulatory region (NRR), which permits cleavage at the S2 site by ADAM/Tumor necrosis factor-a-converting enzyme (TACE) metalloprotease.
  • the ⁇ -secretase complex then mediates S3 cleavage within the transmembrane domain, liberating the Notch intracellular domain (NICD), which translocates to the nucleus and regulates the transcription of Notch target genes.
  • Notch intracellular domain An important mechanism of NICD regulation is protein turnover.
  • the NICD is quickly targeted for degradation by the PEST [rich in proline (P), glutamic acid (E), serine (S) and threonine(T)] domain located at its C-terminus.
  • Notch1 -4 There are four mammalian Notch receptors, Notch1 -4, which bind to five membrane-bound Notch pathway ligands: three Delta-like ligands (DLL1 , DLL3, DLL4) and two Serrate/Jagged-family ligands (Jagged-1 , Jagged-2). Notch activation has been implicated to play a role in tumorogenesis.
  • DLL1 Delta-like ligands
  • DLL3 Delta-like ligands
  • Jagged-1 Serrate/Jagged-family ligands
  • T-ALL T cell acute lymphoblastic leukemia
  • Notch pathway is activated in breast cancer
  • a deeper understanding of the mechanisms of Notch de-regulation may help identify those patients that are most likely to respond to Notch signaling pathway inhibitors, and provide an understanding of heterogeneous responses to GSIs that have been reported in vitro and in vivo. Waiters et al. Cancer Res (2009) 69(23):8949-8957.
  • the present invention provides, in part, methods of identifying and treating patients who will benefit from treatment with a Notch signaling pathway inhibitors, including agents that inhibit Notch receptor cleavages, e.g., GSIs, and agents that interfere with the Notch ligand-receptor interactions, e.g., anti-Notch monoclonal antibodies (mAbs) or antibody-drug conjugates (ADCs) thereof.
  • a Notch signaling pathway inhibitor is a small molecule GSI, as further described herein.
  • Notch PEST domain alterations Patients expected to be responsive to treatment are characterized by, or identified based on, the presence of an activating alteration in the PEST domain of Notch (collectively, “Notch PEST domain alterations") that results in activation of the Notch signaling pathway.
  • the methods provided herein are particularly useful in identifying and treating patients having breast cancer, including TNBC, who are likely to respond to treatment with a Notch signaling pathway inhibitor.
  • the invention provides a method of selecting a patient for treatment with a Notch signaling pathway inhibitor, comprising: (a) detecting a Notch PEST domain alteration in a biological sample from the patient; and (b) selecting the patient for treatment with a Notch signaling pathway inhibitor if a PEST domain alteration is detected.
  • the invention provides a method of determining whether a patient is likely to respond to treatment with a Notch signaling pathway inhibitor, comprising: (a) detecting a Notch PEST domain alteration in a biological sample from the patient; and (b) assigning the patient to a positive responder group if a Notch PEST domain alteration is detected.
  • the method further comprises administering a therapeutically effective amount of a Notch signaling pathway inhibitor to the patient.
  • the method further comprises: administering an amount of a Notch signaling pathway inhibitor to the patient; and administering an amount of at least one additional therapeutic agent to the patient, wherein the amounts of said Notch signaling pathway inhibitor and said at least one additional therapeutic agent are together effective to treat cancer.
  • the invention provides a method of determining the susceptibility of a tumor in a patient to treatment with a Notch signaling pathway inhibitor, comprising detecting a Notch PEST domain alteration in a biological sample from the patient; wherein the presence of a PEST domain alteration is associated with susceptibility of the tumor to treatment with a Notch signaling pathway inhibitor, such as a GSI.
  • the method further comprises administering a therapeutically effective amount of a Notch signaling pathway inhibitor to the patient.
  • the method further comprises: administering an amount of a Notch signaling pathway inhibitor to the patient; and administering an amount of at least one additional therapeutic agent to the patient; wherein the amounts of said Notch signaling pathway inhibitor and said at least one additional therapeutic agent are together effective to treat the patient's tumor.
  • the invention provides a method of selecting a patient for treatment with a Notch signaling pathway inhibitor, comprising: (a) detecting a Notch PEST domain alteration in a biological sample from the patient; (b) selecting the patient for treatment with a Notch signaling pathway inhibitor if a PEST domain alteration is detected; and (c) administering a therapeutically effective amount of a Notch signaling pathway inhibitor to the patient.
  • the invention provides a method of selecting a patient for treatment with a Notch signaling pathway inhibitor, comprising: (a) detecting a Notch PEST domain alteration in a biological sample from the patient; (b) selecting the patient for treatment with a Notch signaling pathway inhibitor if a PEST domain alteration is detected; (c) administering an amount of a Notch signaling pathway inhibitor to the patient; and (d) administering an amount of at least one additional therapeutic agent to the patient, wherein the amounts of said Notch signaling pathway inhibitor and said at least one additional therapeutic agent are together effective to treat cancer.
  • the invention provides a method of determining whether a patient is likely to respond to treatment with a Notch signaling pathway inhibitor, comprising: (a) detecting a Notch PEST domain alteration in a biological sample from the patient; (b) assigning the patient to a positive responder group if a Notch PEST domain alteration is detected; and (c) administering a therapeutically effective amount of a Notch signaling pathway inhibitor to the patient.
  • the invention provides a method of determining whether a patient is likely to respond to treatment with a Notch signaling pathway inhibitor, comprising: (a) detecting a Notch PEST domain alteration in a biological sample from the patient; (b) assigning the patient to a positive responder group if a Notch PEST domain alteration is detected; (c) administering an amount of a Notch signaling pathway inhibitor to the patient; and (d) administering an amount of at least one additional therapeutic agent to the patient, wherein the amounts of said Notch signaling pathway inhibitor and said at least one additional therapeutic agent are together effective to treat cancer.
  • the invention provides a method of determining the susceptibility of a tumor in a patient to treatment with a Notch signaling pathway inhibitor, comprising: (a) detecting a Notch PEST domain alteration in a biological sample from the patient; and (b) administering a therapeutically effective amount of a Notch signaling pathway inhibitor to the patient; wherein the presence of a PEST domain alteration is associated with susceptibility of the tumor to treatment with a Notch signaling pathway inhibitor, such as a GSI.
  • a Notch signaling pathway inhibitor such as a GSI.
  • the invention provides a method of determining the susceptibility of a tumor in a patient to treatment with a Notch signaling pathway inhibitor, comprising: (a) detecting a Notch PEST domain alteration in a biological sample from the patient; (b) administering an amount of a Notch signaling pathway inhibitor to the patient; and (c) administering an amount of at least one additional therapeutic agent to the patient; wherein the amounts of said Notch signaling pathway inhibitor and said at least one additional therapeutic agent are together effective to treat the patient's tumor; and wherein the presence of a PEST domain alteration is associated with susceptibility of the tumor to treatment with a Notch signaling pathway inhibitor, such as a GSI.
  • a Notch signaling pathway inhibitor such as a GSI.
  • patients determined to have a Notch PEST domain alteration in their biological samples are identified as patients suitable for treatment with a Notch signaling pathway inhibitor.
  • patients determined to have a Notch PEST domain alteration in their biological samples are identified as patients likely to respond to treatment with a Notch signaling pathway inhibitor, and are therefore assigned to a positive responder group.
  • patients determined to have a Notch PEST domain alteration in their biological samples are identified as patients whose tumors are susceptible to treatment with a Notch signaling pathway inhibitor.
  • the patient is afflicted with cancer, in particular breast cancer.
  • the patient is afflicted with TNBC or basal-like breast cancer.
  • the patient is afflicted with TNBC.
  • the invention provides a method of predicting the sensitivity of a cancer patient to treatment with a Notch signaling pathway inhibitor, comprising: (a) assaying for a Notch PEST domain alteration in a biological sample obtained from the patient; and (b) comparing the Notch PEST domain alteration with a non-cancerous or normal control sample, wherein the presence of the Notch PEST domain alteration in the biological sample indicates the patient is sensitive to treatment with a Notch signaling pathway inhibitor.
  • the invention provides a method of treating cancer, comprising: (a) detecting a Notch PEST domain alteration in a biological sample from a patient in need of such treatment; (b) selecting the patient for treatment with a Notch signaling pathway inhibitor if a Notch PEST domain alteration is detected; and (c) administering a therapeutically effective amount of a Notch signaling pathway inhibitor to the patient.
  • the invention provides a method of treating cancer, comprising: (a) detecting a Notch PEST domain alteration in a biological sample from a patient in need of such treatment; (b) selecting the patient for treatment with a Notch signaling pathway inhibitor if a Notch PEST domain alteration is detected; (c) administering an amount of a Notch signaling pathway inhibitor to the patient; and (d) administering an amount of at least one additional therapeutic agent to the patient; wherein the amounts of said Notch signaling pathway inhibitor and said at least one additional therapeutic agent are together effective to treat cancer.
  • the invention provides a method of treating breast cancer, comprising: (a) detecting a Notch PEST domain alteration in a biological sample from a patient in need of such treatment; (b) selecting the patient for treatment with a Notch signaling pathway inhibitor if a Notch PEST domain alteration is detected; (c) administering an amount of a Notch signaling pathway inhibitor to the patient; and (d) administering an amount of at least one additional therapeutic agent to the patient; wherein the amounts of said Notch signaling pathway inhibitor and said at least one additional therapeutic agent are together effective to treat breast cancer.
  • the invention provides a method of treating, ameliorating, inhibiting the progression of, or inhibiting the invasiveness of cancer in a patient, comprising administering to the patient a therapeutically effective amount of a Notch signaling pathway inhibitor, wherein the patient has been identified as having a Notch PEST domain alteration.
  • the invention provides a method of treating, ameliorating, inhibiting the progression of, or inhibiting the invasiveness of cancer in a patient, comprising administering to the patient a therapeutically effective amount of PF- 03084014, or a pharmaceutically acceptable salt thereof, wherein the cancer has been characterized as having a Notch PEST domain alteration.
  • the invention provides a method of treating, ameliorating, inhibiting the progression of, or inhibiting the invasiveness of cancer in a patient, comprising administering to the patient an amount of a Notch signaling pathway inhibitor, and administering to the patient an amount of at least one additional therapeutic agent, wherein the amounts of said Notch signaling pathway inhibitor and said at least one additional therapeutic agent are together effective to treat cancer, wherein the patient has been identified as having a Notch PEST domain alteration.
  • the invention provides a method of treating, ameliorating, inhibiting the progression of, or inhibiting the invasiveness of cancer in a patient, comprising administering to the patient an amount of a Notch signaling pathway inhibitor, and administering to the patient an amount of at least one additional therapeutic agent, wherein the amounts of said Notch signaling pathway inhibitor and said at least one additional therapeutic agent are together effective to treat cancer, wherein the cancer has been characterized as having a Notch PEST domain alteration.
  • the invention provides a Notch signaling pathway inhibitor for use in a method of treating cancer, wherein the cancer is characterized as having a Notch PEST domain alteration.
  • the Notch signaling pathway inhibitor is adapted for administration with at least one additional therapeutic agent.
  • the invention provides the use of a Notch signaling pathway inhibitor in a method of treating cancer, wherein the cancer is characterized as having a Notch PEST domain alteration.
  • the use further comprises use in combination with_at least one additional therapeutic agent.
  • the invention provides the use of a Notch signaling pathway inhibitor for the manufacture of a medicament for the treatment of cancer, wherein the cancer is characterized as having a Notch PEST domain alteration.
  • the Notch signaling pathway inhibitor is adapted for administration with at least one additional therapeutic agent.
  • the invention provides a method of treating, ameliorating, inhibiting the progression of, or inhibiting the invasiveness of cancer in a patient, comprising administering to the patient a therapeutically effective amount of PF- 03084014, or a pharmaceutically acceptable salt thereof, wherein the patient has been identified as having a Notch PEST domain alteration.
  • the invention provides a method of treating, ameliorating, inhibiting the progression of, or inhibiting the invasiveness of cancer in a patient, comprising administering to the patient a therapeutically effective amount of PF- 03084014, or a pharmaceutically acceptable salt thereof, wherein the cancer is characterized as having a Notch PEST domain alteration.
  • the invention provides a method of treating, ameliorating, inhibiting the progression of, or inhibiting the invasiveness of cancer in a patient, comprising administering to the patient an amount of PF-03084014, or a pharmaceutically acceptable salt thereof, and administering to the patient an amount of at least one additional therapeutic agent, wherein the amounts of said Notch signaling pathway inhibitor and said at least one additional therapeutic agent are together effective to treat cancer, and wherein the patient has been identified as having a Notch PEST domain alteration.
  • the invention provides PF-03084014, or a pharmaceutically acceptable salt thereof, for use in a method of treating cancer, wherein the cancer is characterized as having a Notch PEST domain alteration.
  • the Notch signaling pathway inhibitor is adapted for administration with at least one additional therapeutic agent.
  • the invention provides the use of PF-03084014, or a pharmaceutically acceptable salt thereof, in a method of treating cancer, wherein the cancer is characterized as having a Notch PEST domain alteration.
  • the use further comprises use in combination with at least one additional therapeutic agent.
  • the invention provides the use of PF-03084014, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of cancer, wherein the cancer is characterized as having a Notch PEST domain alteration.
  • the Notch signaling pathway inhibitor is adapted for administration with at least one additional therapeutic agent.
  • the cancer is breast cancer.
  • the cancer is TNBC or basal-like breast cancer. In some such embodiments, the cancer is TNBC.
  • the patient is in a population of patients being evaluated or selected for treatment with a Notch signaling pathway inhibitor, such as a GSI. In other such embodiments, the patient is in a population of patients being evaluated or tested for responsiveness to a Notch signaling pathway inhibitor, such as a GSI.
  • a Notch signaling pathway inhibitor such as a GSI.
  • the invention provides a method of determining the sensitivity of a cancer cell to a Notch signaling pathway inhibitor, comprising: (a) assaying for a Notch PEST domain alteration in a cancer cell; and (b) comparing the Notch PEST domain alteration with a non-cancerous or normal control cell, wherein the presence of the Notch PEST domain alteration indicates the cancer cell is sensitive to a Notch signaling pathway inhibitor.
  • the invention provides a method of treating a cancer patient with a Notch signaling pathway inhibitor, comprising: (a) assaying for a Notch PEST domain alteration in a cancer cell obtained from the patient; (b) comparing the Notch PEST domain alteration with a non-cancerous or normal control cell, wherein the presence of the Notch PEST domain alteration indicates the cancer cell is sensitive to a Notch signaling pathway inhibitor; and (c) administering a therapeutically effective amount of a Notch signaling pathway inhibitor.
  • the Notch signaling pathway inhibitor is a GSI.
  • the GSI is the compound referred to as PF-03084014, /V-2-[(2S)-6,8-difluoro-1 ,2,3,4-tetrahydro- naphthalen-2-yl]-/V-(1 - ⁇ 2-[(2,2-dimethylpropyl)amino]-1 ,1 -dimethylethyl ⁇ -1 /-/-imidazol-4- yl)-L-norvalinamide, having the structure:
  • PF-03084014 is disclosed in U.S. Patent Nos. 7,342,1 18, 7,795,447 and 7,951 ,958, the contents of which are herein incorporated by reference in their entirety.
  • PF-03084014 may sometimes be referred to as /V-2-[(2S)-(6,8-difluoro- 1 ,2,3,4-tetra-hydronaphthalen-2-ylamino)-pentanoic acid ⁇ 1 -[2-(2,2-dimethyl-propylamino)- 1 ,1 -dimethyl-ethyl]-1 H-imidazoM-yl ⁇ -amide or (S)-2-(((S)-6,8-difluoro-1 ,2,3,4-tetrahydro- naphthalen-2-yl)amino)-/V-(1 -(2-methyl-1 -(neopentylamino)propan-2-yl)-1 /-/-imida
  • the Notch signaling pathway inhibitor is an anti-Notch monoclonal antibody (mAb), an antigen-binding fragment thereof, or an antibody-drug conjugate (ADC) thereof.
  • the Notch signaling pathway inhibitor is selected from the group consisting of an anti-Notchl mAb, an anti-Notch2 mAb, and an anti-Notch3 mAb, an antigen-binding fragment thereof, or an ADC thereof.
  • the detection of a Notch PEST domain alteration in the biological sample indicates the patient is likely to respond to treatment with a Notch signaling pathway inhibitor, such as a GSI.
  • the step of detecting the Notch PEST domain alteration in the biological sample occurs prior to any administration of a Notch signaling pathway inhibitor.
  • the Notch PEST domain alteration comprises a point mutation, a translocation, a partial duplication, an insertion, a deletion (partial or full), or a truncation of the Notch PEST domain.
  • the Notch PEST domain alteration is selected from the group consisting of a Notchi PEST domain alteration, a Notch2 PEST domain alteration, and a Notch3 PEST domain alteration.
  • the Notch PEST domain alteration is a Notchi PEST domain alteration.
  • the Notch PEST domain alteration is a Notch2 PEST domain alteration.
  • the Notch PEST domain alteration is a Notch3 PEST domain alteration.
  • the presence of a Notch PEST domain alteration in a biological sample is detected by sequencing-based methods, including, without limitation, next generation sequencing (NGS), deep sequencing, whole-genome sequencing, whole-exome sequencing, RNAseq or Sanger sequencing.
  • NGS next generation sequencing
  • the Notch PEST domain alteration is detected by next generation sequencing (NGS), deep sequencing, whole-genome sequencing, whole-exome sequencing or RNAseq.
  • Figure 1 shows Notch receptor mutations and focal amplifications in TCGA invasive breast cancer dataset clustered in or near the HD or PEST domains that exhibit pathway activation and are enriched in the triple negative subtype: (a) Lollipop graph depicting simple mutations and complex alterations in NOTCH1; (b) Lollipop graph depicting simple mutations and complex alterations in NOTCH2; (c) Lollipop graph depicting simple mutations and complex alterations in NOTCH3.
  • Figure 2 shows an expression heatmap of Notch pathway genes in 21 Notch altered TN tumors compared to 50 non-altered TN tumors.
  • Figure 3 shows Notch altered tumors are enriched in TNBC and Notch altered TNBC tumors are more likely to exhibit increased Notch pathway activity than other breast cancers.
  • Figure 4 shows the realignment of RNA-seq reads to wildtype sequences or variant sequences for PEST domain alterations in the TCGA breast cancer samples (a) TCGA-A2-A0T0, (b) TCGA-A8-A08X, and (c) TCGA-A8-A0J6.
  • Figure 5 shows whole exome sequencing (WXS) data from the tumor (top panel) and matched normal (bottom panel) samples for each patient (a) TCGA-A2-A0T0, (b) TCGA-A8-A08X, and (c) TCGA-A8-A0J6.
  • WXS whole exome sequencing
  • Figure 6 shows hotspot mutations / alterations in NOTCH1, NOTCH2 or NOTCH3 altered tumors often exhibit increased HES4 and / or HEY2 expression, where each dot represents a breast tumor from the TCGA data set and dotted grey lines indicate twofold above the median expression level for each gene.
  • Figure 7 shows identification of NOTCH2 aberrations in pre-clinical GSI sensitive models: (a) Genomic schematic of the rearranged NOTCH2 gene structures, where the breakpoint within exon 34 of NOTCH2 is denoted by the arrowhead; (b) Sanger sequencing trace image of the junction PCR product using cDNA as template.
  • Figure 8 shows Identification of NOTCH1 gene aberrations in the AA1077 breast cancer PDX model: (a) Genomic schematic of the rearranged NOTCH1 gene structures, where the dotted box indicates the tandem duplicated region spanning from intron 30 to the middle of exon 34; (b) Sanger sequencing trace image of the junction PCR product using cDNA as template.
  • Figure 9 shows identification of NOTCH1 gene aberrations in the HBCx-14 breast cancer PDX model:
  • Figure 10 shows wildtype and genomic alterations in Notch receptors in breast cancer patient-derived xenograft (PDX) models that are sensitive to PF-03084014 and the HCC1599 cell line model: (a) Notch2 wildtype; (b) MAXF1 162 model harbors a fusion (fs) (red lollipop at G2320fs) that disrupts the NOTCH2 PEST domain as well as an amplification of the locus; (c) Notchl wildtype; (d) AA1077 model harbors a partial tandem duplication of NOTCH1 that disrupts the NOTCH1 PEST domain; (e) HBCx-14 model harbors an ECD deletion and a PEST domain frameshifting deletion in NOTCH1; (f) HCC1599 cell line harbors an ECD deletion in NOTCHL
  • Figure 1 1 shows average tumor volume (+ SEM) in Notch altered models that are sensitive to PF-03084014: (a) MAXF1 162 model exhibited 65% tumor regression; (b) AA1077 model exhibited 88% tumor growth inhibition; (c) HBCx-14 model exhibited 60% tumor growth inhibition; and (d) HCC1599 model exhibited 50% tumor regression.
  • Figure 12 shows NOTCH1 mutations alter full length and NICD1 protein and NICD1 half-life: (a) NICD1 Western blot in Notch altered models treated with or without PF-03084014, " * " indicates NICD1 species of lower molecular weight than wt NICD1 ; (b) NICD1 Western blot on the NOTCH1 PEST truncated HBCx-14 model (lane 6) alongside a panel of lysates from TNBC PDX models; (c) NOTCH1 Western blot using an antibody that recognizes the NOTCH1 transmembrane and full length species, where the lower panel is at lighter exposure.
  • Figure 13 shows PEST mutated NICD1 was less diminished after 2 and 12 day treatment with PF-03084014 at 140 mg/kg BID relative to the wt band;
  • Figure 14 shows Notch mutant breast cancer models often exhibit increased Notch pathway expression: (a) Models rank-ordered from left to right using a HES4, HEY2 two gene signature score across an internal in vivo panel, and (b) an external PDX panel,
  • Figure 15 shows highly expressed Notch target genes HES4, HEY1 , HEY2 and HEYL were down-regulated Notch mutant models treated with PF-03084014 for 2 days (HCC1599, MAXF1 162 and AA1077) or 12 days (HBCx-14): (a) Nanostring data for the HCC1599 model; (b) Nanostring data for the MAXF1 162 model; (c) Nanostring data for the AA1077 model; (d) qRT-PCR data were used for the HBCx-14 model. * indicated p- value ⁇ 0.05 by two-sided Student's t-test.
  • the present invention provides methods of identifying and treating patients having Notch PEST domain alterations who will benefit from treatment with Notch signaling pathway inhibitors, such as GSIs.
  • the invention is based on the discovery that certain breast cancer cell lines and models harbouring Notch PEST domain alterations, and in particular TNBC cell lines and models, demonstrated enhanced sensitivity to treatment with a GSI, PF-03084014, either in vitro or in vivo. Without wishing to be being bound by theory, it is believed that Notch PEST domain alterations disrupt the regulation of NICD turnover, leading to an increase in the half-life of the NICD protein and prolongation of Notch signaling.
  • the methods provided herein are useful in identifying and treating patients having breast cancer, and in particular TNBC, who are likely to respond to treatment with a Notch signaling pathway inhibitor, such as a GSI.
  • a Notch signaling pathway inhibitor such as a GSI.
  • Notch refers to one of the four mammalian Notch receptors, Notch1 -4, and in particular, one of the human Notch1 -3 receptors.
  • the domain structures of human Notchl , Notch2 and Notch3 are depicted in Fig. 1 (a)-(c), respectively, which shows the epidermal growth factor-like region (EGF), Lin Notch Repeats (LNR), heterodimerization domain (HD), transmembrane domain (TM), ankyrin domain (ANK) and PEST domain.
  • EGF epidermal growth factor-like region
  • LNR Lin Notch Repeats
  • HD heterodimerization domain
  • TM transmembrane domain
  • ANK ankyrin domain
  • Notch PEST domain refers to the C-terminal PEST [rich in proline (P), glutamic acid (E), serine (S) and threonine (T)] degradation domain of Notch.
  • Notch PEST domain alteration(s) refer to point mutations, translocations, partial duplications, insertions, deletions (partial or complete), or truncations of the PEST domain of Notch, including any of Notch1 -4.
  • an alteration in the PEST domain of Notchl is a "Notchl PEST domain alteration,” etc. Such alterations result in activation of the Notch signaling pathway.
  • Notchl PEST domain alterations are predicted to result in NOTCH signaling gain-of-function:
  • NOTCH1 PEST Domain Any frame-shifting indel or nonsense mutation that lies between chr9: 139,390,613-139,391 , 425 (amino acids p.A2256-p.H2526);
  • NOTCH1 PEST Domain Any NOTCH1-X Rearrangement where one rearrangement position lies between chr9: 139,390,613-139,391 ,425 (amino acids p.A2256-p.H2526) and the other rearrangement position is outside the NOTCH1 gene (does not lie within chr9: 139,388,896-139,440,238;
  • NOTCH1 PEST Domain Any in-frame insertion between chr9: 139,390,613- 139,390,672 (amino acids p.H2507-p.H2526); and
  • NOTCH1 PEST Domain Any in-frame deletion that removes nucleotides between chr9: 139,390,613-9: 139,390,672 (amino acids p.H2507-p.H2526).
  • Notch2 PEST domain alterations are predicted to result in NOTCH signaling gain-of-function:
  • NOTCH2 PEST Domain Any frame-shifting indel or nonsense mutation between chr1 : 120,458,052-120,459,041 (amino acids p.K2102-p.H2431 );
  • NOTCH2 PEST Domain Any NOTCH2-X Rearrangement where one rearrangement lies between chrl : 120,458,052-120,459,041 (amino acids p.K2102- p.H2431 ) and the other rearrangement position is outside the NOTCH2 gene (does not lie within chrl : 120,454, 176-120,612,317);
  • NOTCH2 PEST Domain Any in-frame insertion between chrl : 120,458,052- 120,458, 1 1 1 (amino acids p.H2412-p.H2431 );
  • NOTCH2 PEST Domain Any in-frame deletion that removes nucleotides between chrl : 120,458,052-120,458, 1 1 1 (amino acids between p.H2412-p.H2431 ).
  • Notch3 PEST domain alterations are predicted to result in NOTCH signaling gain-of-function:
  • NOTCH3 PEST Domain Any frame-shifting indel or nonsense mutation between chrl 9: 15,271 , 491 -15,272,363 (amino acids p.Q2026-p.K2316);
  • NOTCH3 PEST Domain Any NOTCH3-X Rearrangement where one rearrangement lies between chrl 9: 15,271 ,491 -15,272,363 (amino acids p.Q2026- p.K2316) and the other rearrangement position is outside the NOTCH3 gene (does not lie between chrl 9: 15,270,444-15,31 1 ,792);
  • NOTCH3 PEST Domain Any in-frame insertion between chrl 9: 15,271 ,491 - 15,271 ,715 (amino acids p.H2242-p.K2316); and
  • NOTCH3 PEST Domain Any in-frame deletion that removes nucleotides between chrl 9:15,271 ,491 -15,271 ,715 (amino acids between p.H2242-p.K2316).
  • Notch signaling pathway inhibitors refer to compounds or agents that inhibit or reduce the biological activity of the molecule to which they bind.
  • Notch signaling pathway inhibitors include, e.g., GSIs, Notch regulator peptides and proteins, and anti-Notch antibodies or fragments thereof that bind to Notch1 -4, which may be optionally conjugated with another molecule to form an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • Such Notch signaling pathway inhibitors may be interchangeably referred to herein as compounds or agents.
  • ⁇ -secretase inhibitor gamma secretase inhibitor
  • GSI gamma secretase inhibitor
  • GSI small molecule GSIs include, e.g., the dipeptide class of GSIs, the sulfonamide class of GSIs, the transition state mimic class of GSIs, the benzocaprolactam class of GSIs, and other GSIs known in the art.
  • the GSI may be selected from the group consisting of PF- 03084014, MK-0752 (Merck), MRK-003 (Merck), RO4929097 (Roche), LY450139 (semagacestat, Eli Lilly), BMS-906024 (Bristol-Myers Squibb) and DAPT, or the pharmaceutically acceptable salts thereof.
  • GSIs include 1 -(S)- endo-N-(1 ,3,3)-Trimethylbicyclo[2.2.1 ]hept-2-yl)-4-fluorophenyl sulfonamide, WPE-III- 31 C, (S)-3-[N'-(3,5-difluorophenyl-alpha-hydroxyacetyl)-L-alaninyl]amino-2,3-dihydro-1 - methyl-5-phenyl-1 H-1 ,4-benzodiazepin-2-one, and (N)-[(S)-2-hydroxy-3-methyl-butyryl]- 1 -(L-alaninyl)-(S)-1 -amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one.
  • Anti-Notch antibodies include both antibodies and antigen-binding fragments thereof, which bind to one or more of the Notch1 -4 receptors, thereby inhibiting the activity of Notch. Such anti-Notch antibodies may be optionally conjugated with another molecule to form an anti-Notch antibody-drug conjugate (anti-Notch-ADC).
  • an “antibody” or “Ab” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
  • antibody encompasses not only intact polyclonal or monoclonal antibodies (mAbs), but also any antigen binding portion (e.g., "antigen- binding fragment") thereof of an intact antibody that retains the ability to specifically bind to a given antigen (e.g., Notch1 -4) or single chain thereof, fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site, for example without limitation, Fab; Fab'; F(ab') 2 ; an Fd fragment; an Fv fragment; a single domain antibody (dAb) fragment; an isolated complementarity determining region (CDR); single chain (scFv) and single domain antibodies (e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology
  • inhibitor or “neutralize” as used herein with respect to bioactivity of an antibody mean the ability of the antibody to substantially antagonize, prohibit, prevent, restrain, slow, disrupt, eliminate, stop, reduce or reverse e.g. progression or severity of that which is being inhibited including, but not limited to, a biological activity.
  • Antibody-drug conjugate refers to an antibody or antigen binding antibody fragment thereof, which is conjugated to a therapeutic agent that exerts a chemotherapeutic, cytotoxic, cytostatic and/or immunomodulatory effect on cancer cells or activated immune cells.
  • suitable therapeutic agent include, without limitation, chemotherapeutic agents, cytotoxic agents, cytostatic agents, and immunomodulatory agents.
  • An “anti-Notch antibody drug conjugate” or “anti-Notch ADC” refers to an anti-Notch antibody or antigen binding fragment thereof, e.g., anti- Notchl or anti-Notch3 antibody or fragment, which is conjugated to such a therapeutic agent.
  • Notch signaling pathway inhibitors include, e.g., Notch regulator peptides and proteins that inhibit Notch signaling.
  • Notch regulators such as antagonists and agonists, may alter Notch signaling activity either directly or indirectly. Indirect modulation of Notch signaling can occur at any step of the NICD cleavage and release process, including at the nucleic acid level, transcriptional level, translational level, protein-folding level, or enzymatic cleavage level.
  • Suitable Notch signaling antagonists are typically capable of preventing or reducing Notch signaling, while Notch signaling agonists typically induce or increase Notch signaling activity.
  • the terms "patient” or “subject” may be used interchangeably herein, and refer to a mammalian subject, and preferably to a human.
  • patients are afflicted with cancer, in particular breast cancer, including TNBC or basal-like breast cancer.
  • Subjects include individuals involved in clinical research trials, epidemiological studies, or subjects used as controls.
  • the subject may have been previously treated with a Notch signaling pathway inhibitor, or not so treated.
  • the subject may have been previously treated with one or more additional therapeutic agents at baseline (i.e., at a set point in time before the administration of a first dose of Notch signaling pathway inhibitor in the methods herein, such as the day of screening the subject before treatment is commenced), or not so treated.
  • treat or “treating”, as used herein, unless otherwise indicated, mean reversing, alleviating, inhibiting the progression of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.
  • treatment refers to the act of treating, as defined immediately above.
  • treating also includes adjuvant and neo-adjuvant treatment of a subject.
  • a “disorder” or “disease” is any condition that would benefit from treatment with a substance/molecule or method of the invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the subject to the disorder in question.
  • disorders to be treated herein include malignant and benign tumors; non-leukemia and lymphoid malignancies; neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, immunologic and other angiogenic disorders.
  • the terms "cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation.
  • the cell proliferative disorder is cancer.
  • the cell proliferative disorder is angiogenesis.
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “cancerous” refer to any malignant and/or invasive growth or tumor caused by abnormal cell growth.
  • cancer includes solid tumors named for the type of cells that form them, cancer of blood, bone marrow, or the lymphatic system.
  • cancer includes, but is not limited to, a primary cancer that originates at a specific site in the body, a metastatic cancer that has spread from the place in which it started to other parts of the body, a recurrence from the original primary cancer after remission, and a second primary cancer that is a new primary cancer in a person with a history of previous cancer of different type from latter one.
  • cancer cancer
  • cancer cancer
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  • cancer cancer
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  • cancer cancer
  • cancer cancer
  • cancer cancer
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  • cancer cancer
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  • cancer cancer
  • cancer examples include, but are not limited to, carcinoma, sarcoma, blastoma, leukemia, lymphoma, and myeloma.
  • the cancer is selected from the group consisting of basal cell cancer, medulloblastoma cancer, liver cancer, rhabdomyosarcoma, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer
  • the methods described herein may be used to identify and/or treat patients afflicted with cancer selected from the group consisting of breast cancer, prostate cancer, pancreatic cancer, colon cancer, ovarian cancer, colorectal cancer, lung cancer and brain cancer.
  • the patient is afflicted with breast cancer, in particular,
  • the patient is afflicted with pancreatic cancer.
  • the patient is afflicted with colon cancer or colorectal cancer.
  • the patient is afflicted with brain cancer, in particular glioma, medulloblastoma or ependymoma.
  • the patient is afflicted with lymphoma or leukemia.
  • the patient is afflicted with lung cancer, in particular non-small cell lung cancer.
  • terapéuticaally effective amount or “effective amount” are used interchangeably herein refer to the amount of a compound being administered that will relieve to some extent one or more of the symptoms of the disorder being treated. Where two or more agents are being administered in combination, the amounts of each agent administered may be adjusted so that the amounts of the two or more agents together are sufficient to provide a therapeutic effect.
  • an effective amount refers to the amount of an agent (or agents) that has the effect of (1 ) reducing the size of the tumor; (2) inhibiting (that is, slowing to some extent, preferably stopping) tumor metastasis; (3) inhibiting to some extent (that is, slowing to some extent, preferably stopping) tumor growth or tumor invasiveness; (4) relieving to some extent (ameliorating, or, preferably, eliminating) one or more signs or symptoms associated with the cancer; (5) inducting apoptosis of cancer cells; and/or (6) inhibiting angiogenesis.
  • a patient's "response” or “responsiveness” to treatment with a Notch signaling pathway inhibitor, or the "susceptibility" of the patient's tumor to treatment with a Notch signaling pathway inhibitor refers to the clinical or therapeutic benefit imparted to the patient from, or as a result of, treatment with a Notch signaling pathway inhibitor, such as a GSI, wherein the Notch signaling pathway inhibitor may be administered alone or in combination with another agent.
  • a Notch signaling pathway inhibitor such as a GSI
  • Such benefit includes cellular or biological responses, a complete response, a partial response, a stable disease (without progression or relapse), or a response with a later relapse of the patient from, or as a result of, the treatment with the Notch signaling pathway inhibitor as described herein.
  • the detection of a Notch PEST domain alteration in a biological sample from the patient indicates that the patient is expected to be responsive to treatment with a Notch signaling pathway inhibitor, or that the patient's tumor is expected to be susceptible to treatment with a Notch signaling pathway inhibitor.
  • sample and “biological sample” may be used interchangeably to refer to any biological sample obtained from an individual subject or patient, including from body tissue, cells, body fluids or other sources.
  • body tissues include, but are not limited to, tumor tissue (e.g., from a tumor biopsy), breast tissue, renal tissue, colonic tissue, brain tissue, muscle tissue, synovial tissue, skin, bone marrow or hair follicles.
  • Cells include, but are not limited to, tumor cell samples (e.g., from a tumor biopsy) or other tissue cell samples or cells cultured therefrom, or cells isolated from body fluids.
  • body fluids include, but are not limited to, blood (e.g., whole fresh blood, peripheral blood mononuclear cells, frozen whole blood), serum, plasma (including fresh or frozen), lymph, ascitic fluid, cystic fluid, amniotic fluid, pleural fluid, peritoneal fluid, pericardial fluid, urine, saliva, sputum, sweat, tears, feces, nasal secretions, bronchial aspirate, semen, vaginal secretions, breast milk, synovial fluid and cerebrospinal fluid.
  • the biological sample may comprise circulating tumor cells (CTCs), circulating nucleic acids (CNAs), or cancer stem cells.
  • Patient samples such as those containing cells, or proteins or nucleic acids, may be taken from a patient who is suspected of having, or is diagnosed as having a proliferative disorder, such as cancer, and hence is likely in need of treatment, or from a normal individual who is not suspected of having any disorder.
  • a proliferative disorder such as cancer
  • the biological sample is preferably a tissue sample, such as a tumor tissue sample.
  • the biological sample comprises a tumor cell sample or cells cultured therefrom.
  • the biological sample comprises cells removed from a solid tumor, e.g., by biopsy.
  • the biological sample comprises circulating tumor cells (CTCs), circulating nucleic acids (CNAs), or cancer stem cells.
  • Alterations of the Notch PEST domain may result in activation of Notch.
  • activation of Notch is determined by measuring the expression level of at least one gene whose expression is regulated by Notch.
  • activation of Notch is determined by an increase in the level of the NICD protein. In other embodiments, activation of Notch is determined by an increase in the half-life of the NICD protein. In still other embodiments, activation of Notch is determined by an increase or decrease in expression of downstream Notch pathway target genes, including receptors, ligands, and regulators such as NUMB, canonical target genes such as the Hes and Hey gene families, and downstream genes that impact cell cycle and apoptosis, including, e.g., MYC, CCND1 , BIRC5, CDKN1A, PTEN, and NOXA1 , which are indicative Notch activation status and can serve as biomarkers.
  • Notch pathway target genes including receptors, ligands, and regulators such as NUMB
  • canonical target genes such as the Hes and Hey gene families
  • downstream genes that impact cell cycle and apoptosis including, e.g., MYC, CCND1 , BIRC5, CDK
  • level of expression or “expression level” are used interchangeably and generally refer to the amount of a polynucleotide or an amino acid product or protein in a biological sample. “Expression” generally refers to the process by which gene-encoded information is converted into the structures present and operating in the cell. Therefore, “expression” of a gene may refer to transcription into a polynucleotide, translation into a protein, or even posttranslational modification of the protein.
  • Fragments of the transcribed polynucleotide, the translated protein, or the post- translationally modified protein are also regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a posttranslational processing of the protein, e.g., by proteolysis.
  • "Expressed genes” include those that are transcribed into a polynucleotide as mRNA and then translated into a protein, and also those that are transcribed into RNA but not translated into a protein (for example, transfer and ribosomal RNAs).
  • activation of Notch is determined by one or more of the following: (1 ) an increase in the level of the NICD protein; (2) an increase in the half- life of the NICD protein; and/or (3) an increase or decrease in expression of one or more downstream Notch pathway target genes (e.g., an increase in expression of the Hes or Hey gene families).
  • the increase (or decrease) is determined relative to an appropriate reference level or value for the particular indicator.
  • Such reference levels or values include, e.g., the median level of the NICD protein, the median NICD protein half-life, or the median expression level of one or more Notch pathway biomarkers in samples from a group or population of patients being assessed for responsiveness to a Notch signaling pathway inhibitor; the level in a sample previously obtained from the patient at a prior time (e.g., pre- or post-treatment); the level in a sample of normal (unaffected) cells, preferably cells of the same tissue or type as the biological test sample obtained from the patient (e.g., pre- or post- treatment); or the level in a sample from a patient who received prior treatment with a Notch signaling pathway inhibitor, such as a GSI, in a primary tumor setting, and who now may be experiencing recurrence or metastasis.
  • a Notch signaling pathway inhibitor such as a GSI
  • each of the aspects described herein may include comparison of the Notch PEST domain alteration in a cancer cell or patient sample with a non-cancerous or normal control sample.
  • the presence of the Notch PEST domain alteration in a cancer cell or patient biological sample indicates the cancer cell or patient is sensitive to treatment with a Notch signaling pathway inhibitor, such as a GSI.
  • the reference or control sample may be a sample of normal (unaffected) cells, preferably cells of the same tissue or type as the biological test sample obtained from the patient; a sample of cells affected by cancer, preferably a cancer of the same type as is in the patient or is suspected to be in the patient.
  • the control sample may be a sample of cells from a tissue type associated with the presence or absence of cancer, and/or from a tumor with good or with poor prognosis.
  • the reference or control sample may be obtained from the patient, from another subject or from a population of subjects. Where a population of subjects is used, the comparison may be made with the average (e.g., mean or median) in samples of cells from said population.
  • One advantage of using a control of normal tissue from the same patient is that it accounts for any individual variation. Where the control is from another patient (either of normal or affected tissue), using a reference value based on a population of patients may be preferred to reduce individual variation.
  • patients identified as having Notch PEST domain alteration are also determined to demonstrate Notch activation, for example by a change in level of one of the indicators above (e.g., NICD protein, half-life or expression of pathway genes) that is greater than or less than the reference level.
  • the change in level of one or more indicators is 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% relative to (i.e., higher or lower than) the reference level, such as the median levels above.
  • a control sample from the patient at an earlier time point, so as to provide a historical record. In some such embodiments, this allows for the assessment or the monitoring of the progression of the condition over time. By comparing the severity of the condition in a patient at two time points, it is possible to determine whether a particular treatment regime is having a positive effect or not, allowing for assessment of the effectiveness of a particular treatment. The effectiveness of any one regime may differ from patient to patient, or during the course of the disease.
  • a gene expression product may be a protein or a transcript (i.e., an RNA molecule expressed by the gene). Determination of protein, gene or transcript level may be made by any of the methods known in the art.
  • suitable methods for assessing protein levels include immunohistochemistry (e.g., immunofluorescence), Western blotting, and solid phase methods such as ELISA (enzyme-linked immunoabsorbant assay).
  • immunohistochemical techniques an assessment of protein level can be made by determining the proportion of cells showing labeling (e.g., staining or fluorescence).
  • Transcript level may be determined by in situ hybridization, e.g., accompanied by assessment of the proportion of cells showing hybridization.
  • quantitative PCR methods may be used, for example, based upon the ABI TaqManTM technology, which is widely used in the art. PCR methods require a primer pair which targets opposite strands of the target gene at a suitable distance apart (typically 50 to 300 bases). Suitable target sequences for the primers may be determined by reference to Genbank sequences.
  • a convenient method is by hybridization of the sample (either directly or after generation of cDNA or cRNA) to a gene chip array and/or micro fluidic card (low density array) based on quantitative PCR methods.
  • gene chip technology the genes may be present in commercially available chips from Affymetrix, which may be used in accordance with protocols from the manufacturer.
  • micro fluidic card technology the genes may be present in commercially available micro fluidic cards from Applied Biosystem, also known as Low Density Arrays. These cards may be used in accordance with protocol from the manufacturer.
  • Gene copy number may be determined using techniques known in the art, including in situ hybridization (ISH) with nucleic acid probes which may be labeled, e.g., with a fluorescent label (FISH), or PCR of genomic DNA.
  • ISH in situ hybridization
  • FISH fluorescent label
  • Therapeutic agents and compounds may be administered by any method that enables their delivery to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), topical, and rectal administration.
  • Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification of dosage unit forms suitable for use in the invention are dictated by and directly dependent on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such agents.
  • the dose and dosing regimen is adjusted in accordance with methods well- known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a patient may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a patient in practicing the present invention.
  • dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values. Thus, the present invention encompasses intra-patient dose-escalation as determined by the skilled artisan. Determining appropriate dosages and regimens for administration of the chemotherapeutic agent are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein.
  • the appropriate dosage of the agent or agents administered will depend on the particular agent being administered, the subject being treated, the severity of the disorder or condition, the type and severity of symptoms to be treated, the patient's clinical history and response to the agent, the rate and mode of administration, the patient's clearance rate for the administered agent, and the discretion of the attending physician.
  • an effective dosage is in the range of about 0.001 to about 100 mg per kg body weight per day, preferably about 0.01 to about 35 mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to about 0.05 to about 7 g/day, preferably about 0.1 to about 2.5 g/day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several small doses for administration throughout the day.
  • the appropriate dosage will depend on the anti-Notch antibody or ADC employed, the severity of the disorder or condition, the type and severity of symptoms to be treated, whether the agent is administered for therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, the rate and mode of administration, the patient's clearance rate for the administered agent, and the discretion of the attending physician.
  • the clinician may administer an anti-Notch antibody or an anti-Notch antibody- drug conjugate until a dosage is reached that achieves the desired result and beyond.
  • Dose and/or frequency can vary over course of treatment, but may stay constant as well. Empirical considerations, such as the half-life, generally will contribute to the determination of the dosage.
  • antibodies that are compatible with the human immune system such as humanized antibodies or fully human antibodies, may be used to prolong half-life of the antibody and to prevent the antibody being attacked by the host's immune system.
  • Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of symptoms, e.g., tumor growth inhibition or delay, etc.
  • sustained continuous release formulations of anti-Notch antibodies or anti-Notch antibody-drug conjugates may be appropriate.
  • Various formulations and devices for achieving sustained release are known in the art.
  • an initial candidate dosage can be about 2 mg/kg.
  • a typical daily dosage might range from about any of 3 g/kg to 30 g/kg to 300 g/kg to 3 mg/kg, to 30 mg/kg, to 100 mg/kg or more, depending on the factors mentioned above.
  • dosage of about 1 mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 10 mg/kg, and about 25 mg/kg may be used.
  • An exemplary dosing regimen comprises administering an initial dose of about 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg of the anti-Notch antibody or anti-Notch antibody- drug conjugate, or followed by a maintenance dose of about 1 mg/kg every other week.
  • Other exemplary dosing regimens comprise administering increasing doses (e.g., initial dose of 1 mg/kg and gradual increase to one or more higher doses every week or longer time period).
  • dosing regimens may also be useful, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve. For example, in some embodiments, dosing from one to four times a week is contemplated. In other embodiments dosing once a month or once every other month or every three months is contemplated as well as weekly, bi-weekly and every three weeks. The progress of this therapy may be monitored by conventional techniques and assays.
  • the dosing regimen (including the anti-Notch antibody or the anti-Notch antibody-drug conjugate used) can vary over time.
  • a Notch signaling pathway inhibitor such as a GSI, or an anti-Notch antibody, fragment or ADC thereof, may be conveniently administered in the form of a pharmaceutical composition, either alone or in combination with another therapeutic agent.
  • a “pharmaceutical composition” refers to a mixture of one or more therapeutic agents as the active ingredient, and at least one pharmaceutically acceptable carrier or excipient, and may comprise two or more pharmaceutically acceptable carriers or excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of an agent to a subject.
  • the pharmaceutical composition may further comprise at least one additional anti-cancer therapeutic agent or palliative agent.
  • a "pharmaceutically acceptable carrier” refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • Pharmaceutical compositions may be administered by any means known in the art that achieve the generally intended purpose to treat proliferative disorders, such as cancer.
  • the pharmaceutical acceptable carrier may comprise any conventional pharmaceutical carrier, diluent or excipient, such as buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents, carriers, and the like.
  • diluent or excipient such as buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents, carriers, and the like.
  • the choice of carrier and/or excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.
  • Suitable pharmaceutical carriers include inert diluents or fillers, water and various organic solvents (such as hydrates and solvates). Excipients may further include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glut
  • tablets containing various excipients such as citric acid may be employed together with various disintegrants such as starch, alginic acid and certain complex silicates and with binding agents such as sucrose, gelatin and acacia.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tableting purposes.
  • Solid compositions of a similar type may also be employed in soft and hard filled gelatin capsules.
  • Non-limiting examples of materials include lactose or milk sugar and high molecular weight polyethylene glycols.
  • the active compound therein may be combined with various sweetening or flavoring agents, coloring matters or dyes and, if desired, emulsifying agents or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof.
  • the pharmaceutical composition may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulation, solution, suspension; for parenteral injection as a sterile solution, suspension or emulsion; for topical administration as an ointment or cream or for rectal administration as a suppository.
  • Exemplary parenteral administration forms include solutions or suspensions of active compound in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms may be suitably buffered, if desired.
  • the pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages.
  • compositions suitable for the delivery of compounds and agents herein, and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in 'Remington's Pharmaceutical Sciences', 19th Edition (Mack Publishing Company, 1995), the disclosure of which is incorporated herein by reference in its entirety.
  • Compounds may be administered orally.
  • Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compound enters the blood stream directly from the mouth.
  • Formulations suitable for oral administration include solid formulations such as tablets, capsules containing particulates, liquids, or powders, lozenges (including liquid- filled), chews, multi- and nan-particulates, gels, solid solution, liposome, films (including much-adhesive), ovules, sprays and liquid formulations.
  • Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be used as fillers in soft or hard capsules and typically include a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.
  • a carrier for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil
  • emulsifying agents and/or suspending agents may also be prepared by the reconstitution of a solid, for example, from a sachet.
  • Compounds may also be used in fast-dissolving, fast-disintegrating dosage forms such as those described in Expert Opinion in Therapeutic Patents, 1J_ (6), 981 -986 by Liang and Chen (2001 ), the disclosure of which is incorporated herein by reference in its entirety.
  • the drug may make up from 1 wt% to 80 wt% of the dosage form, more typically from 5 wt% to 60 wt% of the dosage form.
  • tablets generally contain a disintegrant.
  • disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinized starch and sodium alginate.
  • the disintegrant will comprise from 1 wt% to 25 wt%, preferably from 5 wt% to 20 wt% of the dosage form.
  • Binders are generally used to impart cohesive qualities to a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.
  • lactose monohydrate, spray-dried monohydrate, anhydrous and the like
  • mannitol xylitol
  • dextrose sucrose
  • sorbitol microcrystalline cellulose
  • starch dibasic calcium phosphate dihydrate
  • Tablets may also optionally include surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc.
  • surface active agents such as sodium lauryl sulfate and polysorbate 80
  • glidants such as silicon dioxide and talc.
  • surface active agents are typically in amounts of from 0.2 wt% to 5 wt% of the tablet, and glidants typically from 0.2 wt% to 1 wt% of the tablet.
  • Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate.
  • Lubricants generally are present in amounts from 0.25 wt% to 10 wt%, preferably from 0.5 wt% to 3 wt% of the tablet.
  • compositions include anti-oxidants, colorants, flavoring agents, preservatives and taste-masking agents.
  • Exemplary tablets contain up to about 80 wt% drug, from about 10 wt% to about 90 wt% binder, from about 0 wt% to about 85 wt% diluent, from about 2 wt% to about 10 wt% disintegrant, and from about 0.25 wt% to about 10 wt% lubricant.
  • Tablet blends may be compressed directly or by roller to form tablets. Tablet blends or portions of blends may alternatively be wet-, dry- or melt-granulated, melt congealed, or extruded before tableting.
  • the final formulation may include one or more layers and may be coated or uncoated; or encapsulated.
  • Solid formulations for oral administration may be formulated to be immediate and/or modified release.
  • Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
  • Suitable modified release formulations are described in U.S. Patent No. 6, 106,864. Details of other suitable release technologies such as high energy dispersions and osmotic and coated particles can be found in Verma et al, Pharmaceutical Technology On-line, 25(2), 1 -14 (2001 ). The use of chewing gum to achieve controlled release is described in WO 00/35298. The disclosures of these references are incorporated herein by reference in their entireties.
  • Compounds may also be administered directly into the blood stream, into muscle, or into an internal organ.
  • Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous.
  • Suitable devices for parenteral administration include needle (including micro needle) injectors, needle-free injectors and infusion techniques.
  • Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
  • excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9)
  • a suitable vehicle such as sterile, pyrogen-free water.
  • parenteral formulations under sterile conditions may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.
  • solubility of compounds used in the preparation of parenteral solutions may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents.
  • Formulations for parenteral administration may be formulated to be immediate and/or modified release.
  • Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
  • compounds may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active compound. Examples of such formulations include drug-coated stents and PGLA microspheres.
  • Compounds may also be administered topically to the skin or mucosa, that is, dermally or transdermally.
  • Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, bandages and microemulsions.
  • Liposomes may also be used.
  • Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol.
  • Penetration enhancers may be incorporated; see, for example, J Pharm Sci, 88 (10), 955-958 by Finnin and Morgan (October 1999).
  • topical administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis and micro needle or needle-free (e.g. PowderjectTM, BiojectTM, etc.) injection.
  • electroporation iontophoresis, phonophoresis, sonophoresis and micro needle or needle-free (e.g. PowderjectTM, BiojectTM, etc.) injection.
  • iontophoresis iontophoresis
  • phonophoresis phonophoresis
  • sonophoresis e.g. PowderjectTM, BiojectTM, etc.
  • Formulations for topical administration may be formulated to be immediate and/or modified release.
  • Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
  • Compounds can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer using electrohydrodynamics to produce a fine mist), or nebulizer, with or without the use of a suitable propellant, such as 1 , 1 ,1 ,2-tetrafluoroethane or 1 , 1 , 1 ,2,3,3,3- heptafluoropropane.
  • the powder may include a bioadhesive agent, for example, chitosan or cyclodextrin.
  • the pressurized container, pump, spray, atomizer, or nebulizer contains a solution or suspension of the compound(s) comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilizing, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.
  • a solution or suspension of the compound(s) comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilizing, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.
  • the drug product Prior to use in a dry powder or suspension formulation, the drug product is micronized to a size suitable for delivery by inhalation (typically less than 5 microns). This may be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.
  • comminuting method such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.
  • Capsules made, for example, from gelatin or HPMC
  • blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound or agent, a suitable powder base such as lactose or starch and a performance modifier such as l-leucine, mannitol, or magnesium stearate.
  • the lactose may be anhydrous or in the form of the monohydrate, preferably the latter.
  • Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.
  • a suitable solution formulation for use in an atomizer using electrohydrodynamics to produce a fine mist may contain from 1 g to 20mg of the compound per actuation and the actuation volume may vary from 1 ⁇ _ to 100 ⁇ _.
  • a typical formulation includes a compound or agent, propylene glycol, sterile water, ethanol and sodium chloride.
  • Alternative solvents which may be used instead of propylene glycol include glycerol and polyethylene glycol.
  • Suitable flavors such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations intended for inhaled/intranasal administration.
  • Formulations for inhaled/intranasal administration may be formulated to be immediate and/or modified release using, for example, poly(DL-lactic-coglycolic acid (PGLA).
  • Modified release formulations include delayed- sustained- pulsed-, controlled- targeted and programmed release.
  • the dosage unit is determined by means of a valve which delivers a metered amount.
  • Units in accordance with the invention are typically arranged to administer a metered dose or "puff" containing a desired mount of the compound or agent.
  • the overall daily dose may be administered in a single dose or, more usually, as divided doses throughout the day.
  • Compounds may be administered rectally or vaginally, for example, in the form of a suppository, pessary, or enema. Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate.
  • Formulations for rectal/vaginal administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
  • Compounds may also be administered directly to the eye or ear, typically in the form of drops of a micronized suspension or solution in isotonic, pH-adjusted, sterile saline.
  • Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g. absorbable gel sponges, collagen) and non-biodegradable (e.g. silicone) implants, wafers, lenses and particulate or vesicular systems, such as niosomes or liposomes.
  • Compounds may be further combined with soluble macromolecular entities, such as cyclodextrin and suitable derivatives thereof or polyethylene glycol-containing polymers, in order to improve their solubility, dissolution rate, taste-masking, bioavailability and/or stability for use in any of the aforementioned modes of administration.
  • soluble macromolecular entities such as cyclodextrin and suitable derivatives thereof or polyethylene glycol-containing polymers
  • Drug-cyclodextrin complexes are found to be generally useful for most dosage forms and administration routes. Both inclusion and non-inclusion complexes may be used.
  • the cyclodextrin may be used as an auxiliary additive, i.e. as a carrier, diluent, or solubilizer. Most commonly used for these purposes are alpha-, beta- and gamma-cyclodextrins, which are well-known in the art.
  • kits suitable for coadministration of the compositions.
  • the kit may include two or more separate pharmaceutical compositions, at least one of which contains a a Notch signaling pathway inhibitor (e.g., a GSI such as PF-03084014), or a pharmaceutically acceptable salt thereof, and means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet.
  • a kit is the familiar blister pack used for the packaging of tablets, capsules and the like.
  • the kit of the invention is particularly suitable for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another.
  • the kit typically includes directions for administration and may be provided with a memory aid.
  • kits may also comprise a described cytotoxic conjugate and instructions for the use of the cytotoxic conjugate for killing of particular cell types.
  • the instructions may include directions for using the cytotoxic conjugates in vitro, in vivo or ex vivo.
  • the kit will have a compartment containing the cytotoxic conjugate.
  • the cytotoxic conjugate may be in a lyophilized form, liquid form, or other form amendable to being included in a kit.
  • the kit may also contain additional elements needed to practice the method described on the instructions in the kit, such a sterilized solution for
  • the term “combination therapy” refers to the administration of a Notch signaling pathway inhibitor, such as a GSI or an anti-Notch mAb, fragment or ADC thereof, together with at least one additional therapeutic agent (e.g., an anti-cancer agent).
  • a Notch signaling pathway inhibitor such as a GSI or an anti-Notch mAb, fragment or ADC thereof
  • additional therapeutic agent e.g., an anti-cancer agent
  • the agents may be administered separately, sequentially, simultaneously, concurrently or at chronologically staggered intervals.
  • the combined administration includes coadministration, using separate formulations or a single pharmaceutical formulation (e.g., as combined unit dosage forms, as separate unit dosage forms, as a kit-of-parts or as an admixture), as well as consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.
  • the additional therapeutic agent is administered to a mammal (e.g., a human) prior to administration of the Notch signaling pathway inhibitor.
  • the additional agent is administered to the mammal after administration of the Notch signaling pathway inhibitor.
  • the additional agent is administered to the mammal (e.g., a human) simultaneously with the administration of the Notch signalling pathway inhibitor.
  • the amounts of the Notch signaling pathway inhibitor and the additional therapeutic agent taken together are sufficient to produce a therapeutic effect.
  • Suitable dosages for any of the above coadministered agents are those presently used and may be lowered due to the combined action (synergy) of the newly identified agent and other chemotherapeutic agents or treatments.
  • the use of a combination provides an additive, greater than additive, or synergistic anti-cancer effects.
  • “Synergy” and “synergistic” mean the effect achieved when the two or more agents are used together is greater than the sum of the effects that results from using the compounds separately.
  • a synergistic effect may be attained when the active ingredients are: (1 ) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen.
  • a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g. by different injections in separate syringes.
  • an effective dosage of each active ingredient is administered sequentially, i.e. serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.
  • the other therapeutic agent will be an agent that is standard of care for the specific disease to be treated or is part of a salvage regimen for the specific disease to be treated.
  • the at least one additional therapeutic agent is selected from the group consisting of anti-neoplastic agents, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-tumor agents, anti-angiogenesis agents, anti-apoptotic agents, anti-tubulin agents, signal transduction inhibitors and antiproliferative agents, or a combination thereof.
  • the at least one additional therapeutic agent is selected from the group consisting of anti-neoplastic agents, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, anti-tumor agents, anti-angiogenesis agents, signal transduction inhibitors and antiproliferative agents, or a combination thereof.
  • the additional therapeutic agent comprises a taxane, a nucleoside analog, or a platin.
  • the Notch signaling pathway inhibitor such as a GSI
  • a GSI is administered in combination with one or more anti-cancer agents selected from the group consisting of docetaxel, paclitaxel, abraxane, gemcitabine, capecitabine, cisplatin, carboplatin, trastuzumab, tamoxifen, vinorelbine, exemestane, letrozole, anastrozole, and palbociclib.
  • Anti-cancer agents and chemotherapeutic regimens include, for example, anticancer antibodies, including, for example, anti-CD52 antibodies (e.g. , Alemtuzumab), anti-HER-2 antibodies, anti-CD20 antibodies (e.g.
  • chemotherapeutic regimens including, for example, CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone); CVP (cyclophosphamide, vincristine, and prednisone); RCVP (Rituximab+CVP); RCHOP (Rituximab+CHOP); RICE (Rituximab+ifosamide, carboplatin, etoposide); RDHAP, (Rituximab+dexamethasone, cytarabine, cisplatin); RESHAP (Rituximab+etoposide, methylprednisolone, cytarabine, cisplatin); gemcitabine; combination treatment with vincristine, prednisone, and anthracycline, with or without asparaginase; combination treatment with daunor
  • the additional therapeutic agent used in conjunction with a Notch pathway inhibitor, and pharmaceutical compositions described herein is an anti-angiogenesis agent (e.g., an agent that stops tumors from developing new blood vessels).
  • anti-angiogenesis agents include for example VEGF inhibitors, VEGFR inhibitors, TIE-2 inhibitors, PDGFR inhibitors, angiopoetin inhibitors, ⁇ inhibitors, COX-2 (cyclooxygenase II) inhibitors, integrins (alpha-v/beta-3), MMP-2 (matrix-metalloprotienase 2) inhibitors, and MMP-9 (matrix- metalloprotienase 9) inhibitors.
  • Preferred anti-angiogenesis agents include sunitinib (SutentTM), bevacizumab (AvastinTM), axitinib (AG 13736), SU 14813 (Pfizer), and AG 13958 (Pfizer).
  • Additional anti-angiogenesis agents include vatalanib (CGP 79787), Sorafenib (NexavarTM), pegaptanib octasodium (MacugenTM), vandetanib (ZactimaTM), PF- 0337210 (Pfizer), SU 14843 (Pfizer), AZD 2171 (AstraZeneca), ranibizumab (LucentisTM), NeovastatTM (AE 941 ), tetrathiomolybdata (CoprexaTM), AMG 706 (Amgen), VEGF Trap (AVE 0005), CEP 7055 (Sanofi-Aventis), XL 880 (Exelixis), telatinib (BAY 57- 9352), and CP-868,596 (Pfizer).
  • anti-angiogenesis agents include enzastaurin (LY 317615), midostaurin (CGP 41251 ), perifosine (KRX 0401 ), teprenone (SelbexTM) and UCN 01 (Kyowa Hakko).
  • anti-angiogenesis agents which can be used in conjunction with a compound of the invention and pharmaceutical compositions described herein include celecoxib (CelebrexTM), parecoxib (DynastatTM), deracoxib (SC 59046), lumiracoxib (PreigeTM), valdecoxib (BextraTM), rofecoxib (VioxxTM), iguratimod ⁇ CareramTM), IP 751 (Invedus), SC-58125 (Pharmacia) and etoricoxib (ArcoxiaTM).
  • anti-angiogenesis agents include exisulind (AptosynTM), salsalate (AmigesicTM), diflunisal (DolobidTM), ibuprofen (MotrinTM), ketoprofen (OrudisTM), nabumetone (RelafenTM), piroxicam (FeldeneTM), naproxen (AleveTM, NaprosynTM), diclofenac (VoltarenTM), indomethacin (IndocinTM), sulindac (ClinorilTM), tolmetin (TolectinTM), etodolac (LodineTM), ketorolac (ToradolTM), and oxaprozin (DayproTM).
  • anti-angiogenesis agents include ABT 510 (Abbott), apratastat (TMI 005), AZD 8955 (AstraZeneca), incyclinide (MetastatTM), and PCK 3145 (Procyon).
  • anti-angiogenesis agents include acitretin (NeotigasonTM), plitidepsin (aplidineTM), cilengtide (EMD 121974), combretastatin A4 (CA4P), fenretinide (4 HPR), halofuginone (TempostatinTM), PanzemTM (2-methoxyestradiol), PF-03446962 (Pfizer), rebimastat (BMS 275291), catumaxomab (RemovabTM), lenalidomide (RevlimidTM), squalamine (EVIZONTM), thalidomide (ThalomidTM), UkrainTM (NSC 631570), VitaxinTM (MEDI 522), and zoledronic acid (ZometaTM).
  • acitretin NeotigasonTM
  • plitidepsin aplidineTM
  • cilengtide EMD 121974
  • CA4P
  • the anti-cancer agent is a so called signal transduction inhibitor (e.g., inhibiting the means by which regulatory molecules that govern the fundamental processes of cell growth, differentiation, and survival communicated within the cell).
  • Signal transduction inhibitors include small molecules, antibodies, and antisense molecules.
  • Signal transduction inhibitors include for example kinase inhibitors (e.g., tyrosine kinase inhibitors or serine/threonine kinase inhibitors) and cell cycle inhibitors.
  • More specifically signal transduction inhibitors include, for example, farnesyl protein transferase inhibitors, EGF inhibitor, ErbB-1 (EGFR), ErbB-2, pan erb, IGF1 R inhibitors, MEK, c-Kit inhibitors, FLT-3 inhibitors, K-Ras inhibitors, PI3 kinase inhibitors, JAK inhibitors, STAT inhibitors, Raf kinase inhibitors, Akt inhibitors, mTOR inhibitor, CDK inhibitors, P70S6 kinase inhibitors, inhibitors of the WNT pathway and so called multi-targeted kinase inhibitors.
  • Preferred signal transduction inhibitors include gefitinib (IressaTM), cetuximab (ErbituxTM), erlotinib (TarcevaTM), trastuzumab (HerceptinTM), sunitinib (SutentTM), imatinib (GleevecTM), and PD325901 (Pfizer).
  • signal transduction inhibitors which may be used in conjunction with a compound of the invention and pharmaceutical compositions described herein include BMS 214662 (Bristol-Myers Squibb), lonafarnib (SarasarTM), pelitrexol (AG 2037), matuzumab (EMD 7200), nimotuzumab (TheraCIM h-R3TM), panitumumab (VectibixTM), Vandetanib (ZactimaTM), pazopanib (SB 786034), ALT 1 10 (Alteris Therapeutics), BIBW 2992 (Boehringer lngelheim),and CerveneTM (TP 38).
  • BMS 214662 Bristol-Myers Squibb
  • lonafarnib SarasarTM
  • pelitrexol AG 2037
  • matuzumab EMD 7200
  • nimotuzumab TheraCIM h-R3TM
  • signal transduction inhibitor examples include PF-2341066 (Pfizer), PF- 299804 (Pfizer), PD-0332991 (Pfizer), canertinib (CI 1033), pertuzumab (OmnitargTM), Lapatinib (TycerbTM), pelitinib (EKB 569), miltefosine (MiltefosinTM), BMS 599626 (Bristol-Myers Squibb), Lapuleucel-T (NeuvengeTM), NeuVaxTM (E75 cancer vaccine), OsidemTM (IDM 1 ), mubritinib (TAK-165), CP-724,714 (Pfizer), panitumumab (VectibixTM), lapatinib (TycerbTM), PF-299804 (Pfizer), pelitinib (EKB 569), and pertuzumab (OmnitargTM).
  • signal transduction inhibitors include ARRY 142886 (Array Biopharm), everolimus (CerticanTM), zotarolimus (EndeavorTM), temsirolimus (ToriselTM), AP 23573 (ARIAD), and VX 680 (Vertex).
  • signal transduction inhibitors include XL 647 (Exelixis), sorafenib (NexavarTM), LE-AON (Georgetown University), and GI-4000 (Globelmmune).
  • signal transduction inhibitors include ABT 751 (Abbott), alvocidib (flavopiridol), BMS 387032 (Bristol Myers), EM 1421 (Erimos), indisulam (E 7070), seliciclib (CYC 200), BIO 1 12 (One Bio), BMS 387032 (Bristol-Myers Squibb), PD 0332991 (Pfizer), and AG 024322 (Pfizer).
  • Classical antineoplastic agents include but are not limited to hormonal modulators such as hormonal, anti-hormonal, androgen agonist, androgen antagonist and anti-estrogen therapeutic agents, histone deacetylase (HDAC) inhibitors, gene silencing agents or gene activating agents, ribonucleases, proteosomics, Topoisomerase I inhibitors, Camptothecin derivatives, Topoisomerase II inhibitors, alkylating agents, antimetabolites, poly(ADP-ribose) polymerase-1 (PARP-1 ) inhibitor, microtubulin inhibitors, antibiotics, plant derived spindle inhibitors, platinum- coordinated compounds, gene therapeutic agents, antisense oligonucleotides, vascular targeting agents (VTAs), and statins
  • hormonal modulators such as hormonal, anti-hormonal, androgen agonist, androgen antagonist and anti-estrogen therapeutic agents
  • HDAC histone deacetylase
  • gene silencing agents or gene activating agents ribonuclea
  • antineoplastic agents used in combination therapy with a compound of the invention optionally with one or more other agents include, but are not limited to, glucocorticoids, such as dexamethasone, prednisone, prednisolone, methylprednisolone, hydrocortisone, and progestins such as medroxyprogesterone, megestrol acetate (Megace), mifepristone (RU-486), Selective Estrogen Receptor Modulators (SERMs; such as tamoxifen, raloxifene, lasofoxifene, afimoxifene, arzoxifene, arzoxifene, avaloxifene, ospemifene, tesmilifene, toremifene, trilostane and CHF 4227 (Cheisi)), Selective Estrogen-Receptor Downregulators (SERD'
  • antineoplastic agents used in combination with compounds of the invention include but are not limited to suberolanilide hydroxamic acid (SAHA, Merck Inc./Aton Pharmaceuticals), depsipeptide (FR901228 or FK228), G2M- 777, MS-275, pivaloyloxymethyl butyrate and PXD-101 ; Onconase (ranpirnase),PS-341 (MLN-341 ), Velcade (bortezomib), 9-aminocamptothecin, belotecan, BN-80915 (Roche), camptothecin, diflomotecan, edotecarin, exatecan (Daiichi), gimatecan, 10- hydroxycamptothecin, irinotecan HCI (Camptosar), lurtotecan, Orathecin (rubitecan, Supergen), SN-38, topotecan, camptothecin, 10-hydroxy
  • the invention also contemplates the use of Notch signalling pathway inhibitors together with dihydrofolate reductase inhibitors (such as methotrexate and NeuTrexin (trimetresate glucuronate)), purine antagonists (such as 6-mercaptopurine riboside, mercaptopurine, 6-thioguanine, cladribine, clofarabine (Clolar), fludarabine, nelarabine, and raltitrexed), pyrimidine antagonists (such as 5-fluorouracil (5-FU), Alimta (premetrexed disodium, LY231514, MTA), capecitabine (XelodaTM), cytosine arabinoside, GemzarTM (gemcitabine, Eli Lilly), Tegafur (UFT Orzel or Uforal and including TS-1 combination of tegafur, gimestat and otostat), doxifluridine, carmofur, cytarabine
  • antineoplastic cytotoxic agents for use in combination therapy include, but are not limited to, Abraxane (Abraxis Bioscience, Inc.), Batabulin (Amgen), EPO 906 (Novartis), Vinflunine (Bristol- Myers Squibb Company), actinomycin D, bleomycin, mitomycin C, neocarzinostatin (Zinostatin), vinblastine, vincristine, vindesine, vinorelbine (Navelbine), docetaxel (Taxotere), Ortataxel, paclitaxel (including Taxoprexin a DHA/paciltaxel conjugate), cisplatin, carboplatin, Nedaplatin, oxaliplatin (Eloxatin), Satraplatin, Camptosar, capecitabine (Xeloda), oxaliplatin (Eloxatin), Taxotere alitretinoin, Canfosfamide (Te
  • antineoplastic agents for use in combination therapy include, but are not limited to, as Advexin (ING 201 ), TNFerade (GeneVec, a compound which express TNFalpha in response to radiotherapy), RB94 (Baylor College of Medicine), Genasense (Oblimersen, Genta), Combretastatin A4P (CA4P), Oxi-4503, AVE-8062, ZD-6126, TZT-1027, Atorvastatin (Lipitor, Pfizer Inc.), Pravastatin (Pravachol, Bristol-Myers Squibb), Lovastatin (Mevacor, Merck Inc.), Simvastatin (Zocor, Merck Inc.), Fluvastatin (Lescol, Novartis), Cerivastatin (Baycol, Bayer), Rosuvastatin (Crestor, AstraZeneca), Lovostatin, Niacin (Advicor, Kos Pharmaceuticals), Caduet, Lipitor, to
  • anti-cancer agents include those typically used in adjuvant chemotherapy, such as FOLFOX, a combination of 5-fluorouracil (5-FU) or capecitabine (Xeloda), leucovorin and oxaliplatin (Eloxatin). Further examples of particular anti-cancer agents include those typically used in chemotherapy for metastatic disease, such as FOLFOX or FOLFOX in combination with bevacizumab (Avastin); and FOLFIRI, a combination of 5-FU or capecitabine, leucovorin and irinotecan (Camptosar).
  • FOLFOX a combination of 5-fluorouracil
  • Xeloda capecitabine
  • Eloxatin oxaliplatin
  • anti-cancer agents include those typically used in chemotherapy for metastatic disease, such as FOLFOX or FOLFOX in combination with bevacizumab (Avastin); and FOLFIRI, a combination of 5-FU or capecitabine, leu
  • Further examples include 17-DMAG, ABX-EFR, AMG-706, AMT-2003, ANX-510 (CoFactor), aplidine (plitidepsin, Aplidin), Aroplatin, axitinib (AG-13736), AZD-0530, AZD-2171 , bacillus Calmette-Guerin (BCG), bevacizumab (Avastin), BIO-1 17, BIO-145, BMS-184476, BMS-275183, BMS-528664, bortezomib (Velcade), C-131 1 (Symadex), cantuzumab mertansine, capecitabine (Xeloda), cetuximab (Erbitux), clofarabine (Clofarex), CMD-193, combretastatin, Cotara, CT-2106, CV-247, decitabine (Dacogen), E-7070, E-7820, edotecarin
  • Additional anti-cancer agents include axitinib (AG 13736), capecitabine (Xeloda), interferon alpha, interleukin-2, bevacizumab (Avastin), gemcitabine (Gemzar), thalidomide, cetuximab (Erbitux), vatalanib (PTK-787), sunitinib (SutentTM), AG-13736, SU-1 1248, Tarceva, Iressa, Lapatinib and Gleevec, wherein the amounts of the active agent together with the amounts of the combination anticancer agents is effective in treating renal cell carcinoma.
  • RNA-seq data was obtained from the TCGA breast cancer study and applied a suite of in-house algorithms, collectively called TopNotch, that are based on a local, de novo transcript assembly approach.
  • TopNotch This analysis identified six additional candidate alterations in five tumors predicted to activate the Notch pathway.
  • Four alterations disrupted the PEST domains in NOTCH1, NOTCH2 or NOTCH3 (Tables 1 -3, Fig. 1 ).
  • Tables 1 -3 show somatic NOTCH1, NOTCH2 and NOTCH3 mutations and alterations in the HD and PEST domains identified in the TCGA invasive breast carcinoma cohort.
  • Hes4/Hey2 up-regulation is defined as "yes" when either gene is expressed at 2-fold or more above median expression across the cohort.
  • the PEST domain breakpoints identified from this analysis were further validated in the whole exome sequencing (WXS) data from the same patients, including the matched normal samples to confirm their somatic status (Figs. 4 and 5).
  • This analysis provided evidence for the three PEST domain alterations identified in the TCGA breast cancer dataset, i.e., TCGA-A2-A0T0, TCGA-A8-A08X, and TCGA-A8-A0J6.
  • realignment of RNA-seq reads to either the wildtype sequences or the variant sequences (de novo assembled from the RNA-seq data) 45bp flanking the predicted cDNA breakpoint.
  • RNA-seq data from the matched normal samples of these patients were not available from TCGA. 45bp and 95bp flanking sequences were used for RNA-seq data and WXS data, respectively, due to the fact that the read length of the RNA-seq data is 50bp, and 100bp for the WXS data.
  • Structural rearrangement events for the variant transcripts include the deletion of NOTCH1 exon 21 -27 in TCGA- BH-A1 FC (a TNBC), a 168-bp deletion in NOTCH1 exon 34 of TCGA-A2-A0T0 (a TNBC), an inter-chromosomal translocation involving NOTCH1 exon 34 in TCGA-A8- A08X (a ER-/HER2+ breast cancer), a 62bp deletion in NOTCH2 exon 34 of TCGA-AO- A0J6 (a TNBC), and lastly two events in TCGA-AN-AOAR (a TNBC) including a fusion between exon 1 of BRD4 and exon 26 of NOTCH3, as well as a 1326bp deletion in NOTCH3 exon 33.
  • TNBC TCGA-AN-AOAR
  • RNA-seq reads from the tumor sample were realigned to either the wildtype sequences or the variant sequences (de novo assembled from the RNA-seq data) 45bp flanking the predicted cDNA breakpoint. Results showed that both the wildtype and variant transcript were well supported in either direction.
  • Exon-by-exon copy number ratio for NOTCH1 was derived from the whole exome sequencing (WXS) data available for both the tumor and the matched normal samples from this patient. Data were noisy as the WXS coverage was highly uneven, but a one copy loss appears to have occurred around exon 21 -27, supporting a genomic deletion event rather than an alternative splicing event at the transcript level. The exact genomic breakpoints are likely in the introns thus cannot be determined from the RNA-seq data and the WXS data. The resulting protein was predicted to be missing the S1 and S2 cleavage sites, but with an intact S3 site, and therefore may be sensitive to GSI.
  • WXS whole exome sequencing
  • RNA-seq reads that support either the novel breakpoint identified in exon 33 of NOTCH3, or the wildtype sequence at the breakpoint position were counted by realigning the RNA-seq reads to either the wildtype sequences or the variant sequences (de novo assembled from the RNA-seq data) 45bp flanking the predicted cDNA breakpoint.
  • the exact fusion breakpoints are likely in the introns and thus cannot be determined from the RNA-seq data and the WXS data.
  • the 5' fusion was predicted to produce an in-frame protein utilizing the translation start codon at residue M1583 of the canonical Notch3, which is in between the S1 and S2 cleavage sites, leaving the S3 GS cleavage site intact.
  • the 1326bp deletion in NOTCH3 exon 33 is predicted to truncate the PEST domain at residue P2607.
  • NOTCH3 The mutation and expression pattern is most striking for NOTCH3 where four of seven mutated tumor samples exhibit copy number gain, two of which are focal, and all have either 97 th or 99 th percentile expression of NOTCH3. In contrast to NOTCH1, NOTCH2, and NOTCH3, no compelling mutations or amplifications were found in NOTCH4.
  • 43 breast cancers were identified in the TCGA cohort that carry either somatic mutations in a hotspot domain (ECD/HD or PEST) of NOTCH1-3, or somatic focal amplification of NOTCH2 or NOTCH3 with concomitant increase in receptor expression (collectively referred to as "Notch altered” hereafter).
  • FIG. 2 provides an expression heatmap of Notch pathway genes in 21 Notch altered TN tumors compared to 50 non-altered TN tumors. Un-scaled expression was shown to the right of the heatmap where the circle indicates median expression and the line indicates the range of expression. The dotted red line indicates background expression level.
  • Notch pathway and target genes including NOTCH3, HES1, HEY2, MYC, CCND1, HES4, NRARP and NOTCH1, exhibited significant over-expression (false discovery rate ⁇ 0.05) in the Notch altered tumors, and were among the top 4% most up-regulated genes in Notch altered TNBCs when all genes were queried.
  • the strong up-regulation of the Notch pathway target genes MYC and CCND1 in the Notch altered breast tumors may provide insight into the mechanism whereby Notch activation may lead to an oncogenic phenotype, as well as how these classical breast cancer genes are up-regulated in this subset of breast tumors (see Arnold A, et al.
  • Notch target genes HES4 and HEY2 were differentially expressed between the Notch altered and wild type TN tumors, over-expression of one or both of these target genes was used as an indicator of Notch pathway activation (Fig. 6). Of the 21 TNBCs with Notch alterations of interest, 17 showed evidence of Notch pathway activation. Outside TNBC, however, only 8 of the 22 Notch altered tumors showed evidence of pathway activation (Fig. 3). This suggests Notch alterations are more likely to be functionally relevant in the TNBC subtype, and an estimated 13% of TNBCs fall into this newly identified Notch altered oncogenic driver class.
  • PDX patient-derived xenograft
  • this model also harbored a focal amplification of the NOTCH2 locus (Fig. 7).
  • the AA1077 model harbored a transcript that fused part of the last exon of NOTCH1 with intronic sequence from NOTCH1 between exons 30 and 31 and disrupted the PEST domain at amino acid 2249.
  • WGS data from this model we confirmed that this was due to a tandem duplication between intron 30 and exon 34 of NOTCH1 (Fig. 7).
  • the HBCx-14 PDX model harbored a heterozygous 10 base pair (frameshift) deletion in the PEST domain (disrupting the coding sequence beginning at amino acid 2462) and a homozygous ECD deletion (Fig. 9). While the ECD was deleted, the GS site was still present in the mutated protein. All three models were highly sensitive to PF-03084014, including over 50% tumor regression in the MAXF1 162 model, which was similar to the NOTCH1 ECD deleted HCC1599 cell line xenograft model previously known to be sensitive to GSI (see Zhang CC, et al. Clin Cancer Res (2012) 18(18):5008-19; (Zhang CC, et al. Stem Cels Trans Med (2013) 2(3) ) (Fig. 1 1 ). The MAXF1 162 model was characterized as a HER2 amplified model whereas the other models were triple negative (TN).
  • RNAseq or whole genome sequencing data were analyzed from five PDX models (HBCx-9 (TN), HBCx-5 (HER2+), HBCx-17 (TN), HBCx-12B (TN), and AA0869 (TN)) and seven cell line xenograft models (HCC1937 (TN), HCC1806 (TN), SK-BR-3 (ER lost), MDA-MB-436 (TN), MCF-7 (ER+), BT-474 (HER2+), and MDA-MB- 231 (TN)) which span multiple breast cancer subtypes.
  • No activating Notch hotspot domain alterations were found in these models and responses ranged from less than 50% tumor growth inhibition in seven out of twelve models to slight tumor regression in the AA0869 model.
  • Notch mutant models exhibited 60% or greater tumor growth inhibition including 50% tumor regression in two of the four models (Fig. 1 1 ).
  • the MAXF1 162 model exhibited 65% tumor regression
  • AA1077 model exhibited 88% tumor growth inhibition
  • the HBCx-14 model exhibited 60% tumor growth inhibition
  • the HCC1599 model exhibited 50% tumor regression. All models were statistically significant by the student's t-test (p-value ⁇ 0.05).
  • Notch mutations may serve as a useful biomarker to enrich for patients most likely to respond to treatment with a GSI.
  • mutations in PDX models were identified that are representative of the TCGA mutations in terms of location, predicted functional consequence, spectrum of molecular mechanisms and co-incidence of multiple events. These data therefore confirm and extend the findings from the TCGA analysis and importantly, demonstrate that relevant pre-clinical models harboring Notch alterations are sensitive to the GSI PF-03084014.
  • the functional consequences of the NOTCH1 mutations on NICD1 were explored. Mutations that removed the ECD domain were predicted to result in ligand independent activation of the receptor. NOTCH1 mutations were shown to alter full length and NICD1 protein and NICD1 half-life.
  • Figure 12 shows NOTCH1 mutations alter full length and NICD1 protein and NICD1 half-life.
  • Fig. 12(a) shows the NICD1 Western blot in Notch altered models treated with or without PF-03084014, where " * " indicates NICD1 species of lower molecular weight than wt NICD1 .
  • HPB-ALL is a T-ALL model that is known to generate a NICD1 species of lower molecular weight due to a PEST domain mutation.
  • NICD1 Western blot on the NOTCH1 PEST truncated HBCx-14 model (lane 6) alongside a panel of lysates from TNBC PDX models from the same collection.
  • the HCC1599 cell line xenograft model +/- PF-03084014 for 2 days at 100 mg/kg BID was included as controls.
  • lysate 50 ug lysate was loaded for all lysates except 25 ug for the HCC1599 lysates.
  • the Red arrow indicates the lower molecular weight NICD1 species in the HBCx-14 model.
  • Fig. 12(c) shows the NOTCH1 Western blot using an antibody that recognizes the NOTCH 1 transmembrane and full length species. The lower panel is at lighter exposure.
  • the Red arrows indicate the lower molecular weight species in the HBCx-14 model and the absence of detectable full length protein in either the HBCx-14 model or the HCC1599 model.
  • the same amount of lysate was loaded in a separate gel as in Fig. 12(b) on the same day, and therefore no additional loading control was included for this gel.
  • Notch receptors Activating alterations in Notch receptors were predicted to up-regulate direct transcriptional targets of the pathway including the Hes and Hey family transcription factors. Indeed strong over-expression of HES4, HEY2, HEY1 and/or HEYL was observed in three of the four Notch mutant models compared to xenograft models without Notch alterations.
  • the MAXF1 162 and HBCx-14 models both harbored multiple genetic events in the same Notch receptor and in both cases the mutated receptor itself exhibited the highest mRNA expression among the panel of the xenograft screened (Fig 14). Models are rank-ordered from left to right using a HES4, HEY2 two gene signature score across an in vivo panel (Fig. 14(a)) and an external PDX panel (Fig. 14(b)). Nanostring digital gene expression data were used for the in vivo model panel, and Affymetrix microarray gene expression data were used for the PDX model panel,.
  • PF-03084014 Treatment with PF-03084014 reduced the expression of nearly all target genes in models that exhibited strong over-expression at baseline, demonstrating Notch alterations drive the Notch pathway in these models and that PF-03084014 can effectively repress this hyperactivated transcriptional program (Fig. 15).
  • Highly expressed Notch target genes were down-regulated by PF-03084014 treatment in Notch mutant models.
  • genes that exhibited outlier expression at baseline were down regulated by PF-03084014 treatment.
  • Relative expression was normalized to vehicle which was set at 1 . Two to three tumors per group were analyzed and data is presented in Fig. 15(a)-(d) as average and SEM.
  • HCC1599 and HPB-ALL cells were grown in RPMI-1640 medium and
  • HEPES buffer sodium pyruvate, non-essential amino acids, Pen- Strep, ITS, and glutamine.
  • Tumor-bearing athymic nude mice were dosed twice daily at 140 mg/kg on a 12 day on, 4 day off schedule for 2 cycles.
  • the MAXF1 162 model was run at Oncotest and all experiments were approved by the local authorities and were conducted according to all applicable international, national and local laws and guidelines.
  • Tumor-bearing nude mice were dosed twice daily at 140 mg/kg on a 10 day on 4 day off schedule for 3 cycles.
  • the AA1077 patient-derived and HCC1599 cell line xenograft models were run at Pfizer in SCID-Bg mice.
  • mice were dosed at 1 10 mgs/kg twice daily for 9 days (AA1077) or at 120 mgs/kg twice daily for 12 days (HCC1599).
  • mice with palpable tumor sizes were randomly assigned to different groups, and the mean value of the tumor size was matched between the groups. Differences between the vehicle and PF-03084014-treated groups were statistically significant by the student's t-test. Percent tumor regression was calculated using the following formula 100 * (1 - (Treated final volume/Treated initial volume)) and percent tumor growth inhibition was calculated using the following formula 100 * (1 - (Treated final volume - Treated initial volume)/(Vehicle final volume - Vehicle initial volume)).
  • tumor-bearing mice received 100 - 140 mg/kg PF-03084014 twice daily for two days before terminal collection. Some pharmacodynamic groups received one dose on the second day. The tumors were harvested 4-6 hours after the last dose, snap-frozen and pulverized in a liquid nitrogen- cooled mortar prior to analysis.
  • Genomic DNA and total RNA were prepared from cell pellet or frozen tumor tissue with Qiagen DNeasy Blood and Tissue Kit (Cat. #69504) and Qiagen RNeasy Mini Kit (Cat. 74104), following manufacture's protocol. Junction PCR was then performed to verify the break point of genomic DNA. Primer sequence for each particular sample is listed in the supplementary material.
  • Total RNA were subjected to direct Quantitative RT-PCR (see below), or treated with Calf Intestinal Phosphatase (CIP) and Tobacco Acidic Pyrophosphatase (TAP) and then reverse transcribed to amplify the 5' end messenger RNA sequence of NOTCH1 in HCC1599. CIP and TAP were included in FirstChoice® RLM-RACE Kit from Life Technologies (Cat. # AM1700). Transcriptomic sequencing (RNA-seq) of PDX models
  • RNA-seq was performed on PDX models with 100 bp paired-end reads.
  • Raw RNA-seq reads were filtered using Xenome to remove potential reads from contaminating mouse cells.
  • Non-mouse reads were then aligned to human reference genome using TopHat2. 226.2, 253.9 and 298.3 million mapped reads were generated for HBCx14, AA1077 and MAXF1 162, respectively.
  • Tier-2 mutation, somatic copy number and mRNA expression data (RNA Seq V2 RSEM) from the TCGA invasive breast carcinoma cohort were obtained from the TCGA data portal and Memorial Sloan-Kettering Cancer Center's cBio portal (35).
  • Raw Affymetrix SNP 6.0 array data were also downloaded from the TCGA data portal.
  • Pre- aligned RNA-seq data (in BAM format) were downloaded via The Cancer Genomics Hub (36), dbGaP accession number PHS000178, version phs000178.v8.p7.
  • a total of 956 tumors with complete mutation, copy number and gene expression data were analyzed.
  • Gene expression profiles of the external PDX panel were generated using Affymetrix U133Plus2 arrays.
  • CEL files were provided by the vendor (Xentech).
  • Raw intensity data were processed by GC Robust Multi-array Average (GCRMA) background adjustment, quantile normalization, and median-polish summarization to generate the probe-level data in R. Normalized probe level data were further summarized into gene- level using the GSEA CollapseDataset function.
  • GCRMA GC Robust Multi-array Average
  • Notch pathway genes in the TCGA breast cancer cohort were performed on the RNA-seq based Transcript per Million metric from the RSEM results provided in the TCGA tier-3 data.
  • 12 were selected as Notch altered that include patients with simple mutations or complex alterations in the HD or PEST domain of NOTCH1, NOTCH2 or NOTCH3, and those with focal amplification of NOTCH2 or NOTCH3 (inferred copy number >4).
  • the Notch non-altered group included 41 TNBC tumors with no mutation, nor alteration, nor amplification (inferred copy number ⁇ 2.5) in all Notch receptors.
  • Nanostring technology was used to measure the RNA transcript levels using the nCounter assay according to manufacturer's recommended protocols. Briefly, transcript specific capture and detection probes were designed and manufactured by the Nanostring Technologies and 100 ng of total RNA was hybridized to nCounter probe sets for 16 hours at 65°C. Samples were processed using an automated nCounter Sample Prep Station (NanoString Technologies, Inc., Seattle, WA). Cartridges containing immobilized and aligned reporter complex were subsequently imaged and counted on an nCounter Digital Analyzer (NanoString Technologies, Inc.) set at 1 155 fields of view. Reporter counts were analyzed and normalized using NanoString's nSolver analysis software version 1 .
  • Glyceraldehyde-3 phosphate dehydrogenase (GAPDH) and Peptidylprolyl isomerase E (PPIE) yielded similar results.
  • gDNA from PDX models tumors were chopped into small pieces using a razor blade in a small volume of Accumax (Innovative Cell Technologies, AM105). 20X volume of Accumax was then added and incubated for 30 minutes with shaking at room temperature. The suspension was then passed through a 40 micron cell strainer and the strainer was washed 3X with media. To deplete the mouse cells and debris, cells were incubated with a biotin labeled anti mouse MHC class I antibody and then incubated with anti-biotin MicroBeads (Miltenyi, 130-090-485). Resuspended cells were then subjected to magnetic bead separation and DNA was purified using DNeasy kit (Qiagen).
  • SNP 6.0 arrays were processed using the aroma. affymetrix package in R. Normalized probe intensities in tumor samples were computed relative to a baseline profile of the average of the 128 females from the International HapMap Project. The log2 copy number ratios at all SNP loci were then plotted along each chromosome using the ChromosomeExplorer function from the aroma package to visually inspect the focality of the copy number alteration calls.
  • TopNotch consists of the following three components: ExonFusionFinder, HybridFinder and ExonBreakFinder.
  • ExonFusionFinder is designed to search for fusion or alternative splicing events that join 2 non-adjacent exons, either within the same gene or among different genes.
  • the algorithm takes the aligned RNA-seq reads (e.g. the BAM file) and coordinates of exons of interest as input, and identifies fragments that at least partially overlap both exons for every possible exon pair. These fragments can come from the two ends of a read pair where each end is aligned to a different exon, or a single read that aligns across two exons.
  • neighboring exon pairs can be up to second or third neighbor depending on the exon size, cDNA fragment length and read length) receive the most support.
  • Non-canonical exon-exon junctions in the transcript could also be revealed that may result from intra-gene deletion or alternative splicing.
  • To support such non-canonical exon-exon joining events we require that there must be at least 5 supporting fragments, and that the distance between the two joined exons must be greater than 750bp.
  • the second and third components of the TopNotch suite are both based on local, de novo transcript assembly.
  • gene of interest e.g. NOTCH1, NOTCH2 or NOTCH3 in this study
  • Such reads were then supplied to the Trinity algorithm to assemble into candidate de novo transcript(s) in the tumor sample.
  • HybridFinder module of the TopNotch suite aims to look for hybrid transcripts that encompass both the (partial) candidate gene and any other region in the genome outside the candidate gene. It does so by BLAT search of each de novo transcript in the reference human genome for hits that are at least 50bp long with at least 99% sequence identity. We further required that at least one hit must be within the candidate gene itself and that the total number of chromosomes from all qualifying hits must be less than 5 (to eliminate spurious transcripts that align to many unplaced or random contigs in the reference genome). For de novo transcripts that satisfy these criteria, we then exclude all hits that overlap the candidate gene itself or its family members and close homologs (in this study these include all Notch family receptors and NOTCH2NL).
  • Any de novo transcripts with remaining hits are further scrutinized for the quality of de novo assembly, alignment of the de novo transcript to the reference genome (to determine the actual breakpoints), and reads supporting the predicted breakpoints by re-aligning the original RNA-seq reads (50bp in length) to the 90bp sequences flanking the predicted breakpoint in both the wild-type and mutant transcripts.
  • ExonBreakFinder is specifically designed to search for any breakpoint in the de novo assembled transcript(s) that fall in known "hotspots".
  • This work we focused on the last exon of NOTCH1 -3, which encodes the PEST domain of the respective Notch receptors that are responsible for NICD protein degradation.
  • each candidate transcript was aligned to the reference human genome using BLAT and retained only those: 1 ) that produced separate hits with at least 99% sequence identity to non- overlapping segments on the query transcript (i.e. the de novo Notch transcript), and 2) whose breakpoint within the last Notch exon is not the end of the de novo transcript.
  • Candidate de novo transcripts that pass both criteria were further scrutinized for the quality of the de novo assembly, alignment of the de novo transcript to the reference genome (to determine the actual breakpoints), and reads supporting the predicated breakpoints by re-aligning the original RNA-seq reads (50bp in length) to the 90bp sequences flanking the predicted breakpoint in both the wild-type and mutant transcripts.
  • SV Structural variant
  • Candidate SV calls by CREST were filtered out if they met any of the following criteria: 1 ) if any of the breakpoints coincided with a known germ line event, as describe in the Database of Genomic Variants (7), 2) if any of the breakpoints were located within 1 kb of a known assembly gap region within the reference genome, 3) if both breakpoints fell within a repeat region, as described by repeat masker (A.F.A. Smit, R. Hubley & P.
  • RNA-seq reads from any fragment that partially overlaps the Notch gene locus to both the wildtype and mutant sequences surrounding the predicted breakpoint (45bp on each side of the breakpoint since the RNA-seq read length is 50bp), and counted the number reads that support either the wildtype or the mutant sequence. This confirmed the existence of all breakpoints in the RNA-seq data and also provided an estimate of mutant allele prevalence.
  • the alignment was performed using BLAST. Only alignments that were equal to the read length and had less than two mismatches were counted. In case a read was aligned to both the wildtype and mutant sequences, the alignment with higher score (the bit score in the BLAST output) was taken.
  • RNA-seq or WXS data from the matched normal patient samples we also examined the RNA-seq or WXS data from the matched normal patient samples. For the exon 21 -27 deletion in TCGA-BH-A1 FC, for which the genomic breakpoints are likely in the introns, we ran TopNotch also in the RNA-seq data from its matched normal sample and confirmed that only wildtype NOTCH1 transcript was present in the normal sample. For the other three PEST domain variants, RNA-seq data from the matched normal samples were not available.
  • Stepwise junction PCR protocol is as following:
  • NOTCH2/Hs01050702_m1 NOTCH 3/ Hs01 128541 _m1 , NOTCH4/ Hs00965889_m1 , HES1/Hs00172878_m1 , HES4/Hs00368353_g1 , HEY2/ Hs00232622_m1 ,
  • Notchi cDNA (SEQ ID NO: 15) comprises the DNA sequence of the NCBI reference sequence: NM_017617. Note this includes exons only from the start codon to the stop codon.
  • the Notchi protein sequence (SEQ ID NO: 16) comprises 2555 amino acids, having NCBI Reference Sequence: NP_060087.3. The Notchl PEST domain is indicated in bold in Table 5.
  • Notch2 cDNA (SEQ ID NO: 17) comprises the DNA sequence of the NCBI reference sequence: NM_024408. Note this includes exons only from the start codon to the stop codon.
  • the Notch2 protein sequence (SEQ ID NO: 18) comprises 2471 amino acids, having NCBI Reference Sequence: NP_077719.2.
  • the Notch2 PEST domain is indicated in bold in Table 5.
  • Notch3 cDNA (SEQ ID NO: 19) comprises the DNA sequence of the NCBI reference sequence: NM_000435. Note this includes exons only from the start codon to the stop codon.
  • the Notch3 protein sequence (SEQ ID NO:20) comprises 2321 amino acids, having NCBI Reference Sequence: NP_000426.2.
  • the Notch3 PEST domain is indicated in bold in Table 5.
  • NOTCH1 15 ATGCCGCCGCTCCTGGCGCCCCCCTGCTCTGCCTGGCG cDNA CTGCTGCCCGCGCTCGCCGCACGAGGCCCGCGATGC
  • Notch2 17 ATGCCCGCCCTGCGCCCCGCTCTGCTGTGGGCGCTG cDNA CTGGCGCTCTGGCTGTGCTGCGCGGCCCCCGCGCAT
  • Notch2 18 MPALRPALLWALLALWLCCAAPAHALQCRDGYEPCVNE protein GMCVTYHNGTGYCKCPEGFLGEYCQHRDPCEK
  • Notch3 19 ATGGGGCCGGGGGCCCGTGGCCGCCGCCGCCGCCG cDNA TCGCCCGATGTCGCCGCCACCGCCACCGCCACCCGT

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Abstract

L'invention concerne des méthodes d'identification et de traitement de patients atteints de cancer et présentant des altérations d'activation dans le domaine PEST de Notch1, Notch2 ou Notch3, et dont on estime qu'ils tireront avantage d'un traitement avec un inhibiteur de la voie de signalisation Notch, tel que l'inhibiteur de la gamma-sécrétase, PF-03084014, ou son sel pharmaceutiquement acceptable.
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WO2018029254A1 (fr) 2016-08-09 2018-02-15 Medizinische Universität Wien Génotypage et traitement du cancer, en particulier de la leucémie lymphocytaire chronique
WO2020043736A1 (fr) * 2018-08-27 2020-03-05 Universiteit Maastricht Inhibiteurs sélectifs de la gamma-sécrétase préséniline-2
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