WO2018102736A1 - Methods for the treatment of cancer - Google Patents

Methods for the treatment of cancer Download PDF

Info

Publication number
WO2018102736A1
WO2018102736A1 PCT/US2017/064295 US2017064295W WO2018102736A1 WO 2018102736 A1 WO2018102736 A1 WO 2018102736A1 US 2017064295 W US2017064295 W US 2017064295W WO 2018102736 A1 WO2018102736 A1 WO 2018102736A1
Authority
WO
WIPO (PCT)
Prior art keywords
kdm5a
dna
inhibitor
subject
kdm4a
Prior art date
Application number
PCT/US2017/064295
Other languages
French (fr)
Inventor
Johnathan WHETSTINE
Original Assignee
The General Hospital Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The General Hospital Corporation filed Critical The General Hospital Corporation
Publication of WO2018102736A1 publication Critical patent/WO2018102736A1/en

Links

Classifications

    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • 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
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase

Definitions

  • the present invention relates, at least in part, to methods for controlling DNA amplification, e.g., in cancer cells.
  • Copy number variation is commonly observed during normal development and/or in response to cellular or environmental cues (Mishra and Whetstine, 2016). For example, specific chromosomal regions are amplified in the salivary gland of sciarid flies, which in turn, helps in synthesizing structural proteins during cocoon formation. In Drosophila, the chorion genes are selectively amplified in S phase in the follicle cells that directly impacts egg shell formation (Calvi et al., 1998; Calvi and Spradling, 1999). Similarly, site-specific rereplication and copy gains also have been observed under hypoxic conditions in primary T-cells, cancer cells and tumors (Black et al., 2015).
  • Cancer cells typically have multiple chromosomal aberrations.
  • Genome-wide studies of somatic copy number alterations (SCNAs) across multiple tumor types have demonstrated that specific chromosomal regions exhibit higher frequencies of gains/amplifications, with a concomitant increase in the associated gene expression (Beroukhim et al., 2010; Kim et al., 2013; Zack et al., 2013). These regions frequently harbor pro-survival genes and oncogenes.
  • SCNAs somatic copy number alterations
  • amplification/gain of chromosome lql2-25 are associated with a number of drug resistance-associated oncogenes [e.g, MCLl, CKSIB; (Beroukhim et al., 2010; Dimova et al., 2009; Diskin et al., 2009; Fonseca et al., 2006; Goeze et al., 2002; Inoue et al., 2004; Kendall et al., 2007; Petersen et al., 2000; Weir et al., 2007)].
  • MCLl, CKSIB drug resistance-associated oncogenes
  • DHFR dihydrofolate reductase
  • EGFR epidermal growth factor receptor
  • TSSGs Transient site-specific copy number gains
  • H3K9/36 histone H3 lysine 9/36
  • the present study identified a compendium of H3K4 modifying chromatin regulators that function with H3K9 and H3K36 regulators to orchestrate TSSG formation.
  • H3K4 tri-demethylase KDM5A and specific COMPASS/KMT2 H3K4 methyltransferases modulate specific TSSG loci through H3K4 methylation states, and in turn, KDM4A recruitment.
  • MLL1-KDM4B-KDM5B another network of chromatin modifiers, modulate copy number regulation at a unique genomic locus in a KDM4A-independent manner.
  • This epigenetic addressing system defines site-specific DNA rereplication and amplifications through regulation of lysine methylation balance by histone KMTs and KDMs.
  • the methods include identifying a subject who has a cancer with a KDM5A gene suppression/mutation/deletion.
  • the methods include identifying a subject who has an amplification at lql2h, lql2/21, lq21.2, Iq21.3/CKS1B, or Xql3.1.
  • the methods include identifying a subject who has a cancer with a KDM5B gene suppression/mutation/deletion.
  • the methods include identifying a subject who has an amplification at lp32.3.
  • the methods include administering a cdk4/6 inhibitor to a subject who has an amplification at lp32.3.
  • kits for treating a subject who has a cancer with increased levels or activity of a KMT2 family member by administering to the subject a therapeutically effective amount of an inhibitor of KDM4.
  • the inhibitor of KDM4 inhibits KDM4A, KDM4B, or both KDM4A and KDM4B.
  • the KMT2 family member is KMT2A, KMT2B, KMT2C, KMT2D, SETD1A or SETD1B.
  • the subject has acute myeloid leukemia (AML), the acute lymphoblastic leukemia (ALL), or biphenotypic (mixed lineage) leukemia.
  • the cancer is a carcinoma, sarcoma, myeloma, metastatic disorder, or a hematopoietic neoplastic disorder.
  • FIG. 1 Schematic of siRNA screen in RPE cells against KDM1 through KDM7 family members with two independent siRNAs. DNA FISH was performed post 72hr of knockdown.
  • B Representative DNA FISH images for the indicated KDM5 family siRNAs for chromosome lql2h (green), 8c (red) and merged with DAPI (blue) are shown.
  • C Quantification of FISH in KDM5 siRNA treated RPE for chromosome lql2h and chromosome 8 centromere.
  • D KDM5 A knockdown results in site- specific copy gains of lql2h region in H2591 lung cancer cells.
  • E RPE cells treated with different concentrations of KDM5 inhibitor (KDM5 -C70) for 72hr results in dose dependent increase of copy gains of lql2h.
  • F Neuroblastoma cell line, SK-N- AS treated with different concentrations of KDM5 inhibitor (KDM5 -C70) for 48hr results in dose dependent increase of copy gains of lql2h.
  • G KDM5 A knockdown results in site-specific copy gains of additional regions including lq21.2 and Xql3.1 with no changes in lq23.3, lqTel and Xcen demonstrating the site-specific nature of copy gains rather than global genome instability.
  • ChIP analysis shows significant decrease in the occupancy of KDM5A at copy gained regions upon siRNA depletion (i.e., chrlsat2 and ⁇ q ⁇ 2/2 ⁇ at multiple sites; ⁇ q ⁇ 2/2 ⁇ - ⁇ (chrl : 142,704,000) and lql2/21-2 (chrl : 142,706,000)].
  • Error bars represent the SEM. Any significant differences (P ⁇ 0.05) are indicated with an asterisk by two-tailed Student's t-test. Scale bar corresponds to 5 ⁇ .
  • KDM5A-associated site-specific copy gains are transient, require S phase and are rereplicated.
  • A A schematic of KDM5 inhibitor treatment conditions are shown. Cells were treated with KDM5-C70 for 48hr, followed by drug wash-off with fresh media (24hr wash off). Cells were collected 24h post drug removal.
  • B-C Wash off with fresh media of KDM5i results in significantly diminished copy gains while copy gains are present at 48hr (B) and 72hr (C) post drug treatment.
  • D A schematic representing drug treatment conditions are shown. Cells were treated with hydroxyurea (HU) for 20hr, followed by KDM5 inhibitor (KDM5i) for 24h. HU was replaced with fresh media and cells were harvested for further analyses after 6hr of release.
  • the graph represents heavy-heavy (H:H), heavy-light (H:L) and light-light (L:L) peaks of the fractionated gradients.
  • H Rereplicated pool of heavy-heavy DNA (H:H) was isolated and analyzed by qPCR to determine fold change in rereplication by normalizing to its input DNA. A significant increase was observed at the copy gained regions.
  • FIGS 3A-F KDM5A depletion promotes H3K4me3 and KDM4A recruitment at copy gained loci.
  • KDM4A and KDM5A were co-depleted with siRNAs for 72hr followed by DNA FISH for chrlql2h, chrlq21.2 and chrlq23.3 in RPE cells. Significant rescue of the copy-gained regions was observed after KDM4 A depletion (lql2h and lq21.2), while no change was observed at the negative region, lq23.3.
  • B ChIP for KDM4A demonstrated significant recruitment of KDM4A at the copy-gained locus compared to a non-copy gained site (lq23.3).
  • C ChIP for H3K4me3 mark was performed in control and KDM5 A knockdown cells.
  • H3K4me3 histone 3 lysine 4 trimethylation mark
  • H3K4M histone H3.3 lysine 4 mutation to methionine
  • FIGS 4A-I Site-specific copy gains are H3K4 KMT-dependent.
  • FIG. 1 An ideogram of the chromosome lq arm shows a summary of the specific H3K4 KMTs that rescue copy gains caused by KDM5 A depletion and that are generated upon KMT overexpression.
  • the DNA FISH data summarized in panel A can be found in Figure S5.
  • C Model depicting interplay of KDM5 A-KDM4 A- SETD 1 B at the lql2h locus is shown.
  • D-E Depletion of KDM4A with siRNA followed by KMT2D and SETDIB overexpression abrogates lq21.2 copy gains.
  • KDM5- C70 treatment generates significant lp32.3 copy gains after 48hr. These copy gains returns to baseline after drug wash-off.
  • F Co-overexpression of KMT2A and KDM5B rescues the lp32.3 locus increase but not for lq21.3.
  • G Introduction of H3K4M followed by KDM5B siRNA depletion resulted in decreased copy number gains of lp32.3.
  • H Introduction of H3K4M causes decreased copy number gains at both lp32.3 and 1 q21.3 after KMT2A overexpression compared to KMT2A overexpression alone. Error bars represent the SEM. Any significant differences (P ⁇ 0.05) are indicated with an asterisk by two-tailed Student's t-test. Scale bar corresponds to 10 ⁇ .
  • KDM4B promotes site-specific copy gains and rereplication at lp32.3.
  • A Representative images from DNA FISH of lp32.3 and lq21.3 copy gains after KDM4 family overexpression are depicted. DAPI (in blue), lp32.3 (in green), 1 q21.3 (in red) and merged images are shown.
  • B The histone lysine demethylase 4 family was transiently overexpressed in RPE cells for 24h and assessed for lq21.3 and lp32.3 copy gains. Only KDM4B overexpression resulted in significant copy gains of chrlp32.3 loci.
  • C Copy gains at the lp32.3 locus are recapitulated in RPE cells stably expressing KDM4B.
  • FIG. 1 Schematic depicting genomic primers used for testing rereplicated regions in KDM4B stably expressing cells. The primers labeled as 1 through 6 encompass the regions covered under the FISH probe. Rereplicated DNA was isolated and analyzed by qPCR in control and GFP-KDM4B stable cells for six different lp32.3 genomic regions (marked in the ideogram as 1 through 6). The fold change in rereplication compared to control DNA is shown. A significant increase was observed at the indicated regions as represented by an asterisk.
  • HEK293 cells were co-transfected with NanoLuc-KDM4A and NanoLuc- KDM4B (energy donors) with the following energy acceptors; HaloTag-MCM3, HaloTag-MCM4, HaloTag-MCM5, HaloTag-MCM7, HaloTag-POLA2, HaloTag- POLD1, or HaloTag alone (negative control). Shown are the graphs of corrected BRET ratios obtained for each possible donor-acceptor combination.
  • H3K4M results in a decreased recruitment of KDM4B at the target histones H2B and H4 in vivo.
  • F ChIP for KDM4B recruitment was performed in control and GFP-KDM4B stably expressing cells at the indicated regions 1 ⁇ 32.3-3, 1 ⁇ 32.3-4, lp32.3-5 and lq23.3. The position ofthese loci are noted in Figure 6D.
  • G Quantification of FISH for lq21.2 and lp32.3 in H3.3 lysine 9 to methionine mutation (K9M) and H3.3 lysine 36 to methionine mutation (K36M) are shown.
  • A-D The rescue of KDM5 A depletion gains with co-depletion of H3K4 methyl transferase (KMT2A, KMT2B, KMT2C, KMT2D, SETDlA and SETD1B) at lql2h loci is shown (E-H) The rescue of KDM5 A depletion gains with co-depletion of H3K4 methyl transferase (KMT2A, KMT2B, KMT2C, KMT2D, SETD1 A and
  • SETD1B at lq21.2 loci is shown.
  • I-L The rescue of KDM5A depletion gains with co-depletion of H3K4 methyl transferase (KMT2A, KMT2B, KMT2C, KMT2D, SETDlA and SETD1B) at lp32.3 and lq21.3 locus is shown.
  • M-S FISH analysis on lql2h, chromosome 8 and lq21.2 on samples with KMT2A, KMT2B, KMT2D and SETD1B for transient overexpression for 24h was performed.
  • T-W FISH analysis was performed on lp32.3 and lq21.3 on samples with transient overexpressions of KMT2A, KMT2B, KMT2D, SETDlA and SETD1B for 24h.
  • qPCR and FACS analysis was performed to validate KMT2A and KDM5B overexpressions as shown in the graph.
  • Y western blot for H3K4me3 and histone H3 was performed in samples with control and KDM5B knockdown infected with H3.3 wild type and K4M mutations.
  • Z FACS was done in samples with control and KMT2A overexpression infected with H3.3 wild type and K4M mutations.
  • FIGS 9A-B KDM5 inhibitor generated copy gains are blocked selectively by specific KDM4 members.
  • KDM4 A depletion blocks KDm5 inhibitor generated lql2h, while not impacting the gains of a control region (8c).
  • DNA amplifications are observed in both normal and cancerous cells (Mishra and Whetstine, 2016). These chromosomal changes consist of gains or losses in whole chromosomes, segmental chromosomal arms, and site-specific changes (Mishra and Whetstine, 2016). However, the mechanisms regulating site-specific DNA amplifications are not well understood. In this study, we have identified a
  • H4K20mel methyltransferase SET-domain containing protein 8 promotes pre-replication complex assembly and origin activity (Tardat et al., 2010). Subsequently, Set8 stabilization results in increased H4K20me2/3 through the recruitment and increased function of the H4K20 lysine di/tri -methyl transferases Suv4-20hl/2, resulting in origin firing (Beck et al., 2012; Kuo et al., 2012; Tardat et al., 2010).
  • H3K4 methylation could promote rereplication (Huang et al., 2016; Lu et al., 2016b).
  • depletion of WDR5 which blocks most COMPASS family of H3K4 KMTs, reduced the rereplication and the polyploidy phenotype observed when replication licensing was disrupted (Lu et al., 2016b).
  • histone lysine 4 demethylases have also been suggested to regulate DNA replication. For instance, KDM5C/JARID1C regulates pre-initiation complex assembly and proper firing of the early replication origins (Rondinelli et al., 2015).
  • KDM5A was suggested to regulate ORC recruitment to origin sites and thus regulate appropriate replication (Huang et al., 2016). Instead of causing global genomic instability, the present results suggest that COMPASS H3K4 KMT enzymes and the H3K4 tri-demethylases are controlling rereplication and DNA copy gains at specific loci. For example, the balance between H3K4 KMTs and KDMs "toggles" H3K4 methylation at specific loci, which then regulates their ability to rereplicate and amplify.
  • each KDM5 H3K4 tri-demethylase controls a distinct subset of TSSG sites; KDM5A controls chrlql2h, lq21.2 and 1 q21.3 while KDM5B is involved in the regulation of lp32.3 locus ( Figure 7 J).
  • each COMPASS family H3K4 methyltransferase regulates distinct genomic sites within the subset of known TSSGs being generated ( Figure 7J).
  • interference with H3K4 methylation by introducing H3K4M was able to suppress all the observed TSSGs.
  • suppression of the KDM5 family activity through chemical inhibition allowed all the observed TSSGs to emerge.
  • H3K4M and KDM5 inhibitor experiments emphasize that H3K4 methylation is a critical determinant of rereplication and that there is an extraordinarily specificity in KMTs-KDMs in controlling site-specific amplifications. This underscores the importance of regulating the local chromatin states so that DNA amplifications are prevented or allowed to occur.
  • the present observations also emphasize how distinct sets of DNA amplifications can actually be connected through a common mechanism instead of being a byproduct of random instability.
  • Described herein are methods to reverse or control the appearance of certain DNA copy gains since they are transiently generated in each cell cycle.
  • the ability to therapeutically control DNA amplifications has implications in the area of tumor heterogeneity and drug responses.
  • KDM4 and KDM5 cross-talk generates TSSGs
  • SETD1B, MLL1/KMT2A, MLL2/KMT2B and MLL4/KMT2D modulated subsets of loci that undergo rereplication and copy gains upon KDM5 A depletion and KDM4A overexpression and stabilization.
  • KDM4 targeting is not just limited to KDM4A-dependent TSSGs.
  • KDM4A-dependent TSSGs We have identified, for the first time, a non-KDM4A dependent copy number regulation of chromosome lp32.3 locus by a network of KDM5B-KDM4B-KMT2A hi stone modifying proteins.
  • KDM4B also required H3K4 methylation to generate the lp32.3 TSSG.
  • MLL1/KMT2A was required for the KDM4B copy gains, while KDM5B prevented KDM4B copy gains.
  • disruption of H3K4 methylation caused less KDM4B on the chromatin, illustrating the importance of this modification in recruiting another KDM4 member, and in turn, site-specific rereplication and DNA amplification.
  • KDM4A amplified TSSGs were generated in the presence of both H3K9M and H3K36M; however, the KDM4B copy gained locus was specific to H3K36M. It is possible that the amplifications of specific regions are distinctly controlled by K36 and/or K9 methylation states.
  • H3K36M is a known "onco-histone" associated with certain tumor types (Lu et al., 2016a; Papillon-Cavanagh et al., 2017). KDM5-related TSSG kinetics
  • KDM4A associated with and recruited replication machinery to sites that undergo rereplication and copy gain (Black et al., 2015; Black et al., 2013; Black and Whetstine, 2015)
  • KDM5A depletion would allow this modification to persist and promote KDM4A driven copy gains, whereas resetting the H3K4 methylation would block KDM4A overexpressed protein recruitment. Consistent with this model, we demonstrated that KDM4A was recruited to enriched H3K4me3 sites that rereplicate upon KDM5 A depletion.
  • KDM5 A depletion also impacts an unknown mechanism(s) promoting copy gain removal.
  • Future studies need to elucidate the pathways regulating the removal and regulation of TSSGs. Once the pathway(s) are identified, the relationship between KDM5 enzymes, these pathways and TSSG generation and removal can be explored.
  • this study documents a repertoire of chromatin regulators that can generate transient DNA copy gains. These events are not inherited and require S phase. Given different cellular division rates within a tissue/tumor, the differentially cycling cells could create significant heterogeneity as a consequence of rereplicated fragment generation. The expression/activity/mutation status of the identified KMTs and KDMs could impact the spatial and temporal regulation of the rereplicated fragments. Therefore, cells that are actively dividing and have the appropriate genetic background will be able to execute these alterations, which could provide a basis for DNA copy number heterogeneity within a tissue and/or tumor (Mishra and Whetstine, 2016).
  • the present data identifies an epigenetic addressing system for defining site- specific DNA rereplication and amplifications through modulating methylation states via histone KMT and KDM cross-talk.
  • Different KMTs and KDMs control DNA copy gain of different genomic regions ( Figure 7 J).
  • This work establishes that a network of epigenetic regulators can regulate DNA rereplication in a spatial and temporal fashion.
  • drugs targeting these enzyme pathways can be selectively used to inhibit the amplification of regions associated with cancer progression, resistance and drug responses.
  • the present methods can be used to alter DNA copy numbers in a cell, e.g., in a living mammal, e.g., to treat cancer.
  • KMT2/MLL e.g., KMT2A, KMT2B (also known as MLL2, WBP7, sometimes called MLL4), KMT2C (also known as MLL3, HALR), KMT2D (also known as MLL4, ALR, sometimes called MLL2), KMT2E (also known as MLL5), KMT2F (also known as hSETIA, SETD1A), and KMT2G (also known as hSETIB, SETD1B) genes, to a subject in need thereof.
  • KMT2A, KMT2B also known as MLL2, WBP7, sometimes called MLL4
  • KMT2C also known as MLL3, HALR
  • KMT2D also known as MLL4, ALR, sometimes called MLL2
  • KMT2E also known as MLL5
  • KMT2F also known as hSETIA, SETD1A
  • KMT2G also known as hSETIB, SETD1B
  • cancers include, e.g., carcinoma, sarcoma, myeloma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias.
  • a metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.
  • cancer refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth.
  • hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state.
  • pathologic i.e., characterizing or constituting a disease state
  • non-pathologic i.e., a deviation from normal but not associated with a disease state.
  • the term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • Pathologic hyperproliferative occur in disease states
  • hyperproliferative cells include proliferation of cells associated with wound repair.
  • cancer or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • carcinoma is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas,
  • the disease is renal carcinoma or melanoma.
  • Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
  • the term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • sarcoma is art recognized and refers to malignant tumors of mesenchymal derivation.
  • proliferative disorders include hematopoietic neoplastic disorders.
  • hematopoietic neoplastic disorders includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
  • the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia.
  • Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, Crit Rev. in Oncol./Hemotol.
  • APML acute promyeloid leukemia
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and ALL
  • WM Waldenstrom's macroglobulinemia
  • Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T- cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed- Sternberg disease.
  • the cancer is related to H3.3-K36M mutations, e.g., leukemia, neuroblastoma, or sarcoma. See, e.g., Pfister and Ashworth, Nature Reviews Drug Discovery 16, 241-263 (2017).
  • an “effective amount” is an amount sufficient to effect beneficial or desired results.
  • a therapeutic amount is one that achieves the desired
  • an effective amount can be administered in one or more administrations, applications or dosages.
  • a therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected.
  • the compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
  • treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments.
  • Dosage, toxicity and therapeutic efficacy of the therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds that exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • an inhibitor of KDM4A can be used to treat a subject who has a cancer with reduced levels or activity of KDM5A, e.g., as a result of a suppressive gene mutation or deletion, e.g., as known in the art or shown in Table 1, or who is being treated with a KDM5 A inhibitor.
  • KDM5 A depletion or inhibition causes DNA amplification associated with aggressive cancers and drug resistance (e.g., amplifications at lql2h, lql2/21, lq21.2, Iq21.3/CKS1B, and Xql3.1).
  • Cancers that are associated with inhibitory KDM5A mutations include penile cancer, stomach cancer, melanoma, brain tumors, skin cancer, colorectal adenocarcinoma, ampullary carcinoma, cervical cancer, small cell lung cancer, bladder cancer, breast cancer, esaphogogastric cancer, and anal cancer.
  • the methods can include identifying a subject who has a cancer with a KDM5A gene suppression/mutation/deletion, e.g., as shown in Table 1, using methods known in the art, or identifying a subject who is being treated with a KDM5A inhibitor.
  • the methods include identifying a subject who has (e.g., by detecting the presence of) a KDM5A DNA mutation or amplification at lql2h, lql2/21, lq21.2, Iq21.3/CKS1B, or Xql3.1, using methods known in the art.
  • An exemplary human KDM5A reference sequence is at NG 046993.1 , RefSeqGene Range 5001-114399.
  • the methods include administering to these identified subjects an inhibitor of KDM (including KDM4), or an inhibitor of KDM4, e.g., an agent that inhibits KDM4A (preferably inhibiting levels or activity by at least 25%, or at least 50%) and does not significantly inhibit KDM5, or only inhibits KDM5 at a different (by at least 50% or at least 1 order of magnitude) concentration or dose, and/or is separate from any inhibitor of KDM5A that is being administered.
  • the agent specifically inhibits KDM4A (i.e., inhibits KDM4A and does not significantly inhibit other KDM4 family members, or only inhibits other KDM4 family members at a different (by at least 50% or at least 1 order of magnitude) concentration or dose).
  • an inhibitor of KDM4B can be used to treat a subject who has a cancer with reduced levels or activity of KDM5B, e.g., as a result of a suppressive gene mutation or deletion, e.g., as known in the art or Table 2, or who is being treated with a KDM5B inhibitor.
  • KDM5B depletion or inhibition causes DNA amplification associated with aggressive cancers and drug resistance (e.g., amplifications at lp32.3 locus).
  • Cancers that are associated with inhibitory KDM5B mutations include breast, ovarian, oral, neuroblastoma, esophageal, hepatocellular carcinoma, glioma, cervical, and colorectal cancer.
  • the methods can include identifying a subject who has a cancer with a
  • KDM5AB gene suppression/mutation/deletion e.g., as shown in Table 2, using methods known in the art, or identifying a subject who is being treated with a
  • the methods include identifying a subject who has (e.g., by detecting the presence of) a KDM5B DNA mutation or an
  • KDM5B reference sequence is at NG_050659.1, RefSeqGene Range 4938-89223.
  • the methods include administering to these identified subjects an inhibitor of KDM (including KDM4), or an inhibitor of KDM4, e.g., an agent that inhibits KDM4A (preferably inhibiting levels or activity by at least 25%, or at least 50%) and does not significantly inhibit KDM5, or only inhibits KDM5 at a different (by at least 50% or at least 1 order of magnitude) concentration or dose, and/or is separate from any inhibitor of KDM5A that is being administered.
  • the agent specifically inhibits KDM4B (i.e., inhibits KDM4B and does not significantly inhibit other KDM4 family members, or only inhibits other KDM4 family members at a different (by at least 50% or at least 1 order of magnitude) concentration or dose).
  • the methods include administration of a cdk4/6 inhibitor (e.g., Voruciclib (P1446A-05) (Eliades et al., Cancer Biol Ther.
  • a cdk4/6 inhibitor e.g., Voruciclib (P1446A-05) (Eliades et al., Cancer Biol Ther.
  • KDM4B and associated KMT2A inhibitors e.g., chemical, RNA, peptide, etc
  • KDM4B and associated KMT2A inhibitors could be used to sensitize to cdk4/6 inhibitors or other therapeutics that the cdkn2c tumor suppressor will sensitize to upon normal or increased expression (e.g., DNA damaging agents or apoptotic agents).
  • the KMT family includes KMT2A-G, SETD1 A, and SETD1B, and are among the most frequently mutated genes in human cancer. See Rao and Dou, Nat Rev Cancer. 2015 Jun; 15(6): 334-346.
  • the mixed-lineage leukemia (MLL1) gene (chromosome 1 lq23, also known as KMT2A) is often rearranged in acute myeloid leukemias (AML), the acute lymphoblastic leukemias (ALL), and the biphenotypic (mixed lineage) leukemias (Morera et al. Clinical Epigenetics (2016) 8:57).
  • MLL1 When MLL1 is translocated, the catalytic methyltransferase SET domain is lost and the remaining MLL1 protein is fused with a variety of partners (e.g., AF4, AF9, AF10, and ENL). These fusion partners recruit DOTIL, increasing H3K79 methylation and the expression of proleukemogenic genes (like HOXA9 and MEISl) resulting in development of leukemia.
  • partners e.g., AF4, AF9, AF10, and ENL.
  • An inhibitor of KDM4 can be used to treat a subject who has a cancer with increased KMT activity or levels, e.g., due to KMT gene amplification or
  • KMT2A mutations are associated with cancers of the large intestine, lung, bladder, endometrium, and breast.
  • KMT2B and C mutations are associated with cancers of the large intestine, lung, bladder, endometrium, and brain.
  • KMT2D mutations are associated with cancers of the blood/lymph nodes, large intestine, lung, endometrium, and brain.
  • KMT2E mutations are associated with cancers of the large intestine, lung, endometrium, skin (melanoma), and liver.
  • KMT2F mutations are associated with cancers of the large intestine, kidney, and endometrium. See Rao and Dou, Nat Rev Cancer. 2015 Jun; 15(6): 334-346.
  • the methods can include identifying a subject who has a cancer (e.g., a leukemia) with a KMT gene translocation, or a KMT mutation, e.g., as shown in Table 3, using methods known in the art.
  • the methods include identifying a subject who has (e.g., by detecting the presence of) a mutation or DNA translocation or KMT using methods known in the art.
  • An exemplary human KMT2A reference sequence is NC_000011.10, Reference GRCh38.p7 Primary Assembly Range 118436490- 118526832.
  • the methods include administering to these identified subjects an inhibitor of KDM (including KDM4), or an inhibitor of KDM4, e.g., an agent that inhibits KDM4A (preferably inhibiting levels or activity by at least 25%, or at least 50%) and does not significantly inhibit KDM5, or only inhibits KDM5 at a different (by at least 50% or at least 1 order of magnitude) concentration or dose, and/or is separate from any inhibitor of KDM5A that is being administered.
  • the agent specifically inhibits KDM4A (i.e., inhibits KDM4A and does not significantly inhibit other KDM4 family members, or only inhibits other KDM4 family members at a different (by at least 50% or at least 1 order of magnitude) concentration or dose).
  • the methods rely on detection of a sequence or a plurality of sequences associated with a particular disease state or disease susceptibility.
  • the methods include obtaining a sample from a subject, and evaluating the sequence, e.g., by determining the sequence, and/or the presence of a mutation, gene translocation, and/or level of gene amplification or deletion in the sample, and comparing the presence and/or level with one or more references, e.g., a control reference that represents a normal sequence, e.g., a sequence in a non-cancerous or normal control cell (preferably from the same tissue as the cancer cell) from the same subject or an unaffected subject, and/or a disease reference that represents a sequence associated with cancer.
  • sample when referring to the material to be tested for the presence of a biological marker using the method of the invention, includes inter alia tissue, whole blood, plasma, serum, bone marrow, urine, sweat, saliva, breath, exosome or exosome-like microvesicles (U.S. Patent No. 8.901.284), lymph, feces, cerebrospinal fluid, ascites, bronchoalveolar lavage fluid, pleural effusion, seminal fluid, sputum, nipple aspirate, post-operative seroma or wound drainage fluid.
  • tissue whole blood, plasma, serum, bone marrow, urine, sweat, saliva, breath, exosome or exosome-like microvesicles
  • lymph feces, cerebrospinal fluid, ascites, bronchoalveolar lavage fluid, pleural effusion, seminal fluid, sputum, nipple aspirate, post-operative seroma or wound drainage fluid.
  • the sample is or includes blood, serum, and/or plasma, or a portion or subfraction thereof, e.g., free RNA in serum or RNA within exosomes in blood.
  • the sample comprises (or is suspected of comprising) CTCs.
  • the sample is or includes urine or a portion or subfraction thereof.
  • the sample includes known or suspected tumor cells, e.g., is a biopsy sample, e.g., a fine needle aspirate (FNA), endoscopic biopsy, or core needle biopsy; in some embodiments the sample comprises cells from the bone marrow, pancreas, lung, breast, prostate, kidney, liver, ovary, skin, or colon of the subject.
  • FNA fine needle aspirate
  • the sample comprises lung cells obtained from a sputum sample or from the lung of the subject by brushing, washing, bronchoscopic biopsy, transbronchial biopsy, or FNA, e.g., bronchoscopic, fluoroscopic, or CT-guided FNA (such methods can also be used to obtain samples from other tissues as well).
  • the sample is frozen, fixed and/or permeabilized, e.g., is an formalin-fixed paraffin-embedded (FFPE) sample.
  • FFPE formalin-fixed paraffin-embedded
  • an “isolated” or “purified” biological marker is substantially free of cellular material or other contaminants from the cell or tissue source from which the biological marker is derived i.e. partially or completely altered or removed from the natural state through human intervention.
  • nucleic acids contained in the sample are first isolated according to standard methods, for example using lytic enzymes, chemical solutions, or isolated by nucleic acid-binding resins following the manufacturer's instructions.
  • the presence and/or level of a nucleic acid can be evaluated using methods known in the art, e.g., using polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative or semi-quantitative real-time RT- PCR, digital PCR i.e. BEAMing ((Beads, Emulsion, Amplification, Magnetics) Diehl (2006) Nat Methods 3 :551-559) ; RNAse protection assay; Northern blot; various types of nucleic acid sequencing (Sanger, pyrosequencing, NextGeneration
  • RNA in situ hybridization RNA expression assays, e.g., microarray analysis; multiplexed gene expression analysis methods, e.g., RT-PCR, RNA-sequencing, and oligo hybridization assays including RNA expression microarrays; hybridization based digital barcode quantification assays such as the nCounter ® System
  • MLPA Luminex/XMAP
  • branched DNA analysis methods See, e.g., International Publication No. WO 2012/048113, which is incorporated herein by reference in its entirety. See also Lehninger Biochemistry (Worth Publishers, Inc., current addition; Sambrook, et al, Molecular Cloning: A Laboratory Manual (3. Sup.rd Edition, 2001); Bernard (2002) Clin Chem 48(8): 1178-1185; Miranda (2010) Kidney International 78: 191-199; Bianchi (2011) EMBO Mol Med 3 :495-503; Taylor (2013) Front. Genet. 4: 142; Yang (2014) PLOS One 9(1 l):el 10641); Nordstrom (2000) Biotechnol. Appl. Biochem.
  • high throughput methods e.g., gene chips as are known in the art (see, e.g., Ch. 12, Genomics, in Griffiths et al., Eds. Modern genetic Analysis, 1999,W. H. Freeman and Company; Ekins and Chu, Trends in
  • RNAs and DNAs can be used to detect the presence and/or level of a sequence or genomic alteration as described herein. Measurement can be direct or indirect. For example, the sequence or copy number of a gene can be directly determined or quantitated. Alternatively, the amount of a biomarker can be determined indirectly by measuring abundance levels of cDNA, amplified RNAs or DNAs, or by measuring quantities or activities of RNAs, or other molecules that are indicative of the expression level of the biomarker. In some embodiments, a technique suitable for the detection of alterations in the structure or sequence of nucleic acids, such as the presence of deletions, amplifications, or substitutions, can be used.
  • RT-PCR can be used to determine the expression profiles of biomarkers (U.S. Patent No. 2005/0048542A1).
  • the first step in expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction (Ausubel et al (1997) Current Protocols of Molecular Biology, John Wiley and Sons).
  • RT-PCR is usually performed using an internal standard, which is expressed at constant level among tissues, and is unaffected by the experimental treatment. Housekeeping genes are most commonly used.
  • Gene arrays are typically prepared by selecting probes that comprise a polynucleotide sequence, and then immobilizing such probes to a solid support or surface.
  • the probes may comprise DNA sequences, RNA sequences, copolymer sequences of DNA and RNA, DNA and/or RNA analogues, or combinations thereof.
  • the probe sequences can be synthesized either enzymatically in vivo, enzymatically in vitro (e.g. by PCR), or non-enzymatically in vitro.
  • KDM and KMT inhibitors are known in the art; exemplary agents include small molecules as well as inhibitory nucleic acids.
  • KDM4 KDM4
  • KDM4A, 4B, 4C and 4D The human KDM4 family consists of four members, KDM4A, 4B, 4C and 4D, and two pseudo-genes (KDM4E and KDM4F); KDM4A, 4B, and 4C contain a catalytic histone demethylase domain (jumanji domain), and double PHD and Vietnamese domains. KDM4D contains a catalytic domain and no PHD or Vietnamese domains.
  • KDM4 inhibitors include NCDM-32B (IC50 values of 3.0 and 1.0 ⁇ for KDM4A and KDM4C, see Ye et al., Am J Cancer Res 2015;5(4): 1519-1530); l,5-bis[(E)-2-(3,4- dichlorophenyl ) ethenyl ] - 2 , 4 - d i nitrobenzene (NSC636819) (Chu et al., J. Med. Chem.
  • H3K4M peptide mimics e.g., a synthetic peptide of the Histone H3 tail (residues 1-21), can also be used as inhibitors of KDM4; see, e.g., Zhang et al., Cell Research (2013) 23, 225-241 (2013). See also WO2014/197835, which is incorporated herein in its entirety.
  • KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4).
  • Inhibitors of KDM5A include Compound 15e (Wu et al., Bioorganic & Medicinal Chemistry Letters 26 (2016) 2284-2288); compound 20 (Gehling et al., Bioorganic & Medicinal Chemistry Letters 26 (2016) 4350-4354); compound 50 (Liang et al., Bioorganic & Medicinal Chemistry Letters 27 (2017) 2974-2981); N3, N4, N8, N10, Nl 1, N12, N16, and N19 (Horton et al., 2016, Cell Chemical Biology 23, 769-781); KDM5-C49 and its cell-permeable ethyl ester derivative, KDM5-C70 (WO2014053491, Johansson et al., Nature Chemical Biology 12, 539-545 (2016)); YUKA1 (4-([2-(allyloxy)-3
  • Inhibitors of KDM5B include KDOAM-25 (Tumber et al., 2017, Cell
  • JIB 04 (E,Z)-N-(5-chloro-pyridin-2-yl)-N'-(phenyl-pyridin-2-yl-methylene)- hydrazine) inhibits the Jumonji family of KDMs (KDM5A and KDM5D-E are more sensitive than KDM4D/4B/4A/6B/4C (Wang et al., Nat. Commun. 4:2035 doi:
  • KDM5 inhibitors include compounds described in McAllister et al., J. Med. Chem., 2016, 59 (4), pp 1308-1329 and Thinnes et al., Biochimica et Biophysica Acta 1839 (2014) 1416-1432;
  • KMT/MLL inhibitors include WDR5 inhibitors including WDR5-0101 [N-(2- (4-methylpiperazin-l-yl)-5 nitrophenyl)benzamide], WDR5-0102 [2-chloro-N-(2-(4- methylpiperazin-l-yl)-5-nitrophenyl) benzamide] and WDR5-0103 [methyl-3-(3- methoxybenzamido)-4-(4-methylpiperazin-l-yl)benzoate] (Senisterra et al., Biochem. J. (2013) 449, 151-159), as well as RBC-1, -2, -3, and -4 (Ferry et al., Assay Drug Dev Technol.
  • DOT1L inhibitors include EPZ004777 and its derivative EPZ-5676 (ClinicalTrials.gov Identifier: NCT02141828; Morera et al. Clinical Epigenetics (2016) 8:57). See also Ferry et al., Assay and Drug Dev. Tevh 13(4)221 (2015).
  • Inhibitory nucleic acids useful in the present methods and compositions include anti sense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases/locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimetics that hybridize to at least a portion of the target nucleic acid and modulate its function.
  • Exemplary human sequences are provided in Table 4. In some cases, other isoforms of the same transcript may be targeted.
  • KMT2A/MLL lysine methyltransferase 2A
  • the inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof.
  • RNAi interference RNA
  • siRNA short interfering RNA
  • miRNA micro, interfering RNA
  • shRNA small, temporal RNA
  • shRNA short, hairpin RNA
  • small RNA-induced gene activation RNAa
  • small activating RNAs saRNAs
  • the inhibitory nucleic acids are 10 to 50, 10 to 20, 10 to 25, 13 to 50, or 13 to 30 nucleotides in length.
  • the inhibitory nucleic acids are 15 nucleotides in length.
  • the inhibitory nucleic acids are 12 or 13 to 20, 25, or 30 nucleotides in length.
  • inhibitory nucleic acids having complementary portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or any range therewithin (complementary portions refers to those portions of the inhibitory nucleic acids that are complementary to the target sequence).
  • the inhibitory nucleic acids useful in the present methods are sufficiently complementary to the target RNA, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
  • “Complementary” refers to the capacity for pairing, through hydrogen bonding, between two sequences comprising naturally or non-naturally occurring bases or analogs thereof. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a RNA, then the bases are considered to be
  • Routine methods can be used to design an inhibitory nucleic acid that binds to the target sequence with sufficient specificity.
  • the methods include using bioinformatics methods known in the art to identify regions of secondary structure, e.g., one, two, or more stem-loop structures, or pseudoknots, and selecting those regions to target with an inhibitory nucleic acid.
  • bioinformatics methods known in the art to identify regions of secondary structure, e.g., one, two, or more stem-loop structures, or pseudoknots, and selecting those regions to target with an inhibitory nucleic acid.
  • "gene walk" methods can be used to optimize the inhibitory activity of the nucleic acid; for example, a series of oligonucleotides of 10-30 nucleotides spanning the length of a target RNA can be prepared, followed by testing for activity.
  • gaps e.g., of 5-10 nucleotides or more, can be left between the target sequences to reduce the number of oligonucleotides synthesized and tested.
  • GC content is preferably between about 30-60%). Contiguous runs of three or more Gs or Cs should be avoided where possible (for example, it may not be possible with very short (e.g., about 9-10 nt) oligonucleotides).
  • the inhibitory nucleic acid molecules can be designed to target a specific region of the RNA sequence.
  • a specific functional region can be targeted, e.g., a region comprising a known RNA localization motif (i.e., a region complementary to the target nucleic acid on which the RNA acts).
  • highly conserved regions can be targeted, e.g., regions identified by aligning sequences from disparate species such as primate (e.g., human) and rodent (e.g., mouse) and looking for regions with high degrees of identity.
  • Percent identity can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), e.g., using the default parameters.
  • BLAST programs Basic local alignment search tools
  • inhibitory nucleic acid compounds are chosen that are sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient specificity (i.e., do not substantially bind to other non-target RNAs), to give the desired effect.
  • hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • Complementary refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a RNA molecule, then the inhibitory nucleic acid and the RNA are considered to be complementary to each other at that position.
  • the inhibitory nucleic acids and the RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
  • RNA target a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a RNA, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.
  • a complementary nucleic acid sequence need not be 100% complementary to that of its target nucleic acid to be specifically
  • a complementary nucleic acid sequence for purposes of the present methods is specifically hybridisable when binding of the sequence to the target RNA molecule interferes with the normal function of the target RNA to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target RNA sequences under conditions in which specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency.
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C.
  • Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
  • concentration of detergent e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ⁇ g/ml denatured salmon sperm DNA (ssDNA).
  • hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C.
  • wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
  • Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
  • the inhibitory nucleic acids useful in the methods described herein have at least 80% sequence complementarity to a target region within the target nucleic acid, e.g., 90%, 95%, or 100% sequence complementarity to the target region within an RNA.
  • a target region within the target nucleic acid e.g. 90%, 95%, or 100% sequence complementarity to the target region within an RNA.
  • an antisense compound in which 18 of 20 nucleobases of the antisense oligonucleotide are complementary, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity.
  • Percent complementarity of an inhibitory nucleic acid with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al., J. Mol.
  • Inhibitory nucleic acids that hybridize to an RNA can be identified through routine experimentation. In general the inhibitory nucleic acids must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of, transcripts other than the intended target.
  • inhibitory nucleic acids please see:
  • US2010/0317718 antisense oligos
  • US2010/0249052 double-stranded ribonucleic acid (dsRNA)
  • US2009/0181914 and US2010/0234451 LNAs
  • US2007/0191294 siRNA analogues
  • US2008/0249039 modified siRNA
  • WO2010/129746 and WO2010/040112 inhibitor nucleic acids
  • the inhibitory nucleic acids are antisense
  • Antisense oligonucleotides are typically designed to block expression of a DNA or RNA target by binding to the target and halting expression at the level of transcription, translation, or splicing.
  • Antisense oligonucleotides of the present invention are complementary nucleic acid sequences designed to hybridize under stringent conditions to an RNA. Thus, oligonucleotides are chosen that are sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient specificity, to give the desired effect.
  • the nucleic acid sequence that is complementary to a target RNA can be an interfering RNA, including but not limited to a small interfering RNA ("siRNA”) or a small hairpin RNA (“shRNA").
  • interfering RNAs include a small interfering RNA (“siRNA”) or a small hairpin RNA (“shRNA”).
  • siRNA small interfering RNA
  • shRNA small hairpin RNA
  • Methods for constructing interfering RNAs are well known in the art.
  • the interfering RNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self- complementary (i.e., each strand comprises nucleotide sequence that is
  • the antisense strand and sense strand form a duplex or double stranded structure
  • the antisense strand comprises nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (i.e., an undesired gene)
  • the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • interfering RNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions are linked by means of nucleic acid based or non-nucleic acid-based linker(s).
  • the interfering RNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the interfering can be a circular single- stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNA interference.
  • the interfering RNA coding region encodes a self- complementary RNA molecule having a sense region, an antisense region and a loop region.
  • a self- complementary RNA molecule having a sense region, an antisense region and a loop region.
  • Such an RNA molecule when expressed desirably forms a "hairpin" structure, and is referred to herein as an "shRNA.”
  • the loop region is generally between about 2 and about 10 nucleotides in length. In some embodiments, the loop region is from about 6 to about 9 nucleotides in length.
  • the sense region and the antisense region are between about 15 and about 20 nucleotides in length.
  • the small hairpin RNA is converted into a siRNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family.
  • Dicer which is a member of the RNase III family.
  • the siRNA is then capable of inhibiting the expression of a gene with which it shares homology.
  • Dicer a member of the RNase III family.
  • the siRNA is then capable of inhibiting the expression of a gene with which it shares homology.
  • siRNAs The target RNA cleavage reaction guided by siRNAs is highly sequence specific.
  • siRNA containing a nucleotide sequences identical to a portion of the target nucleic acid are preferred for inhibition.
  • 100% sequence identity between the siRNA and the target gene is not required to practice the present invention.
  • the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence.
  • siRNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition.
  • siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition.
  • the siRNAs must retain specificity for their target, i.e., must not directly bind to, or directly
  • transcripts other than the intended target significantly affect expression levels of, transcripts other than the intended target.
  • Trans-cleaving enzymatic nucleic acid molecules can also be used; they have shown promise as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037).
  • Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the RNA non- functional.
  • enzymatic nucleic acids with RNA cleaving activity act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.
  • the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
  • ribozymes that are optimal for catalytic activity would contribute significantly to any strategy that employs RNA- cleaving ribozymes for the purpose of regulating gene expression.
  • the hammerhead ribozyme functions with a catalytic rate (kcat) of about 1 min "1 in the presence of saturating (10 rnM) concentrations of Mg 2+ cofactor.
  • RNA ligase ribozyme has been shown to catalyze the corresponding self-modification reaction with a rate of about 100 min "1 .
  • certain modified hammerhead ribozymes that have substrate binding arms made of DNA catalyze RNA cleavage with multiple turn-over rates that approach 100 min "1 .
  • the inhibitory nucleic acids used in the methods described herein are modified, e.g., comprise one or more modified bonds or bases.
  • a number of modified bases include phosphorothioate, methylphosphonate, peptide nucleic acids, or locked nucleic acid (LNA) molecules.
  • LNA locked nucleic acid
  • Some inhibitory nucleic acids are fully modified, while others are chimeric and contain two or more chemically distinct regions, each made up of at least one nucleotide.
  • inhibitory nucleic acids typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • Chimeric inhibitory nucleic acids of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides,
  • the oligonucleotide is a gapmer (contain a central stretch (gap) of DNA monomers sufficiently long to induce RNase H cleavage, flanked by blocks of LNA modified nucleotides; see, e.g., Stanton et al., Nucleic Acid Ther. 2012. 22: 344-359; Nowotny et al., Cell, 121 : 1005-1016, 2005; Kurreck, European Journal of
  • the oligonucleotide is a mixmer (includes alternating short stretches of LNA and DNA; Naguibneva et al., Biomed Pharmacother. 2006 Nov;
  • the inhibitory nucleic acid comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-0-alkyl, 2'-0- alkyl-O-alkyl or 2'-fluoro-modified nucleotide.
  • RNA modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA. Such modifications are routinely incorporated into oligonucleotides and these
  • oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than; 2'-deoxyoligonucleotides against a given target.
  • modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH2 -NH-0-CH2,
  • CH, ⁇ N(CH3) ⁇ 0 ⁇ CH2 (known as a methylene(methylimino) or MMI backbone], CH2 --0-N (CH3)-CH2, CH2 -N (CH3)-N (CH3)-CH2 and O-N (CH3)- CH2 -CH2 backbones, wherein the native phosphodiester backbone is represented as O- P— O- CH,); amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366- 374); morpholino backbone structures (see Summerton and Weller, U.S. Pat. No.
  • PNA peptide nucleic acid
  • Phosphorus- containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters,
  • aminoalkylphosphotriesters methyl and other alkyl phosphonates comprising
  • thionophosphoramidates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see US patent nos. 3,687,808; 4,469,863;
  • Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.
  • Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc, 2000, 122, 8595-8602.
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl
  • internucleoside linkages mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones;
  • One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, OCH 3 OCH 3 , OCH 3 0(CH 2 )n CH 3 , 0(CH 2 )n H 2 or 0(CH 2 )n CH 3 where n is from 1 to about 10; Ci to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF3 ; OCF3; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH3; S02 CH3; ON02; N02; N3; H2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino;
  • a preferred modification includes 2'- methoxyethoxy [2'-0-CH 2 CH 2 OCH 3 , also known as 2'-0-(2-methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486).
  • Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
  • Inhibitory nucleic acids can also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base”) modifications or
  • nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me- C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino)adenine, 2- (imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines
  • both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • PNA compounds comprise, but are not limited to, US patent nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference . Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.
  • Inhibitory nucleic acids can also include one or more nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases comprise other synthetic and natural nucleobases such as 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5-sub
  • nucleobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in 'The Concise Encyclopedia of Polymer Science And Engineering', pages 858-859, Kroschwitz, J. I, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandle Chemie, International Edition', 1991, 30, page 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications', pages 289- 302, Crooke, S.T. and Lebleu, B. ea., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • 5-substituted pyrimidines 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6- 1.2 ⁇ 0>C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds, Antisense Research and Applications', CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications. Modified nucleobases are described in US patent nos.
  • the inhibitory nucleic acids are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
  • moieties comprise but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S- tritylthiol (Manoharan et al, Ann. N. Y.
  • Acids Res., 1992, 20, 533- 538 an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49- 54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O- hexadecyl- rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl.
  • a phospholipid e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O- hexadecyl
  • conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No.
  • Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5- tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexy
  • LNAs Locked Nucleic Acids
  • the modified inhibitory nucleic acids used in the methods described herein comprise locked nucleic acid (LNA) molecules, e.g., including [alpha]-L-LNAs.
  • LNAs comprise ribonucleic acid analogues wherein the ribose ring is "locked" by a methylene bridge between the 2'-oxgygen and the 4'- carbon - i.e., oligonucleotides containing at least one LNA monomer, that is, one 2'- O,4'-C-methylene- ?-D-ribofuranosyl nucleotide.
  • LNA bases form standard Watson- Crick base pairs but the locked configuration increases the rate and stability of the basepairing reaction (Jepsen et al., Oligonucleotides, 14, 130-146 (2004)).
  • LNAs also have increased affinity to base pair with RNA as compared to DNA. These properties render LNAs especially useful as probes for fluorescence in situ hybridization (FISH) and comparative genomic hybridization, as knockdown tools for miRNAs, and as antisense oligonucleotides to target mRNAs or other RNAs, e.g., RNAs as described herien.
  • the LNA molecules can include molecules comprising 10-30, e.g., 12-24, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to a target region in the RNA.
  • the LNA molecules can be chemically synthesized using methods known in the art.
  • the LNA molecules can be designed using any method known in the art; a number of algorithms are known, and are commercially available (e.g., on the internet, for example at exiqon.com). See, e.g., You et al., Nuc. Acids. Res. 34:e60 (2006); McTigue et al., Biochemistry 43 :5388-405 (2004); and Levin et al., Nuc. Acids. Res. 34:el42 (2006).
  • "gene walk” methods can be used to optimize the inhibitory activity of the LNA; for example, a series of oligonucleotides of 10-30 nucleotides spanning the length of a target RNA can be prepared, followed by testing for activity.
  • gaps e.g., of 5-10 nucleotides or more, can be left between the LNAs to reduce the number of oligonucleotides synthesized and tested.
  • GC content is preferably between about 30-60%).
  • General guidelines for designing LNAs are known in the art; for example, LNA sequences will bind very tightly to other LNA sequences, so it is preferable to avoid significant complementarity within an LNA. Contiguous runs of more than four LNA residues, should be avoided where possible (for example, it may not be possible with very short (e.g., about 9-10 nt) oligonucleotides). In some
  • the LNAs are xylo-LNAs.
  • RNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/ generated recombinantly.
  • Recombinant nucleic acid sequences can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including e.g. in vitro, bacterial, fungal, mammalian, yeast, insect or plant cell expression systems.
  • Nucleic acid sequences of the invention can be inserted into delivery vectors and expressed from transcription units within the vectors.
  • the recombinant vectors can be DNA plasmids or viral vectors.
  • Generation of the vector construct can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al.
  • RNA Viruses A Practical Approach
  • suitable vectors are available for transferring nucleic acids of the invention into cells.
  • Viral vectors comprise a nucleotide sequence having sequences for the production of recombinant virus in a packaging cell.
  • Viral vectors expressing nucleic acids of the invention can be constructed based on viral backbones including, but not limited to, a retrovirus, lentivirus, adenovirus, adeno-associated virus, pox virus or alphavirus.
  • the recombinant vectors capable of expressing the nucleic acids of the invention can be delivered as described herein, and persist in target cells (e.g., stable transformants).
  • Nucleic acid sequences used to practice this invention can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440- 3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33 :7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; U.S. Patent No. 4,458,066.
  • nucleic acid sequences of the invention can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification.
  • nucleic acid sequences of the invention includes a phosphorothioate at least the first, second, or third internucleotide linkage at the 5' or 3' end of the nucleotide sequence.
  • the nucleic acid sequence can include a 2'-modified nucleotide, e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'- O-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0- dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0 ⁇ N-methylacetamido (2'-0- NMA).
  • a 2'-modified nucleotide e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'- O-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'
  • the nucleic acid sequence can include at least one 2'-0- methyl-modified nucleotide, and in some embodiments, all of the nucleotides include a 2'-0-methyl modification.
  • the nucleic acids are "locked," i.e., comprise nucleic acid analogues in which the ribose ring is "locked” by a methylene bridge connecting the 2'-0 atom and the 4'-C atom (see, e.g., Kaupinnen et al., Drug Disc. Today 2(3):287-290 (2005); Koshkin et al., J. Am. Chem. Soc, 120(50): 13252-13253 (1998)).
  • For additional modifications see US 20100004320, US 20090298916, and US 20090143326.
  • nucleic acids used to practice this invention such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature. See, e.g., Sambrook et al., Molecular Cloning; A Laboratory Manual 3d ed. (2001); Current Protocols in Molecular Biology, Ausubel et al., eds. (John Wiley & Sons, Inc., New York 2010); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); Laboratory Techniques In Biochemistry And Molecular Biology: Hybridization With Nucleic Acid Probes, Parti. Theory and Nucleic Acid Preparation, Tijssen, ed.
  • the methods described herein can include the administration of
  • compositions and formulations comprising inhibitors as described herein, e.g., inhibitory nucleic acid sequences designed to target an RNA.
  • compositions are formulated with a
  • compositions and formulations can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally.
  • the pharmaceutical compositions and formulations can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally.
  • compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005.
  • the inhibitors can be administered alone or as a component of a
  • composition The compounds may be formulated for administration, in any convenient way for use in human or veterinary medicine.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • Formulations of the compositions of the invention include those suitable for intradermal, inhalation, oral/ nasal, topical, parenteral, rectal, and/or intravaginal administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient (e.g., nucleic acid sequences of this invention) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect, e.g., an antigen specific T cell or humoral response.
  • compositions can be prepared according to any method known to the art for the manufacture of pharmaceuticals.
  • Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents.
  • a formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture.
  • Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient.
  • Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores.
  • Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen.
  • Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Push-fit capsules can contain active agents mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
  • Aqueous suspensions can contain an active agent (e.g., nucleic acid sequences of the invention) in admixture with excipients suitable for the manufacture of aqueous suspensions, e.g., for aqueous intradermal injections.
  • an active agent e.g., nucleic acid sequences of the invention
  • Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono
  • the aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin.
  • preservatives such as ethyl or n-propyl p-hydroxybenzoate
  • coloring agents such as a coloring agent
  • flavoring agents such as aqueous suspension
  • sweetening agents such as sucrose, aspartame or saccharin.
  • Formulations can be adjusted for osmolality.
  • oil-based pharmaceuticals are used for administration of nucleic acid sequences of the invention.
  • Oil-based suspensions can be formulated by suspending an active agent in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. See e.g., U.S. Patent No. 5,716,928 describing using essential oils or essential oil components for increasing bioavailability and reducing inter- and intra-individual variability of orally administered hydrophobic pharmaceutical compounds (see also U.S. Patent No. 5,858,401).
  • the oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid.
  • an injectable oil vehicle see Minto (1997) J. Pharmacol. Exp. Ther. 281 :93-102.
  • compositions can also be in the form of oil-in-water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these.
  • Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate.
  • the emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent.
  • these injectable oil-in-water emulsions of the invention comprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitan monooleate and/or an ethoxylated sorbitan trioleate.
  • the pharmaceutical compounds can also be administered by in intranasal, intraocular and intravaginal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see e.g., Rohatagi (1995) J. Clin. Pharmacol. 35: 1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75: 107- 111).
  • Suppositories formulations can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug.
  • suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug.
  • Such materials are cocoa butter and polyethylene glycols.
  • the pharmaceutical compounds can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
  • the pharmaceutical compounds can also be delivered as microspheres for slow release in the body.
  • microspheres can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.
  • the pharmaceutical compounds can be parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or lumen of an organ.
  • IV intravenous
  • These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier.
  • Acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride.
  • sterile fixed oils can be employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter.
  • These formulations may be sterilized by conventional, well known sterilization techniques.
  • the formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs.
  • the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation can also be a suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3- butanediol.
  • the administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time).
  • the pharmaceutical compounds and formulations can be lyophilized.
  • Stable lyophilized formulations comprising an inhibitory nucleic acid can be made by lyophilizing a solution comprising a pharmaceutical of the invention and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof.
  • a process for preparing a stable lyophilized formulation can include lyophilizing a solution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. 20040028670.
  • compositions and formulations can be delivered by the use of liposomes.
  • liposomes particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Patent Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13 :293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46: 1576-1587.
  • liposome means a vesicle composed of amphiphilic lipids arranged in a bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered.
  • Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.
  • Liposomes can also include "sterically stabilized" liposomes, i.e., liposomes comprising one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes with enhanced circulation lifetimes relative to liposomes lacking such specialized lipids.
  • sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
  • PEG polyethylene glycol
  • compositions of the invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a subject who is need of reduced triglyceride levels, or who is at risk of or has a disorder described herein, in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the disorder or its
  • compositions of the invention are administered in an amount sufficient to decrease serum levels of triglycerides in the subject.
  • the amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose.
  • the dosage schedule and amounts effective for this use i.e., the dosing regimen, will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into
  • the dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51 :337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84: 1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24: 103-108; Remington: The Science and Practice of Pharmacy, 21st ed., 2005).
  • pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Bio
  • formulations can be given depending on for example: the dosage and frequency as required and tolerated by the patient, the degree and amount of therapeutic effect generated after each administration (e.g., effect on tumor size or growth), and the like.
  • the formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate conditions, diseases or symptoms.
  • administration are in a daily amount of between about 1 to 100 or more mg per kilogram of body weight per day.
  • Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ.
  • Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation.
  • Actual methods for preparing parenterally or non-parenterally administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, 21st ed., 2005.
  • LNAs locked nucleic acids
  • the methods described herein can include coadministration with other drugs or pharmaceuticals, e.g., compositions for providing cholesterol homeostasis.
  • the inhibitors can be co-administered with drugs for treating or reducing risk of a disorder described herein.
  • transfections were performed using Lipofectamine 3000 transfection reagent (Life Technologies) in OPTI-MEM medium (Life Technologies). Transfections were changed to complete media after 4hr of transfection, and cells were collected at 72hr. Transient overexpression transfections were performed using Lipofectamine 3000 transfection reagent and P3000 reagent (Life Technologies) in OPTI-MEM medium for 4hr. Silencer select negative controls and siRNAs were purchased from Life Technologies. At least two different siRNAs against every gene were used for every experiment.
  • FISH DNA Fluorescence in situ hybridization
  • FISH protocol was performed as described previously in (Black et al., 2013). Imaging was performed with multiple planes of fields of nuclei. At least 100 cells were counted from each experiment. All FISH experiments include at least two biological replicates. Extended methods can be found in supplemental methods. RNA extraction and quantitative real-time PCR
  • Asynchronized and synchronized cells were fixed and processed as performed in (Black et al., 2010).
  • Cell cycle was analyzed by Propium iodide (PI) or EdU staining with a Click-IT EdU flow cytometry kit (Life Technologies). Cells were stained with 10 ⁇ EdU for lh prior to cell cycle analyses with EdU staining. Cell cycle distribution was analyzed with a flow cytometer LSRII or Fortessa.
  • KDM4B and lysates were prepared were processing as described previously in (Black et al., 2013).
  • Halo tag-purified KDM4B complexes were analyzed and processed as described previously in (Black et al., 2013).
  • Chromatin IPs was performed as in (Black et al., 2013). Data presented are the averages from at least two independent siRNAs or two independent stable cell lines. RPE cells were treated with 2mM HU for 20h before cross-linking and assessment for KDM4A, KDM4B and H3K4me3. DNA Polymerase alpha (DNA Pol a) ChIP was performed after Ro-3306 treatment. Detailed list of procedure and antibodies are provided in the supplemental methods and table.
  • RPE Retinal pigment epithelial
  • FBS fetal bovine serum
  • H2591 lung cancer cells were cultured in RPMI (Sigma) with 10% fetal bovine serum, 1% penicillin/streptomycin, and L- glutamine.
  • SK-N-AS cells were maintained in DMEM/F12 (Gibco) with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% L-glutamine.
  • Transient siRNA transfections were performed using Lipofectamine 3000 transfection reagent (Invitrogen) in OPTI-MEM medium (Life Technologies). Transfections were changed to complete media after 4hr of transfection, and cells were collected at 72hr.
  • Transient overexpression transfections were performed using Lipofectamine 3000 transfection reagent and P3000 reagent (Life Technologies) in OPTI-MEM medium for 4hr, followed by change to complete media.
  • Silencer select negative controls and siRNAs were purchased from Life Technologies. At least two different siRNAs against every gene were used for every experiment.
  • FISH DNA Fluorescence in situ hybridization
  • FISH probes for chromosome 1 classical satellite (lql2h), chromosome 8 centromere (alpha satellite), CKS1B/CDKN2C and chromosome X alpha satellite were purchased from Cytocell (Oxford Gene Technologies).
  • Probes for lq21.2, lp32.3 and lq23.3 were purchased from Agilent Technologies. The lp32.3 probe shown in the figure by itself is performed using probe from Agilent Technologies.
  • FISH protocol was performed as described previously in Black et al. (2013).
  • BACS for Xql3.1 were prepared utilizing PureLink HiPure Plasmid Filter Maxiprep kit (Life Technologies) using the recommended modified wash buffer.
  • Probes were nick translated (Abbot Molecular Kit) in the presence of fluorescently labeled dTTP (Enzo Life Science). Imaging was performed with multiple planes of fields of nuclei. Images were acquired with Olympus 1X81 Spinning Disk Microscope and Olympus 1X83 and analyzed using Slidebook 5.0 and Slidebook 6.0 softwares. Copy number gains were scored in RPE cells as more than or equal to three foci. For SK-N-AS cells, copy gain was scored for any cell with 5 or more foci. For H2591, copy gain was scored for any cell with 6 or more foci. At least 100 cells were counted from each experiment. All FISH experiments include at least two biological replicates. Extended list of probes used are provided in the table.
  • KDM5-C70 was purchased from Xcessbio [Fisher Scientific; (Johansson et al., 2016)] and treated at the indicated concentrations for 48hr or 72hr. Cesium Chloride gradient centrifugation
  • RPE cells were grown and transfected with KDM5 A siRNA for a total of 72hr including BrdU treatment. Cells were labeled with BrdU for 12 hours and 30 minutes. Genomic DNA was purified and ultracentrifuged as was described previously ((Black et al., 2013). Heavy:heavy fractions were pooled together and purified. The re- replicated samples were analyzed by qPCR on a Roche LC480 using FastStart Universal SYBR Green Master Mix (Roche) following the manufacturer's
  • Cytoplasmic, nuclear and chromatin fractions were prepared from RPE cells. Cell pellets were washed twice in ice cold PBS and resuspended in ice cold hypotonic buffer (lOmM HEPES pH 7.9, lOmM KCl, 0.1M EDTA, 0.5M EGTA) and incubated on ice for 15 minutes. Swollen cells were lysed by addition of NP-40 to 10% with 10 seconds of vortexing. Lysed cells were centrifuged and the supernatant kept as cytoplasm.
  • ice cold hypotonic buffer lOmM HEPES pH 7.9, lOmM KCl, 0.1M EDTA, 0.5M EGTA
  • the nuclear pellet was resuspended in high salt buffer (lOmM HEPES pH 7.9, 400mM NaCl, ImM EDTA, 5mM EGTA) and incubated at 4°C for 15min with rotation. Extracts were centrifuged and the supernatant was kept as nuclear extract. The pellets were resuspended in N-Buffer (20mM Trish pH 7.5, lOOmM KCl, 2mM MgC12, ImM CaC12, 0.3M Sucrose, 0.1% Triton X-100, 3U per ml micrococcal nuclease).
  • Antibody against KDM4A was purchased from Neuro mAb, 75-189, KDM5A from abeam (ab70892), ⁇ -actin from Millipore, actinin from Santa Cruz
  • KDM4B was immunoprecipitated from whole- cell lysates using antibody (abeam abl91434) and probed for MCM2 (ab6153) and MCM5 (ab6154).
  • Chromatin IPs was performed as in Black et al. (2013). Data presented are the averages from two independent siRNAs.
  • 0.3x10 6 RPE were seeded in 10cm plates and transfected with Lipofectamine 3000 (Life Technology) following the supplier instructions. Cells were harvested after 72h of transfection.
  • Antibodies used for ChIP are as follows: KDM5A (abeam ab70892), KDM5A (Bethyl A300-897 A), KDM4A (P006, Structural Genomic Consortium), KDM4A (P014, Structural Genomic Consortium), KDM4B (abeam abl91434), H3K4me3 (Millipore 07-473), H3 (abeam abl791), Polymerase alpha (ab31777-100) and H3K36me3 (Abeam, ab9050).
  • KDM5A and KDM4A ChlPs were performed with two antibodies, the average of both are presented.
  • HEK293 cells (4 x 10 5 ) were plated in each well of a 12-well plate and co- transfected various NanoLuc donor and HaloTag acceptor pairs including; HaloTag- MCM3 (P25205), HaloTag-MCM4 (P33991), HaloTag-MCM5 (P33992), HaloTag- MCM7 (P33993), HaloTag-POLA2 (Q14181), HaloTag -POLD1 (P28340), NanoLuc- KDM4A (075164), NanoLuc-KDM4B (094953) or HaloTag alone (Promega G6591).
  • All HaloTag and NanoLuc fusion vectors utilized pFN21 A and pFN3 IK backbones respectively (Promega) and were transfected at 1 : 100 fold dilution (2ug HaloTag acceptor: 0.02ug NL donor) or in varying ratios of 1 : 100, 33.3, 11.1 3.7, 1.2, 0.41, 0.14, .0.046, and 0 for donor saturation assays. Twenty hours post-transfection cells were collected, washed with PBS, and exchanged into phenol red-free Opti- MEM in the absence (control sample) or the presence (experimental sample) of 100 nM NanoBRET 618 fluorescent ligand (Promega).
  • NanoBRET furimazine substrate (Promega) was added to both control and experimental samples at a final concentration of 10 ⁇ . Readings were performed within 5 minutes using the GloMax Discover (Promega) equipped with NanoBRET 450/8 nm bandpass and 600 nm longpass filters with a 0.3 second reading setting. A corrected BRET ratio was calculated and is defined as the ratio of the emission at 600 nm/450 nm for experimental samples (i.e.
  • BRET ratios are expressed as milliBRET units (mBU), where 1 mBU corresponds to the corrected BRET ratio multiplied by 1000.
  • Histone lysine demethylase KDM4A generates TSSGs of regions on chromosomes 1 and X (Black et al., 2013). It was hypothesized that additional histone lysine demethylases might also be involved in regulating TSSGs.
  • an unbiased siRNA screen was conducted against all lysine demethylase families (KDMl through KDM7) with at least two independent siRNAs (Figure 1A). These were performed in immortalized retinal pigment epithelial cells (RPEs) that have a nearly diploid genome (Black et al., 2013; Jiang et al., 1999).
  • KDM5A depletion was sufficient to cause other previously identified TSSGs to undergo copy number gains.
  • KDM5A depletion caused significant copy number gains for other TSSGs ( Figure 1G; lq21.2 and Xql3.1), while control regions did not have copy number changes- lq23.3, lqtel and X centromere ( Figure 1G).
  • chromatin immunoprecipitation (ChIP) analyses demonstrated a significant reduction of KDM5A at TSSG sites upon KDM5A siRNA treatment
  • TSSGs are transient and occur in an S phase-dependent manner (Black et al., 2015; Black et al., 2013).
  • KDM5i KDM5-C70
  • KDM5 inhibitor Treatment with KDM5 inhibitor causes a global change in H3K4me3 level as observed previously (Johansson et al., 2016).
  • RPE cells generated copy gains after 48h or 72h of drug exposure (no wash off); however, DNA copy gains significantly diminished 24h after the KDM5 inhibitor was removed (24h wash off, Figure 2A-C).
  • Rereplication could be one mechanism underlying TSSG generation (Black et al., 2015; Black et al., 2013).
  • KDM5A depletion induced rereplication we first performed cesium chloride gradient ultracentrifugation to isolate rereplicated DNA (termed heavy-heavy, H:H; Figure 2G) for both control and KDM5A knockdown cells. The heavy-heavy fractions for each cellular condition were pooled together before the genomic DNA was purified and quantified by polymerase chain reaction (qPCR) ( Figure 2G).
  • KDM5A demethylates H3K4me3/2/l, which associates with active gene expression (Christensen et al., 2007; Iwase et al., 2007; Klose et al., 2007). Since KDM4A overexpression and stabilization generate TSSGs (Mishra and Whetstine, 2016), we tested whether KDM4A protein levels increased after KDM5A depletion. KDM4A protein levels did not change upon KDM5A depletion ( Figure 3F). Another possibility is that KDM5A depletion targets KDM4A to sites undergoing TSSG. The Mathematics domains within KDM4A recognize H3K4me3 and H4K20me3 (Huang et al., 2006; Spektor and Rice, 2009) and are required for TSSGs (Black et al., 2013).
  • KMTs methyltransferases
  • siRNA depletion of H3K4 KMTs KMT2A, KMT2B, KMT2C, KMT2D, SETD1 A and SETD1B
  • KMT2A, KMT2B, KMT2C, KMT2D, SETD1 A and SETD1B siRNA depletion of H3K4 KMTs
  • Each KMT was depleted with at least two independent siRNAs.
  • the single and co-depletions for each KMT siRNA were verified for knockdown and assessed for major alterations in cell cycle profiles by FACS. Minor yet significant cell cycle arrests were observed with KMT siRNAs.
  • SETD1 A depletion alone resulted in an increase in Gl/S as previously reported (Tajima et al., 2015). Additional cell cycle changes occurred upon
  • KMT2A overexpression resulted in DNA copy gains at lp32.3 ( Figure 4G).
  • This region has been used as a control region for TSSG experiments and was not regulated by KDM4A overexpression or stabilization (Black et al., 2015; Black et al., 2013; Black et al., 2016) or KDM5A depletion (Figure 5A- B). Therefore, KMT2A overexpression generated a site-specific copy gain through another set of chromatin regulators, specifically KDMs associated with H3K4 methylation.
  • KDMs related to H3K4 could balance KMT2A-dependent DNA copy gains at lp32.3.
  • An siRNA screen against KDM5 family members identified KDM5B as a significant regulator of lp32.3 DNA copy gains (Figure 5A- B).
  • chemical inhibition of the KDM5 family members also generated copy gains of both lp32.3 and 1 q21.3 regions ( Figure 5C).
  • Figure 5D To determine if the lp32.3 copy gains are transient, the KDM5i drug was washed off (Figure 5D) and the copy gains returned to baseline levels, suggesting that the lp32.3 copy gains are transiently generated (Figure 5E).
  • KDM5B overexpression could block KMT2A-generated lp32.3 copy gains (Figure 5F and Figure 8X), while not interfering with the other KMT2A copy gained site (lq21.3) under KDM5A regulation ( Figure 5F).
  • introduction of H3K4M abrogated KDM5B and KMT2A-dependent lp32.3 copy gains ( Figure 5G-H and Figures 8Y-A').
  • H3K4M blocked both the copy gained regions generated by KMT2A (lp32.3 and lq21.3) even though specific KDM5 members controlled these TSSGs.
  • KDM4A KDM4B and KDM4C also have Tale domains that are involved in their recruitment to chromatin (Pedersen et al., 2016; Su et al., 2016). Therefore, we hypothesized that additional KDM4 enzymes could be involved in regulating the KDM4A-independent lp32.3 DNA copy gains.
  • KDM4A-C family members and assessed whether copy gains occurred at lp32.3 locus. Only KDM4B overexpression resulted in a significant lp32.3 copy gains ( Figure 6A-B). Furthermore, lp32.3 copy gains required KDM4B catalytic activity (Figure 6C).
  • KDM4B overexpression caused rereplication at the lp32.3 locus. Upon KDM4B overexpression, rereplication was observed within the lp32.3 region covered by the DNA FISH probe ( Figure 6D).
  • KDM4B depletion abrogated KMT2A-generated lp32.3 copy gains, demonstrating that KDM4B was essential for the copy gain generation by KMT2A ( Figure 7A and Figure S7A-B).
  • KDM4B was required for the copy gain generated with KDM5B depletion, while KDM4A depletion did not have any effect on lp32.3 copy gains ( Figure 7B).
  • RPE cells were transfected with two independent siRNA (life technologies) to either KDM4A (sl8635 and sl8636) or KDM4B (s22867 and s229325) before being treated with the KDM5 inhibitor KDM5 C-70 (10 ⁇ ).
  • KDM4A sl8635 and sl8636
  • KDM4B s22867 and s229325
  • KDM4A depletion illustrating the importance of KDM4A in the KDM5 inhibitor generated copy gains.
  • KDM4B depletion blocked the same KDM5 inhibitor's ability to generate lp32.3 rereplication.
  • KDM4A lysine demethylase induces site-specific copy gain and rereplication of regions amplified in tumors. Cell 154, 541-555.
  • RBP2 belongs to a family of demethylases, specific for tri-and dimethylated lysine 4 on histone 3. Cell 128, 1063-1076.
  • Diskin S.J., Hou, C, Glessner, J.T., Attiyeh, E.F., Laudenslager, M., Bosse, K., Cole, K., Mosse, Y.P., Wood, A., Lynch, J.E., et al. (2009). Copy number variation at 1 q21.1 associated with neuroblastoma. Nature 459, 987-991.
  • retinoblastoma binding protein RBP2 is an H3K4 demethylase.
  • Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape. Science 352, 844-849.
  • H3K4me3 demethylation by the histone demethylase KDM5C/JARID1C promotes DNA replication origin firing. Nucleic Acids Res 43, 2560-2574.
  • SETD1 A modulates cell cycle progression through a miRNA network that regulates p53 target genes. Nat Commun 6, 8257.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The present invention relates, at least in part, to methods for controlling DNA amplification, e.g., in cancer cells.

Description

METHODS FOR THE TREATMENT OF CANCER
CLAIM OF PRIORITY
This application claims the benefit of U.S. Provisional Application Serial No. 62/428,941, filed on 1 December 2016. The entire contents of the foregoing are incorporated herein by reference. FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with Government support under Grant No.
GM097360 awarded by the National Institutes of Health. The Government has certain rights in the invention.
TECHNICAL FIELD
The present invention relates, at least in part, to methods for controlling DNA amplification, e.g., in cancer cells.
BACKGROUND
Copy number variation (CNV) is commonly observed during normal development and/or in response to cellular or environmental cues (Mishra and Whetstine, 2016). For example, specific chromosomal regions are amplified in the salivary gland of sciarid flies, which in turn, helps in synthesizing structural proteins during cocoon formation. In Drosophila, the chorion genes are selectively amplified in S phase in the follicle cells that directly impacts egg shell formation (Calvi et al., 1998; Calvi and Spradling, 1999). Similarly, site-specific rereplication and copy gains also have been observed under hypoxic conditions in primary T-cells, cancer cells and tumors (Black et al., 2015). In the case of hypoxia, at least one of the regions resulted in the increased expression of the drug resistant oncogene CKSIB (Black et al., 2015), which associated with reduced drug response (Black et al., 2016). These observations illustrate that site-specific rereplication and amplification occur during development and upon environmental cues. However, the molecular mechanisms that control these processes are just beginning to be understood.
Cancer cells typically have multiple chromosomal aberrations. Genome-wide studies of somatic copy number alterations (SCNAs) across multiple tumor types have demonstrated that specific chromosomal regions exhibit higher frequencies of gains/amplifications, with a concomitant increase in the associated gene expression (Beroukhim et al., 2010; Kim et al., 2013; Zack et al., 2013). These regions frequently harbor pro-survival genes and oncogenes. For example, the
amplification/gain of chromosome lql2-25 (chr lql2-25) are associated with a number of drug resistance-associated oncogenes [e.g, MCLl, CKSIB; (Beroukhim et al., 2010; Dimova et al., 2009; Diskin et al., 2009; Fonseca et al., 2006; Goeze et al., 2002; Inoue et al., 2004; Kendall et al., 2007; Petersen et al., 2000; Weir et al., 2007)]. These studies illustrate that certain tumors may express different genes from consistently observed amplified regions. In other cases, drug selection can result in the appearance of integrated or extrachromosomal gene amplification as seen for dihydrofolate reductase (DHFR) when cells are treated with methotrexate (Alt et al., 1978; Biedler and Spengler, 1976a, b; Haber and Schimke, 1981) or the negative selection of extrachromosomal amplification for epidermal growth factor receptor (EGFR) gene amplifications in glioblastoma patients (Nathanson et al., 2014). A recent study illustrated that -50% of the tumors evaluated contained
extrachromosomal amplifications that impact genetic heterogeneity and tumor adaptation (Turner et al., 2017). Even though DNA copy gains are frequent in cancer, little knowledge exists about the mechanisms that produce site-specific DNA copy gains de novo or under selective pressure.
SUMMARY
Acquired chromosomal DNA amplifications are features of many tumors. Mechanisms directly controlling site-specific DNA copy gains are not well defined. Transient site-specific copy number gains (TSSGs) occur upon overexpression and stabilization of the histone H3 lysine 9/36 (H3K9/36) tri-demethylase KDM4A. The present study identified a compendium of H3K4 modifying chromatin regulators that function with H3K9 and H3K36 regulators to orchestrate TSSG formation.
Specifically, the H3K4 tri-demethylase KDM5A and specific COMPASS/KMT2 H3K4 methyltransferases modulate specific TSSG loci through H3K4 methylation states, and in turn, KDM4A recruitment. Furthermore, another network of chromatin modifiers, MLL1-KDM4B-KDM5B, modulate copy number regulation at a unique genomic locus in a KDM4A-independent manner. This epigenetic addressing system defines site-specific DNA rereplication and amplifications through regulation of lysine methylation balance by histone KMTs and KDMs. Thus, provided herein are methods for treating a subject who has a cancer with reduced levels or activity of KDM5A, or who is being treated with a KDM5A inhibitor, by administering to the subject a therapeutically effective amount of an inhibitor of KDM4A.
In some embodiments, the methods include identifying a subject who has a cancer with a KDM5A gene suppression/mutation/deletion.
In some embodiments, the methods include identifying a subject who has an amplification at lql2h, lql2/21, lq21.2, Iq21.3/CKS1B, or Xql3.1.
Also provided herein are methods for treating a subject who has a cancer with reduced levels or activity of KDM5B, or who is being treated with a KDM5B inhibitor, by administering to the subject a therapeutically effective amount of an inhibitor of KDM4B.
In some embodiments, the methods include identifying a subject who has a cancer with a KDM5B gene suppression/mutation/deletion.
In some embodiments, the methods include identifying a subject who has an amplification at lp32.3.
In some embodiments, the methods include administering a cdk4/6 inhibitor to a subject who has an amplification at lp32.3.
In addition, provided herein are methods for treating a subject who has a cancer with increased levels or activity of a KMT2 family member, by administering to the subject a therapeutically effective amount of an inhibitor of KDM4.
In some embodiments, the inhibitor of KDM4 inhibits KDM4A, KDM4B, or both KDM4A and KDM4B.
In some embodiments, the KMT2 family member is KMT2A, KMT2B, KMT2C, KMT2D, SETD1A or SETD1B.
Also provided herein are methods for treating a subject who has a mixed- lineage leukemia (MLL) gene (KMT2A) translocation, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of KDM4. In some embodiments, the subject has acute myeloid leukemia (AML), the acute lymphoblastic leukemia (ALL), or biphenotypic (mixed lineage) leukemia.
Further provided herein are methods for treating a subject who has cancer, by administering to the subject (i) an inhibitor of KDM4A and an inhibitor of KDM5A; or (ii) an inhibitor of KDM4b and an inhibitor of KDM5B. In some embodiments of the methods described herein, the cancer is a carcinoma, sarcoma, myeloma, metastatic disorder, or a hematopoietic neoplastic disorder.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
DESCRIPTION OF DRAWINGS
Figures 1 A-H. KDM5A inhibition promotes site-specific copy gains.
(A) Schematic of siRNA screen in RPE cells against KDM1 through KDM7 family members with two independent siRNAs. DNA FISH was performed post 72hr of knockdown. (B) Representative DNA FISH images for the indicated KDM5 family siRNAs for chromosome lql2h (green), 8c (red) and merged with DAPI (blue) are shown. (C) Quantification of FISH in KDM5 siRNA treated RPE for chromosome lql2h and chromosome 8 centromere. (D) KDM5 A knockdown results in site- specific copy gains of lql2h region in H2591 lung cancer cells. (E) RPE cells treated with different concentrations of KDM5 inhibitor (KDM5 -C70) for 72hr results in dose dependent increase of copy gains of lql2h. (F) Neuroblastoma cell line, SK-N- AS treated with different concentrations of KDM5 inhibitor (KDM5 -C70) for 48hr results in dose dependent increase of copy gains of lql2h. (G) KDM5 A knockdown results in site-specific copy gains of additional regions including lq21.2 and Xql3.1 with no changes in lq23.3, lqTel and Xcen demonstrating the site-specific nature of copy gains rather than global genome instability. (H) ChIP analysis shows significant decrease in the occupancy of KDM5A at copy gained regions upon siRNA depletion (i.e., chrlsat2 and \q\2/2\ at multiple sites; \q\2/2\-\ (chrl : 142,704,000) and lql2/21-2 (chrl : 142,706,000)]. Error bars represent the SEM. Any significant differences (P<0.05) are indicated with an asterisk by two-tailed Student's t-test. Scale bar corresponds to 5 μιη.
Figures 2A-K. KDM5A-associated site-specific copy gains are transient, require S phase and are rereplicated.
(A) A schematic of KDM5 inhibitor treatment conditions are shown. Cells were treated with KDM5-C70 for 48hr, followed by drug wash-off with fresh media (24hr wash off). Cells were collected 24h post drug removal. (B-C) Wash off with fresh media of KDM5i results in significantly diminished copy gains while copy gains are present at 48hr (B) and 72hr (C) post drug treatment. (D) A schematic representing drug treatment conditions are shown. Cells were treated with hydroxyurea (HU) for 20hr, followed by KDM5 inhibitor (KDM5i) for 24h. HU was replaced with fresh media and cells were harvested for further analyses after 6hr of release. (E) KDM5 inhibition (KDM5 -C70) after HU arrest results in the attenuation of copy gains. (F) The copy gains are restored when cells are released from 6hr from HU arrest. (G) A cesium chloride density gradient centrifugation was performed in control and
KDM5 A knockdown RPE cells treated with 100 μΜ BrdU for 12hr prior to harvest. The graph represents heavy-heavy (H:H), heavy-light (H:L) and light-light (L:L) peaks of the fractionated gradients. (H) Rereplicated pool of heavy-heavy DNA (H:H) was isolated and analyzed by qPCR to determine fold change in rereplication by normalizing to its input DNA. A significant increase was observed at the copy gained regions. (I) A schematic for CDK1 inhibitor (Ro-3306) treatment conditions are shown. KDM5 A was depleted for 48h with siRNA followed by a cell cycle arrest at G2 using Ro-3306 for 20hr. (J) Copy gains persist after siKDM5 A and Ro-3306 treatment using DNA-FISH analyses. (K) ChIP qPCR for DNA Polymerase alpha (Pol a) was performed in cells with siKDM5A and Ro-3306 treatment. Significant enrichment of Pol a was observed at the copy gained locus, sat2 and lql2/21 but not at chrlq23.3, a negative region that is not copy gained after KDM5A depletion. Error bars represent the SEM. Significant differences (P<0.05) are indicated with an asterisk by two-tailed Student's t-test.
Figures 3A-F. KDM5A depletion promotes H3K4me3 and KDM4A recruitment at copy gained loci.
(A) KDM4A and KDM5A were co-depleted with siRNAs for 72hr followed by DNA FISH for chrlql2h, chrlq21.2 and chrlq23.3 in RPE cells. Significant rescue of the copy-gained regions was observed after KDM4 A depletion (lql2h and lq21.2), while no change was observed at the negative region, lq23.3. (B) ChIP for KDM4A demonstrated significant recruitment of KDM4A at the copy-gained locus compared to a non-copy gained site (lq23.3). (C) ChIP for H3K4me3 mark was performed in control and KDM5 A knockdown cells. A significant increase in the histone 3 lysine 4 trimethylation mark (H3K4me3) at the copy-gained chrl sat2 and chrlql2/21 genomic locus was observed in siKDM5A cells. (D) Introduction of a histone H3.3 lysine 4 mutation to methionine (H3K4M) causes decreased copy number gains of lql2h and lq21.2 in siKDM5A cells, while having no impact on lq23.3. (E) H3K4M mutation rescued lq21.2 copy gains generated with KDM4 A overexpression
(KDM4A OE). (F) Western blot shows decreased KDM5A and KDM4A protein level in samples treated with KDM4A siRNA, KDM5A siRNA and both for 72h. Actinin is used as a loading control. Error bars represent the SEM. Any significant differences (P<0.05) are indicated with an asterisk by two-tailed Student's t-test.
Figures 4A-I. Site-specific copy gains are H3K4 KMT-dependent.
(A) An ideogram of the chromosome lq arm shows a summary of the specific H3K4 KMTs that rescue copy gains caused by KDM5 A depletion and that are generated upon KMT overexpression. The DNA FISH data summarized in panel A can be found in Figure S5. (B) Depletion of KDM4A with siRNA followed by SETDIB overexpression abrogates SETD IB-dependent lql2h copy gains. (C) Model depicting interplay of KDM5 A-KDM4 A- SETD 1 B at the lql2h locus is shown. (D-E) Depletion of KDM4A with siRNA followed by KMT2D and SETDIB overexpression abrogates lq21.2 copy gains. (F) Model depicting interplay of KDM5 A-KDM4 A- KMT2D and SETDIB at the lq21.2 locus is shown. (G) Depletion of KDM4A with siRNA followed by KMT2A overexpression only abrogates lq21.3 copy gains but not lp32.3. (H) siRNA depletion of KDM4A followed by KMT2D overexpression abrogates chrl q21.3 copy gains. (I) Model depicting interplay of KDM5A-KDM4A- KMT2A and KMT2D on lq21.3 locus is shown. Error bars represent the SEM.
Significant differences (P<0.05) are indicated with an asterisk by two-tailed Student's t-test.
Figures 5A-H. KDM5B regulates the lp32.3 TSSG.
(A) Images from lp32.3 and lq21.3 DNA-FISH are shown for control, siKDM5A, siKDM5B and siKDM5C. DAPI (in blue), lp32.3 (in green), lq21.3 (in red) and merged images are shown. (B) KDM5B depletion results in a significant increase in lp32.3 locus, while KDM5 A depletion promotes lq21.2. (C) lp32.3 and 1 q21.3 copy gains are observed after 72hr of KDM5-C70 inhibitor treatment in RPE cells. (D) Schematics showing KDM5-C70 treatment for 48hr, followed by drug wash-off with fresh media. Cells were collected 24h post drug removal. (E) KDM5- C70 treatment generates significant lp32.3 copy gains after 48hr. These copy gains returns to baseline after drug wash-off. (F) Co-overexpression of KMT2A and KDM5B rescues the lp32.3 locus increase but not for lq21.3. (G) Introduction of H3K4M followed by KDM5B siRNA depletion resulted in decreased copy number gains of lp32.3. (H) Introduction of H3K4M causes decreased copy number gains at both lp32.3 and 1 q21.3 after KMT2A overexpression compared to KMT2A overexpression alone. Error bars represent the SEM. Any significant differences (P<0.05) are indicated with an asterisk by two-tailed Student's t-test. Scale bar corresponds to 10 μπι.
Figures 6A-F. KDM4B promotes site-specific copy gains and rereplication at lp32.3.
(A) Representative images from DNA FISH of lp32.3 and lq21.3 copy gains after KDM4 family overexpression are depicted. DAPI (in blue), lp32.3 (in green), 1 q21.3 (in red) and merged images are shown. (B) The histone lysine demethylase 4 family was transiently overexpressed in RPE cells for 24h and assessed for lq21.3 and lp32.3 copy gains. Only KDM4B overexpression resulted in significant copy gains of chrlp32.3 loci. (C) Copy gains at the lp32.3 locus are recapitulated in RPE cells stably expressing KDM4B. Inhibition of catalytic activity by HI 89A mutation abrogates lp32.3 copy gains. (D) Schematic depicting genomic primers used for testing rereplicated regions in KDM4B stably expressing cells. The primers labeled as 1 through 6 encompass the regions covered under the FISH probe. Rereplicated DNA was isolated and analyzed by qPCR in control and GFP-KDM4B stable cells for six different lp32.3 genomic regions (marked in the ideogram as 1 through 6). The fold change in rereplication compared to control DNA is shown. A significant increase was observed at the indicated regions as represented by an asterisk. (E) NanoBRET assay showing interaction of KDM4A (top panel) and KDM4B (bottom panel) with MCM3, MCM4, MCM5, MCM7, POLA2, POLD1 and HaloTag alone (negative control). HEK293 cells were co-transfected with NanoLuc-KDM4A and NanoLuc- KDM4B (energy donors) with the following energy acceptors; HaloTag-MCM3, HaloTag-MCM4, HaloTag-MCM5, HaloTag-MCM7, HaloTag-POLA2, HaloTag- POLD1, or HaloTag alone (negative control). Shown are the graphs of corrected BRET ratios obtained for each possible donor-acceptor combination. (F) NanoBRET donor saturation assay showing the specificity of the interaction of KDM4B with MCM3, MCM4, MCM5, MCM7, POLA2, and POLD1. HEK293 cells were co- transfected with a fixed low level of NanoLuc-KDM4B in the presence of increasing amounts of acceptor DNA of HaloTag-MCM3, HaloTag-MCM4, HaloTag-MCM5, HaloTag-MCM7, HaloTag-POLA2, HaloTag-POLD 1 , or HaloTag alone (negative control). Error bars represent the SEM. Any significant differences (P<0.05) are indicated with an asterisk by two-tailed Student's t-test. Scale bar corresponds to 10 μπι.
Figures 7A-J. KDM4B recruitment promotes lp32.3 TSSG formation through reduced H3K36me3.
(A) Depletion of KDM4B with siRNA followed by KMT2A overexpression only abrogates lp32.3 copy gains but not lq21.3. (B) Knockdown of KDM4B rescues siKDM5B associated chrlp32.3 copy gains by DNA FISH. (C) Introduction of H3K4M caused decreased lp32.3 copy gains after KDM4B overexpression compared to KDM4B alone. (D-E) RPE cells transduced with either wild type H3 or H3K4M before being transiently transfected with NanoLuc-KDM4B (KDM4B OE) along with HALO-H2B or HALO-H4 in 12- well plates. H3K4M results in a decreased recruitment of KDM4B at the target histones H2B and H4 in vivo. (F) ChIP for KDM4B recruitment was performed in control and GFP-KDM4B stably expressing cells at the indicated regions 1ρ32.3-3, 1ρ32.3-4, lp32.3-5 and lq23.3. The position ofthese loci are noted in Figure 6D. (G) Quantification of FISH for lq21.2 and lp32.3 in H3.3 lysine 9 to methionine mutation (K9M) and H3.3 lysine 36 to methionine mutation (K36M) are shown. (H) ChIP for H3 and H3K36me3 was performed in control and KDM4B stably expression cells. Ratio of H3K36me3/H3 is plotted for control and GFP-KDM4B cells at the indicated genomic regions. (I) A model depicting interplay of KDM5B-KDM4B-KMT2A on chrlp32.3 locus is shown. (J) A model showing KMT-KDM cross-talk at representative locus on chromosome 1. Error bars represent the SEM in all the panels. Any significant differences (P<0.05) are indicated with an asterisk by two-tailed Student's t-test. Figures 8 A- A'. Site-specific amplifications are H3K4 methylation dependent. (A-D) The rescue of KDM5 A depletion gains with co-depletion of H3K4 methyl transferase (KMT2A, KMT2B, KMT2C, KMT2D, SETDlA and SETD1B) at lql2h loci is shown (E-H) The rescue of KDM5 A depletion gains with co-depletion of H3K4 methyl transferase (KMT2A, KMT2B, KMT2C, KMT2D, SETD1 A and
SETD1B) at lq21.2 loci is shown. (I-L) The rescue of KDM5A depletion gains with co-depletion of H3K4 methyl transferase (KMT2A, KMT2B, KMT2C, KMT2D, SETDlA and SETD1B) at lp32.3 and lq21.3 locus is shown. (M-S) FISH analysis on lql2h, chromosome 8 and lq21.2 on samples with KMT2A, KMT2B, KMT2D and SETD1B for transient overexpression for 24h was performed. (T-W) FISH analysis was performed on lp32.3 and lq21.3 on samples with transient overexpressions of KMT2A, KMT2B, KMT2D, SETDlA and SETD1B for 24h. (X) qPCR and FACS analysis was performed to validate KMT2A and KDM5B overexpressions as shown in the graph. (Y) western blot for H3K4me3 and histone H3 was performed in samples with control and KDM5B knockdown infected with H3.3 wild type and K4M mutations. (Z) FACS was done in samples with control and KMT2A overexpression infected with H3.3 wild type and K4M mutations. (A) Western blot for FLAG and histone H3 was done in samples with control and KMT2A overexpression infected with H3.3 wild type and K4M mutations. Error bars represent the SEM. Any significant differences (P<0.05) are indicated with an asterisk by two-tailed Student's t test.
Figures 9A-B. KDM5 inhibitor generated copy gains are blocked selectively by specific KDM4 members. (A) KDM4 A depletion blocks KDm5 inhibitor generated lql2h, while not impacting the gains of a control region (8c). (B) KDm4B depletion blocked KDM5 inhibitor lp32.3 copy gains. Error bars represent the SEM and the * refers to P<0.05 based on Student T-test.
DETAILED DESCRIPTION
DNA amplifications are observed in both normal and cancerous cells (Mishra and Whetstine, 2016). These chromosomal changes consist of gains or losses in whole chromosomes, segmental chromosomal arms, and site-specific changes (Mishra and Whetstine, 2016). However, the mechanisms regulating site-specific DNA amplifications are not well understood. In this study, we have identified a
compendium of epigenetic factors, specifically lysine methyltransferases and demethylases, that directly impact locus-specific DNA copy gains (Figure 13). These chromatin modulators control histone lysine 4 methylation state, and in turn, coordinate specific H3K9 and K36 tri -demethylases to regulate rereplication and DNA amplifications of specific genomic loci. Described herein is a mechanism for achieving site-specific copy gains through the cross-talk of eight chromatin regulators and their modulation of localized chromatin states, and methods for governing DNA amplifications within certain genomic loci.
H3K4 methylation and TSSGs
In recent years, there is an increasing awareness that chromatin and chromosomal architecture play an important role in DNA replication, and in turn, genome stability (Dileep et al., 2015; Donley and Thayer, 2013; MacAlpine and Almouzni, 2013; Rivera et al., 2014; Smith and Aladjem, 2014). One key mechanism that regulates various aspects of DNA replication is dependent on histone post- translational modifications (PTMs). Chromatin modification states, such as acetylation and methylation of histones play an important role in regulating origin recognition complex (ORC) protein recruitment and defining replication origins (MacAlpine and Almouzni, 2013). For example, the H4K20mel methyltransferase SET-domain containing protein 8 (PR-Set7/Set8) promotes pre-replication complex assembly and origin activity (Tardat et al., 2010). Subsequently, Set8 stabilization results in increased H4K20me2/3 through the recruitment and increased function of the H4K20 lysine di/tri -methyl transferases Suv4-20hl/2, resulting in origin firing (Beck et al., 2012; Kuo et al., 2012; Tardat et al., 2010). In addition to H4K20 methylation, recent reports have also suggested that H3K4 methylation could promote rereplication (Huang et al., 2016; Lu et al., 2016b). Specifically, depletion of WDR5, which blocks most COMPASS family of H3K4 KMTs, reduced the rereplication and the polyploidy phenotype observed when replication licensing was disrupted (Lu et al., 2016b). Consistent with this observation, histone lysine 4 demethylases have also been suggested to regulate DNA replication. For instance, KDM5C/JARID1C regulates pre-initiation complex assembly and proper firing of the early replication origins (Rondinelli et al., 2015). Additionally, KDM5A was suggested to regulate ORC recruitment to origin sites and thus regulate appropriate replication (Huang et al., 2016). Instead of causing global genomic instability, the present results suggest that COMPASS H3K4 KMT enzymes and the H3K4 tri-demethylases are controlling rereplication and DNA copy gains at specific loci. For example, the balance between H3K4 KMTs and KDMs "toggles" H3K4 methylation at specific loci, which then regulates their ability to rereplicate and amplify. Consistent with this hypothesis, each KDM5 H3K4 tri-demethylase controls a distinct subset of TSSG sites; KDM5A controls chrlql2h, lq21.2 and 1 q21.3 while KDM5B is involved in the regulation of lp32.3 locus (Figure 7 J). Similarly, each COMPASS family H3K4 methyltransferase regulates distinct genomic sites within the subset of known TSSGs being generated (Figure 7J). However, interference with H3K4 methylation by introducing H3K4M was able to suppress all the observed TSSGs. In a similar fashion, suppression of the KDM5 family activity through chemical inhibition allowed all the observed TSSGs to emerge. The H3K4M and KDM5 inhibitor experiments emphasize that H3K4 methylation is a critical determinant of rereplication and that there is an exquisite specificity in KMTs-KDMs in controlling site-specific amplifications. This underscores the importance of regulating the local chromatin states so that DNA amplifications are prevented or allowed to occur. The present observations also emphasize how distinct sets of DNA amplifications can actually be connected through a common mechanism instead of being a byproduct of random instability.
Described herein are methods to reverse or control the appearance of certain DNA copy gains since they are transiently generated in each cell cycle. The ability to therapeutically control DNA amplifications has implications in the area of tumor heterogeneity and drug responses.
KDM4 and KDM5 cross-talk generates TSSGs
Previously, it was demonstrated that overexpressed KDM4A required the Tudor domains to cause TSSGs (Black et al., 2013). The Tudor domain has been shown to recognize H4K20me2/3 and H3K4me3 (Huang et al., 2006; Mallette et al., 2012; Spektor and Rice, 2009). However, the significance of either set of
modifications in modulating KDM4A driven biology remains an unanswered question. In this study, an unbiased KDM depletion screen uncovered that H3K4 methylation was critical for the KDM4A-dependent regulation of site-specific rereplication and DNA copy gains. This was demonstrated herein through direct manipulation of the H3K4 methylation as well as establishing that specific KMT2 and KDM5 family members were important in generating KDM4A-dependent copy gains (Figure 73). For example, KDM5A depletion resulted in rereplication and TSSG formation for specific genomic loci that were dependent on increased H3K4 methylation and KDM4A recruitment. Furthermore, SETD1B, MLL1/KMT2A, MLL2/KMT2B and MLL4/KMT2D modulated subsets of loci that undergo rereplication and copy gains upon KDM5 A depletion and KDM4A overexpression and stabilization. These data highlight how the enrichment of H3K4 methylation can be used as a honing beacon for the endogenous recruitment of KDM4A. Without wishing to be bound by theory, it is believed that increased H3K4me3 helps in recruiting KDM4A, through its Tudor domain, which further removes H3K9me3 and H3K36me3, eventually making the chromatin more permissive for DNA rereplication.
KDM4 targeting is not just limited to KDM4A-dependent TSSGs. We have identified, for the first time, a non-KDM4A dependent copy number regulation of chromosome lp32.3 locus by a network of KDM5B-KDM4B-KMT2A hi stone modifying proteins. We demonstrated that KDM4B also required H3K4 methylation to generate the lp32.3 TSSG. MLL1/KMT2A was required for the KDM4B copy gains, while KDM5B prevented KDM4B copy gains. Furthermore, disruption of H3K4 methylation caused less KDM4B on the chromatin, illustrating the importance of this modification in recruiting another KDM4 member, and in turn, site-specific rereplication and DNA amplification. These results suggest the existence of a complex molecular network of KMTs and KDMs that intricately regulate site-specific copy gains in the genome. This highlights the importance of KDM4 members for TSSG generation. Therefore, chemical (Black et al., 2015) or RNAbased strategies (Black et al., 2016) to target the KDM4 family could have a profound effect on DNA copy number heterogeneity driven by the H3K4 modifying enzymes in diseases such as cancer.
KDM4A amplified TSSGs were generated in the presence of both H3K9M and H3K36M; however, the KDM4B copy gained locus was specific to H3K36M. It is possible that the amplifications of specific regions are distinctly controlled by K36 and/or K9 methylation states. H3K36M is a known "onco-histone" associated with certain tumor types (Lu et al., 2016a; Papillon-Cavanagh et al., 2017). KDM5-related TSSG kinetics
Depletion of KDM5 A results in TSSGs that occur outside of S phase, which was consistent with a previous report suggesting replication occurred in other cell cycle phases (Huang et al., 2016). However, the appearance of these TSSGs was in sharp contrast to KDM4A overexpression alone (Black et al., 2013). These data suggest that the H3K4 methylation state provides a permissive chromatin for allowing recruitment and rereplication outside of S phase. Since KDM4A associated with and recruited replication machinery to sites that undergo rereplication and copy gain (Black et al., 2015; Black et al., 2013; Black and Whetstine, 2015), KDM5A depletion would allow this modification to persist and promote KDM4A driven copy gains, whereas resetting the H3K4 methylation would block KDM4A overexpressed protein recruitment. Consistent with this model, we demonstrated that KDM4A was recruited to enriched H3K4me3 sites that rereplicate upon KDM5 A depletion. DNA
polymerase was also enriched on these regions in late G2 when TSSGs are observed. Furthermore, H3K4M blocked the TSSGs generated upon KDM4A overexpression and KDM5 A depletion. These data highlight the importance of the localized chromatin environment in controlling site-specific rereplication and copy gains.
However, we cannot rule out the possibility that KDM5 A depletion also impacts an unknown mechanism(s) promoting copy gain removal. Future studies need to elucidate the pathways regulating the removal and regulation of TSSGs. Once the pathway(s) are identified, the relationship between KDM5 enzymes, these pathways and TSSG generation and removal can be explored.
In summary, this study documents a repertoire of chromatin regulators that can generate transient DNA copy gains. These events are not inherited and require S phase. Given different cellular division rates within a tissue/tumor, the differentially cycling cells could create significant heterogeneity as a consequence of rereplicated fragment generation. The expression/activity/mutation status of the identified KMTs and KDMs could impact the spatial and temporal regulation of the rereplicated fragments. Therefore, cells that are actively dividing and have the appropriate genetic background will be able to execute these alterations, which could provide a basis for DNA copy number heterogeneity within a tissue and/or tumor (Mishra and Whetstine, 2016). These transiently generated copy gains could be generated in each cycle and then resolved by an as of yet undiscovered mechanism. These pathways may work together with epigenetic dysregulation to control tumor heterogeneity and drug responses (Burrell and Swanton, 2014; Mishra and Whetstine, 2016), providing diagnostics and therapeutic options.
Methods of Treatment
The present data identifies an epigenetic addressing system for defining site- specific DNA rereplication and amplifications through modulating methylation states via histone KMT and KDM cross-talk. Different KMTs and KDMs control DNA copy gain of different genomic regions (Figure 7 J). This work establishes that a network of epigenetic regulators can regulate DNA rereplication in a spatial and temporal fashion. Hence, drugs targeting these enzyme pathways can be selectively used to inhibit the amplification of regions associated with cancer progression, resistance and drug responses. In contrast to blocking amplifications, these findings provide a basis to reestablish cellular sensitivities when tumor suppressors are lost or contain mutations, especially in the context of loss of heterozygosity (LOH) at tumor suppressors or genes critical for development. Being able to control DNA copy number has implications across numerous diseases.
As described herein, the present methods can be used to alter DNA copy numbers in a cell, e.g., in a living mammal, e.g., to treat cancer. The methods can include administration of therapeutically effective amounts of one or more inhibitors of a KDM, e.g., a small molecule or inhibitory nucleic acid that inhibits activity or expression of a KDM, e.g., KDM4A, KDM4B, KDM5A, or KDM5B, or of a
KMT2/MLL, e.g., KMT2A, KMT2B (also known as MLL2, WBP7, sometimes called MLL4), KMT2C (also known as MLL3, HALR), KMT2D (also known as MLL4, ALR, sometimes called MLL2), KMT2E (also known as MLL5), KMT2F (also known as hSETIA, SETD1A), and KMT2G (also known as hSETIB, SETD1B) genes, to a subject in need thereof.
Examples of cancers include, e.g., carcinoma, sarcoma, myeloma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.
As used herein, the terms "cancer," "hyperproliferative," and "neoplastic" refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. "Pathologic hyperproliferative" cells occur in disease states
characterized by malignant tumor growth. Examples of non-pathologic
hyperproliferative cells include proliferation of cells associated with wound repair.
The terms "cancer" or "neoplasms" include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas,
gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the disease is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
The term "sarcoma" is art recognized and refers to malignant tumors of mesenchymal derivation.
Additional examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term "hematopoietic neoplastic disorders" includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, Crit Rev. in Oncol./Hemotol. 11:267-297, 1991); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and
Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T- cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed- Sternberg disease.
In some embodiments, the cancer is related to H3.3-K36M mutations, e.g., leukemia, neuroblastoma, or sarcoma. See, e.g., Pfister and Ashworth, Nature Reviews Drug Discovery 16, 241-263 (2017).
An "effective amount" is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired
therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments.
Dosage, toxicity and therapeutic efficacy of the therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
KDM4A/KDM5A
As one example, an inhibitor of KDM4A can be used to treat a subject who has a cancer with reduced levels or activity of KDM5A, e.g., as a result of a suppressive gene mutation or deletion, e.g., as known in the art or shown in Table 1, or who is being treated with a KDM5 A inhibitor. Often, KDM5 A depletion or inhibition causes DNA amplification associated with aggressive cancers and drug resistance (e.g., amplifications at lql2h, lql2/21, lq21.2, Iq21.3/CKS1B, and Xql3.1). Cancers that are associated with inhibitory KDM5A mutations include penile cancer, stomach cancer, melanoma, brain tumors, skin cancer, colorectal adenocarcinoma, ampullary carcinoma, cervical cancer, small cell lung cancer, bladder cancer, breast cancer, esaphogogastric cancer, and anal cancer.
Table 1. Frequent Mutations in KDM5A
Impact
DNA Change Type Consequences (VEP)
chrl2:g.283549delT Deletion 3 Prime UTR KDM5A MODIFI ER
Frameshift KDM5A
chrl2:g.307787delT Deletion G1200Dfs*9 HIGH
chrl2:g.307900T>G Substitution Missense KDM5A K1162Q MODERATE Table 1. Frequent Mutations in KDM5A
Impact
DNA Change Type Consequences (VEP) chrl2 :g.355238G>A Substitution Stop Gained KDM5A R264* HIGH chrl2 :g.318325C>T Substitution Missense KDM5A R893Q MODERATE chrl2 :g.323202G>A Substitution Missense KDM5A R719C MODERATE chrl2 :g.355237C>T Substitution Missense KDM5A R264Q MODERATE chrl2 :g.354159C>A Substitution Missense KDM5A D316Y MODERATE chrl2 :g.307603G>A Substitution Missense KDM5A R1261W MODERATE chrl2 :g.309946C>T Substitution Missense KDM5A D1079N MODERATE chrl2 :g.355208C>T Substitution Missense KDM5A A274T MODERATE
Frameshift KDM5A
chrl2 :g.323083delT Deletion D759lfs*2 HIGH chrl2 :g.284141A>T Substitution 3 Prime UTR KDM5A MODIFI ER chrl2 :g.321063C>A Substitution Stop Gained KDM5A E825* HIGH chrl2 :g.284336A>T Substitution 3 Prime UTR KDM5A MODIFI ER chrl2 :g.323664G>A Substitution Missense KDM5A R696W MODERATE chrl2 :g.307669G>A Substitution Missense KDM5A R1239W MODERATE chrl2 :g.328891C>A Substitution Stop Gained KDM5A E638* HIGH chrl2 g.309905delT Deletion Frameshift KDM5A V1093* HIGH
Synonymous KDM5A
chrl2 g.310921C>T Substitution T1060T LOW chrl2 g.365973G>C Substitution Synonymous KDM5A L166L LOW
Synonymous KDM5A
chrl2 g.285585A>T Substitution A1648A LOW chrl2 g.334255G>C Substitution Missense KDM5A I492M MODERATE chrl2 g.323663C>T Substitution Missense KDM5A R696Q MODERATE chrl2 g.284324delTT Deletion 3 Prime UTR KDM5A MODIFI ER chrl2 g.328860C>T Substitution Missense KDM5A R648Q MODERATE chrl2 g.307728C>T Substitution Missense KDM5A R1219Q MODERATE chrl2 g.334383del Frameshift KDM5A
CAGGCATGaC | Deletion N447Sfs* 17 HIGH chrl2 g.297054C>A Substitution Missense KDM5A K1407N MODERATE chrl2 g.333592C>A Substitution Missense KDM5A E516D MODERATE chrl2 g.310922G>A Substitution Missense KDM5A T1060M MODERATE chrl2 g.354117C>T Substitution Missense KDM5A V330M MODERATE chrl2 g.293162C>A Substitution Missense KDM5A S1488I MODERATE
Synonymous KDM5A
chrl2 g.307823G>A Substitution S1187S LOW chrl2 g.334325G>T Substitution Missense KDM5A P469Q MODERATE chrl2 g.323780C>T Substitution Missense KDM5A G657D MODERATE chrl2 g.352296C>T Substitution Missense KDM5A G353E MODERATE chrl2 g.363006C>A Substitution Missense KDM5A R210M MODERATE chrl2 g.388938G>A Substitution Missense KDM5A R52W MODERATE chrl2 g.306993T>C Substitution Missense KDM5A T1343A MODERATE chrl2 g.284466C>T Substitution 3 Prime UTR KDM5A MODIFI ER
Synonymous KDM5A
chrl2 g.292795G>A Substitution C1610C LOW Table 1. Frequent Mutations in KDM5A
Impact
DNA Change Type Consequences (VEP) chrl2: g.328983C>T Substitution Missense KDM5A R607H MODERATE
Splice Acceptor KDM5A
chrl2: g.329030C>T Substitution X592_splice HIGH chrl2: g.297193G>C Substitution Missense KDM5A A1361G MODERATE chrl2: g.307476G>A Substitution Missense KDM5A A1303V MODERATE chrl2: g.297172C>A Substitution Missense KDM5A S1368I MODERATE chrl2: g.307699G>A Substitution Missense KDM5A L1229F MODERATE chrl2: g.322488C>T Substitution Synonymous KDM5A R785R LOW chrl2: g.307496G>C Substitution Missense KDM5A I 1296M MODERATE chrl2: g.318173C>T Substitution Missense KDM5A E944K MODERATE
Frameshift KDM5A
chrl2: g.389069delC Deletion G8Afs*58 HIGH chrl2: g.389238C>A Substitution 5 Prime UTR KDM5A MODIFI ER chrl2: g.285339C>A Substitution 3 Prime UTR KDM5A MODIFI ER chrl2: g.352321A>C Substitution Missense KDM5A C345G MODERATE chrl2: g.323611C>A Substitution Missense KDM5A K713N MODERATE chrl2: g.292817G>C Substitution Missense KDM5A S1603C MODERATE chrl2: g.350647G>A Substitution Missense KDM5A R428W MODERATE chrl2: g.286189C>T Substitution Intron KDM5A MODIFI ER chrl2: g.295579G>A Substitution Synonymous KDM5A 114831 LOW chrl2: g.293079G>T Substitution Missense KDM5A L1516I MODERATE chrl2: g.352240T>G Substitution Missense KDM5A N372H MODERATE chrl2: g.286205T>A Substitution Intron KDM5A MODIFI ER chrl2: g.366037C>T Substitution Missense KDM5A R145H MODERATE chrl2: g.307813G>A Substitution Missense KDM5A L1191F MODERATE chrl2: g.389118C>T Substitution 5 Prime UTR KDM5A MODIFI ER chrl2: g.389361G>A Substitution 5 Prime UTR KDM5A MODIFIER chrl2: g.293006A>C Substitution Stop Gained KDM5A L1540* HIGH chrl2: g.356475C>T Substitution Synonymous KDM5A G245G LOW chrl2: g.356497T>G Substitution Missense KDM5A K238T MODERATE
Synonymous KDM5A
chrl2: g.309947G>A Substitution T1078T LOW chrl2: g.354131G>A Substitution Missense KDM5A P325L MODERATE chrl2: g.333594C>T Substitution Missense KDM5A E516K MODERATE chrl2: g.295769G>A Substitution Missense KDM5A P1420L MODERATE chrl2: g.352278C>A Substitution Missense KDM5A R359L MODERATE chrl2: ¾.276541G>A Substitution Downstream Gene KDM5A MODIFI ER chrl2:j i.350635G>C Substitution Missense KDM5A L432V MODERATE chrl2:j ?.283195delT Deletion 3 Prime UTR KDM5A MODIFI ER chrl2:j ?.283318G>A Substitution 3 Prime UTR KDM5A MODIFI ER chrl2:j ?.354132G>A Substitution Missense KDM5A P325S MODERATE chrl2:j ?.322498C>T Substitution Missense KDM5A R782Q MODERATE chrl2:f >.283152T>G Substitution 3 Prime UTR KDM5A MODIFI ER chrl2:{ >.293166C>T Substitution Missense KDM5A D1487N MODERATE Table 1. Frequent Mutations in KDM5A
Impact
DNA Change Type Consequences (VEP)
chrl2 g.334289_ Frameshift KDM5A
334290insA Insertion C481Lfs*5 HIGH
chrl2 g.363023G>T Substitution Synonymous KDM5A S204S LOW
chrl2 g.292850G>A Substitution Missense KDM5A P1592L MODERATE chrl2 g.354150G>C Substitution Missense KDM5A H319D MODERATE
Frameshift KDM5A
chrl2 g.355230delTC Deletion R266Qfs*10 HIGH
Frameshift KDM5A
chrl2 g.318248delAAGT Deletion T918Wfs*4 HIGH
chrl2 g.283651G>A Substitution 3 Prime UTR KDM5A MODIFI ER chrl2 g.309898C>A Substitution Stop Gained KDM5A E1095* HIGH
chrl2 g.352264G>T Substitution Missense KDM5A Q364K MODERATE chrl2 g.321082C>A Substitution Synonymous KDM5A R818R LOW
chrl2 g.356536G>C Substitution Stop Gained KDM5A S225* HIGH
chrl2 g.352254C>T Substitution Missense KDM5A G367E MODERATE
Synonymous KDM5A
chrl2 g.309911C>T Substitution R1090R LOW
chrl2 g.355209G>A Substitution Synonymous KDM5A D273D LOW
chrl2 g.293040_ Frameshift KDM5A
293041insAACC Insertion K1529Gfs*2 HIGH
chrl2 g.350661G>A Substitution Missense KDM5A P423L MODERATE chrl2 g.310950G>A Substitution Missense KDM5A R1051W MODERATE
Thus, the methods can include identifying a subject who has a cancer with a KDM5A gene suppression/mutation/deletion, e.g., as shown in Table 1, using methods known in the art, or identifying a subject who is being treated with a KDM5A inhibitor. In some embodiments, the methods include identifying a subject who has (e.g., by detecting the presence of) a KDM5A DNA mutation or amplification at lql2h, lql2/21, lq21.2, Iq21.3/CKS1B, or Xql3.1, using methods known in the art. An exemplary human KDM5A reference sequence is at NG 046993.1 , RefSeqGene Range 5001-114399. The methods include administering to these identified subjects an inhibitor of KDM (including KDM4), or an inhibitor of KDM4, e.g., an agent that inhibits KDM4A (preferably inhibiting levels or activity by at least 25%, or at least 50%) and does not significantly inhibit KDM5, or only inhibits KDM5 at a different (by at least 50% or at least 1 order of magnitude) concentration or dose, and/or is separate from any inhibitor of KDM5A that is being administered. In some embodiments, the agent specifically inhibits KDM4A (i.e., inhibits KDM4A and does not significantly inhibit other KDM4 family members, or only inhibits other KDM4 family members at a different (by at least 50% or at least 1 order of magnitude) concentration or dose).
KDM4B/KDM5B
In addition, an inhibitor of KDM4B (or KDM4B/C/D) can be used to treat a subject who has a cancer with reduced levels or activity of KDM5B, e.g., as a result of a suppressive gene mutation or deletion, e.g., as known in the art or Table 2, or who is being treated with a KDM5B inhibitor. In some embodiments, KDM5B depletion or inhibition causes DNA amplification associated with aggressive cancers and drug resistance (e.g., amplifications at lp32.3 locus). Cancers that are associated with inhibitory KDM5B mutations include breast, ovarian, oral, neuroblastoma, esophageal, hepatocellular carcinoma, glioma, cervical, and colorectal cancer.
Table 2. Frequent Mutations in KDM5A
Impact
DNA Change Type Consequences (VEP) chrl: g.202729013delG Deletion 3 Prime UTR KDM5B MODI FIER chrl: g.202729823G>A Substitution Missense KDM5B R1461C MODERATE chrl: g.202735497delT Deletion Frameshift KDM5B S1119Afs*4 HIGH chrl: g.202750771C>T Substitution Missense KDM5B R570Q MODERATE chrl: g.202728776_202728777insA Insertion 3 Prime UTR KDM5B MODI FIER chrl: g.202758449C>T Substitution Missense KDM5B R380H MODERATE chrl: g.202729071G>A Substitution Missense KDM5B R1534C MODERATE chrl: g.202733552C>T Substitution Missense KDM5B R1253Q MODERATE chrl: g.202777042C>T Substitution Missense KDM5B R86H MODERATE chrl: g.202762711delT Deletion Frameshift KDM5B A303Pfs*37 HIGH
Thus, the methods can include identifying a subject who has a cancer with a
KDM5AB gene suppression/mutation/deletion, e.g., as shown in Table 2, using methods known in the art, or identifying a subject who is being treated with a
KDM5B inhibitor. In some embodiments, the methods include identifying a subject who has (e.g., by detecting the presence of) a KDM5B DNA mutation or an
amplification at lp32.3, using methods known in the art. An exemplary human
KDM5B reference sequence is at NG_050659.1, RefSeqGene Range 4938-89223.
The methods include administering to these identified subjects an inhibitor of KDM (including KDM4), or an inhibitor of KDM4, e.g., an agent that inhibits KDM4A (preferably inhibiting levels or activity by at least 25%, or at least 50%) and does not significantly inhibit KDM5, or only inhibits KDM5 at a different (by at least 50% or at least 1 order of magnitude) concentration or dose, and/or is separate from any inhibitor of KDM5A that is being administered. In some embodiments, the agent specifically inhibits KDM4B (i.e., inhibits KDM4B and does not significantly inhibit other KDM4 family members, or only inhibits other KDM4 family members at a different (by at least 50% or at least 1 order of magnitude) concentration or dose). lp32.3 is near the cdkn2c locus and amplification of this region may suppress expression of the tumor suppressor; thus, in some embodiments, where the subject has an amplification at lp32.3, the methods include administration of a cdk4/6 inhibitor (e.g., Voruciclib (P1446A-05) (Eliades et al., Cancer Biol Ther. 2016 Jul 2; 17(7):778- 84; Trilaciclib (G1T28), palbociclib, abemaciclib and nbociclib, see Xu et al., Journal of Hematology & Oncology 2017 10:97), optionally with plus one or more of a KDM5B inhibitor and/or a KMT inhibitor. In cases where the amplification suppresses cdkn2c expssion or function, KDM4B and associated KMT2A inhibitors (e.g., chemical, RNA, peptide, etc) could be used to sensitize to cdk4/6 inhibitors or other therapeutics that the cdkn2c tumor suppressor will sensitize to upon normal or increased expression (e.g., DNA damaging agents or apoptotic agents).
KDM4/KMTs
The KMT family includes KMT2A-G, SETD1 A, and SETD1B, and are among the most frequently mutated genes in human cancer. See Rao and Dou, Nat Rev Cancer. 2015 Jun; 15(6): 334-346. The mixed-lineage leukemia (MLL1) gene (chromosome 1 lq23, also known as KMT2A) is often rearranged in acute myeloid leukemias (AML), the acute lymphoblastic leukemias (ALL), and the biphenotypic (mixed lineage) leukemias (Morera et al. Clinical Epigenetics (2016) 8:57). When MLL1 is translocated, the catalytic methyltransferase SET domain is lost and the remaining MLL1 protein is fused with a variety of partners (e.g., AF4, AF9, AF10, and ENL). These fusion partners recruit DOTIL, increasing H3K79 methylation and the expression of proleukemogenic genes (like HOXA9 and MEISl) resulting in development of leukemia.
An inhibitor of KDM4 can be used to treat a subject who has a cancer with increased KMT activity or levels, e.g., due to KMT gene amplification or
rearrangement, e.g., a translocation, or a mutation, e.g., as known in the art or shown in Table 3, that stabilize the protein; these subjects can also be treated with a KMT inhibitor. TABLE 3. Frequent Mutations in MLL1/KMT
DNA Change Type Consequences
chrll g 118473471delC Deletion Frameshift KMT2A P773 fs*8 chrll g 1184737430T Substitution Stop Gained KMT2A R862*
chrll g 1184731980T Substitution Missense KMT2A S680L
chrll g 1184723780T Substitution Stop Gained KMT2A R407*
chrll g 118478083OT Substitution Stop Gained KMT2A 1151* chrll g 118472788G>A Substitution Synonymous KMT2A T543T
chrll g 118481870OT Substitution Stop Gained KMT2A R1264* chrll g 118491843delA Deletion Frameshift KMT2A E1639Sfs*6 chrll g 118505200delA Deletion Frameshift KMT2A l3102Sfs*3 chrll g 118502463OT Substitution Stop Gained KMT2A R2188*
For example, KMT2A mutations are associated with cancers of the large intestine, lung, bladder, endometrium, and breast. KMT2B and C mutations are associated with cancers of the large intestine, lung, bladder, endometrium, and brain. KMT2D mutations are associated with cancers of the blood/lymph nodes, large intestine, lung, endometrium, and brain. KMT2E mutations are associated with cancers of the large intestine, lung, endometrium, skin (melanoma), and liver. KMT2F mutations are associated with cancers of the large intestine, kidney, and endometrium. See Rao and Dou, Nat Rev Cancer. 2015 Jun; 15(6): 334-346. Thus, the methods can include identifying a subject who has a cancer (e.g., a leukemia) with a KMT gene translocation, or a KMT mutation, e.g., as shown in Table 3, using methods known in the art. In some embodiments, the methods include identifying a subject who has (e.g., by detecting the presence of) a mutation or DNA translocation or KMT using methods known in the art. An exemplary human KMT2A reference sequence is NC_000011.10, Reference GRCh38.p7 Primary Assembly Range 118436490- 118526832. The methods include administering to these identified subjects an inhibitor of KDM (including KDM4), or an inhibitor of KDM4, e.g., an agent that inhibits KDM4A (preferably inhibiting levels or activity by at least 25%, or at least 50%) and does not significantly inhibit KDM5, or only inhibits KDM5 at a different (by at least 50% or at least 1 order of magnitude) concentration or dose, and/or is separate from any inhibitor of KDM5A that is being administered. In some embodiments, the agent specifically inhibits KDM4A (i.e., inhibits KDM4A and does not significantly inhibit other KDM4 family members, or only inhibits other KDM4 family members at a different (by at least 50% or at least 1 order of magnitude) concentration or dose). Methods of Selecting Subjects
Included herein are methods for selecting subjects for treatment using a method described herein. The methods rely on detection of a sequence or a plurality of sequences associated with a particular disease state or disease susceptibility. The methods include obtaining a sample from a subject, and evaluating the sequence, e.g., by determining the sequence, and/or the presence of a mutation, gene translocation, and/or level of gene amplification or deletion in the sample, and comparing the presence and/or level with one or more references, e.g., a control reference that represents a normal sequence, e.g., a sequence in a non-cancerous or normal control cell (preferably from the same tissue as the cancer cell) from the same subject or an unaffected subject, and/or a disease reference that represents a sequence associated with cancer.
As used herein the term "sample", when referring to the material to be tested for the presence of a biological marker using the method of the invention, includes inter alia tissue, whole blood, plasma, serum, bone marrow, urine, sweat, saliva, breath, exosome or exosome-like microvesicles (U.S. Patent No. 8.901.284), lymph, feces, cerebrospinal fluid, ascites, bronchoalveolar lavage fluid, pleural effusion, seminal fluid, sputum, nipple aspirate, post-operative seroma or wound drainage fluid. In some embodiments of the present methods, the sample is or includes blood, serum, and/or plasma, or a portion or subfraction thereof, e.g., free RNA in serum or RNA within exosomes in blood. In some embodiments, the sample comprises (or is suspected of comprising) CTCs. In some embodiments, the sample is or includes urine or a portion or subfraction thereof. In some embodiments, the sample includes known or suspected tumor cells, e.g., is a biopsy sample, e.g., a fine needle aspirate (FNA), endoscopic biopsy, or core needle biopsy; in some embodiments the sample comprises cells from the bone marrow, pancreas, lung, breast, prostate, kidney, liver, ovary, skin, or colon of the subject. In some embodiments, the sample comprises lung cells obtained from a sputum sample or from the lung of the subject by brushing, washing, bronchoscopic biopsy, transbronchial biopsy, or FNA, e.g., bronchoscopic, fluoroscopic, or CT-guided FNA (such methods can also be used to obtain samples from other tissues as well). In some embodiments, the sample is frozen, fixed and/or permeabilized, e.g., is an formalin-fixed paraffin-embedded (FFPE) sample. The type of sample used may vary depending upon the identity of the biological marker to be tested and the clinical situation in which the method is used. Various methods are known in the art for the identification and/or isolation and/or purification of a biological marker from a sample. An "isolated" or "purified" biological marker is substantially free of cellular material or other contaminants from the cell or tissue source from which the biological marker is derived i.e. partially or completely altered or removed from the natural state through human intervention. For example, nucleic acids contained in the sample are first isolated according to standard methods, for example using lytic enzymes, chemical solutions, or isolated by nucleic acid-binding resins following the manufacturer's instructions.
The presence and/or level of a nucleic acid can be evaluated using methods known in the art, e.g., using polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative or semi-quantitative real-time RT- PCR, digital PCR i.e. BEAMing ((Beads, Emulsion, Amplification, Magnetics) Diehl (2006) Nat Methods 3 :551-559) ; RNAse protection assay; Northern blot; various types of nucleic acid sequencing (Sanger, pyrosequencing, NextGeneration
Sequencing); fluorescent in-situ hybridization (FISH); or gene array/chips) RNA in situ hybridization (RNA ISH); RNA expression assays, e.g., microarray analysis; multiplexed gene expression analysis methods, e.g., RT-PCR, RNA-sequencing, and oligo hybridization assays including RNA expression microarrays; hybridization based digital barcode quantification assays such as the nCounter® System
(NanoString Technologies, Inc., Seattle, WA; Kulkarni (2011) Curr. Protoc. Mol. Biol. Chapter 25: Unit25B.10), and lysate based hybridization assays utilizing branched DNA signal amplification such as the QuantiGene 2.0 Single Plex and Multiplex Assays (Affymetrix, Inc., Santa Clara, CA; see, e.g., Linton et al. (2012) J. Mol. Diagn. 14(3): 223-32); SAGE, high-throughput sequencing, multiplex PCR,
MLPA, Luminex/XMAP, or branched DNA analysis methods. See, e.g., International Publication No. WO 2012/048113, which is incorporated herein by reference in its entirety. See also Lehninger Biochemistry (Worth Publishers, Inc., current addition; Sambrook, et al, Molecular Cloning: A Laboratory Manual (3. Sup.rd Edition, 2001); Bernard (2002) Clin Chem 48(8): 1178-1185; Miranda (2010) Kidney International 78: 191-199; Bianchi (2011) EMBO Mol Med 3 :495-503; Taylor (2013) Front. Genet. 4: 142; Yang (2014) PLOS One 9(1 l):el 10641); Nordstrom (2000) Biotechnol. Appl. Biochem. 31(2): 107-112; Ahmadian (2000) Anal Biochem 280: 103-110. In some embodiments, high throughput methods, e.g., gene chips as are known in the art (see, e.g., Ch. 12, Genomics, in Griffiths et al., Eds. Modern genetic Analysis, 1999,W. H. Freeman and Company; Ekins and Chu, Trends in
Biotechnology, 1999, 17:217-218; MacBeath and Schreiber, Science 2000,
289(5485): 1760- 1763 ; Simpson, Proteins and Proteomics: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 2002; Hardiman, Microarrays Methods and Applications: Nuts & Bolts, DNA Press, 2003), can be used to detect the presence and/or level of a sequence or genomic alteration as described herein. Measurement can be direct or indirect. For example, the sequence or copy number of a gene can be directly determined or quantitated. Alternatively, the amount of a biomarker can be determined indirectly by measuring abundance levels of cDNA, amplified RNAs or DNAs, or by measuring quantities or activities of RNAs, or other molecules that are indicative of the expression level of the biomarker. In some embodiments, a technique suitable for the detection of alterations in the structure or sequence of nucleic acids, such as the presence of deletions, amplifications, or substitutions, can be used.
RT-PCR can be used to determine the expression profiles of biomarkers (U.S. Patent No. 2005/0048542A1). The first step in expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction (Ausubel et al (1997) Current Protocols of Molecular Biology, John Wiley and Sons). To minimize errors and the effects of sample-to- sample variation, RT-PCR is usually performed using an internal standard, which is expressed at constant level among tissues, and is unaffected by the experimental treatment. Housekeeping genes are most commonly used.
Gene arrays are typically prepared by selecting probes that comprise a polynucleotide sequence, and then immobilizing such probes to a solid support or surface. For example, the probes may comprise DNA sequences, RNA sequences, copolymer sequences of DNA and RNA, DNA and/or RNA analogues, or combinations thereof. The probe sequences can be synthesized either enzymatically in vivo, enzymatically in vitro (e.g. by PCR), or non-enzymatically in vitro. KDM/KMT Inhibitors
KDM and KMT inhibitors are known in the art; exemplary agents include small molecules as well as inhibitory nucleic acids. KDM4
The human KDM4 family consists of four members, KDM4A, 4B, 4C and 4D, and two pseudo-genes (KDM4E and KDM4F); KDM4A, 4B, and 4C contain a catalytic histone demethylase domain (jumanji domain), and double PHD and Tudor domains. KDM4D contains a catalytic domain and no PHD or Tudor domains. KDM4 inhibitors include NCDM-32B (IC50 values of 3.0 and 1.0 μΜ for KDM4A and KDM4C, see Ye et al., Am J Cancer Res 2015;5(4): 1519-1530); l,5-bis[(E)-2-(3,4- dichlorophenyl ) ethenyl ] - 2 , 4 - d i nitrobenzene (NSC636819) (Chu et al., J. Med. Chem. 2014, 57, 5975-5985); 8-hydroxyquinoline (8HQ) and derivatives thereof (B3, Al, and 19, which have nanomolar IC50s for inhibiting KDM4B demethylase activityXDuan et al., Chem Biol. 2015 September 17; 22(9): 1185-1196); PKF118- 310 (KDM4A, Franci et al., Epigenetics 2017, Vol. 12, No. 3, 198-205); QC6352 (Metzger et al., Cancer Res; 77(21) (2017); 1-13; Chen et al., ACS Med. Chem. Lett., 2017, 8 (8), pp 869-874); 4-biphenylalanine- and 3 -phenyl tyrosine-derived hydroxamic acids (e.g., compounds 16p, 16r, 161, 16n, and 19 (Morera et al.,
ChemMedChem 2016, 11, 2063 - 2083); and LDD2269 and derivatives thereof as described in Lee et al., Invest New Drugs (2017) 35: 733. Cyclic peptide inhibitors of KDM4A including CP2, CP2(polyR), and CP2.3 are described in Kawamura et al., Nature Communications 8, 14773 doi: 10.1038/ncommsl4773 (2017). Compounds 8, 17 and 18 (Hamada et al., J. Med. Chem. 2010, 53, 5629-5638) and 3-((furan-2- ylmethyl)amino)pyridine-4-carboxylic acid (compound 34) and 3-(((3- methylthiophen-2-yl)methyl)amino)pyridine-4-carboxylic acid (compound 39) (Westaway et al., J. Med. Chem. 2016, 59, 1357-1369) also inhibit KDM4A.
8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one derivatives that inhibit KDM4A are described in Bavetsias et al., J. Med. Chem. 2016, 59, 1388-1409, including compounds 15, 58, 54j, 54k, and 54i described therein. Additional KDM4 inhibitors are described in Chin and Han, Expert Opin Ther Pat. 2015 Feb;25(2): 135-44, and patents and patent applications described therein. Other KDM4 inhibitors include compounds described in McAllister et al., J. Med. Chem., 2016, 59 (4), pp 1308-1329 and Thinnes et al., Biochimica et Biophysica Acta 1839 (2014) 1416-1432.
H3K4M peptide mimics, e.g., a synthetic peptide of the Histone H3 tail (residues 1-21), can also be used as inhibitors of KDM4; see, e.g., Zhang et al., Cell Research (2013) 23, 225-241 (2013). See also WO2014/197835, which is incorporated herein in its entirety.
KDM5
The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4). Inhibitors of KDM5A include Compound 15e (Wu et al., Bioorganic & Medicinal Chemistry Letters 26 (2016) 2284-2288); compound 20 (Gehling et al., Bioorganic & Medicinal Chemistry Letters 26 (2016) 4350-4354); compound 50 (Liang et al., Bioorganic & Medicinal Chemistry Letters 27 (2017) 2974-2981); N3, N4, N8, N10, Nl 1, N12, N16, and N19 (Horton et al., 2016, Cell Chemical Biology 23, 769-781); KDM5-C49 and its cell-permeable ethyl ester derivative, KDM5-C70 (WO2014053491, Johansson et al., Nature Chemical Biology 12, 539-545 (2016)); YUKA1 (4-([2-(allyloxy)-3-methoxybenzyl]amino)-4H-l,2,4- triazole-3 -thiol) and YUKA2 (N-[(4-allyl-5-mercapto-4H-l,2,4-triazol-3-yl)methyl]- 3-methylbenzamide) (Gale et al., Oncotarget, Vol. 7, No. 26, 39931- 39944 (2016)); compounds 6a, 6i, 6j, and 7j (Itoh et al., ACS Med. Chem. Lett. 2015, 6, 665-670). Pyridine derivative KDM5 A and KDM5B inhibitors are also described in WO
2014100463 and WO 2014151945).
Inhibitors of KDM5B include KDOAM-25 (Tumber et al., 2017, Cell
Chemical Biology 24, 371-380); EPT-103182 (EpiTherapeutics/Gilead); PBIT and its analogue ebselen, and 2,4-PDCA (Sayegh et al. J. Biological Chemistry Vol. 288, No. 13, pp. 9408-9417, March 29, 2013).
JIB 04 ((E,Z)-N-(5-chloro-pyridin-2-yl)-N'-(phenyl-pyridin-2-yl-methylene)- hydrazine)) inhibits the Jumonji family of KDMs (KDM5A and KDM5D-E are more sensitive than KDM4D/4B/4A/6B/4C (Wang et al., Nat. Commun. 4:2035 doi:
10.1038/ncomms3035; Banelli et al., Oncotarget, 2017, Vol. 8, (No. 21), pp: 34896- 34910; Cascella et al., Chem Commun (Camb). Chem. Commun., 2017, 53, 2174). GSK-J1 inhibits KDM5 and KDM6 family members (Horton et al., J. Biol. Chem. 2016 291 : 2631). CPI-455 and CPI-4203 inhibit KDM5A and KDM5B (Vinogradova et al., Nat Chem Biol. 2016 Jul; 12(7):531-8). Other KDM5 inhibitors include compounds described in McAllister et al., J. Med. Chem., 2016, 59 (4), pp 1308-1329 and Thinnes et al., Biochimica et Biophysica Acta 1839 (2014) 1416-1432;
WO2014144850; WO 2012/007007; and WO 2012/007008.. KMT MLL
KMT/MLL inhibitors include WDR5 inhibitors including WDR5-0101 [N-(2- (4-methylpiperazin-l-yl)-5 nitrophenyl)benzamide], WDR5-0102 [2-chloro-N-(2-(4- methylpiperazin-l-yl)-5-nitrophenyl) benzamide] and WDR5-0103 [methyl-3-(3- methoxybenzamido)-4-(4-methylpiperazin-l-yl)benzoate] (Senisterra et al., Biochem. J. (2013) 449, 151-159), as well as RBC-1, -2, -3, and -4 (Ferry et al., Assay Drug Dev Technol. 2015 May; 13(4):221-34); menin inhibitors such as MI-2, MI-2-2, and MI-3 (Shi et al., Blood. 2012;120(23):4461-4469; Grembecka et al., Nat Chem Biol. 2012 Jan 29;8(3):277-84) and US 2014/0371239, MI-463, MI-503, MM-401 and MM408 inhibit MLL1 H3K4 methyltransferase activity (Cao et al., Mol Cell. 2014 January 23; 53(2): 247-261, Liu et al., Cell Discovery (2016) 2, 16036; Borkin et al., 2015, Cancer Cell 27, 589-602). DOT1L inhibitors include EPZ004777 and its derivative EPZ-5676 (ClinicalTrials.gov Identifier: NCT02141828; Morera et al. Clinical Epigenetics (2016) 8:57). See also Ferry et al., Assay and Drug Dev. Tevh 13(4)221 (2015).
Inhibitory Nucleic Acids
Inhibitory nucleic acids useful in the present methods and compositions include anti sense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases/locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimetics that hybridize to at least a portion of the target nucleic acid and modulate its function. Exemplary human sequences are provided in Table 4. In some cases, other isoforms of the same transcript may be targeted.
Figure imgf000031_0001
lysine methyltransferase 2A (KMT2A/MLL)
histone-lysine N-methyl transferase 2 A
isoform 1 precursor
NM_001197104.1 NP_001184033.1 histone-lysine N-methyl transferase 2 A
isoform 2 precursor NM_005933.3 NP_005924.2
KMT2B NM_014727.2 NP_055542.1
KMT2C NM_170606.2 NP_733751.2
KMT2D NM_003482.3 NP_003473.3 histone-lysine N-methyl transferase SETD1 A NM_014712.2 NP_055527.1 (SETD1A)
histone-lysine N-methyl transferase SETD1B NM_001353345.1 NP_001340274.1 (SETD1B)
In some embodiments, the inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof. See, e.g., WO 2010040112.
In some embodiments, the inhibitory nucleic acids are 10 to 50, 10 to 20, 10 to 25, 13 to 50, or 13 to 30 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies inhibitory nucleic acids having complementary portions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or any range therewithin. In some embodiments, the inhibitory nucleic acids are 15 nucleotides in length. In some embodiments, the inhibitory nucleic acids are 12 or 13 to 20, 25, or 30 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies inhibitory nucleic acids having complementary portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or any range therewithin (complementary portions refers to those portions of the inhibitory nucleic acids that are complementary to the target sequence).
The inhibitory nucleic acids useful in the present methods are sufficiently complementary to the target RNA, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. "Complementary" refers to the capacity for pairing, through hydrogen bonding, between two sequences comprising naturally or non-naturally occurring bases or analogs thereof. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a RNA, then the bases are considered to be
complementary to each other at that position. 100% complementarity is not required.
Routine methods can be used to design an inhibitory nucleic acid that binds to the target sequence with sufficient specificity. In some embodiments, the methods include using bioinformatics methods known in the art to identify regions of secondary structure, e.g., one, two, or more stem-loop structures, or pseudoknots, and selecting those regions to target with an inhibitory nucleic acid. For example, "gene walk" methods can be used to optimize the inhibitory activity of the nucleic acid; for example, a series of oligonucleotides of 10-30 nucleotides spanning the length of a target RNA can be prepared, followed by testing for activity. Optionally, gaps, e.g., of 5-10 nucleotides or more, can be left between the target sequences to reduce the number of oligonucleotides synthesized and tested. GC content is preferably between about 30-60%). Contiguous runs of three or more Gs or Cs should be avoided where possible (for example, it may not be possible with very short (e.g., about 9-10 nt) oligonucleotides).
In some embodiments, the inhibitory nucleic acid molecules can be designed to target a specific region of the RNA sequence. For example, a specific functional region can be targeted, e.g., a region comprising a known RNA localization motif (i.e., a region complementary to the target nucleic acid on which the RNA acts). Alternatively or in addition, highly conserved regions can be targeted, e.g., regions identified by aligning sequences from disparate species such as primate (e.g., human) and rodent (e.g., mouse) and looking for regions with high degrees of identity.
Percent identity can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), e.g., using the default parameters.
Once one or more target regions, segments or sites have been identified, e.g., within a target sequence known in the art or provided herein, inhibitory nucleic acid compounds are chosen that are sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient specificity (i.e., do not substantially bind to other non-target RNAs), to give the desired effect. In the context of this invention, hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Complementary, as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a RNA molecule, then the inhibitory nucleic acid and the RNA are considered to be complementary to each other at that position. The inhibitory nucleic acids and the RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridisable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the inhibitory nucleic acid and the RNA target. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a RNA, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.
It is understood in the art that a complementary nucleic acid sequence need not be 100% complementary to that of its target nucleic acid to be specifically
hybridisable. A complementary nucleic acid sequence for purposes of the present methods is specifically hybridisable when binding of the sequence to the target RNA molecule interferes with the normal function of the target RNA to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target RNA sequences under conditions in which specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency. For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
In general, the inhibitory nucleic acids useful in the methods described herein have at least 80% sequence complementarity to a target region within the target nucleic acid, e.g., 90%, 95%, or 100% sequence complementarity to the target region within an RNA. For example, an antisense compound in which 18 of 20 nucleobases of the antisense oligonucleotide are complementary, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity. Percent complementarity of an inhibitory nucleic acid with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). Inhibitory nucleic acids that hybridize to an RNA can be identified through routine experimentation. In general the inhibitory nucleic acids must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of, transcripts other than the intended target.
For further disclosure regarding inhibitory nucleic acids, please see
US2010/0317718 (antisense oligos); US2010/0249052 (double-stranded ribonucleic acid (dsRNA)); US2009/0181914 and US2010/0234451 (LNAs); US2007/0191294 (siRNA analogues); US2008/0249039 (modified siRNA); and WO2010/129746 and WO2010/040112 (inhibitory nucleic acids).
Antisense
In some embodiments, the inhibitory nucleic acids are antisense
oligonucleotides. Antisense oligonucleotides are typically designed to block expression of a DNA or RNA target by binding to the target and halting expression at the level of transcription, translation, or splicing. Antisense oligonucleotides of the present invention are complementary nucleic acid sequences designed to hybridize under stringent conditions to an RNA. Thus, oligonucleotides are chosen that are sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient specificity, to give the desired effect. siRNA/shRNA
In some embodiments, the nucleic acid sequence that is complementary to a target RNA can be an interfering RNA, including but not limited to a small interfering RNA ("siRNA") or a small hairpin RNA ("shRNA"). Methods for constructing interfering RNAs are well known in the art. For example, the interfering RNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self- complementary (i.e., each strand comprises nucleotide sequence that is
complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure); the antisense strand comprises nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (i.e., an undesired gene) and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, interfering RNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions are linked by means of nucleic acid based or non-nucleic acid-based linker(s). The interfering RNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The interfering can be a circular single- stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNA interference.
In some embodiments, the interfering RNA coding region encodes a self- complementary RNA molecule having a sense region, an antisense region and a loop region. Such an RNA molecule when expressed desirably forms a "hairpin" structure, and is referred to herein as an "shRNA." The loop region is generally between about 2 and about 10 nucleotides in length. In some embodiments, the loop region is from about 6 to about 9 nucleotides in length. In some embodiments, the sense region and the antisense region are between about 15 and about 20 nucleotides in length. Following post-transcriptional processing, the small hairpin RNA is converted into a siRNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family. The siRNA is then capable of inhibiting the expression of a gene with which it shares homology. For details, see Brummelkamp et al., Science 296:550-553, (2002); Lee et al, Nature Biotechnol., 20, 500-505, (2002); Miyagishi and Taira, Nature Biotechnol 20:497-500, (2002); Paddison et al. Genes & Dev. 16:948-958, (2002); Paul, Nature Biotechnol, 20, 505-508, (2002); Sui, Proc. Natl. Acad. Sd. USA, 99(6), 5515-5520, (2002); Yu et al. Proc NatlAcadSci USA 99:6047- 6052, (2002).
The target RNA cleavage reaction guided by siRNAs is highly sequence specific. In general, siRNA containing a nucleotide sequences identical to a portion of the target nucleic acid are preferred for inhibition. However, 100% sequence identity between the siRNA and the target gene is not required to practice the present invention. Thus the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence. For example, siRNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Alternatively, siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition. In general the siRNAs must retain specificity for their target, i.e., must not directly bind to, or directly
significantly affect expression levels of, transcripts other than the intended target.
Ribozymes
Trans-cleaving enzymatic nucleic acid molecules can also be used; they have shown promise as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the RNA non- functional.
In general, enzymatic nucleic acids with RNA cleaving activity act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
Several approaches such as in vitro selection (evolution) strategies (Orgel, 1979, Proc. R. Soc. London, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing a variety of reactions, such as cleavage and ligation of phosphodiester linkages and amide linkages, (Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992, Scientific American 267, 90-97;
Breaker et al, 1994, TIBTECH 12, 268; Bartel et al, 1993, Science 261 : 1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al, 1995, FASEB J., 9, 1183; Breaker, 1996, Curr. Op. Biotech., 1, 442). The development of ribozymes that are optimal for catalytic activity would contribute significantly to any strategy that employs RNA- cleaving ribozymes for the purpose of regulating gene expression. The hammerhead ribozyme, for example, functions with a catalytic rate (kcat) of about 1 min"1 in the presence of saturating (10 rnM) concentrations of Mg2+ cofactor. An artificial "RNA ligase" ribozyme has been shown to catalyze the corresponding self-modification reaction with a rate of about 100 min"1. In addition, it is known that certain modified hammerhead ribozymes that have substrate binding arms made of DNA catalyze RNA cleavage with multiple turn-over rates that approach 100 min"1.
Modified Inhibitory Nucleic Acids
In some embodiments, the inhibitory nucleic acids used in the methods described herein are modified, e.g., comprise one or more modified bonds or bases. A number of modified bases include phosphorothioate, methylphosphonate, peptide nucleic acids, or locked nucleic acid (LNA) molecules. Some inhibitory nucleic acids are fully modified, while others are chimeric and contain two or more chemically distinct regions, each made up of at least one nucleotide. These inhibitory nucleic acids typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimeric inhibitory nucleic acids of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides,
oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. In some embodiments, the oligonucleotide is a gapmer (contain a central stretch (gap) of DNA monomers sufficiently long to induce RNase H cleavage, flanked by blocks of LNA modified nucleotides; see, e.g., Stanton et al., Nucleic Acid Ther. 2012. 22: 344-359; Nowotny et al., Cell, 121 : 1005-1016, 2005; Kurreck, European Journal of
Biochemistry 270: 1628-1644, 2003; FLuiter et al., Mol Biosyst. 5(8):838-43, 2009). In some embodiments, the oligonucleotide is a mixmer (includes alternating short stretches of LNA and DNA; Naguibneva et al., Biomed Pharmacother. 2006 Nov;
60(9):633-8; 0rom et al., Gene. 2006 May 10; 372(): 137-41). Representative United States patents that teach the preparation of such hybrid structures comprise, but are not limited to, US patent nos. 5,013,830; 5, 149,797; 5, 220,007; 5,256,775;
5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference.
In some embodiments, the inhibitory nucleic acid comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-0-alkyl, 2'-0- alkyl-O-alkyl or 2'-fluoro-modified nucleotide. In other preferred embodiments, RNA modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA. Such modifications are routinely incorporated into oligonucleotides and these
oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than; 2'-deoxyoligonucleotides against a given target.
A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide; these modified oligos survive intact for a longer time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH2 -NH-0-CH2,
CH,~N(CH3)~0~CH2 (known as a methylene(methylimino) or MMI backbone], CH2 --0-N (CH3)-CH2, CH2 -N (CH3)-N (CH3)-CH2 and O-N (CH3)- CH2 -CH2 backbones, wherein the native phosphodiester backbone is represented as O- P— O- CH,); amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366- 374); morpholino backbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506); peptide nucleic acid (PNA) backbone (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497). Phosphorus- containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising
3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see US patent nos. 3,687,808; 4,469,863;
4,476,301; 5,023,243; 5, 177, 196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321, 131; 5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677;
5,476,925; 5,519, 126; 5,536,821; 5,541,306; 5,550, 111; 5,563, 253; 5,571,799;
5,587,361; and 5,625,050.
Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.
Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc, 2000, 122, 8595-8602.
Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl
internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones;
methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts; see US patent nos. 5,034,506; 5, 166,315; 5,185,444; 5,214,134; 5,216, 141; 5,235,033; 5,264, 562; 5, 264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;
5,489,677; 5,541,307; 5,561,225; 5,596, 086; 5,602,240; 5,610,289; 5,602,240;
5,608,046; 5,610,289; 5,618,704; 5,623, 070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.
One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3 OCH3, OCH3 0(CH2)n CH3, 0(CH2)n H2 or 0(CH2)n CH3 where n is from 1 to about 10; Ci to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF3 ; OCF3; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH3; S02 CH3; ON02; N02; N3; H2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino;
substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A preferred modification includes 2'- methoxyethoxy [2'-0-CH2CH2OCH3, also known as 2'-0-(2-methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486). Other preferred modifications include 2'- methoxy (2'-0-CH3), 2'-propoxy (2'-OCH2 CH2CH3) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
Inhibitory nucleic acids can also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base") modifications or
substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me- C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino)adenine, 2- (imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5- hydroxymethyluracil, 8- azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and 2,6- diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, 1980, pp75- 77; Gebeyehu, G., et al. Nucl. Acids Res. 1987, 15:4513). A "universal" base known in the art, e.g., inosine, can also be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2<0>C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions.
It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.
In some embodiments, both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds comprise, but are not limited to, US patent nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference . Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.
Inhibitory nucleic acids can also include one or more nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases comprise other synthetic and natural nucleobases such as 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine.
Further, nucleobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in 'The Concise Encyclopedia of Polymer Science And Engineering', pages 858-859, Kroschwitz, J. I, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandle Chemie, International Edition', 1991, 30, page 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications', pages 289- 302, Crooke, S.T. and Lebleu, B. ea., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6- 1.2<0>C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds, Antisense Research and Applications', CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications. Modified nucleobases are described in US patent nos.
3,687,808, as well as 4,845,205; 5, 130,302; 5,134,066; 5, 175, 273; 5, 367,066;
5,432,272; 5,457, 187; 5,459,255; 5,484,908; 5,502, 177; 5,525,711; 5,552,540;
5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941, each of which is herein incorporated by reference.
In some embodiments, the inhibitory nucleic acids are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties comprise but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S- tritylthiol (Manoharan et al, Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533- 538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49- 54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O- hexadecyl- rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). See also US patent nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552, 538;
5,578,717, 5,580,731; 5,580,731; 5,591,584; 5, 109,124; 5,118,802; 5, 138,045;
5,414,077; 5,486, 603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762, 779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582;
4,958,013; 5,082, 830; 5,112,963; 5,214, 136; 5,082,830; 5,112,963; 5,214,136; 5, 245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;
5,371,241, 5,391, 723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5, 565,552; 5,567,810; 5,574, 142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;
5,599,923; 5,599, 928 and 5,688,941, each of which is herein incorporated by reference.
These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No.
PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference. Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5- tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino- carbonyl-oxy cholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,580,731; 5,591,584; 5, 109,124; 5, 118,802; 5,138,045; 5,414,077; 5,486,603;
5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;
4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;
5, 112,963; 5,214, 136; 5,082,830; 5, 112,963; 5,214, 136; 5,245,022; 5,254,469;
5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;
5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;
5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.
Locked Nucleic Acids (LNAs)
In some embodiments, the modified inhibitory nucleic acids used in the methods described herein comprise locked nucleic acid (LNA) molecules, e.g., including [alpha]-L-LNAs. LNAs comprise ribonucleic acid analogues wherein the ribose ring is "locked" by a methylene bridge between the 2'-oxgygen and the 4'- carbon - i.e., oligonucleotides containing at least one LNA monomer, that is, one 2'- O,4'-C-methylene- ?-D-ribofuranosyl nucleotide. LNA bases form standard Watson- Crick base pairs but the locked configuration increases the rate and stability of the basepairing reaction (Jepsen et al., Oligonucleotides, 14, 130-146 (2004)). LNAs also have increased affinity to base pair with RNA as compared to DNA. These properties render LNAs especially useful as probes for fluorescence in situ hybridization (FISH) and comparative genomic hybridization, as knockdown tools for miRNAs, and as antisense oligonucleotides to target mRNAs or other RNAs, e.g., RNAs as described herien.
The LNA molecules can include molecules comprising 10-30, e.g., 12-24, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to a target region in the RNA. The LNA molecules can be chemically synthesized using methods known in the art.
The LNA molecules can be designed using any method known in the art; a number of algorithms are known, and are commercially available (e.g., on the internet, for example at exiqon.com). See, e.g., You et al., Nuc. Acids. Res. 34:e60 (2006); McTigue et al., Biochemistry 43 :5388-405 (2004); and Levin et al., Nuc. Acids. Res. 34:el42 (2006). For example, "gene walk" methods, similar to those used to design antisense oligos, can be used to optimize the inhibitory activity of the LNA; for example, a series of oligonucleotides of 10-30 nucleotides spanning the length of a target RNA can be prepared, followed by testing for activity. Optionally, gaps, e.g., of 5-10 nucleotides or more, can be left between the LNAs to reduce the number of oligonucleotides synthesized and tested. GC content is preferably between about 30-60%). General guidelines for designing LNAs are known in the art; for example, LNA sequences will bind very tightly to other LNA sequences, so it is preferable to avoid significant complementarity within an LNA. Contiguous runs of more than four LNA residues, should be avoided where possible (for example, it may not be possible with very short (e.g., about 9-10 nt) oligonucleotides). In some
embodiments, the LNAs are xylo-LNAs.
For additional information regarding LNAs see U.S. Pat. Nos. 6,268,490; 6,734,291; 6,770,748; 6,794,499; 7,034, 133; 7,053,207; 7,060,809; 7,084,125; and 7,572,582; and U.S. Pre-Grant Pub. Nos. 20100267018; 20100261175; and
20100035968; Koshkin et al. Tetrahedron 54, 3607-3630 (1998); Obika et al.
Tetrahedron Lett. 39, 5401-5404 (1998); Jepsen et al., Oligonucleotides 14: 130-146 (2004); Kauppinen et al., Drug Disc. Today 2(3):287-290 (2005); and Ponting et al., Cell 136(4): 629-641 (2009), and references cited therein. Making and Using Inhibitory Nucleic Acids
The nucleic acid sequences used to practice the methods described herein, whether RNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof, can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/ generated recombinantly. Recombinant nucleic acid sequences can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including e.g. in vitro, bacterial, fungal, mammalian, yeast, insect or plant cell expression systems.
Nucleic acid sequences of the invention can be inserted into delivery vectors and expressed from transcription units within the vectors. The recombinant vectors can be DNA plasmids or viral vectors. Generation of the vector construct can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al.
Molecular Cloning: A Laboratory Manual. (1989)), Coffin et al. (Retroviruses.
(1997)) and "RNA Viruses: A Practical Approach" (Alan J. Cann, Ed., Oxford University Press, (2000)). As will be apparent to one of ordinary skill in the art, a variety of suitable vectors are available for transferring nucleic acids of the invention into cells. The selection of an appropriate vector to deliver nucleic acids and optimization of the conditions for insertion of the selected expression vector into the cell, are within the scope of one of ordinary skill in the art without the need for undue experimentation. Viral vectors comprise a nucleotide sequence having sequences for the production of recombinant virus in a packaging cell. Viral vectors expressing nucleic acids of the invention can be constructed based on viral backbones including, but not limited to, a retrovirus, lentivirus, adenovirus, adeno-associated virus, pox virus or alphavirus. The recombinant vectors capable of expressing the nucleic acids of the invention can be delivered as described herein, and persist in target cells (e.g., stable transformants).
Nucleic acid sequences used to practice this invention can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440- 3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33 :7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; U.S. Patent No. 4,458,066.
Nucleic acid sequences of the invention can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification. For example, nucleic acid sequences of the invention includes a phosphorothioate at least the first, second, or third internucleotide linkage at the 5' or 3' end of the nucleotide sequence. As another example, the nucleic acid sequence can include a 2'-modified nucleotide, e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'- O-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0- dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0~N-methylacetamido (2'-0- NMA). As another example, the nucleic acid sequence can include at least one 2'-0- methyl-modified nucleotide, and in some embodiments, all of the nucleotides include a 2'-0-methyl modification. In some embodiments, the nucleic acids are "locked," i.e., comprise nucleic acid analogues in which the ribose ring is "locked" by a methylene bridge connecting the 2'-0 atom and the 4'-C atom (see, e.g., Kaupinnen et al., Drug Disc. Today 2(3):287-290 (2005); Koshkin et al., J. Am. Chem. Soc, 120(50): 13252-13253 (1998)). For additional modifications see US 20100004320, US 20090298916, and US 20090143326.
Techniques for the manipulation of nucleic acids used to practice this invention, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature. See, e.g., Sambrook et al., Molecular Cloning; A Laboratory Manual 3d ed. (2001); Current Protocols in Molecular Biology, Ausubel et al., eds. (John Wiley & Sons, Inc., New York 2010); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); Laboratory Techniques In Biochemistry And Molecular Biology: Hybridization With Nucleic Acid Probes, Parti. Theory and Nucleic Acid Preparation, Tijssen, ed.
Elsevier, N.Y. (1993). Pharmaceutical Compositions
The methods described herein can include the administration of
pharmaceutical compositions and formulations comprising inhibitors as described herein, e.g., inhibitory nucleic acid sequences designed to target an RNA.
In some embodiments, the compositions are formulated with a
pharmaceutically acceptable carrier. The pharmaceutical compositions and formulations can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally. The pharmaceutical
compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005.
The inhibitors can be administered alone or as a component of a
pharmaceutical formulation (composition). The compounds may be formulated for administration, in any convenient way for use in human or veterinary medicine.
Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Formulations of the compositions of the invention include those suitable for intradermal, inhalation, oral/ nasal, topical, parenteral, rectal, and/or intravaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient (e.g., nucleic acid sequences of this invention) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect, e.g., an antigen specific T cell or humoral response. Pharmaceutical formulations can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.
Pharmaceutical formulations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores. Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen. Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Push-fit capsules can contain active agents mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
Aqueous suspensions can contain an active agent (e.g., nucleic acid sequences of the invention) in admixture with excipients suitable for the manufacture of aqueous suspensions, e.g., for aqueous intradermal injections. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolality.
In some embodiments, oil-based pharmaceuticals are used for administration of nucleic acid sequences of the invention. Oil-based suspensions can be formulated by suspending an active agent in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. See e.g., U.S. Patent No. 5,716,928 describing using essential oils or essential oil components for increasing bioavailability and reducing inter- and intra-individual variability of orally administered hydrophobic pharmaceutical compounds (see also U.S. Patent No. 5,858,401). The oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid. As an example of an injectable oil vehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281 :93-102.
Pharmaceutical formulations can also be in the form of oil-in-water emulsions.
The oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent. In alternative embodiments, these injectable oil-in-water emulsions of the invention comprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitan monooleate and/or an ethoxylated sorbitan trioleate.
The pharmaceutical compounds can also be administered by in intranasal, intraocular and intravaginal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see e.g., Rohatagi (1995) J. Clin. Pharmacol. 35: 1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75: 107- 111). Suppositories formulations can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug. Such materials are cocoa butter and polyethylene glycols.
In some embodiments, the pharmaceutical compounds can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
In some embodiments, the pharmaceutical compounds can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.
In some embodiments, the pharmaceutical compounds can be parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or lumen of an organ. These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IV administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3- butanediol. The administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time).
In some embodiments, the pharmaceutical compounds and formulations can be lyophilized. Stable lyophilized formulations comprising an inhibitory nucleic acid can be made by lyophilizing a solution comprising a pharmaceutical of the invention and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof. A process for preparing a stable lyophilized formulation can include lyophilizing a solution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. 20040028670.
The compositions and formulations can be delivered by the use of liposomes. By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Patent Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13 :293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46: 1576-1587. As used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered.
Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.
Liposomes can also include "sterically stabilized" liposomes, i.e., liposomes comprising one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes with enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860.
The formulations of the invention can be administered for prophylactic and/or therapeutic treatments. In some embodiments, for therapeutic applications, compositions are administered to a subject who is need of reduced triglyceride levels, or who is at risk of or has a disorder described herein, in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the disorder or its
complications; this can be called a therapeutically effective amount. For example, in some embodiments, pharmaceutical compositions of the invention are administered in an amount sufficient to decrease serum levels of triglycerides in the subject.
The amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose. The dosage schedule and amounts effective for this use, i.e., the dosing regimen, will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into
consideration.
The dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51 :337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84: 1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24: 103-108; Remington: The Science and Practice of Pharmacy, 21st ed., 2005). The state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent and disease or condition treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e., dose schedule and dosage levels, administered practicing the methods of the invention are correct and appropriate.
Single or multiple administrations of formulations can be given depending on for example: the dosage and frequency as required and tolerated by the patient, the degree and amount of therapeutic effect generated after each administration (e.g., effect on tumor size or growth), and the like. The formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate conditions, diseases or symptoms.
In alternative embodiments, pharmaceutical formulations for oral
administration are in a daily amount of between about 1 to 100 or more mg per kilogram of body weight per day. Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ. Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation. Actual methods for preparing parenterally or non-parenterally administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, 21st ed., 2005.
Various studies have reported successful mammalian dosing using
complementary nucleic acid sequences. For example, Esau C, et al., (2006) Cell Metabolism, 3(2):87-98 reported dosing of normal mice with intraperitoneal doses of miR-122 antisense oligonucleotide ranging from 12.5 to 75 mg/kg twice weekly for 4 weeks. The mice appeared healthy and normal at the end of treatment, with no loss of body weight or reduced food intake. Plasma transaminase levels were in the normal range (AST ¾ 45, ALT ¾ 35) for all doses with the exception of the 75 mg/kg dose of miR-122 ASO, which showed a very mild increase in ALT and AST levels. They concluded that 50mg/kg was an effective, non-toxic dose. Another study by
Kriitzfeldt J., et al., (2005) Nature 438, 685-689, injected anatgomirs to silence miR- 122 in mice using a total dose of 80, 160 or 240 mg per kg body weight. The highest dose resulted in a complete loss of miR-122 signal. In yet another study, locked nucleic acids ("LNAs") were successfully applied in primates to silence miR-122. Elmen J., et al., (2008) Nature 452, 896-899, report that efficient silencing of miR- 122 was achieved in primates by three doses of 10 mg kg-1 LNA-antimiR, leading to a long-lasting and reversible decrease in total plasma cholesterol without any evidence for LNA-associated toxicities or histopathological changes in the study animals.
In some embodiments, the methods described herein can include coadministration with other drugs or pharmaceuticals, e.g., compositions for providing cholesterol homeostasis. For example, the inhibitors can be co-administered with drugs for treating or reducing risk of a disorder described herein.
EXAMPLES
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
MATERIALS AND METHODS
The following materials and methods were usd in the
Cell Culture and Transfection Conditions
The generation of stable cells, plasmids, antibodies and chemicals used can be found in the extended experimental procedure and table. Transient siRNA
transfections were performed using Lipofectamine 3000 transfection reagent (Life Technologies) in OPTI-MEM medium (Life Technologies). Transfections were changed to complete media after 4hr of transfection, and cells were collected at 72hr. Transient overexpression transfections were performed using Lipofectamine 3000 transfection reagent and P3000 reagent (Life Technologies) in OPTI-MEM medium for 4hr. Silencer select negative controls and siRNAs were purchased from Life Technologies. At least two different siRNAs against every gene were used for every experiment.
DNA Fluorescence in situ hybridization (FISH)
FISH protocol was performed as described previously in (Black et al., 2013). Imaging was performed with multiple planes of fields of nuclei. At least 100 cells were counted from each experiment. All FISH experiments include at least two biological replicates. Extended methods can be found in supplemental methods. RNA extraction and quantitative real-time PCR
Cells were washed and collected by trypsinization, followed by washing in PBS two times. Cell pellet was resuspended in Trizol reagent (Qiagen) and stored at - 80°C before further processing. Quantitative real-time PCR was performed using FastStart Universal SYBR Green Master mix (Roche) according to the manufacturer's instructions on a LightCycler 480 PCR machine. Extended methods can be found in supplemental methods.
Cell Cycle Analyses
Asynchronized and synchronized cells were fixed and processed as performed in (Black et al., 2010). Cell cycle was analyzed by Propium iodide (PI) or EdU staining with a Click-IT EdU flow cytometry kit (Life Technologies). Cells were stained with 10μΜ EdU for lh prior to cell cycle analyses with EdU staining. Cell cycle distribution was analyzed with a flow cytometer LSRII or Fortessa.
HaloTag Mammalian Pulldown for Mass spectrometry
293 T cells were seeded and transfected with HALO-Control or HALO-
KDM4B and lysates were prepared were processing as described previously in (Black et al., 2013). Halo tag-purified KDM4B complexes were analyzed and processed as described previously in (Black et al., 2013).
Cesium Chloride gradient centrifugation
Cells were labeled with BrdU and genomic DNA was purified and
ultracentrifuged as was described previously (Black et al., 2013). Heavy:heavy fractions were pooled together and purified. The rereplicated samples were analyzed by qPCR on a Roche LC480 using FastStart Universal SYBR Green Master Mix (Roche) following the manufacturer's instructions. Extended methods can be found in supplemental methods.
Chromatin Immunoprecipitation (ChIP)
Chromatin IPs was performed as in (Black et al., 2013). Data presented are the averages from at least two independent siRNAs or two independent stable cell lines. RPE cells were treated with 2mM HU for 20h before cross-linking and assessment for KDM4A, KDM4B and H3K4me3. DNA Polymerase alpha (DNA Pol a) ChIP was performed after Ro-3306 treatment. Detailed list of procedure and antibodies are provided in the supplemental methods and table. Cell Culture and Transfection Conditions
The generation of stable cells, plasmids, antibodies and chemicals used can be found in the table. Retinal pigment epithelial (RPE) and 293T cells were cultured in DMEM (Sigma) media with 10% fetal bovine serum (FBS), 1%
penicillin/streptomycin, and 1% L-glutamine. H2591 lung cancer cells were cultured in RPMI (Sigma) with 10% fetal bovine serum, 1% penicillin/streptomycin, and L- glutamine. SK-N-AS cells were maintained in DMEM/F12 (Gibco) with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% L-glutamine. Transient siRNA transfections were performed using Lipofectamine 3000 transfection reagent (Invitrogen) in OPTI-MEM medium (Life Technologies). Transfections were changed to complete media after 4hr of transfection, and cells were collected at 72hr. Transient overexpression transfections were performed using Lipofectamine 3000 transfection reagent and P3000 reagent (Life Technologies) in OPTI-MEM medium for 4hr, followed by change to complete media. Silencer select negative controls and siRNAs were purchased from Life Technologies. At least two different siRNAs against every gene were used for every experiment.
Plasmids and Constructs
Extended list of plasmids used are provided in the table.
DNA Fluorescence in situ hybridization (FISH)
FISH probes for chromosome 1 classical satellite (lql2h), chromosome 8 centromere (alpha satellite), CKS1B/CDKN2C and chromosome X alpha satellite were purchased from Cytocell (Oxford Gene Technologies). Probes for lq21.2, lp32.3 and lq23.3 were purchased from Agilent Technologies. The lp32.3 probe shown in the figure by itself is performed using probe from Agilent Technologies. FISH protocol was performed as described previously in Black et al. (2013). BACS for Xql3.1 were prepared utilizing PureLink HiPure Plasmid Filter Maxiprep kit (Life Technologies) using the recommended modified wash buffer. Probes were nick translated (Abbot Molecular Kit) in the presence of fluorescently labeled dTTP (Enzo Life Science). Imaging was performed with multiple planes of fields of nuclei. Images were acquired with Olympus 1X81 Spinning Disk Microscope and Olympus 1X83 and analyzed using Slidebook 5.0 and Slidebook 6.0 softwares. Copy number gains were scored in RPE cells as more than or equal to three foci. For SK-N-AS cells, copy gain was scored for any cell with 5 or more foci. For H2591, copy gain was scored for any cell with 6 or more foci. At least 100 cells were counted from each experiment. All FISH experiments include at least two biological replicates. Extended list of probes used are provided in the table.
Infection with Histone H3.3 variants
Plasmids for H3.3 K4M, K9M and K36M mutants provided by Peter Lewis
(University of Wisconsin). They were used to generate virus and infect RPE cells in the presence of 8μg/ml polybrene for 8 hours (Lewis et al., 2013). Cells were washed 2 times with DMEM. For K4M experiments, cells were infected for 24hr followed by a total of 72hr of siRNA transfections. For H3 3K9M and H3,3K36M, cells were collected 48hr post infections for analysis by FISH and western blot. Incorporation of FLAG tag for histone variants was confirmed by subcellular chromatin fractionation and western blotting.
RNA extraction and quantitative real-time PCR
Cells were washed and collected by trypsinization, followed by washing in PBS two times. Cell pellet was resuspended in Trizol reagent (Qiagen) and stored at - 80°C before further processing. Total RNA was extracted using miRNAeasy Mini Kit (Qiagen) with an on-column DNase digestion according to the manufacturer's instructions. RNA was quantified using Nanodrop 1000D. Single strand cDNA was prepared using Super Script IV first strand synthesis kit (Invitrogen) using oligo dT primers. Expression levels were analyzed using FastStart Universal SYBR Green Master mix (Roche) according to the manufacturer's instructions on a LightCycler 480 PCR machine. Samples were normalized to beta-actin. Primers are available upon request.
Drug treatment Conditions
Cells were synchronized in Gl/S using hydroxyurea (HU; Sigma) at 2mM final concentration for 20hr in RPE..Cells were released from HU by washing two times with fresh media followed by culturing in complete media for the indicated time points. Synchronization at G2/M was performed with Ro-3306 [Enzo
LifeSciences,(Black et al., 2013)] at 10μΜ final concentration for 20hr. KDM5-C70 was purchased from Xcessbio [Fisher Scientific; (Johansson et al., 2016)] and treated at the indicated concentrations for 48hr or 72hr. Cesium Chloride gradient centrifugation
RPE cells were grown and transfected with KDM5 A siRNA for a total of 72hr including BrdU treatment. Cells were labeled with BrdU for 12 hours and 30 minutes. Genomic DNA was purified and ultracentrifuged as was described previously ((Black et al., 2013). Heavy:heavy fractions were pooled together and purified. The re- replicated samples were analyzed by qPCR on a Roche LC480 using FastStart Universal SYBR Green Master Mix (Roche) following the manufacturer's
instructions at 5.0 ng DNA per well including the input DNA. Fold change was determined after normalization of each sample to its input DNA. Primers will be provided upon request.
Cell Fractionation
Cytoplasmic, nuclear and chromatin fractions were prepared from RPE cells. Cell pellets were washed twice in ice cold PBS and resuspended in ice cold hypotonic buffer (lOmM HEPES pH 7.9, lOmM KCl, 0.1M EDTA, 0.5M EGTA) and incubated on ice for 15 minutes. Swollen cells were lysed by addition of NP-40 to 10% with 10 seconds of vortexing. Lysed cells were centrifuged and the supernatant kept as cytoplasm. The nuclear pellet was resuspended in high salt buffer (lOmM HEPES pH 7.9, 400mM NaCl, ImM EDTA, 5mM EGTA) and incubated at 4°C for 15min with rotation. Extracts were centrifuged and the supernatant was kept as nuclear extract. The pellets were resuspended in N-Buffer (20mM Trish pH 7.5, lOOmM KCl, 2mM MgC12, ImM CaC12, 0.3M Sucrose, 0.1% Triton X-100, 3U per ml micrococcal nuclease). Samples were sonicated for 15 minutes at 70% amplitude in a Q700 cup horn (QSonica) and then incubated at room temperature for 15 minutes for MNase digestion. Reactions were stopped by addition of 5mM EDTA and centrifuged to clear. Supernatant was kept as chromatin extract.
Antibodies
Antibody against KDM4A was purchased from Neuro mAb, 75-189, KDM5A from abeam (ab70892), β-actin from Millipore, actinin from Santa Cruz
Biotechnology (sc- 17829), histone H3 from abeam (abl791), H3K4me3 from
Millipore (07-473) and H3K36me3 from abeam (ab9050). Complete list of antibodies are provided in the table. Western Blot
Cells were trypsinized and washed two times with PBS before resuspending in RIPA lysis buffer [50mM Tris pH 7.4, 150mM NaCl, 0.25% Sodium Deoxycholate, 1% P40, ImM EDTA, 10% Glycerol] supplemented with protease inhibitor and PhosSTOP phosphatase inhibitor cocktails (Roche). Cells were lysed on ice for 15min and stored at -80°C until further processing. Lysates were sonicated for 15min at 70% amplitude in QSonica Q700 sonicator followed by centrifugation at 12,000rpm for 15min. Cell supernatant was transferred to a fresh tube and protein estimations were performed with BCA reagent. Western blot analyses were performed as described previously in Black et al. (2010).
Immunoprecipitation
Immunoprecipitation was carried out as described previously in (Van Rechem et al., 2011; Van Rechem et al., 2015). KDM4B was immunoprecipitated from whole- cell lysates using antibody (abeam abl91434) and probed for MCM2 (ab6153) and MCM5 (ab6154).
Chromatin Immunoprecipitation (ChIP)
Chromatin IPs was performed as in Black et al. (2013). Data presented are the averages from two independent siRNAs. For the transfections 0.3x106 RPE were seeded in 10cm plates and transfected with Lipofectamine 3000 (Life Technology) following the supplier instructions. Cells were harvested after 72h of transfection.
Antibodies used for ChIP are as follows: KDM5A (abeam ab70892), KDM5A (Bethyl A300-897 A), KDM4A (P006, Structural Genomic Consortium), KDM4A (P014, Structural Genomic Consortium), KDM4B (abeam abl91434), H3K4me3 (Millipore 07-473), H3 (abeam abl791), Polymerase alpha (ab31777-100) and H3K36me3 (Abeam, ab9050). KDM5A and KDM4A ChlPs were performed with two antibodies, the average of both are presented.
NanoBRET assays
HEK293 cells (4 x 105) were plated in each well of a 12-well plate and co- transfected various NanoLuc donor and HaloTag acceptor pairs including; HaloTag- MCM3 (P25205), HaloTag-MCM4 (P33991), HaloTag-MCM5 (P33992), HaloTag- MCM7 (P33993), HaloTag-POLA2 (Q14181), HaloTag -POLD1 (P28340), NanoLuc- KDM4A (075164), NanoLuc-KDM4B (094953) or HaloTag alone (Promega G6591). All HaloTag and NanoLuc fusion vectors utilized pFN21 A and pFN3 IK backbones respectively (Promega) and were transfected at 1 : 100 fold dilution (2ug HaloTag acceptor: 0.02ug NL donor) or in varying ratios of 1 : 100, 33.3, 11.1 3.7, 1.2, 0.41, 0.14, .0.046, and 0 for donor saturation assays. Twenty hours post-transfection cells were collected, washed with PBS, and exchanged into phenol red-free Opti- MEM in the absence (control sample) or the presence (experimental sample) of 100 nM NanoBRET 618 fluorescent ligand (Promega). Cell density was adjusted to 2 x 105 cells/ml and then re-plated in a 96-well assay white plate (Corning Costar #3917) and let recover for 20 hours at 37°C in the presence of 5% CO2. NanoBRET furimazine substrate (Promega) was added to both control and experimental samples at a final concentration of 10μΜ. Readings were performed within 5 minutes using the GloMax Discover (Promega) equipped with NanoBRET 450/8 nm bandpass and 600 nm longpass filters with a 0.3 second reading setting. A corrected BRET ratio was calculated and is defined as the ratio of the emission at 600 nm/450 nm for experimental samples (i.e. those treated with NanoBRET fluorescent ligand) subtracted by the emission at 600 nm/450 nm for control samples (not treated with NanoBRET fluorescent ligand). BRET ratios are expressed as milliBRET units (mBU), where 1 mBU corresponds to the corrected BRET ratio multiplied by 1000.
Example 1. siRNA screen demonstrated KDM5A depletion promotes site- specific copy gains
Histone lysine demethylase KDM4A generates TSSGs of regions on chromosomes 1 and X (Black et al., 2013). It was hypothesized that additional histone lysine demethylases might also be involved in regulating TSSGs. In order to address this hypothesis, an unbiased siRNA screen was conducted against all lysine demethylase families (KDMl through KDM7) with at least two independent siRNAs (Figure 1A). These were performed in immortalized retinal pigment epithelial cells (RPEs) that have a nearly diploid genome (Black et al., 2013; Jiang et al., 1999). Each set of siRNAs were validated and assessed for major cell cycle defects by flow cytometry analysis before being assayed by DNA fluorescent in situ hybridization (DNA FISH). Cell cycle was evaluated because complete cell cycle arrest could contribute to altered TSSG formation (Black et al., 2013). Evaluation of FACS profiles revealed that only KDMl A, KDMIB, KDM3A, KDM5A, KDM5B, KDM6B and KDM7B resulted in modest yet significant changes in cell cycle distribution. Therefore, we conducted DNA FISH for the chromosome lql2h region that undergoes TSSG and a negative control region on chromosome 8 (chr 8 centromere, 8c) on all KDM siRNA samples (Figure 1A-C).
Only the depletion of KDM5A caused a significant increase in lql2h copy gains (Figure 1B,C), while spectral karyotyping (SKY) of KDM5A siRNA depleted cells showed no major karyotype issues. We further validated the lql2h copy gains upon KDM5A siRNA in an additional cancer cell line (Figure ID). Consistent with this observation, pharmacologic inhibition with the KDM5 enzyme family inhibitor [KDM5i, KDM5-C70 (Johansson et al., 2016)] caused a dose-dependent increase in lql2h copy gains in multiple cell lines (Figure 1E,F). We further tested whether KDM5A depletion was sufficient to cause other previously identified TSSGs to undergo copy number gains. KDM5A depletion caused significant copy number gains for other TSSGs (Figure 1G; lq21.2 and Xql3.1), while control regions did not have copy number changes- lq23.3, lqtel and X centromere (Figure 1G).
Furthermore, chromatin immunoprecipitation (ChIP) analyses demonstrated a significant reduction of KDM5A at TSSG sites upon KDM5A siRNA treatment
(Figure 1H), which suggests that KDM5 A directly binds to these regions and prevents copy number gains. Taken together, these data demonstrate that KDM5 A depletion and inhibition is sufficient to directly generate copy number gains of specific loci.
Example 2. KDM5A-dependent copy gains are transient, require S phase and are derived from rereplication
A key characteristic of TSSGs is that they are transient and occur in an S phase-dependent manner (Black et al., 2015; Black et al., 2013). In order to address these points, we took advantage of a KDM5 family inhibitor that generated TSSGs (KDM5i, KDM5-C70 (Johansson et al., 2016)]. Treatment with KDM5 inhibitor causes a global change in H3K4me3 level as observed previously (Johansson et al., 2016). RPE cells generated copy gains after 48h or 72h of drug exposure (no wash off); however, DNA copy gains significantly diminished 24h after the KDM5 inhibitor was removed (24h wash off, Figure 2A-C). These experiments highlight the transient nature of the KDM5A-dependent copy gains.
In order to determine whether the copy gains are generated during S phase, RPE cells were arrested in Gl/S with hydroxyurea (HU) treatment before receiving KDM5 inhibitor (Figure 2D). The pretreatment with HU blocked KDM5i from generating DNA copy gains (Figure 2E). However, KDM5i treated cells released from HU generated copy gains (Figure 2F). These data demonstrated that KDM5 inhibition results in S phase-dependent copy gains.
Rereplication could be one mechanism underlying TSSG generation (Black et al., 2015; Black et al., 2013). In order to determine whether KDM5A depletion induced rereplication, we first performed cesium chloride gradient ultracentrifugation to isolate rereplicated DNA (termed heavy-heavy, H:H; Figure 2G) for both control and KDM5A knockdown cells. The heavy-heavy fractions for each cellular condition were pooled together before the genomic DNA was purified and quantified by polymerase chain reaction (qPCR) (Figure 2G). We observed significant enrichment of heavy-heavy DNA at copy gained regions lql2h, lql2/21, lq21.2 and Xql3.1 upon KDM5A depletion (Figure 2H); however, no enrichment was observed at the lq23.3 control region. This suggested that reduced KDM5A levels promote rereplication at sites undergoing TSSG. Example 3. KDM5A depletion promotes TSSG generation post S phase
TSSGs are cleared by the end of S phase of cell cycle (Black et al., 2013). We tested whether the copy gains generated by KDM5 A depletion follow the classical TSSG kinetics. The cells were arrested in late G2 with a CDKl/cyclin inhibitor (CDKli, Ro-3306) before copy gain analyses by DNA FISH (Figure 21). We observed that the TSSGs generated by KDM5A depletion occurred after Ro-3306 treatment (Figure 2J), which demonstrated that copy gains occurred or persisted outside of S phase. Therefore, either the mechanism(s) that remove copy number gains are altered in KDM5A depleted cells or the copy gains are still being generated during G2 in KDM5A depleted cells. Since these copy gains were transient, we hypothesized that KDM5A depletion created a permissive chromatin state that allowed replication to occur during late G2. To test this possibility, we determined whether DNA polymerase (i.e., DNA pola) was present during late G2 at rereplicated regions. Upon KDM5A depletion, cells arrested in late G2 had enriched DNA Pola occupancy at rereplicated and copy gained loci compared to control cells, while a non-copy gained locus did not have DNA Pola enrichment (Figure 2K). Our observations support the hypothesis that KDM5A depletion alters the chromatin state so that rereplication and copy gains occur during S phase and continues into late G2. This hypothesis and set of observations are also consistent with a previous report suggesting that KDM5A impacts origin recognition (Huang et al., 2016).
Example 4. KDM5A-dependent copy gains require KDM4A
KDM5A demethylates H3K4me3/2/l, which associates with active gene expression (Christensen et al., 2007; Iwase et al., 2007; Klose et al., 2007). Since KDM4A overexpression and stabilization generate TSSGs (Mishra and Whetstine, 2016), we tested whether KDM4A protein levels increased after KDM5A depletion. KDM4A protein levels did not change upon KDM5A depletion (Figure 3F). Another possibility is that KDM5A depletion targets KDM4A to sites undergoing TSSG. The Tudor domains within KDM4A recognize H3K4me3 and H4K20me3 (Huang et al., 2006; Spektor and Rice, 2009) and are required for TSSGs (Black et al., 2013).
Therefore, we hypothesized that the depletion of KDM5A promotes DNA copy gains by increasing H3K4me3 and enriching KDM4A at rereplicated and DNA copy gained sites. To address this hypothesis, we conducted DNA FISH on cells co-depleted for KDM4A and KDM5A (Figure 3A). Depletion of KDM4A blocked the DNA copy gains observed with KDM5A depletion (Figure 3A). Consistent with this observation, there was an increase in KDM4A occupancy at regions undergoing amplification upon KDM5A depletion (Figure 3B) and increased levels of H3K4me3 (Figure 3C). However, the non-copy gained region (lq23.3) did not have KDM4A binding or an increased H3K4me3 (Figure 3B-). In addition, introduction of a histone H3.3 lysine 4 mutant (H3K4M) abrogated the TSSG generated by KDM5A depletion and KDM4A overexpression (Figure 3D-E). These observations illustrate a mechanism for recruiting KDM4A to sites so that site-specific rereplication and copy gains are generated. Example 5. H3K4 KMTs regulate specific sites undergoing TSSG
Our results emphasized the importance of H3K4 methylation balance in regulating TSSGs. Therefore, we determined whether the H3K4 lysine
methyltransferases (KMTs) regulate site-specific copy gains. First, we conducted co- depletion experiments upon KDM5A siRNA treatment to establish the enzymes balancing KDM5 A. The KMTs that rescued the TSSGs generated by KDM5 A depletion were then assessed for their ability to generate TSSGs when overexpressed alone. By requiring the KMT to score in both assays, there was an increased probability that the enzyme was directly involved in modulating site-specific copy gains (Figure 4A).
Specifically, we assessed whether siRNA depletion of H3K4 KMTs (KMT2A, KMT2B, KMT2C, KMT2D, SETD1 A and SETD1B) would block the TSSGs generated by KDM5 A depletion. Each KMT was depleted with at least two independent siRNAs. The single and co-depletions for each KMT siRNA were verified for knockdown and assessed for major alterations in cell cycle profiles by FACS. Minor yet significant cell cycle arrests were observed with KMT siRNAs. For example, SETD1 A depletion alone resulted in an increase in Gl/S as previously reported (Tajima et al., 2015). Additional cell cycle changes occurred upon
KMT2B/MLL2 and KMT2D/MLL4 depletion with KDM5A. We then evaluated copy gains upon the depletion of individual KMTs (Figure 8A-L). Only SETD 1 A appeared to cause copy gains at most sites tested (Figures 8C, G, K), which likely suggests a global chromosomal instability in SETD1 A depleted cells.
Even though depletion of individual KMTs did not significantly impact copy gains, the depletion of select KMTs altered copy gains caused by KDM5 A
knockdown (Figures 8A-L). For example, depletion of KMT2B, KMT2C, KMT2D and SETD1B rescued lql2h and lq21.2 copy gains caused by KDM5A depletion (Figures 8A, B, D, E, F, H). On the other hand, KMT2A/MLL1 and KMT2D/MLL4 depletion rescued lq21.3 DNA copy gains (Figure 81). This suggests a higher degree of specificity for KMTs in generating site-specific copy gains in the genome;
however, an interplay between KMTs could explain why a collection of KMTs impact certain TSSGs (e.g., Iql2h and lq21.2).
To ensure that each KMT was truly impacting the sites undergoing TSSG, we overexpressed individual H3K4 KMTs, with the exception of KMT2C because of our inability to generate full-length expression vectors. Overexpression was validated and FACS profiled before being assayed by DNA FISH for copy gains (Figures 8M-W). Upon overexpression, an even higher degree of specificity for the KMTs was observed (Figure 4A and Figures 8M-W). For example, SETD1B was the only KMT that generated lql2h upon overexpression (Figure 4A and Figures 8M). The other KMTs did not generate lql2h copy gains (Figures 8N-P) even though some of them rescued the copy gains observed in KDM5A depleted cells (Figures 8A-D). In the case of lq21.2, DNA copy gains were caused by KMT2B, KMT2D and SETD1B (Figures 8Q-S), while only KMT2A and KMT2D overexpression caused lq21.3 DNA copy gains (Figures 8T-W). These observations illustrate that specific subsets of H3K4 KMTs are maintaining a balance with KDM5A at sites undergoing TSSG (Figure 4A)
Since KMT2 members balance KDM5A generation of TSSGs and KDM4A was required for KDM5 A-associated copy gains, we hypothesized that individual KMTs generate their associated TSSG via KDM4A. To test this hypothesis, we depleted KDM4A before overexpressing individual H3K4 KMTs and assessing copy gains by DNA FISH. Since KMT2B expression was decreased after KDM4A depletion, we did not assess the copy gain relationship for this pair. However, TSSGs generated by overexpression of the other H3K4 KMTs were either completely or partially rescued upon KDM4A depletion (Figure 4B-I). For example, depletion of KDM4A suppressed SETD1B generated lql2h and lq21.2 gains (Figure 4B,C and 4E-F), but only partially rescuing lq21.2 copy gains generated by KMT2D overexpression (Figure 4D). The lq21.3 TSSG generated by either KMT2A or KMT2D overexpression were rescued upon KDM4A depletion (Figure 4G-I). This emphasizes the need to balance KMT/KDM expression so that site-specific DNA amplification is kept in check. Taken together, these experiments reveal that multiple chromatin factors are regulating different TSSGs within the human genome, which suggests the probability that other KMT/KDM networks are modulating TSSGs at other sites in the genome.
Example 6. Identification of a KDM4A-independent TSSG
While testing whether KDM4A depletion could suppress each KMT driven copy gain, we observed that KMT2A overexpression resulted in DNA copy gains at lp32.3 (Figure 4G). This region has been used as a control region for TSSG experiments and was not regulated by KDM4A overexpression or stabilization (Black et al., 2015; Black et al., 2013; Black et al., 2016) or KDM5A depletion (Figure 5A- B). Therefore, KMT2A overexpression generated a site-specific copy gain through another set of chromatin regulators, specifically KDMs associated with H3K4 methylation.
We tested whether KDMs related to H3K4 could balance KMT2A-dependent DNA copy gains at lp32.3. An siRNA screen against KDM5 family members identified KDM5B as a significant regulator of lp32.3 DNA copy gains (Figure 5A- B). In fact, chemical inhibition of the KDM5 family members also generated copy gains of both lp32.3 and 1 q21.3 regions (Figure 5C). To determine if the lp32.3 copy gains are transient, the KDM5i drug was washed off (Figure 5D) and the copy gains returned to baseline levels, suggesting that the lp32.3 copy gains are transiently generated (Figure 5E). We then demonstrated that KDM5B overexpression could block KMT2A-generated lp32.3 copy gains (Figure 5F and Figure 8X), while not interfering with the other KMT2A copy gained site (lq21.3) under KDM5A regulation (Figure 5F). Furthermore, introduction of H3K4M abrogated KDM5B and KMT2A-dependent lp32.3 copy gains (Figure 5G-H and Figures 8Y-A'). In fact, H3K4M blocked both the copy gained regions generated by KMT2A (lp32.3 and lq21.3) even though specific KDM5 members controlled these TSSGs. Taken together, these data underscore an important role for H3K4 KMTs and KDMs in regulating TSSGs at other genomic loci that are independent of KDM4A regulation.
Example 7. Ip32.3 DNA copy gains require KDM4B
Besides KDM4A, KDM4B and KDM4C also have Tudor domains that are involved in their recruitment to chromatin (Pedersen et al., 2016; Su et al., 2016). Therefore, we hypothesized that additional KDM4 enzymes could be involved in regulating the KDM4A-independent lp32.3 DNA copy gains. To test this hypothesis, we overexpressed KDM4A-C family members and assessed whether copy gains occurred at lp32.3 locus. Only KDM4B overexpression resulted in a significant lp32.3 copy gains (Figure 6A-B). Furthermore, lp32.3 copy gains required KDM4B catalytic activity (Figure 6C). We then tested whether KDM4B overexpression caused rereplication at the lp32.3 locus. Upon KDM4B overexpression, rereplication was observed within the lp32.3 region covered by the DNA FISH probe (Figure 6D).
Consistent with KDM4B overexpression causing rereplication and copy gains, mass spectrometry analyses identified an association of KDM4B with replication machinery proteins (i.e., MCM3, MCM5, MCM7, RFC3 and DNA J A3) that was verified by co-immunoprecipitation analyses. These interactions were also confirmed in vivo. We used NanoBRET (Nano-luciferase Bioluminescent Resonance Energy Transfer), a proximity based protein: protein interaction live cell assay, to confirm the interactions between KDM4B, MCM components and DNA polymerases. We observed an interaction of KDM4A with the MCM proteins and DNA polymerases (Figure 6E, top panel) that is consistent with our previous discovery that KDM4A associated with the replication machinery components (Black et al., 2013). Energy transfer between KDM4B and the MCMs and DNA polymerase subunits were observed to varying degrees across multiple subunits within these larger complexes, which indicates association with these protein complexes (Figure 6E, bottom panel). As a negative control, HaloTag alone was used and did not result in significant energy transfer to KDM4B. In addition, NanoBRET donor saturation assays were performed with KDM4B to demonstrate specificity of the NanoBRET signal (Figure 6F).
Interactions of KDM4B with either the MCM or DNA polymerase proteins showed expected saturation of signal after increasing acceptor levels relative to a low amount of fixed donor. The linear and flat response of KDM4B with the HaloTag alone is an example of a response when there is no specific interaction (Figure 6F). Taken together, these results suggest that KDM4B directly associates with replication machinery to promote rereplication and copy gain of the lp32.3 region.
Since KMT2A promoted lp32.3 DNA copy gain in a KDM4A-independent manner (Figure 4G), we tested whether KDM4B was required for KMT2A generated copy gains of lp32.3. KDM4B depletion abrogated KMT2A-generated lp32.3 copy gains, demonstrating that KDM4B was essential for the copy gain generation by KMT2A (Figure 7A and Figure S7A-B). We further demonstrated that KDM4B was required for the copy gain generated with KDM5B depletion, while KDM4A depletion did not have any effect on lp32.3 copy gains (Figure 7B). Furthermore, the introduction of the H3K4M mutant abrogated the lp32.3 copy gain generated by KDM4B overexpression (Figure 7C), which suggests KDM4B recruitment on chromatin occurs via H3K4 methylation. Consistent with this possibility, we demonstrated with the Nano-BRET assay that cells expressing H3K4M had reduced KDM4B associated with chromatin in vivo (Figure 7D-E).
Our data suggest that KDM4B recruitment is required for the rereplication and lp32.3 copy gains. Using ChIP, we observed an enrichment of KDM4B at the rereplication sites upon stable overexpression (Figure 7F). Furthermore, siRNA depletion of KDM4B in KDM4B overexpressing cells reduced the occupancy of KDM4B to the baseline level at the rereplicated regions (lp32.3-4 and lp32.3-5). Since KDM4B catalyzes demethylation of histone lysine 9 and lysine 36 residues, we tested whether direct methylation interference by introducing H3K9M and H3K36M histone mutations would induce lp32.3 copy gains (Black et al., 2013). Introduction of both K9M and K36M caused copy gains of lq21.2 region (Figure 7G), which was consistent with our previous report (Black et al., 2013). However, only H3K36M caused the lp32.3 locus to undergo copy gains (Figure 7G), which suggests specificity for H3K36 methylation in causing copy gains of lp32.3 region. Since KDM4B is recruited to the rereplicated and copy-gained region and the copy gains are catalytically dependent, we assessed the impact of KDM4B overexpression on H3K36me3 at lp32.3 locus. A significant decrease in the H3K36me3 levels occurred with KDM4B overexpression at the rereplicated region (i.e., lp32.3-3, -4 and -5), while a non-rereplicated region (lp32.3-6) remained unaffected (Figure 7H). Taken together, these data suggest that KDM4B recruitment and catalytic activity is essential for reducing H3K36me3 at the rereplicated and copy gained lp32.3 locus (Figure 71). These findings emphasize the need to maintain an appropriate KMT-KDM balance to preserve proper DNA copy number within the genome. In addition, these results highlight the importance of localized chromatin states and their impact on site- specific rereplication and DNA copy gains.
Example 8. KDM5 inhibitor generated copy gains are blocked selectively by specific KDM4 members
RPE cells were transfected with two independent siRNA (life technologies) to either KDM4A (sl8635 and sl8636) or KDM4B (s22867 and s229325) before being treated with the KDM5 inhibitor KDM5 C-70 (10 μΜ). The lql2h region
rereplication that was caused with KDM5 inhibitor was rescued with KDM4A depletion, illustrating the importance of KDM4A in the KDM5 inhibitor generated copy gains. Similarly, KDM4B depletion blocked the same KDM5 inhibitor's ability to generate lp32.3 rereplication. These data collectively illustrate the importance in targeting KDM4 members to control the generation of amplifications downstream of global KDM5 inhibition.
REFERENCES
Alt, F.W., Kellems, R.E., Bertino, J.R., and Schimke, R.T. (1978). Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. The Journal of biological chemistry 253, 1357-1370. Beck, D.B., Burton, A., Oda, H., Ziegler-Birling, C, Torres-Padilla, M.E., and Reinberg, D. (2012). The role of PR-Set7 in replication licensing depends on Suv4- 20h. Genes Dev 26, 2580-2589.
Beroukhim, R., Mermel, C.H., Porter, D., Wei, G., Raychaudhuri, S.,
Donovan, J., Barretina, J., Boehm, J.S., Dobson, J., Urashima, M., et al. (2010). The landscape of somatic copy-number alteration across human cancers. Nature 463, 899- 905.
Biedler, J.L., and Spengler, B.A. (1976a). Metaphase chromosome anomaly: association with drug resistance and cell-specific products. Science 191, 185-187.
Biedler, J.L., and Spengler, B.A. (1976b). A novel chromosome abnormality in human neuroblastoma and antifolate-resistant Chinese hamster cell lives in culture. Journal of the National Cancer Institute 57, 683-695.
Black, J.C., Allen, A., Van Rechem, C, Forbes, E., Longworth, M., Tschop, K., Rinehart, C, Quiton, J., Walsh, R., Smallwood, A., et al. (2010). Conserved antagonism between JMJD2A/KDM4A and HPlgamma during cell cycle
progression. Molecular cell 40, 736-748.
Black, J.C., Atabakhsh, E., Kim, J., Biette, K.M., Van Rechem, C, Ladd, B., Burrowes, P.D., Donado, C, Mattoo, H., Kleinstiver, B.P., et al. (2015). Hypoxia drives transient site-specific copy gain and drug-resistant gene expression. Genes & development 29, 1018-1031.
Black, J.C., Manning, A.L., Van Rechem, C, Kim, J., Ladd, B., Cho, J., Pineda, CM., Murphy, N., Daniels, D.L., Montagna, C, et al. (2013). KDM4A lysine demethylase induces site-specific copy gain and rereplication of regions amplified in tumors. Cell 154, 541-555.
Black, J.C., and Whetstine, J.R. (2015). Too little 02 Too much gain. Cell cycle 14, 2869-2870.
Black, J.C., Zhang, H., Kim, J., Getz, G, and Whetstine, J.R. (2016).
Regulation of Transient Site-specific Copy Gain by microRNA. The Journal of biological chemistry.
Burrell, R.A., and Swanton, C. (2014). Tumour heterogeneity and the evolution of polyclonal drug resistance. Mol Oncol 8, 1095-1111.
Calvi, B.R., Lilly, M.A., and Spradling, A.C. (1998). Cell cycle control of chorion gene amplification. Genes Dev 12, 734-744. Calvi, B.R., and Spradling, A.C. (1999). Chorion gene amplification in Drosophila: A model for metazoan origins of DNA replication and S-phase control. Methods 18, 407-417.
Christensen, J., Agger, K., Cloos, P. A., Pasini, D., Rose, S., Sennels, L., Rappsilber, J., Hansen, K.H., Salcini, A.E., and Helin, K. (2007). RBP2 belongs to a family of demethylases, specific for tri-and dimethylated lysine 4 on histone 3. Cell 128, 1063-1076.
Dileep, V., Rivera-Mulia, J.C., Sima, J., and Gilbert, D M. (2015). Large- Scale Chromatin Structure-Function Relationships during the Cell Cycle and
Development: Insights from Replication Timing. Cold Spring Harb Symp Quant Biol.
Dimova, I, Orsetti, B., Theillet, C, Dimitrov, R., and Toncheva, D. (2009). Copy Number Changes in 1 q21.3 and lq23.3 have Different Clinical Relevance in Ovarian Tumors. Balkan Journal of Medical Genetics 12, 29-37 '.
Diskin, S.J., Hou, C, Glessner, J.T., Attiyeh, E.F., Laudenslager, M., Bosse, K., Cole, K., Mosse, Y.P., Wood, A., Lynch, J.E., et al. (2009). Copy number variation at 1 q21.1 associated with neuroblastoma. Nature 459, 987-991.
Donley, N., and Thayer, M.J. (2013). DNA replication timing, genome stability and cancer: late and/or delayed DNA replication timing is associated with increased genomic instability. Semin Cancer Biol 23, 80-89.
Fonseca, R., Van Wier, S.A., Chng, W.J., Ketterling, R., Lacy, M.Q.,
Dispenzieri, A., Bergsagel, P.L., Rajkumar, S.V., Greipp, P.R., Litzow, M.R., et al. (2006). Prognostic value of chromosome lq21 gain by fluorescent in situ
hybridization and increase CKS1B expression in myeloma. Leukemia 20, 2034-2040.
Goeze, A., Schluns, K., Wolf, G., Thasler, Z., Petersen, S., and Petersen, I. (2002). Chromosomal imbalances of primary and metastatic lung adenocarcinomas. J Pathol 196, 8-16.
Haber, D.A., and Schimke, R.T. (1981). Unstable amplification of an altered dihydrofolate reductase gene associated with double-minute chromosomes. Cell 26, 355-362.
Huang, C, Cheng, J., Bawa-Khalfe, T., Yao, X., Chin, Y.E., and Yeh, E T.
(2016). SUMOylated ORC2 Recruits a Histone Demethylase to Regulate Centromeric Histone Modification and Genomic Stability. Cell Rep 15, 147-157. Huang, Y., Fang, J., Bedford, M.T., Zhang, Y., and Xu, R.M. (2006).
Recognition of histone H3 lysine-4 methylation by the double tudor domain of JMJD2A. Science 312, 748-751.
Inoue, J., Otsuki, T., Hirasawa, A., Imoto, I, Matsuo, Y., Shimizu, S., Taniwaki, M., and Inazawa, J. (2004). Overexpression of PDZKl within the Iql2-q22 Amplicon Is Likely To Be Associated with Drug-Resistance Phenotype in Multiple Myeloma. The American Journal of Pathology 165, 71-81.
Iwase, S., Lan, F., Bayliss, P., de la Torre-Ubieta, L., Huarte, M., Qi, H.H., Whetstine, J.R., Bonni, A., Roberts, T.M., and Shi, Y. (2007). The X-linked mental retardation gene SMCX/JARIDIC defines a family of histone H3 lysine 4
demethylases. Cell 128, 1077-1088.
Jiang, X.R., Jimenez, G., Chang, E., Frolkis, M., Kusler, B., Sage, M., Beeche, M., Bodnar, A.G., Wahl, G.M., Tlsty, T.D., et al. (1999). Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat Genet 21, 111-114.
Johansson, C, Velupillai, S., Tumber, A., Szykowska, A., Hookway, E.S., Nowak, R.P., Strain-Damerell, C, Gileadi, C, Philpott, M., Burgess-Brown, N., et al. (2016). Structural analysis of human KDM5B guides histone demethylase inhibitor development. Nat Chem Biol 12, 539-545.
Kendall, J., Liu, Q., Bakleh, A., Krasnitz, A., Nguyen, K.C., Lakshmi, B.,
Gerald, W.L., Powers, S., and Mu, D. (2007). Oncogenic cooperation and
coamplification of developmental transcription factor genes in lung cancer. Proc Natl Acad Sci U S A 104, 16663-16668.
Kim, T.M., Xi, R., Luquette, L.J., Park, R.W., Johnson, M.D., and Park, P.J. (2013). Functional genomic analysis of chromosomal aberrations in a compendium of 8000 cancer genomes. Genome Res 23, 217-227.
Klose, R.J., Yan, Q., Tothova, Z., Yamane, K., Erdjument-Bromage, H., Tempst, P., Gilliland, D.G., Zhang, Y., and Kaelin, W.G, Jr. (2007). The
retinoblastoma binding protein RBP2 is an H3K4 demethylase. Cell 128, 889-900.
Kuo, A.J., Song, J., Cheung, P., Ishibe-Murakami, S., Yamazoe, S., Chen,
J.K., Patel, D.J., and Gozani, O. (2012). The BAH domain of ORCl links H4K20me2 to DNA replication licensing and Meier-Gorlin syndrome. Nature 484, 115-119. Lawrence, M.S., Stojanov, P., Mermel, C.H., Robinson, J.T., Garraway, L.A., Golub, T.R., Meyerson, M, Gabriel, S.B., Lander, E.S., and Getz, G. (2014).
Discovery and saturation analysis of cancer genes across 21 tumour types. Nature 505, 495-501.
Lawrence, M.S., Stojanov, P., Polak, P., Kryukov, G.V., Cibulskis, K.,
Sivachenko, A., Carter, S.L., Stewart, C, Mermel, C.H., Roberts, S.A., et al. (2013). Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature s, 214-218.
Lu, C, Jain, S.U., Hoelper, D., Bechet, D., Molden, R.C., Ran, L., Murphy, D., Venneti, S., Hameed, M., Pawel, B.R., et al. (2016a). Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape. Science 352, 844-849.
Lu, F., Wu, X., Yin, F., Chia-Fang Lee, C, Yu, M., Mihaylov, I S., Yu, J., Sun, H., and Zhang, H. (2016b). Regulation of DNA replication and chromosomal polyploidy by the MLL-WDR5-RBBP5 methyltransferases. Biol Open 5, 1449-1460.
MacAlpine, D.M., and Almouzni, G. (2013). Chromatin and DNA replication. Cold Spring Harbor perspectives in biology 5, aO 10207.
Mallette, F.A., Mattiroli, F., Cui, G., Young, L.C., Hendzel, M.J., Mer, G., Sixma, T.K., and Richard, S. (2012). RNF8- and RNF168-dependent degradation of KDM4A/JMJD2A triggers 53BP1 recruitment to DNA damage sites. EMBO J 31, 1865-1878.
Mishra, S., and Whetstine, J.R. (2016). Different Facets of Copy Number Changes: Permanent, Transient, and Adaptive. Mol Cell Biol.
Morgan, M.A., and Shilatifard, A. (2015). Chromatin signatures of cancer. Genes Dev 29, 238-249.
Nathanson, D.A., Gini, B., Mottahedeh, J., Visnyei, K., Koga, T., Gomez, G., Eskin, A., Hwang, K., Wang, J., Masui, K., et al. (2014). Targeted therapy resistance mediated by dynamic regulation of extrachromosomal mutant EGFR DNA. Science 343, 72-76.
Papillon-Cavanagh, S., Lu, C, Gayden, T., Mikael, L.G., Bechet, D.,
Karamboulas, C, Allies, L., Karamchandani, J., Marchione, D.M., Garcia, B.A., et al. (2017). Impaired H3K36 methylation defines a subset of head and neck squamous cell carcinomas. Nat Genet 49, 180-185. Pedersen, M.T., Kooistra, S.M., Radzisheuskaya, A., Laugesen, A., Johansen, J. V., Hayward, D.G., Nilsson, J., Agger, K., and Helin, K. (2016). Continual removal of H3K9 promoter methylation by Jmjd2 demethylases is vital for ESC self-renewal and early development. EMBO J 35, 1550-1564.
Petersen, S., Aninat-Meyer, M., Schluns, K., Gellert, K., Dietel, M., and
Petersen, I. (2000). Chromosomal alterations in the clonal evolution to the metastatic stage of squamous cell carcinomas of the lung. Br J Cancer 82, 65-73.
Rivera, C, Gurard-Levin, Z.A., Almouzni, G., and Loyola, A. (2014). Histone lysine methylation and chromatin replication. Biochim Biophys Acta 1839, 1433- 1439.
Rondinelli, B., Schwerer, H., Antonini, E., Gaviraghi, M., Lupi, A.,
Frenquelli, M., Cittaro, D., Segalla, S., Lemaitre, J.M., and Tonon, G. (2015).
H3K4me3 demethylation by the histone demethylase KDM5C/JARID1C promotes DNA replication origin firing. Nucleic Acids Res 43, 2560-2574.
Smith, O.K., and Aladjem, M.I. (2014). Chromatin structure and replication origins: determinants of chromosome replication and nuclear organization. J Mol Biol 426, 3330-3341.
Spektor, T.M., and Rice, J.C. (2009). Identification and characterization of posttranslational modification-specific binding proteins in vivo by mammalian tethered catalysis. Proc Natl Acad Sci U S A 106, 14808-14813.
Su, Z., Wang, F., Lee, J.H., Stephens, K.E., Papazyan, R., Voronina, E., Krautkramer, K.A., Raman, A., Thorpe, J. J., Boersma, M.D., et al. (2016). Reader domain specificity and lysine demethylase-4 family function. Nat Commun 7, 13387.
Tajima, K., Yae, T., Javaid, S., Tarn, O., Comaills, V., Morris, R., Wittner, B.S., Liu, M., Engstrom, A., Takahashi, F., et al. (2015). SETD1 A modulates cell cycle progression through a miRNA network that regulates p53 target genes. Nat Commun 6, 8257.
Tardat, M., Brustel, J., Kirsh, O., Lefevbre, C, Callanan, M., Sardet, C, and ien, E. (2010). The histone H4 Lys 20 methyltransferase PR-Set7 regulates replication origins in mammalian cells. Nat Cell Biol 12, 1086-1093.
Turner, K.M., Deshpande, V., Beyter, D., Koga, T., Rusert, J., Lee, C, Li, B., Arden, K., Ren, B., Nathanson, D.A., et al. (2017). Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity. Nature 543, 122- 125.
Van Rechem, C, and Whetstine, J.R. (2014). Examining the impact of gene variants on histone lysine methylation. Biochim Biophys Acta 1839, 1463-1476.
Weir, B.A., Woo, M.S., Getz, G., Perner, S., Ding, L., Beroukhim, R., Lin, W.M., Province, M.A., Kraja, A., Johnson, L.A., et al. (2007). Characterizing the cancer genome in lung adenocarcinoma. Nature 450, 893-898.
Zack, T.I., Schumacher, S.E., Carter, S.L., Cherniack, A.D., Saksena, G., Tabak, B., Lawrence, M.S., Zhsng, C.Z., Wala, J., Mermel, C.H., et al. (2013). Pan- cancer patterns of somatic copy number alteration. Nat Genet 45, 1134-1140.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. A method of treating a subject who has a cancer with reduced levels or activity of KDM5 A, or who is being treated with a KDM5 A inhibitor, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of KDM4A.
2. The method of claim 1, further comprising identifying a subject who has a cancer with a KDM5A gene suppression/mutation/deletion.
3. The method of claim 1, further comprising identifying a subject who has an
amplification at lql2h, lql2/21, lq21.2, Iq21.3/CKS1B, or Xql3.1.
4. A method of treating a subject who has a cancer with reduced levels or activity of KDM5B, or who is being treated with a KDM5B inhibitor, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of KDM4B.
5. The method of claim 4, further comprising identifying a subject who has a cancer with a KDM5B gene suppression/mutation/deletion.
6. The method of claim 4, further comprising identifying a subject who has an
amplification at lp32.3.
7. The method of claim 6, further comprising administering a cdk4/6 inhibitor to a subject who has an amplification at lp32.3.
8. A method of treating a subject who has a cancer with increased levels or activity of a KMT2 family member, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of KDM4.
9. The method of claim 8, wherein the inhibitor of KDM4 inhibits KDM4A,
KDM4B, or both KDM4A and KDM4B.
10. The method of claim 8, wherein the KMT2 family member is KMT2A, KMT2B, KMT2C, KMT2D, SETDlA or SETD1B.
11. A method of treating a subject who has a mixed-lineage leukemia (MLL) gene (KMT2A) translocation, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of KDM4.
12. The method of claim 11, wherein the subject has acute myeloid leukemia (AML), the acute lymphoblastic leukemia (ALL), or biphenotypic (mixed lineage) leukemia.
13. A method of treating a subject who has cancer, the method comprising
administering to the subject:
(i) an inhibitor of KDM4A and an inhibitor of KDM5A; or
(ii) an inhibitor of KDM4b and an inhibitor of KDM5B.
14. The method of any of the preceding claims, wherein the cancer is a carcinoma, sarcoma, myeloma, metastatic disorder, or a hematopoietic neoplastic disorder.
PCT/US2017/064295 2016-12-01 2017-12-01 Methods for the treatment of cancer WO2018102736A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201662428941P 2016-12-01 2016-12-01
US62/428,941 2016-12-01

Publications (1)

Publication Number Publication Date
WO2018102736A1 true WO2018102736A1 (en) 2018-06-07

Family

ID=62241977

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2017/064295 WO2018102736A1 (en) 2016-12-01 2017-12-01 Methods for the treatment of cancer

Country Status (1)

Country Link
WO (1) WO2018102736A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023244870A1 (en) * 2022-06-17 2023-12-21 INSTITUTE FOR CANCER RESEARCH d.b.a THE RESEARCH INSTITUTE OF FOX CHASE CANCER CENTER Compositions and methods for control of transient site-specific copy gains, genomic insertions, and rearrangements associated with mixed lineage leukemia

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160113911A1 (en) * 2013-06-06 2016-04-28 The General Hospital Corporation Methods and compositions for the treatment of cancer
WO2016186949A1 (en) * 2015-05-15 2016-11-24 Whetstine Johnathan R Methods relating to the prevention and treatment of drug resistance
WO2017099829A1 (en) * 2015-12-11 2017-06-15 The General Hospital Corporation Compositions and methods for treating drug-tolerant glioblastoma

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160113911A1 (en) * 2013-06-06 2016-04-28 The General Hospital Corporation Methods and compositions for the treatment of cancer
WO2016186949A1 (en) * 2015-05-15 2016-11-24 Whetstine Johnathan R Methods relating to the prevention and treatment of drug resistance
WO2017099829A1 (en) * 2015-12-11 2017-06-15 The General Hospital Corporation Compositions and methods for treating drug-tolerant glioblastoma

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BLACK ET AL.: "Histone Lysine Methylation Dynamics: Establishment, Regulation, and Biological Impact", MOLECULAR CELL, vol. 48, no. 4, 30 November 2012 (2012-11-30), pages 491 - 507, XP055492085, Retrieved from the Internet <URL:doi:10.1016/j.molcel.2012.11.006> *
HOPP ET AL.: "Epigenetic Heterogeneity of B- Cell Lymphoma: Chromatin Modifiers", GENES, vol. 6, no. 4, 21 October 2015 (2015-10-21), pages 1076 - 1112, XP055492084, ISSN: 2073-4425, Retrieved from the Internet <URL:doi:10.3390/genes6041076> *
KLEIN ET AL.: "The Histone H3K4-Specific Demethylase KDM5B Binds to its Substrate and Product Through Distinct PHD Fingers", CELL REPORTS, vol. 6, no. 2, 30 January 2014 (2014-01-30), pages 325 - 335, XP055492077, Retrieved from the Internet <URL:doi:10.1016/j.celrep.2013.12.021> *
KRESSE ET AL.: "DNA Copy Number Changes in Human Malignant Fibrous Histiocytomas by Array Comparative Genomic Hybridisation", PLOS ONE, vol. 5, no. 11, 9 November 2010 (2010-11-09), pages 1 - 10, XP055492081, Retrieved from the Internet <URL:https://doi.org/10.1371/journal.pone.0015378> *
ZHANG ET AL.: "Synchronized Targeting of Notch and ERBB Signaling Suppresses Melanoma Tumor Growth through Inhibition of Notch1 and ERBB3", THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, vol. 136, no. 2, 18 January 2015 (2015-01-18), pages 464 - 472, XP055492053, Retrieved from the Internet <URL:DOI:10.1016/j.jid.2015.11.006> *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023244870A1 (en) * 2022-06-17 2023-12-21 INSTITUTE FOR CANCER RESEARCH d.b.a THE RESEARCH INSTITUTE OF FOX CHASE CANCER CENTER Compositions and methods for control of transient site-specific copy gains, genomic insertions, and rearrangements associated with mixed lineage leukemia

Similar Documents

Publication Publication Date Title
US20230348908A1 (en) RNA Interactome of Polycomb Repressive Complex 1 (PRC1)
US11066673B2 (en) Polycomb-associated non-coding RNAs
DK2655621T3 (en) Polycomb-associated non-coding RNAS
US9322015B2 (en) Methods of using microRNA-26a to promote angiogenesis
US20220152081A1 (en) Agents and methods for treating pancreatic ductal adenocarcinomas
EP3283502A1 (en) Methods for reactivating genes on the inactive x chromosome
WO2022086935A1 (en) Targeting xist and rna methylation for x reactivation therapy
US11459568B2 (en) Targeting microRNA-101-3p in cancer therapy
WO2018160356A1 (en) Genetic and pharmacological transcriptional upregulation of the repressed fxn gene as a therapeutic strategy for friedreich ataxia
WO2020081585A1 (en) The long non-coding rna inca1 and homo sapiens heterogeneous nuclear ribonucleoprotein h1 (hnrnph1) as therapeutic targets for immunotherapy
US20210222168A1 (en) Methods for reactivating genes on the inactive x chromosome
WO2018102736A1 (en) Methods for the treatment of cancer
US20220340908A1 (en) Methods for depletion of deleterious mitochondrial genomes
US20210380988A1 (en) Reducing Prominin2-Mediated Resistance to Ferroptotic Cell Death
US20230374505A1 (en) Human XIST Antisense Oligonucleotides for X Reactivation Therapy
US20230287427A1 (en) Inhibition of lncExACT1 to Treat Heart Disease
US20230374508A1 (en) Compositions and Methods Targeting circ2082 for the Treatment of Cancer
WO2017096053A2 (en) Methods of reducing expression of x-inactivation escapee genes and autosomal genes
EP3946629A1 (en) Therapeutic targets for oncogenic kras-dependent cancers
WO2015038593A1 (en) Targeting microrna-26a/b for the treatment of neurodegenerative disease

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17877369

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17877369

Country of ref document: EP

Kind code of ref document: A1