US20180021331A1 - Hdac1,2 inhibitors and methods of using the same - Google Patents

Hdac1,2 inhibitors and methods of using the same Download PDF

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US20180021331A1
US20180021331A1 US15/541,012 US201515541012A US2018021331A1 US 20180021331 A1 US20180021331 A1 US 20180021331A1 US 201515541012 A US201515541012 A US 201515541012A US 2018021331 A1 US2018021331 A1 US 2018021331A1
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Srividya BHASKARA
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University of Utah Research Foundation UURF
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5748—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncogenic proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to an agent that selectively inhibits HDAC1 and HDAC2, a method of treating a cancer, a method of sensitizing a cancer to a chemotherapeutic agent, a method for determining if a cancer is sensitive to an agent that selectively inhibits HDAC1 and HDAC2, and a method for monitoring the efficacy of a treatment for a cancer.
  • Histone deacetylases remove acetyl groups from certain amino acid side chains of histone and non-histone proteins. The presence or absence of these acetyl groups often results in the regulation of the target protein and its cognate genetic or biochemical pathway. Aberrant activity or recruitment of HDACs to the target protein results in misregulation and in some instances, promotes the formation and/or growth of cancer.
  • SAHA and Depsipeptide are pan HDAC inhibitors that have been used in cancer treatment. However, given their lack of selectivity towards specific HDACs, SAHA and Depsipeptide inhibit multiple HDACs and thus, affect multiple cellular functions and thus, result in numerous side effects.
  • the present disclosure provides methods of treating cancer characterized by BCR-ABL expression or BBAP overexpression in a subject in need thereof comprising administering to the subject an agent that selectively inhibits HDAC1 and HDAC2.
  • the present disclosure also provides methods of sensitizing a cancer characterized by BCR-ABL expression or BBAP overexpression to a chemotherapeutic agent in a subject in need thereof, the method comprising administering an agent that selectively inhibits HDAC1 and HDAC2 to the subject.
  • the methods may further comprise administering doxorubicin to the subject.
  • the present disclosure further provides methods for determining if a cancer is sensitive to an agent that selectively inhibits HDAC1 and HDAC2 comprising: (a) obtaining a sample from a subject suffering from the cancer; (b) measuring a level of one or more markers in the sample, wherein the one or more markers are selected from the group consisting of BCR-ABL and BBAP; (c) comparing the measured level of the one or more markers in the sample to a level of the one or more markers in a control sample; and (d) determining that the cancer is sensitive to the agent when the measured level of the one or more markers in the sample is increased relative to the level of the one or more markers in the control sample.
  • the present disclosure further provides methods for monitoring the efficacy of a treatment for a cancer that includes administration of an agent that selectively inhibits HDAC1 and HDAC2, the method comprising: (a) obtaining a first sample from the subject before the treatment and a second sample from the subject during or after the treatment; (b) measuring a first level of one or more markers in the first sample and a second level of the one or more markers in the second sample, wherein the one or more markers are selected from the group consisting of 53BP1 and ⁇ H2AX; (c) comparing the first level of the one or more markers and the second level of the one or more markers; and (d) determining that the treatment is effective when the second level of the one or more markers is higher than the first level of the one or more markers.
  • FIG. 1 shows the levels of histone H3K27me3 in EZH2 gain-of-function mutant DLBCL cells when compared to other cancer cell lines: Western blot analysis of whole cell lysates prepared from Karpas-422, SUDHL4, NALM6, HeLa and mouse fibrosarcoma cells was performed with anti-H3K27me3 and anti-EZH2 antibodies. Histone H3 served as a loading control.
  • FIG. 2 shows characterization of selective HDAC1,2 inhibitor in EZH2 gain-of-function mutant DLBCL cells:
  • the compounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 ⁇ M tris(2-carboxyethyl)phosphine) to 6 fold their final concentration.
  • the HDAC enzymes purchased from BPS Biosciences
  • the tripeptide substrate (synthesized in house) and trypsin at 0.05 ⁇ M final concentration were diluted in assay buffer at 6 fold their final concentration.
  • Five ⁇ l compounds and 20 ⁇ l of enzyme were added to wells of a black, opaque 384 well plate in duplicate. Enzyme and compound were incubated together at room temperature for 10 minutes.
  • FIG. 3 shows that HDAC1,2 inhibition caused apoptosis in Karpas-422 cells and a G1 arrest in SUDHL4 cells:
  • A SUDHL4 and Karpas-422 were treated with DMSO, 2 ⁇ M ACY-957, 0.5 ⁇ M DZNEP, 0.5 ⁇ M GSK126 or a combination of drugs for 72 h prior to cell cycle analysis by propidium-iodide staining. Representative plots from one experiment out of three independent experiments are shown in the figure.
  • FIG. 4 shows that selective HDAC1,2 inhibition increased global chromatin-associated H3K27ac without altering H3K27me3 in EZH2 gain-of-function mutant DLBCL cells:
  • Karpas-422 (A) and SUDHL4 (B) were treated with DMSO, 2 M ACY957, 2 ⁇ M ACY1044, 0.5 ⁇ M DZNEP, 0.5 ⁇ M GSK126 or combinations of these drugs for 48 hr prior to chromatin extraction.
  • Western blot analysis was done with anti-H3K27ac, H3K27me3 or EZH2 and histone H3 served as a loading control.
  • FIG. 5 shows that inhibition of HDAC1,2 activity increased the expression of DNA damage response genes and H3K27ac at these promoters:
  • A Total RNA was isolated from Karpas-422 cells following a 24 h treatment with 2 ⁇ M ACY-957 for 24 h. RNA samples obtained from three independent treatments were subjected to RiboZero stranded sequencing using the Illumina Hiseq2000 sequencer. RNA-seq data from ACY-957-treated samples was compared to the publically available RNA seq data set from Karpas-422 cells following GSK126 treatment of Karpas-422 cells. Venn diagrams were made using the R package VennDiagram. Genes with adjusted p-value ⁇ 0.05 and absolute log 2 fold change >0.585 were used in this analysis.
  • the threshold cycles from qRT-PCR (C T values) obtained for the rabbit IgG control were between 31-33 cycles, and the signal obtained from using the H3K27ac or H3K27me3 antibodies ranged between 22-28.
  • C T values the threshold cycles from qRT-PCR
  • To calculate fold change in the levels of H3K27 modification at a given target locus we normalized the C T value obtained from H3K27me3 or H3K27ac ChIP to the C T value obtained from histone H3 ChIP.
  • the ChIP signal obtained from the antibodies recognizing HDAC1 or HDAC2 antibodies was normalized to the C T values obtained from the input DNA.
  • FIG. 6 shows that selective inhibition of HDAC1,2 activated DNA damage response and impaired DSB repair in EZH2 gain-of-function mutant DLBCL cells:
  • A SUDHL4 and Karpas-422 cells were treated with DMSO, 2 ⁇ M ACY-957, 0.5 ⁇ M DZNEP or 0.5 ⁇ M GSK126 for 48 hr and immunofluorescence staining with ⁇ H2AX was performed. The percentage of cells with 6 or greater ⁇ H2AX foci were counted in three independent experiments and at least 100 cells were counted in each experiment. The average with standard errors calculated from three independent experiments is shown in the figure.
  • FIG. 7 shows that HDAC1,2 activity did not change global chromatin-associated H3K27me3 following irradiation and are critical for H3K27me3 enrichment at defined laser-induced break sites in chemoresistant DLBCL cells:
  • FIG. 8 shows that EZH2 gain-of-function mutant DLBCL cells also over expressed BBAP E3 ligase, HDAC1,2 target H4K91ac and inhibition of HDAC1,2 activity decreased H4 monoubiquitination following doxorubicin treatment:
  • Whole cell lysate of HeLa cells was prepared following transfection of cells with either non-targeting or two different BBAP siRNAs. These extracts served as negative controls for the signal from BBAP antibody.
  • FIG. 9 shows that HDAC1,2 inhibition delayed the kinetics of 53BP1 foci formation in refractory EZH2-mutant DLBCL cells:
  • FIG. 10 shows that HDAC1,2 inhibition sensitized chemoresistant Karpas-422 cells to doxorubicin induced cell death:
  • A Karpas-422 cells were treated with DMSO, 2 ⁇ M ACY-957, 50 nM doxorubicin or ACY-957 plus 50 nM doxorubicin for 48 h. Cell cycle analysis of propidium-iodide stained cells was performed with fixed cells. Representative plots from five independent experiments are shown in the figure.
  • FIG. 11 shows a model for the mechanism of action of HDAC1,2-selective inhibitor in EZH2 gain-of-function mutant DLBCL cells:
  • A HDAC1,2 selective inhibition decreased H3K27me3 at break sites to impair DSB repair and activated DNA damage response.
  • B HDAC1,2 inhibition increased H3K27ac at DNA damage response genes to increase their transcription.
  • a and B together overcame the survival advantage provided by increased H3K27me3 in these EZH2 gain-of-function DLBCL cells.
  • C HDAC1,2 inhibition increased H4K91ac and decreased H4K91 monoubiquitination following doxorubicin treatment.
  • Hdac1,2 inhibition overcame BBAP-mediated chemoresistance following doxorubicin treatment in DLBCL cells with EZH2 hyperactive mutation.
  • FIG. 12 shows SUDHL4 (A) and Karpas-422 (B) cells were treated with increasing concentrations of ACY1044 and whole cell lysates were prepared following a 24 hr treatment. Western blot analysis of H3K9K14ac and H4K5ac was performed. Histone H3 and H4 were used as controls.
  • FIG. 13 shows in (A): SUDHL4 and Karpas-422 were treated with DMSO, 2 ⁇ M ACY-957, 0.504 DZNEP, 0.5 ⁇ M GSK or a combination of drugs for 24 or 48 hr and cell cycle analysis of propidium-iodide stained cells was performed. Representative plots are shown in the figure.
  • FIG. 14 shows HeLa cells that were laser micro-irradiated and allowed to recover for 15 minutes before fixation and immunofluorescence staining was performed with anti- ⁇ H2AX and anti-H3K27ac as described in Example 1.
  • FIG. 15 shows a model for the mode-of-action of the HDAC1,2 agent in Pre-B-ALL leukemic cells.
  • FIG. 16 shows a model for HDAC1,2 selective inhibitor action in Pre-B-ALL cells.
  • WRN interacted with FEN1 via lysine (K) 375 residue.
  • BCR-ABL stimulated WRN and FEN1 activities for hyperactive DNA repair and protected leukemic cells.
  • HDAC1,2 inhibition increased FEN1-K375 acetylation and disrupted FEN1 interaction with WRN. This overrided BCR-ABL-mediated stimulation resulting in DNA breaks and cell death.
  • FIG. 17 shows confirmation of the selectivity of HDAC1,2 inhibitors.
  • A For IC 50 determination, recombinant HDAC enzymes were treated with HDAC1,2 or HDAC3 inhibitors in vitro HDAC assays.
  • B Western blots for H3K23ac using extracts from Pre-B-ALL SupB15 cells treated with increasing dose of HDAC1,2 inhibitor (ACY1035).
  • C Hdac1 F1/F1 ,2 F1/F1 cells were infected with Ad-Cre virus to delete HDAC1 and HDAC2 (HDAC1,2 null) or uninfected (WT).
  • D Hdac3 F1/F1 cells were either uninfected (WT) or infected with Ad-Cre virus to delete Hdac3.
  • HDAC3-null cells were treated with DMSO or the HDAC1,2 selective inhibitor.
  • FIG. 18 in (A) shows a model for how HDAC1,2 regulated inter- and intra-nucleosomal interactions by targeting H4K16ac and H4K91ac, respectively.
  • FIG. 19 shows FEN1 was a non-histone target of HDAC1,2.
  • A Extracted ion chromatogram of peptide TGAAGK[Ac]FK (m/z 411.230) attributed to protein FEN1_HUMAN. Chromatograms showed increased abundance of this peptide in samples treated with HDAC1,2 inhibitor (HD12IN).
  • B Pre-B-ALL SupB15 cells were treated with DMSO or ACY1035 for 24 h before lysate preparation for immunoprecipitation (IP) using an antibody recognizing pan-acetyl lysine. FEN1 present in the immunoprecipitate was detected using ⁇ FEN1 antibody.
  • FIG. 20 shows in (A) five BCR-ABL expressing cell lines that were treated with DMSO, HDAC3 inhibitor (1044) or three HDAC1,2 selective inhibitors for 96 h. FACS was performed following propidium-iodide staining (PI).
  • B BCR-ABL transformed or non-transformed murine Baf3 cells were treated with increasing dose of ACY1035 or ACY1071. Cells in subG1 phase (dead cells) were measured using FACS after PI.
  • C Wild-type mice injected with 50 mg/kg body weight of ACY1035 or cyclodextrin carrier every day for 5 days. FACS was used to measure percentage B220+ B-cells in total bone marrow cells isolated from these mice.
  • D D).
  • % B220+ B-cells in the peripheral blood of mice treated with ACY1035 for 5 days or after a week of recovery without inhibitor treatment % B220+ B-cells from bone marrow and spleen after recovery are also shown.
  • C carrier
  • T HDAC1,2 inhibitor.
  • E FACS profiles for B220+CD43+ population isolated from bone marrow of mice with or without recovery from inhibitor treatment.
  • arrows indicated decrease in B-cells upon inhibitor treatment and their reemergence following recovery from inhibitor treatment. Mice treated with ACY1035 as described for panel (C).
  • FACS profiles for LSK+ stem and progenitor cells Lineage negative, cKit+, Sca-1+ isolated from bone marrow of control or HDAC1,2 inhibitor treated mice.
  • G SupB15 cells were treated with HDAC1,2 inhibitor for 24 h and molecular combing was done to measure fork velocity. Average fiber length were shown on the side.
  • H Representative IF image for SupB15 cells, Ph+mononuclear cells and Ph+stem/progenitor cells was shown in panels (H-J). Merged IF images for ⁇ H2AX foci and Hoechst, nuclear stain were shown.
  • K Quantitation of DNA damage in CD34+ stem/progenitor cells isolated from normal bone marrow and Ph+ patient bone marrow. DNA damage was measured using immunofluorescence (IF) staining for ⁇ H2AX.
  • FIG. 21 shows Repli-seq of Ph+Pre-B-ALL SupB15 cells, in particular, representative snapshots of Illumina sequencing data. Signal strengths (number of reads) for BrdU-labeled loci that fire in early or in late S-phase cells treated with either DMSO or the HDAC1,2 inhibitor are shown. Reads obtained for the same loci without BrdU labeling (background) are also shown. Replication timing was changed at these loci in the absence of HDAC1,2 activities.
  • FIG. 22 shows that HDAC1,2 function in DNA repair.
  • siRNA-mediated knockdown of HDAC1,2 or HDAC3 induced DNA damage, as seen by ⁇ H2AX foci formation.
  • Loss of HDAC1,2, but not HDAC3, in NIH3T3 cells impaired 53BP1 foci formation.
  • NTsi non-targeting siRNA (negative control).
  • FIG. 23 shows that (A) FEN1 interacted with PCNA at replication forks, where it was involved in removing the 5′-flap structures as part of the Okazaki fragment maturation process. The RNA primer is also shown. (B). Upon replication stress, FEN1 was recruited by WRN, the helicase, to process aberrant replication intermediates. (C). DNA damage in SupB15 cells following shRNA-mediated knockdown of endogenous FEN1 and re-expression of wild-type FEN1 or FEN1-K375 mutants. Merged IF images were shown. (D). Homologous recombination (HR) repair in the presence of DMSO or the HDAC1,2 inhibitor was measured using cells expressing BCR-ABL and containing a stably integrated DR-GFP reporter cassette. HR quantitation was done.
  • HR Homologous recombination
  • FIG. 24 shows DNA damage activation in Ph+PreB-ALL cells.
  • SupB15 cells were treated with 2 ⁇ M ACY1035, ACY1071, ACY957, or solvent (DMSO) for 48 hours.
  • A Immunofluorescence with ⁇ H2AX was performed on treated SupB15 cells. The number of ⁇ H2AX foci was counted in around 100 cells for each treatment and classified into cells with no foci, 1-5, 6-10, and greater than 10 foci.
  • SupB15 cells treated with DMSO or ACY1035 for 48 hours were tested via neutral comet assay to measure the amount of double-strand breaks (DSBs), indicated by the tail.
  • DSBs double-strand breaks
  • FIG. 25 shows mass spectrometry analysis in Ph+BCR-ABL expressing cells following treatment with doxorubicin, HDAC1,2 inhibitor, or doxorubicin plus HDAC1,2 inhibitor.
  • A Schematic diagram of quantitative one-dimensional liquid chromatography and tandem mass spectrometry (1D-LC-MS/MS) analysis in BCR-ABL containing cells following ACY1035 treatment for 48 hours or 0.104 doxorubicin for 10 hours or ACY1035+ doxorubicin for 36 hours+10 hours. Proteins that showed a differential binding to chromatin were filtered following an ANOVA test across all the four treatment groups. An ANOVA value of 0.01 or less than 0.01 cut-off was used to filter statistically significant proteins.
  • FIG. 26 shows validation of data obtained from mass spectrometry analysis using western blot analysis.
  • A Western blots targeting various proteins were performed with at least five independent isolates of chromatin from cells treated with solvent (DMSO), HDAC1,2 inhibitor ACY1035 (1035), doxorubicin (Dox), or doxorubicin plus ACY1035 (1035+Dox).
  • B Quantitation of the western blot signal for various repair proteins after normalization to total H3.
  • FIG. 27 shows that BAF180 knockdown leads to increased DNA damage in cells expressing BCR-ABL.
  • HAP1 adherent cells that contain the BCR-ABL translocation were transfected with either non-targeting or BAF180 siRNA pool and immunofluorescence for ⁇ H2AX was performed at 72 hours post-siRNA transfection. Increased DNA damage is seen in BAF180 knockdown cells when compared to the control non-targeting siRNA knockdown cells.
  • FIG. 28 shows that HDAC1,2-selective inhibitors cause cytotoxic or cytostatic effects and impair DNA repair in Ph+CD34+ stem/progenitor cells.
  • A FACS analysis of propidium iodide stained-cells following treatment of cells with solvent (DMSO, ACY957, ACY1035, or ACY1071, showing the percentage of cells determined to be in the SubG1 stage following each treatment.
  • B Neutral comet assays show increased DNA damage and decreased DNA repair in Ph+CD34+ cells upon HDAC1,2 inhibitor treatment. Comet analysis was performed with two different Ph+ALL CD34+ samples and representative pictures from one experiment are shown.
  • FIG. 29 shows (A) proteins that decreased on the chromatin following ACY1035+ doxorubicin treatment when compared to ACY1035 treatment alone in mass spectrometry analysis; (B) Panther analysis demonstrating that the majority of proteins identified in mass spectrometry analysis that are affected upon doxorubicin treatment belong to DNA replication; and (C) increased DNA damage on nascent chromatin in SupB15 cells as identified by BrdU-ChIP-slot assay.
  • FIG. 30 shows that (A) HDAC1,2-selective inhibitors cause no cytotoxic or cytostatic effects normal mononuclear cells (MNC) but (B) do cause cytotoxic or cytostatic effects in Ph+mononuclear cells. (C) Neutral comet assay shows increased DNA damage and decreased DNA repair in Ph+MNC upon HDAC1,2 inhibitor treatment for 48 hours. Comet analysis was performed with four different Ph+ALL patient samples and the cumulative tail moment from this analysis is shown, along with representative comet assay images.
  • FIG. 31 shows hematoxylin and eosin staining of bone marrow (BM) and spleen (SPL) sections from normal NOD-SCID-Gamma mice injected with solvent, 25 mg/kg ACY1035 (fifteen doses), or 0.5 mg/kg doxorubicin (six doses). These tests showed no abnormalities or changes in the cell morphology in bone marrow or spleens of mice after ACY1035 or doxorubicin administration. ACY1035 does not have toxic effects in vivo.
  • FIG. 32 shows in (A). FACS profiles for human (h) CD45 B-cell marker in spleen (SPL) and bone marrow (BM) 5-wk post SupB15 cell engraftment into NSG mice.
  • B Representative TdT staining of a bone cross-section from a Supb15 xenograft showing patchy leukemic infiltrate in the bone marrow (arrow). Inset showed normal bone from a control mouse (c) and pale bone from a xenograft (x) mouse.
  • C CD19 immunohistochemistry showing membrane positivity in leukemia cells with negative signal in adjacent normal spleen cells in a SupB15 xenograft.
  • Inset showed normal spleen from a control mouse and an enlarged spleen from a xenograft mouse.
  • D FACS data for human (h) CD45 cells in peripheral blood (PB), bone marrow (BM) and spleen (SPL) from a primary patient-derived xenograft mouse (1 0 PDX). Analysis was done 4-wk post engraftment of Ph+CD34+ leukemic cells from a newly-diagnosed patient into NSG mouse. Normal bone from a control mouse (Con.) and pale bone from a primary PDX mouse were shown.
  • PB peripheral blood
  • BM bone marrow
  • SPL spleen
  • FIG. 33 shows that HDAC1,2 inhibition has therapeutic benefits in a Ph+ALL patient-derived xenograft mouse model.
  • Patient-derived xenograft mice were treated with either cyclodextrin (solvent) or 25 mg/kg ACY1035 every other day for a total of 21 doses.
  • A The level of human CD19, CD45, and CD34 markers were assessed in the bone marrow of these mice by FACS analysis. For each FACS marker, the bars represent (from left to right): solvent, ACY1035, doxorubicin, ACY1035+ doxorubicin.
  • Solvent-treated mice have bones infiltrated with white blood cells.
  • FIG. 34 shows that HDAC1, 2 inhibition affects DNA repair and induces DNA damage in EZH2 GOF DLBCL cells.
  • A Measurement of repair efficiency in SUDHLS (EZH2 WT ) and Karpas-422 (EZH2 GOF ) cells using neutral comet assay that detects only double-strand breaks (DSBs).
  • DSBs double-strand breaks
  • the head (H) represents intact DNA
  • the tail (T) represents the number of DSBs in a single cell.
  • Tail moment is a measure of length and intensity of DNA in the tail, and the average tail moments from 100 comets are indicated within the figure for each cell/treatment combination.
  • Doxo indicates doxorubicin; H12IN indicates HDAC1,2 inhibitor.
  • NT indicates non-targeting control siRNA;
  • HDAC1,2 siRNA indicates combined treatment with HDAC1 siRNA and HDAC2 siRNA.
  • (C) Karpas-422 cells treated with DMSO solvent or HDAC1,2 inhibitor were subjected to microlaser irradiation. H3K27me3, ⁇ H2AX, Bmi-1, and serine2-phosphorylated RNA Polymerase II (engaged in transcriptional elongation) were monitored at laser break sites (arrows) after 15 minutes of recovery time. Numbers overlaying the images indicate percentages of cells with ⁇ H2AX lines that co-localized with H3K27me3, Bmi-1, or Pol2Ser2-P lines from at least four independent experiments (p value ⁇ 0.01).
  • FIG. 35 shows that H2AK119ac is a novel target of HDAC1,2 and that HDAC1,2 inhibition alters the H2AK119ac-ub switch.
  • A Western blot showing that treatment with an HDAC1,2 inhibitor (H12IN) in EZH2 GOF DLBCL cells leads to increased global H2AK119ac.
  • B Western blot showing that treatment with an HDAC1,2 inhibitor in EZH2 GOF cells leads to decreased chromatin-associated H2AK119ub.
  • FIG. 36 shows chemoresistant and chemosensitive DLBCL xenograft mouse models.
  • A Xenografted chemoresistant EZH2 GOF DLBCL Karpas-422 cells home to spleen in NOD-SCID-Gamma mice. Spleens are enlarged in Karpas-422 NSG mice compared to control NSG mice.
  • B Xenografted chemosensitive EZH2WT DLBCL SUDHL8 cells home to lymph nodes in NOD-SCID-Gamma mice.
  • C FACS analyses of human CD19 and IgM markers in peripheral blood of Karpas-422/NSG xenograft mice treated with solvent or doxorubicin (Doxo) or the HDAC1,2 inhibitor ACY1035 (H12IN). Seventeen doses of HDAC1,2 inhibitor and five doses of doxorubicin were administered.
  • FIG. 37 shows FACS analyses of cell cycle progression in U-87 and U-251 glioma cells treated with the HDAC1,2 inhbitor ACY957 (957) or solvent (DMSO).
  • Cells were treated with ACY957 or DMSO for 72 hours, stained with propidium iodide, and assessed by FACS. The percentage of cells in Sub-G1, G1, S, and G2-M phases were quantified for each sample.
  • the methods of the present disclosure generally include administration of an agent that selectively inhibits HDAC1 and HDAC2 (i.e., an HDAC1,2 inhibiting agent).
  • an agent that selectively inhibits HDAC1 and HDAC2 i.e., an HDAC1,2 inhibiting agent.
  • This selective inhibition of HDAC1,2 was found to decrease double-stranded break (DSB) repair and to activate the DNA damage response in cancer cells. This caused damaged DNA to accumulate in the cancer cells, thereby causing cytotoxic or cytostatic effects in the cancer cells. This inhibition also was found to further sensitize the cancer cells to a chemotherapeutic agent.
  • the present disclosure provides methods of treating a cancer and methods of sensitizing a cancer to the chemotherapeutic agent, each comprising administering the agent that selectively inhibits HDAC1 and HDAC2 to a subject in need thereof.
  • the present disclosure further provides methods for determining if a cancer may be sensitive to an agent that selectively inhibits HDAC1 and HDAC2, as well as methods for monitoring the efficacy of a treatment for a cancer, where the treatment includes administering the agent, which selectively inhibits HDAC1 and HDAC2.
  • These methods each include measuring a level of one or more markers in a sample(s) obtained from a subject suffering from the cancer.
  • the one or more markers may include BCR-ABL, BBAP, 53BP1, ⁇ H2AX, EZH2 gain-of-function mutation, or any combination thereof.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • C x-y The number of carbon atoms in a hydrocarbyl substituent can be indicated by the prefix “C x-y ,” where x is the minimum and y is the maximum number of carbon atoms in the substituent.
  • a C x chain means a hydrocarbyl chain containing x carbon atoms.
  • alkyl refers to saturated, straight- or branched-chain hydrocarbon moieties containing, in certain embodiments, between one and six, or one and eight carbon atoms, respectively.
  • Examples of C 1-6 alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl moieties; and examples of C 1-8 alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl, heptyl, and octyl moieties.
  • alkenyl denotes a monovalent group derived from a hydrocarbon moiety containing, in certain embodiments, from two to six, or two to eight carbon atoms having at least one carbon-carbon double bond. The double bond may or may not be the point of attachment to another group.
  • Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl, octenyl and the like.
  • alkynyl denotes a monovalent group derived from a hydrocarbon moiety containing, in certain embodiments, from two to six, or two to eight carbon atoms having at least one carbon-carbon triple bond.
  • the alkynyl group may or may not be the point of attachment to another group.
  • Representative alkynyl groups include, but are not limited to, for example, ethynyl, 1-propynyl, 1-butynyl, heptynyl, octynyl and the like.
  • aryl refers to a mono- or poly-cyclic carbocyclic ring system having one or more aromatic rings, fused or non-fused, including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like.
  • aryl groups have 6 carbon atoms.
  • aryl groups have from six to ten carbon atoms.
  • aryl groups have from six to sixteen carbon atoms.
  • cycloalkyl denotes a monovalent group derived from a monocyclic or polycyclic saturated or partially unsaturated carbocyclic ring compound.
  • Examples of C.sub.3-8-cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl and cyclooctyl; and examples of C.sub.3-C.sub.12-cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl, and bicyclo[2.2.2]octyl.
  • cycloalkyl groups have from three to six carbon atoms. In some embodiments, cycloalkyl groups have from three to eight carbon atoms.
  • halo and “halogen,” as used herein, refer to an atom selected from fluorine, chlorine, bromine and iodine.
  • heteroaryl refers to a mono- or poly-cyclic (e.g., bi-, or tri-cyclic or more) fused or non-fused, moieties or ring system having at least one aromatic ring, having from five to ten ring atoms of which one ring atom is selected from oxygen, sulfur, and nitrogen; zero, one or two ring atoms are additional heteroatoms independently selected from oxygen, sulfur, and nitrogen; and the remaining ring atoms are carbon.
  • the heteroaryl group has from about one to six carbon atoms, and in further embodiments from one to fifteen carbon atoms.
  • the heteroaryl group contains five to sixteen ring atoms of which one ring atom is selected from oxygen, sulfur, and nitrogen; zero, one, two, or three ring atoms are additional heteroatoms independently selected from oxygen, sulfur, and nitrogen; and the remaining ring atoms are carbon.
  • Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.
  • aryls, substituted aryls, heteroaryls and substituted heteroaryls described herein can be any aromatic group.
  • Aromatic groups can be substituted or unsubstituted.
  • heterocycloalkyl refers to a non-aromatic 3-, 4-, 5-, 6- or 7-membered ring or a bi- or tri-cyclic group fused of non-fused system, where (i) each ring contains between one and three heteroatoms independently selected from oxygen, sulfur, and nitrogen, (ii) each 5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0 to 2 double bonds, (iii) the nitrogen and sulfur heteroatoms may optionally be oxidized, (iv) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above rings may be fused to a benzene ring.
  • heterocycloalkyl groups include, but are not limited to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.
  • the heterocycloalkyl group is a 4-7, e.g., 4-6, membered ring.
  • subject or “patient” as used herein interchangeably, means any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc)) and a human.
  • a mammal e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse
  • a non-human primate for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc
  • the subject or patient may be a human or a non-human.
  • the subject or patient may be undergoing
  • sample means a sample or isolate of blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes, can be used directly as obtained from a subject or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.
  • the term also means any biological material being tested for and/or suspected of containing an analyte of interest.
  • the sample may be any tissue sample taken or derived from the subject.
  • the sample from the subject may comprise protein.
  • the sample from the subject may comprise nucleic acid. Any cell type, tissue, or bodily fluid may be utilized to obtain a sample.
  • Such cell types, tissues, and fluid may include sections of tissues such as biopsy (such as muscle biopsy) and autopsy samples, frozen sections taken for histological purposes, blood (such as whole blood), plasma, serum, sputum, stool, tears, mucus, saliva, hair, skin, red blood cells, platelets, interstitial fluid, ocular lens fluid, cerebral spinal fluid, sweat, nasal fluid, synovial fluid, menses, amniotic fluid, semen, etc.
  • Cell types and tissues may also include muscle tissue or fibres, lymph fluid, ascetic fluid, gynecological fluid, urine, peritoneal fluid, cerebrospinal fluid, a fluid collected by vaginal rinsing, or a fluid collected by vaginal flushing.
  • a tissue or cell type may be provided by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose). Archival tissues, such as those having treatment or outcome history, may also be used. Protein or nucleotide isolation and/or purification may not be necessary.
  • test sample can comprise further moieties in addition to the analyte of interest, such as antibodies, antigens, haptens, hormones, drugs, enzymes, receptors, proteins, peptides, polypeptides, oligonucleotides or polynucleotides.
  • the sample can be a whole blood sample obtained from a subject. It can be necessary or desired that a test sample, particularly whole blood, be treated prior to immunoassay as described herein, e.g., with a pretreatment reagent.
  • pretreatment of the sample is an option that can be performed for mere convenience (e.g., as part of a protocol on a commercial platform).
  • the sample may be used directly as obtained from the subject or following pretreatment to modify a characteristic of the sample. Pretreatment may include extraction, concentration, inactivation of interfering components, and/or the addition of reagents.
  • Treat”, “treating” or “treatment” are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progress of a disease, or one or more symptoms of such disease, to which such term applies.
  • the term also refers to preventing a disease, and includes preventing the onset of a disease, or preventing the symptoms associated with a disease.
  • a treatment may be either performed in an acute or chronic way.
  • the term also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease.
  • prevention or reduction of the severity of a disease prior to affliction refers to administration of an antibody or pharmaceutical composition of the present invention to a subject that is not at the time of administration afflicted with the disease. “Preventing” also refers to preventing the recurrence of a disease or of one or more symptoms associated with such disease. “Treatment” and “therapeutically,” refer to the act of treating, as “treating” is defined above.
  • the disclosed compounds may optionally be substituted with one or more substituents, such as are illustrated generally above, or as exemplified by particular classes, subclasses, and species. It will be appreciated that the phrase “optionally substituted” is used interchangeably with the phrase “substituted or unsubstituted.” In general, the term “substituted,” whether preceded by the term “optionally” or not, refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent.
  • an optionally substituted group may have a substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • the terms “optionally substituted” and any other optionally substituted group as used herein, refer to groups that are substituted or unsubstituted by independent replacement of one, two, or three or more of the hydrogen atoms thereon with substituents including, but not limited to:
  • alkyl alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl, heteroaryl,
  • the optionally substituted groups include the following: C 1-12 -alkyl, C 2-12 -alkenyl, C 2-12 -alkynyl, C 3-12 -cycloalkyl, C 3-12 -aryl, C 3-12 -heterocycloalkyl, or C 3-12 -heteroaryl.
  • aryls, heteroaryls, alkyls, and the like can be further substituted.
  • HDAC histone deacetylase
  • HDAC1 and HDAC2 inhibiting agents which include any compound or composition that selectively inhibits the deacetylase activity of HDAC1, HDAC2, or both HDAC1 and HDAC2.
  • HDAC1 and HDAC2 may kill cancer cells, or may inhibit or prevent growth of cancer cells, as described in more detail below.
  • the HDAC1,2 agent also may sensitize cancer cells to a chemotherapeutic agent.
  • HDAC1,2 inhibiting agents that may be used for the methods of the present disclosure may include, but are not limited to, those disclosed in U.S. Patent Application Publication No. 2014/0128391 and U.S. Pat. No. 9,145,412, the complete disclosures of which are hereby incorporated by reference for all purposes.
  • Specific examples of HDAC1,2 inhibiting agents may include, but are not limited to, ACY-957, ACY-1035, ACY-1071, pharmaceutically acceptable salts thereof, and combinations thereof. Each of these compounds is described in more detail below.
  • HDAC1,2 inhibiting agent selectively inhibits HDAC1 and HDAC2, HDAC1 and HDAC2 each belong to the class I HDAC family and interact with the polycomb repression complex (PRC), which contains EZH2 as the catalytic subunit.
  • PRC polycomb repression complex
  • Aberrant HDAC1 and HDAC2 activity and/or recruitment to genomic loci or target proteins may contribute to misregulated gene expression, and thus, cancer.
  • HDAC1,2 inhibiting agents caused cytotoxic or cytostatic effects in DLBCL cells that had a gain-of-function mutation in EZH2 (EZH2 GOF ).
  • Blocking the activities of HDAC1,2 increased global H3K27ac without causing a concomitant global decrease in H3K27me3 levels.
  • inhibition with HDAC1,2 inhibiting agents was sufficient to decrease H3K27me3 present at double-stranded breaks (DSBs), decrease DSB repair and activate the DNA damage response in these DLBCL cells.
  • selective inhibition with HDAC1,2 inhibiting agents in B-cell malignant cells led to increased numbers of foci containing ⁇ H2AX; such foci mark DNA damage.
  • selective inhibition with HDAC1,2 inhibiting agents increased H4K91ac, decreased BBAP-mediated H4K91 monoubiquitination, impaired BBAP-dependent double strand break (DSB) repair and sensitized the refractory EZH2 GOF DLBCL cells to treatment with the chemotherapeutic agent (e.g., doxorubicin).
  • chemotherapeutic agent e.g., doxorubicin
  • acetylation of FEN1 was increased upon selective inhibition of HDAC1,2, which in turn, prevented DNA repair that was mediated by BCR-ABL and WRN ( FIG. 16 ).
  • This inhibition of DNA repair caused DNA damage to accumulate and death of Pre-B-acute-lymphoblastic leukemia (Pre-B-ALL) cells ( FIG. 15 ).
  • a compounds according to the following Formula I, or a pharmaceutically acceptable salt thereof may be used as an HDAC1, 2 inhibiting agent:
  • Y 1 is CR 7 or NR 7 ;
  • Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 are each independently CH, CH 2 , N, or C(O), wherein at least one of Y 2 , Y 3 , Y 4 , and Y 5 are CH;
  • R 1 is mono-, bi-, or tri-cyclic aryl or heteroaryl, wherein the mono-, bi-, or tri-cyclic aryl or heteroaryl is optionally substituted;
  • R 2 and R 3 are each independently selected from C 2-6 -alkenyl, C 2-6 -alkynyl, C 3-6 -cycloalkyl, C 1-6 -alkyl-C 3-6 -cycloalkyl, heterocyclo alkyl, C 1-6 -alkyl-heterocycloalkyl, NR 4 R 5 , O—C 1-6 -alkyl-OR 6 , C 1-6 -alkyl-OR 6 , aryl, heteroaryl, C(O)N(H)-heteroaryl, C(O)-heteroaryl, C(O)-heterocycloalkyl, C(O)-aryl, C(O)—C 1-6 -alkyl, CO 2 -C 1-6 -alkyl, or C(O)—C 1-6 -alkyl-heterocycloalkyl, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl
  • R 4 is H, C 1-6 -alkyl, or C 1-6 -alkyl-OR 6 ;
  • R 5 is CO 2 R 6 , C 1 -C 6 -alkyl-aryl, or C 1-6 -alkyl-OR 6 ;
  • R 6 is H or C 1-6 -alkyl
  • R 7 is null, H, C 1-6 -alkyl, C 3-6 -cycloalkyl, C 1-6 -alkyl-C 3-6 -cycloalkyl, heterocycloalkyl, or C 1-6 -alkyl-heterocycloalkyl;
  • a line denotes an optionally double bond
  • n 0 or 1
  • n 0 or 1
  • morn 1
  • a compounds according to the following Formula II, or a pharmaceutically acceptable salt thereof may be used as an HDAC1, 2 inhibiting agent:
  • R 1 and R 2 are independently H, mono-, bi-, or tri-cyclic aryl or heteroaryl, wherein the mono-, bi-, or tri-cyclic aryl or heteroaryl is optionally substituted;
  • R 1 and R 2 are linked together to form a group as shown below:
  • R 3 and R 4 are independently selected from H, C 1-6 -alkyl, C 2-6 -alkenyl, C 2-6 -alkynyl, C 3-6 -cycloalkyl, C 1-6 -alkyl- C 3-6 -cycloalkyl, heterocyclo alkyl, C 1-6 alkyl-heterocycloalkyl, NR 5 R 6 , O—C 1-6 -alkyl-OR 7 , aryl, heteroaryl, C(O)N(H)-heteroaryl, C(O)-heteroaryl, C(O)-heterocycloalkyl, C(O)-aryl, C(O)—C 1-6 -alkyl, CO.sub.2—C 1-6 -alkyl, or C(O)—C 1-6 -alkyl-heterocycloalkyl, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally
  • R 5 is H, C 1-6 -alkyl, CO 2 R 7 or C 1-6 -alkyl-OR 7 ;
  • R 6 is H, C 1-6 -alkyl, CO 2 R 7 or C 1-6 -alkyl-OR 7 ;
  • R 7 is H or C 1-6 -alkyl
  • X 1 , X 2 , and X 3 are each independently CH, N, or S, wherein at least one of X 1 or X 2 is N or S;
  • a line denotes an optionally double bond
  • p 0 or 1.
  • ACY-957 is an exemplary HDAC1,2 inhibiting agent that may be used in connection with the methods disclosed herein.
  • ACY-957 also may be known herein as N-(2-amino-5-(thiophen-2-yl)phenyl)-2-(piperazin-1-yl)quinoline-6-carboxamide and have the following chemical structure:
  • ACY-1035 is another exemplary HDAC1,2 inhibiting agent that also may be used in connection with the methods disclosed herein.
  • ACY-1035 may also be known herein as of N-(2-amino-5-(thiophen-2-yl)phenyl)-2-cyclopropyl-1-(2-morpholinoethyl)-1H-indole-5-carboxamide and have the following chemical structure:
  • ACY-1071 is another exemplary HDAC1,2 inhibiting agent that also may be used in connection with the methods disclosed herein.
  • ACY-1071 may also be known herein as N-(2-amino-5-(pyridin-4-yl)phenyl)-2-cyclopropyl-1-(2-morpholinoethyl)-1H-indole-5-carboxamide and have the following chemical structure:
  • the HDAC1,2 inhibiting agent may be incorporated into pharmaceutical compositions suitable for administration to a subject (such as a patient, which may be a human or non-human).
  • the pharmaceutical compositions may include a “therapeutically effective amount” or a “prophylactically effective amount” of the HDAC1,2 agent.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount of the composition may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • the pharmaceutical composition may include one or more pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carrier means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;
  • HDAC1,2 inhibiting agents may selectively inhibit HDAC1 and HDAC2, thereby killing a cancer cell, or inhibiting or preventing the growth of the cancer.
  • the cancer may characterized by BCR-ABL expression, BBAP overexpression, by a gain-of-function mutation in Enhancer of Zeste Homologue 2 (EZH2), as being dependent upon a double-stranded break repair pathway, increased H3K27me3, or any combination thereof.
  • the double-stranded break repair pathway may include FEN1.
  • the cancer may be a B cell malignancy.
  • the B cell malignancy may be characterized by BCR-ABL expression, BBAP overexpression, dependency upon the double-stranded break repair pathway, increased H3K27me3, or any combination thereof.
  • the B cell malignancy may be diffuse large B cell lymphoma (DLBCL) or pre-B cell derived acute lymphoblastic leukemia (Pre-B-ALL).
  • the DLBCL may contain a gain-of-function mutation in Enhancer of Zeste Homologue 2 (EZH2), a gain-of-function mutation in a histone H3K27 methyltransferase, a loss-of-function mutation in a H3K27 demethylase, or any combination thereof.
  • EZH2 Enhancer of Zeste Homologue 2
  • the Pre-B-ALL may contain a chromosomal translocation, for example, but not limited to, t(9:22); such a Pre-B-ALL may also be referred to as Ph+Pre-B-ALL.
  • the chromosomal translocation t(9;22) may encode the oncogenic protein BCR-ABL.
  • the method of treating may apply the method for determining if the cancer is sensitive to the HDAC1,2 agent, which is described below in more detail. Accordingly, the method of treating may include detecting a level of BCR-ABL and a level of BBAP in a sample obtained from the subject. The method of treating may also include comparing the detected levels of BCR-ABL and BBAP to levels of BCR-ABL and BBAP in a control sample. If the detected level of BCR-ABL is increased relative to the control level of BCR-ABL, then the cancer may be characterized by BCR-ABL expression.
  • the cancer may not be characterized by BCR-ABL expression. If the detected level of BBAP is increased relative to the control level of BBAP, then the cancer may be characterized by BBAP overexpression. If the detected level of BBAP is not increased relative to the control level of BBAP, then the cancer may not be characterized by BBAP overexpression.
  • chemotherapeutic agent may be doxorubicin or other components of the CHOP chemotherapy regimen (cyclophosphamide, vincistrine, Prednisone).
  • the method may include administering the HDAC1,2 inhibiting agent to the subject.
  • HDAC1,2 inhibiting agent and the types of cancer that may be treated with are described above in more detail.
  • the method may also include administering the chemotherapeutic agent to the subject.
  • the HDAC1,2 inhibiting agent and chemotherapeutic agent may be administered together to the subject.
  • the HDAC1,2 inhibiting agent may be administered to the subject before the chemotherapeutic agent is administered to the subject.
  • the HDAC1,2 inhibiting agent may be administered at least about 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 90 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks
  • the method of sensitizing may apply the method for determining if the cancer is sensitive to the HDAC1,2 agent, which is described below in more detail.
  • the method of treating may include detecting a level of BCR-ABL and a level of BBAP in a sample obtained from the subject.
  • the method of treating may also include comparing the detected levels of BCR-ABL and BBAP to levels of BCR-ABL and BBAP in a control sample. If the detected level of BCR-ABL is increased relative to the control level of BCR-ABL, then the cancer may be characterized by BCR-ABL expression.
  • the cancer may not be characterized by BCR-ABL expression. If the detected level of BBAP is increased relative to the control level of BBAP, then the cancer may be characterized by BBAP overexpression. If the detected level of BBAP is not increased relative to the control level of BBAP, then the cancer may not be characterized by BBAP overexpression.
  • the methods may include obtaining a sample from the subject suffering from the cancer and measuring a level of one or more markers in the subject.
  • the one or more markers may be BCR-ABL or BBAP or EZH2 mutation.
  • Measuring the level of the one or more markers may include an immunoassay, fluorescence in situ hybridization (FISH), or polymerase chain reaction (PCR).
  • the method may also include comparing the measured level of the one or more markers to a level of the one or more markers in a control sample.
  • the method may further include determining the cancer is sensitive to the HDAC1,2 inhibiting agent when the measured level of the one or more markers is increased relative to the level of the one or more markers in the control sample.
  • the method may further include administering the HDAC1,2 agent to the subject.
  • the treatment may include administering the HDAC1,2 inhibiting agent.
  • the HDAC1,2 inhibiting agent and the cancer are described above in more detail.
  • the method may include obtaining a first sample from the subject before the treatment and a second sample from the subject during or after the treatment.
  • the method may also include measuring a first level of one or more markers in the first sample and a second level of the one or more markers in the second sample.
  • the one or more markers may be 53BP1 or ⁇ H2AX. Measuring the first and second levels of the one or more markers may include measuring foci formation of the one or more markers.
  • the method may further include comparing the first level of the one or more markers and the second level of the one or more markers, and determining that the treatment is effective when the second level of the one or more markers is higher than the first level of the one or more markers.
  • the method may include continuing to administer the treatment to the subject, administering the chemotherapeutic agent to the subject, or a combination thereof.
  • kits for use with the methods disclosed herein may include reagents for detecting the one or more markers, which are described above in more detail.
  • the reagents may be any of those reagents known in the art for immunoassays (e.g., ELISA, western blotting, immunoprecipitation (IP), immunohistochemistry, etc.) to detect the one or more markers.
  • the reagents may also be any of those reagents known in the art for detecting nucleic acids, for example, polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), quantitative RT-PCR (qRT-PCR), and so forth.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase-PCR
  • qRT-PCR quantitative RT-PCR
  • the kit may include one or more positive controls and/or one or more negative controls.
  • the kit may further include other material(s), which may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and/or other material useful in sample processing, washing, or conducting any other step of the methods described herein.
  • the kit according to the present disclosure may include instructions for carrying out the methods of the invention. Instructions included in the kit of the present disclosure may be affixed to packaging material or may be included as a package insert. While instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, and chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” may include the address of an internet site which provides instructions.
  • the present invention has multiple aspects, illustrated by the following non-limiting examples.
  • HeLa cells were cultured in DMEM containing 10% fetal bovine serum (Hyclone, Logan, Utah, USA), 1% penicillin-streptomycin and 1% glutamine.
  • Karpas, SUDHL4 and SUDHL8 cells were cultured in RPMI supplemented with 20% heat inactivated fetal bovine serum (Hyclone, Logan, Utah, USA), 1% penicillin-streptomycin, 1% L-glutamine, 0.002% HEPES, 0.1% amphotericin B.
  • NIH3T3 cells were cultured in DMEM (CellgroTM, Tewksbury, Mass., USA) containing 10% fetal calf serum, 1% penicillin-streptomycin and 1% glutamine.
  • HDAC1,2 and HDAC3 conditional knockout fibrosarcoma cells were cultured and infected with Ad-Cre.
  • DZnep was purchased from Cayman Chemicals, and GSK126 was purchased from Xcess Bio.
  • Antibodies The following antibodies were purchased from Abcam: H4, H4K5ac, H4K9lac, BBAP, ⁇ H2AX (for western), H4K16ac. H3K23ac, H4K16ac, H2AX and H3K27ac antibodies were purchased form Active Motif 53BP1 antibody was purchased from Bethyl Laboratories. Anti-H3K27me3 and EZH2 were purchased from Cell Signaling. ⁇ H2AX (for immunofluorescence), H3K9K14ac and H3 antibodies were purchased from Millipore.
  • HeLa cells were seeded in 8-well LabTek II chamber dishes. After three hours, the media was removed and replaced with fresh media containing HDAC inhibitors in the described concentrations. After 24 hours of drug treatment, cells were pre-sensitized with 1 ⁇ g/ml Hoechst 33342 for ten minutes.
  • Laser micro-irradiation was performed on an inverted confocal microscope (A1 Confocal System, Nikon), using a 405 nm laser focused through a 60x oil objective. Laser output was set to 100%, with twelve regions of interest (ROIs) at a scan speed of 1/16 and pixel dwell of 56.7, which was sufficient to produce detectable damage without noticeable cytotoxicity.
  • ROIs regions of interest
  • Micro-irradiation was performed along parallel lines (ROIs) that spanned each field of view.
  • ROIs parallel lines
  • Cells were allowed to recover for 15 minutes, at which time they were washed once with PBS and fixed for ten minutes in 10% formalin, followed by immunofluorescence staining after permeabilization with 0.5% Triton-X for 4 minutes.
  • HDAC inhibitor treated cells were then added to wells and allowed to settle for 30 minutes, with Hoechst 33342 being added 20 minutes into this incubation.
  • DLBCL cells used in these experiments were treated with HDAC inhibitor for 24 hours prior in 6-well dishes.
  • Laser output was set to 100%, with twenty-four ROIs, a scan speed of 1/24 and pixel dwell of 86.3.
  • the autofocus ability (Perfect Focus System) of the microscope was also utilized in order to keep the smaller Karpas-422 cells in focus for the laser. A special stage was created to hold the 8-well chamber dishes and to keep the cells in focus. Recovery and fixation were the same as in HeLa cells, but permeabilization during immunofluorescence staining was modified. Permeabilization with either 0.1% Triton-X for 4 minutes or with ice-cold acetone for 10 minutes at ⁇ 20° C. was used for DLBCL cells.
  • DLBCL cells were treated with ACY957 in 25cm 2 flasks. Following treatment, cells were pelleted, washed with PBS and the pellet was re-suspended in 400 ⁇ l of lysis buffer with protease inhibitors (200 ⁇ l 0.5M HEPES.KOH, pH 7.9, 15 ⁇ l 1M MgCl2, 50 ⁇ l 2M KCl, 50 ⁇ l 0.1M DTT, 100 ⁇ l 0.4M NEM, 10 ⁇ l 1000 ⁇ Aprotinin/Leupeptin, 10 ⁇ l 1000 ⁇ Pepstatin A, and water to a total volume of 10 mL plus Roche protease inhibitor cocktail).
  • protease inhibitors 200 ⁇ l 0.5M HEPES.KOH, pH 7.9, 15 ⁇ l 1M MgCl2, 50 ⁇ l 2M KCl, 50 ⁇ l 0.1M DTT, 100 ⁇ l 0.4M NEM, 10 ⁇ l 1000 ⁇ Aprotinin/Leupeptin
  • the histone pellets were centrifuged at the same settings but for 5 minutes then washed again with cold acetone. After this wash the pellets were centrifuged for another 5 minutes and the acetone was discarded. Pellets were dried in an incubator at 37° C. for 5 minutes then re-suspended in 50 ⁇ l 2 ⁇ SDS sample buffer+ ⁇ -mercaptoethanol and 8 ⁇ l 1M Tris HCl pH 8 to neutralize. The samples were then boiled for 8 minutes at 95° C. and if there was still visible pellet they were either boiled longer, sonicated, had more sample buffer added or a combination thereof.
  • Nuclear and chromatin cell extracts were prepared according to methods known in the art, for example, as described in Bhaskara, S. et al. Epigenetics and Chromatin (2013), 6:27, the complete disclosure of which is hereby incorporated by reference for all purposes.
  • ChIP assays were performed according to methods known in the art, for example, as described in Bhaskara, S. et al. Molecular Cell (2008) 30(1):61-72, the complete disclosure of which is hereby incorporated by reference for all purposes.
  • RNA-Seq Analysis Total RNA was isolated from Karpas cells that were treated with DMSO or 2 ⁇ M ACY-957 for 24 h using the Versagene RNA isolation kit (5 Prime). The treatments were done in triplicate and sequenced using the Illumina Hiseq2000 sequencer. Illumina TruSeq Stranded sequencing following RiboZero treatment was performed.
  • Propidiurn-iodide Cell Cycle Analysis Propidium-iodide cell cycle analysis was performed according to methods known in the art, for example, as described in Bhaskara, S. et al. Molecular Cell (2008) 30(1):61-72, the complete disclosure of which is hereby incorporated by reference for all purposes.
  • H3K27me3 is Increased in DLBCL Cells Expressing Gain-of-Function Mutation of EZH2 Compared to Other Cancer Cells
  • the Karpas-422 line was established from the pleural effusion of a patient with chemotherapy-resistant non-Hodgkin's lymphoma (NHL) and the SUDHL4 line was derived from the peritoneal effusion of a 38-year male NHL patient.
  • Karpas-422 and SUDHL4 lines expressed mutant EZH2 with an amino acid substitution in the catalytic SET domain: the Karpas-422 line contained the Y641N mutation and the SUDHL4 line expressed the Y641S mutation. These changes in EZH2 were gain-of-function mutations resulting in a hyperactive enzyme.
  • H3K27me3 EZH2 catalyzed H3K27 trimethylation
  • ACY-957 is a Selective Small Molecule Inhibitor of HDAC1 and HDAC2 Enzymatic Activities
  • H3K27me3 is linked to transcriptional repression and it is enriched at the transcription start sites of inactive genes. H3K27me3 contributes to the proliferation and chemoresistance in a subset of DLBCL cells. In addition to methylation, the H3K27 residue also undergoes acetylation, which is dynamically regulated by the action of histone acetyltransferases (HATs) and deacetylases (HDACs). HDAC1,2 interacts with the EZH2-containing PRC2 complex and may function to remove the acetyl group from the lysine residue in order for EZH2 catalyzed methylation to occur.
  • HATs histone acetyltransferases
  • HDACs deacetylases
  • inhibition of HDAC1,2 activities alone or in combination with inhibition of EZH2 activity could be a strategy to overcome lymphomagenesis in DLBCL cells by blocking the gain-of-function mutant EZH2 mediated H3K27 hypertrimethylation via increased histone acetylation.
  • HDAC1,2 activities in DLBCL cells using a novel small molecule, ACY-957.
  • Hdac1,2 share a 50% sequence homology with Hdac3. Therefore, we first determined the selectivity of ACY-957 towards HDAC1 and HDAC2 in vitro enzyme assays using recombinant enzymes.
  • ACY-1044 a selective small molecule inhibitor of HDAC3, as a control in our characterization studies.
  • biochemical IC 50 values obtained using in vitro HDAC assays showed that a 7.4-fold and 3.3-fold higher concentration of ACY-1044 (the HDAC3-selective inhibitor) was required to inhibit HDAC1 and HDAC2, respectively, when compared to HDAC3 ( FIG. 2A ).
  • ACY-957 was 185-fold selective for HDAC1 and 72-fold more selective for HDAC2 when compared to HDAC3 ( FIG. 2A ).
  • Hdac1, 2 or Hdac3 knockout cells using Western blotting to identify a histone acetylation mark that is targeted by HDAC1,2 and not HDAC3.
  • Western analysis of histone acetylation marks altered in Hdacs1,2 ⁇ / ⁇ or Hdac3 ⁇ / ⁇ fibrosarcoma cells revealed that global H3K23ac levels were increased only upon loss of HDAC1,2 and not in cells lacking HDAC3 ( FIG. 2D ). Therefore, this result identified H3K23ac as a substrate that was specifically targeted by HDAC1,2 and not HDAC3.
  • H3K23ac we used changes in H3K23ac as a readout to test the ability of ACY957 to specifically inhibit HDAC1,2 activities in DLBCL cells.
  • SAHA Vorinostat
  • Hdac1,2 were the primary class I HDACs involved in deacetylating H3K23ac.
  • Addition of ACY-957, but not ACY-1044, to Karpas-422 or SUDHL4 cells resulted in an increase in H3K23ac levels compared to the DMSO treated control cells ( FIG. 2F ).
  • FIG. 2F Taken together, these results demonstrated that ACY-957 specifically inhibited HDAC1,2 activities in DLBCL cells.
  • HDAC1,2 activity was required for the proliferation and/or survival of the EZH2 GOF DLBCL cells. Since these DLBCL cells expressed a hyperactive form of EZH2, pharmacological inhibition of EZH2 activity was considered a therapeutic strategy. Therefore, we treated Karpas-422 and SUDHL4 cells with ACY-957 for 24 h , 48 h and 72 h, and performed cell cycle analysis to measure the extent of cell death (cytotoxic) and/or cell cycle arrest (cytostatic) triggered as a result of inhibiting HDAC1,2 activity in these cells.
  • cytotoxic cytotoxic
  • cytostatic cell cycle arrest
  • EZH2 inhibitor DZNep or GSK126
  • GSK126 was a S-adenosyl-methionine-competitive inhibitor of EZH2 methyltransferase activity
  • DZNep was a S-adenosylhomocysteine hydrolase inhibitor that disrupted the components of the PRC2 complex, thereby causing reduced chromatin-associated EZH2 levels.
  • H3K27ac was Increased in the EZH2 Gain-of-Function Mutant DLBCL Cells Upon Selective Inhibition of HDAC1,2 Activities
  • the histone H3K27 residue was subjected to reversible acetylation and methylation.
  • the aberrantly increased H3K27me3 resulting from a hyperactive EZH2 in DLBCL cells may contribute to both lymphomagenesis and chemoresistance.
  • HDAC1,2 inhibition While selective inhibition of HDAC1,2 activity did not reduce global H3K27me3 in the EZH2 gain-of-function DLBCL cells ( FIG. 4 ), the cell cycle arrest and/or death triggered in these cells by HDAC1,2 inhibition may be due to increased H3K27ac with a concomitant decrease in H3K27me3 locally at select target loci, such as, genes involved in cell cycle regulation, DNA repair or DNA damage signaling and apoptosis, which could also be the targets of EZH2.
  • target loci such as, genes involved in cell cycle regulation, DNA repair or DNA damage signaling and apoptosis
  • RNA-seq analysis showed that expression of 492 genes was up regulated following inhibition of HDAC1,2 activity in Karpas-422 cells compared to the DMSO control ( FIG. 5A ).
  • BMF BCL2-modifying factor, a pro-apoptotic gene
  • SAHA a pan-HDAC inhibitor
  • TP63 tumor protein 63
  • MEFs mouse embryo fibroblasts
  • HDAC1 and HDAC2 play a direct role at these genes and regulate H3K27ac at their promoter regions.
  • ChIP analysis was performed with HDAC1 and HDAC2 antibodies.
  • HDAC1,2 inhibition triggers death and cell cycle defects in the refractory DLBCL cells without requiring a reduction in the repressive H3K27me3 at the target genes.
  • data from our gene expression analyses indicated that the death and/or cell cycle arrest in chemoresistant/refractory DLBCL cells triggered by selective inhibition of HDAC1,2 activity was from the activation of DNA damage response resulting from endogenous DNA breaks.
  • HDAC1,2 Activity were Required for the Enrichment of H3K27me3 at Break Sites During DNA Repair in EZH2 Gain-of-Function Mutant DLBCL Cells
  • H3K27me3 was enriched at laser induced break sites in DLBCL cells following a 15 min recovery period following exposure to laser ( FIGS. 7B-D ). Additionally, H3K27me3 was not enriched at laser induced break sites in DLBCL cells after a 5 min recovery (data not shown).
  • ACY-957 treatment reduced the extent of H3K27me3 present at laser-induced break sites when compared to that present at the break sites in the DMSO treated control cells ( FIG. 7 ).
  • WSU-DLCL2 has an Y641F mutation in the SET domain.
  • HeLa independent cell line
  • FIG. 7D Similar to that observed in the refractory DLBCL cells, decreased H3K27me3 was observed at laser induced break sites in HeLa cells ( FIG. 7D ).
  • HDAC1,2 activity was required for EZH2-mediated H3K27me3 to occur at break sites during DNA repair.
  • H3K27ac forms ‘anti-stripe’ and was excluded from the laser-induced break sites.
  • a pan-nuclear staining for H3K27ac was observed in DMSO treated control Karpas-422 ( FIG. 7J ) or HeLa cells ( FIG. 14 ), which was increased following inhibition of HDAC1,2 activities using ACY-957 ( FIGS. 7J and 14 ).
  • H3K27ac was neither enriched at nor excluded from laser-induced break sites in DMSO or ACY-957 treated cells ( FIGS. 7J and 14 ).
  • HDAC1,2 did not remove all the H3K27ac marks at break sites; but they targeted a fraction of H3K27ac (specific or stochastic) in order for the enrichment of H3K27me3 catalyzed by EZH2 to occur at the break sites during DNA repair.
  • these findings indicated that selective inhibition of HDAC1,2 activity impaired DNA repair in the EZH2 gain-of-function mutant DLBCL cells in part by blocking EZH2-mediated H3K27me3.
  • GSK126 was a highly selective and potent inhibitor of EZH2 activity, and it targeted the catalytic SET domain.
  • GSK126 treatment of SUDHL4 and Karpas-422 cells led to a significant decrease in H3K27me3 following 48 h treatment of cells with this EZH2 inhibitor ( FIG. 4 ), but viability or cell cycle progression of these chemoresistant DLBCL cells was not compromised at this time point or even after 72 h post-incubation with GSK126 ( FIG. 3 ).
  • a genetically diverse set of DLBCL cell lines were classified as either sensitive or resistant to chemotherapy based on responsiveness to CHOP chemotherapy regime, which included doxorubicin.
  • Karpas-422 was a chemoresistant DLBCL cell line and SUDHL4 DLBCL cell line was partially resistant to chemotherapy drugs when compared to the sensitive SUDHL8 DLBCL cell line.
  • Deltex (DTX)-3-like E3 ubiquitin ligase (DTX3L), also known as B-lymphoma and BAL-associated protein (BBAP) was overexpressed in high risk, chemotherapy-resistant aggressive form of DLBCL.
  • BBAP was required for the monoubiquitination of histone H4K91 (H4K91ub1) and may protect cells from death when exposed to DNA-damaging agents.
  • BBAP and H4K91ub1 may also contribute to the chemoresistance and/or survival of the DLBCL cells.
  • chemoresistant DLBCL cells with gain-of-function mutation in EZH2 also expressed higher levels of BBAP compared to the chemosensitive DLBCL cells.
  • BBAP was required for the efficient repair of double strand DNA breaks, and BBAP-mediated H4K91ub1 was increased upon exposure of cells to doxorubicin (a DNA-damaging chemotherapy drug).
  • doxorubicin a DNA-damaging chemotherapy drug.
  • the increased BBAP levels in the refractory DLBCL cells may promote repair of the DNA damage induced by chemotherapy drugs via increased H4K91ub1 to confer chemoresistance and support the proliferation/survival of these cells.
  • H4K91 residue is involved in DNA repair in yeast and mammalian cells. H4K91 undergoes chemically exclusive modifications, acetylation and ubiquitination, in mammalian cells. Therefore, we hypothesized that selective inhibition of HDAC1,2 activity may increase H4K91ac to block BBAP-mediated H4K91ub1 and impair DNA repair in the chemoresistant DLBCL cells.
  • H4K16ac ( FIG. 8B ), which is a target of HDAC1,2.
  • H4K16ac occurred at the N-terminal tail region and disrupted chromatin packaging by preventing inter-nucleosomal interactions.
  • the H4K91 residue was present at the interface between the H3-H4 tetramer core and the H2A-H2A dimer within the nucleosome, and acetylation at this residue may weaken nucleosome stability by preventing the salt bridge formation and adversely affect chromatin assembly. Therefore, these results together indicated that Hdacs1, 2 play a role in nucleosome and chromatin dynamics in the chemoresistant DLBCL cells.
  • H4K91ub1 was impacted upon selective inhibition of HDAC1,2 activity in DLBCL cells.
  • Covalent addition of a bulky 7.6-kDa ubiquitin moiety onto the H4K91 residue retarded gel migration, resulting in slower migrating species of H4.
  • H4K91R a Flag-Myc epitope-tagged H4 or Flag-Myc epitope-tagged H4 mutant harboring a lysine to arginine substitution at position 91 (H4K91R) and exposed to doxorubicin.
  • H4K91ub1 induced by doxorubicin was reduced by H4K91R mutation.
  • H4K91ub1 An antibody recognizing H4K91ub1 was not available.
  • this short duration of inhibitor treatment enabled us to bypass the DNA repair events triggered by endogenous DNA damage in the absence of HDAC1,2 activity and directly follow the kinetics of doxorubicin-mediated DNA repair upon inhibiting HDAC1,2.
  • Immunofluorescence showed co-localization of 53BP1 with ⁇ H2AX at 4 h and 8 h following addition of doxorubicin to the control DMSO-treated Karpas-422 cells ( FIG. 9 ).
  • the percentage of cells containing 0, 1-5, 6-10 and greater than 10 ⁇ H2AX and 53BP1 foci were determined in at least 100 cells from three independent experiments. Quantitation of the percentage of cells with 53BP1 foci co-localizing with ⁇ H2AX foci revealed a decrease in the number of 53BP1 foci in ACY-957-treated Karpas-422 cells at 4 h post-doxorubicin treatment when compared to DMSO-treated controls ( FIG. 9 ). Following an 8 h treatment with doxorubicin, the percentage of cells with 1-5, 6-10, and greater than 10 53BP1 foci in ACY-957-treated cells increased to the level that was observed in the control DMSO-treated cells.
  • Hdac1,2 inhibition could sensitize the chemorefractory EZH2 GOF Karpas-422 cells to doxorubicin treatment and overcome drug resistance.
  • Karpas-422 cells showed a 1.5 to 2-fold increase in the percentage of cells in G2/M phase with a modest increase in cell death upon doxorubicin treatment ( FIG. 10A ). Additionally, a sub-G1 population (i.e., dead cells) was observed following ACY-957 treatment of Karpas-422 cells ( FIG. 10A ). However, combined treatment of Karpas-422 cells with doxorubicin and ACY-957 resulted in an increased sensitivity of cells to death, as the sub-G1 population increased by about 5-fold when compared to DMSO control ( FIG. 10A ). BrdU-PI analysis also showed a consistent increase in sub-G1 population upon combined application of ACY-957 and doxorubicin to Karpas-422 cells ( FIG.
  • Examples 2-12 demonstrated that selective inhibition of histone deacetylase 1,2 (HDAC1,2) activity using a small molecule inhibitor caused cytotoxic or cytostatic effects in EZH2 gain-of-function mutant (EZH2 GOF ) DLBCL cells.
  • HDAC1,2 histone deacetylase 1,2
  • Our results showed that blocking the activities of HDAC1,2 increased global H3K27ac without causing a concomitant global decrease in H3K27me3 levels.
  • Our data showed that inhibition of HDAC1,2 was sufficient to decrease H3K27me3 present at double-stranded breaks (DSBs), decrease DSB repair and activate the DNA damage response in these cells.
  • IC50 values obtained from in vitro enzyme assays showed that compounds ACY1035, ACY957, ACY1071 inhibited Hdac1,2 activities very selectively compared to Hdac3 ( FIG. 17A ).
  • Hdac1,2 inhibitors were rigorously validated the selectivity of all the Hdac1,2 inhibitors in hand using conditional Hdac1,2 or Hdac3 knockout cell lines and by examining changes in H4K5ac (a mark targeted by Hdacs1,2 and 3).
  • Hdac1,2 when Hdac1,2 are deleted from cells, an Hdac1,2 selective inhibitor will lack its target enzymes and therefore it cannot change H4K5ac levels any further. If however H4K5ac levels are increased in Hdac1,2-null cells upon adding an Hdac1,2 inhibitor, then it would mean that this compound has an off-target effect and inhibits Hdac3 activity.
  • our Hdac1,2 inhibitors did not increase H4K5ac when added to Hdac1,2 knockout cells (compare lanes 4-6 to lane 3 in FIG. 17C ).
  • MS275 is a benzamide-class inhibitor of Hdacs1, 2 and 330, and it caused a greater increase in H4K5ac than the deletion of just Hdac1,2 (compare lane 3 to 1 in FIG. 17C ). If an Hdac1,2 selective inhibitor targeted Hdac3, then it would be functionally equivalent to MS275; but H4K5ac levels following Hdac1,2 inhibition were not similar to that seen in MS275 treatment (compare lanes 4-6 to 1 in FIG. 17C ).
  • Hdac1,2 inhibitors in hand were designed to hit the zinc-binding pocket that was not present in Class III sirtuin HDACs. Also, these Hdac1,2 and Hdac3 inhibitors in hand belonged to amino-benzamide class of inhibitors (similar to MS-275) that only inhibited Hdacsl-344. Also, biochemical data showed that Hdac1,2 or Hdac3 inhibitors did not inhibit Class II Hdacs.
  • Hdac1,2 inhibitors did not increase Smc3 acetylation, a target of Hdac8, another Class I HDAC. Therefore, all our results together confirmed that the ACY compounds were highly selective inhibitors of Hdac1,2. Further endorsing the utility of these compounds for our studies, phenotypes obtained using these Hdac1,2 selective inhibitors agreed very well with those obtained using genetic systems to delete Hdac1,2. We also have pharmacokinetic (PK) data for these compounds for our in vivo experiments in mice.
  • PK pharmacokinetic
  • H4K91ac is a Histone Target of HDAC1,2 and Involved in Nucleosome Assembly and DNA Repair
  • Hdac1,2 may affect nucleosome assembly in addition to chromatin packaging. How may Hdac1,2 affect both chromatin packaging and nucleosome assembly? H4K16ac increased chromatin accessibility to MNase digestion by inhibiting the inter-nucleosomal interactions mediated by the H4 ‘tail’ domain to promote chromatin decompaction ( FIG. 18A ). H4K91ac influenced nucleosome assembly by regulating H3-H4 tetramer core interaction with the H2A-H2B dimers48 ( FIG. 18A ).
  • Hdac1,2 inhibition caused an increase in global H4K91ac in Ph+Pre-B-ALL cells ( FIG. 18B ).
  • H4K91ac as a histone target of Hdac1,2.
  • Increased H4K91ac following Hdac1,2 inhibition may prevent H2A-H2B deposition onto H3-H4 tetramer and disrupt nucleosome assembly, which in turn may result in an unstable nucleosome/chromatin that was prone to DNA damage in cancer cells, including Pre-B-ALL, and thus providing another mechanism for Hdac1,2 inhibitor action.
  • FEN1 is a Nuclease Involved in Processing DNA During Replication and Repair, and is a Non-Histone Target of HDAC1,2
  • HDAC1,2 are Therapeutic Targets for Early B-Cell Derived Acute Lymphoblastic Leukemia
  • Acute lymphoblastic leukemia is a fast-growing cancer of the lymphocyte-forming cells (the lymphoblasts) with>3200 new cases reported in the United States per year; 80-85% of these cases are precursor B-cell ALL (Pre-B-ALL).
  • Pre-B-ALL is characterized by the presence of oncogenic BCR-ABL fusion protein, which increased homologous recombination based DNA repair in cycling leukemic cells to prevent apoptosis.
  • Hdac1,2 inhibition impaired BCR-ABL mediated hyperactive DNA repair to cause DNA damage and death in Ph+Pre-B-ALL cells.
  • Hdac1,2 inhibitors caused significant cell death in three Ph+Pre-B-ALL cell lines (Nalm1, Tom1 and SupB15) ( FIG. 20A ). This effect was specific for Hdac1,2 inhibition, as treatment with a Hdac3 inhibitor (ACY 1044 ) did not cause any cell death ( FIG. 20A ).
  • Hdac3 inhibitor ACY 1044
  • Hdac1,2 selective inhibitors caused a significant dose-dependent cell death in BCR-ABL transformed, but not in the control non-transformed, mouse IL-3 dependent immortalized Pro-B Baf3 cells ( FIG. 20B ). Overall, these results showed that BCR-ABL expressing cells were sensitive to Hdac1,2 inhibition.
  • Hdac1,2 Deletion of Hdac1,2 in mice led to a block in B-cell development.
  • Treatment of mice with Hdac1,2 inhibitor decreased B220+ B-cells in the bone marrow ( FIG. 20C ), peripheral blood and spleen (data not shown).
  • normal B220+B-cell population reappeared in within 7 days after stopping Hdac1,2 inhibitor treatment ( FIG. 20D ).
  • Normal B220+CD43+ (early, immature) and B220+CD43 ⁇ (late, mature) B-cell population were also observed in the bone marrow following recovery from Hdac1,2 inhibition ( FIG. 20E ).
  • Hdac1,2 inhibition reduced replication fork velocity, activated DNA damage response in Ph+Pre-B-ALL SupB15 cells ( FIGS. 20G and 20H ) and in mononuclear cells obtained from a Ph+Pre-B-ALL patient ( FIG. 201 ). These results showed that Hdac1,2 inhibitors were effective in cycling Ph+ leukemic cells.
  • Hdac1,2 inhibition did not cause any significant DNA damage in normal stem/progenitor cells obtained from a healthy individual, but it triggered DNA damage in a vast majority of Ph+ stem/progenitor cells ( FIGS. 20J and 20K ).
  • Ph+LSCs have reduced quiescence, increased cell division and higher level of HR repair activity compared to normal hematopoietic stem cells, and given the link between Hdac1,2 and DNA replication, we therefore propose that Hdac1,2 inhibition induced DNA damage in these relatively cycling/replicating LSCs, likely via its direct adverse effects on the nascent chromatin and FEN1 repair activity at replication forks.
  • Hdac1,2 selective inhibition of Hdac1,2 was an effective therapeutic strategy for treating Ph+Pre-B acute lymphoblastic leukemia, as inhibiting Hdac1,2 activities caused replication stress in cycling cells to induce DNA damage in addition to impairing DNA repair.
  • Hdac1,2 affects chromatin structure in S-phase
  • the ‘open’ euchromatic regions replicate early in S-phase and the condensed heterochromatin replicates late in S-phase. Therefore, use of genome-wide approaches will enable us to obtain a global view of Hdac1,2 functions at and around replication forks present in different chromatin environments.
  • bioinformatics tools e.g., Useq or IGB, see attached letter
  • Hdac1,2 activities made nascent mononucleosomes sensitive to MNase digestion in addition to polynucleosomes. This indicated a role for Hdac1,2 in regulating intra-nucleosomal interactions in addition to inter-nucleosomal interactions.
  • H4K91ac a modification in the nucleosome core, as a target of Hdac1,2 ( FIG. 18C ).
  • H4K91 influenced nucleosome assembly by regulating interactions between the H3-H4 tetramer core and the H2A-H2B dimers ( FIG. 18A ). Proper nucleosome assembly was important for fork progression. During DNA replication, parental nucleosomes are disassembled to facilitate fork movement and reassembled after DNA synthesis.
  • the E3 ligase BBAP (B-cell lymphoma & BAL-associated protein) catalyzes H4K91 monoubiquitylation (H4K91ub1) during DNA repair.
  • H4K91ub1 is essential for establishing H4K20 methylation (me), which in turn is involved in the recruitment of 53BP1 (p53-binding protein) to the break sites.
  • 53BP1 is an important factor promoting chromatin dynamics during double-strand break (DSB) repair. Loss or inhibition of Hdac1,2 led to a defective 53BP1 recruitment to break sites ( FIG. 22 ).
  • H4K91ac upon Hdac1,2 inhibition blocks H4K91ub1 resulting in reduced H4K2Ome and impaired 53BP1 recruitment to the break sites. Therefore, our studies allude to the presence of a hitherto unknown ‘H4K91 acetyl-ubiquityl switch’ at double-strand break sites regulated by Hdac1,2 during DNA repair.
  • I-Ppo1 a homing endonuclease
  • Ph+Pre-B-ALL cells DMSO or Hdac1,2 inhibitor treatment.
  • I-Ppo1 cleaves at a 15 bp recognition sequence present at multiple sites within the human genome.
  • H4K91ub1 we will also examine the levels of 53BP1 and other above-mentioned histone marks around specific I-Ppo1 break sites in Ph+PreB-ALL cells using ChIP.
  • FEN1 (Flap endonuclease 1), a nuclease involved in DNA replication and replication-stress induced repair ( FIG. 23 ), as a target of Hdac1,2 ( FIG. 19 ).
  • WRN interacts with FEN1 to remove DNA intermediates formed during replication stress.
  • Acetylation-mimetic mutation in K375 of FEN1 disrupts its binding to WRN.
  • Hdac1,2 impairs FEN1 repair activity during replication stress, and makes it unresponsive to the increased WRN helicase activity stimulated by the oncogenic BCR-ABL ( FIG. 16 ).
  • Hdac1,2 inhibition impairs FEN1 functions during DNA replication (Example 23) and in replication stress-induced repair (Example 24) in Pre-B-ALL cells.
  • FEN1 interacts with 34 different proteins involved in various aspects of DNA metabolism, and many of these interactions occur at the C-terminal region of FEN1. Consistent with its role in Okazaki fragment maturation, FEN1 interacts with DNA polymerase ⁇ , RPA (the single-strand DNA binding protein) and DNA ligase 1. We will test if acetylation targeted by Hdac1,2 plays any role in regulating FEN1 interaction with these proteins involved in DNA replication/Okazaki fragment maturation using co-IPs following Hdac1,2 inhibitor treatment of Ph+Pre-B-ALL cells.
  • FEN1 site-specifically acetylated at K375. This involves expressing acetyl-lysyl-tRNA synthetase and the cognate tRNACUA in bacterial cells, which will direct the incorporation of NE-acetyl-lysine in response to an amber codon (TAG) introduced for residue 375 of FEN1.
  • TAG amber codon
  • Inhibiting Hdac1,2 activities during hydroxyurea-induced replication fork arrest increases the formation of collapsed forks, which suggests a role for Hdac1,2 in DNA repair and/or restart of stalled forks following replication stress.
  • Stalled replication forks can be converted into a four-way structure resembling a Holliday junction.
  • FEN1 interacts with WRN to form a complex.
  • WRN stimulates FEN1 nuclease activity to process aberrant DNA replication intermediates formed during replication stress in order to enable repair and fork restart.
  • WRN interacts with the C-terminal region of FEN1 and mutating the K375 residue in FEN1 to alanine severely disrupted its interaction with WRN. Changing lysine to alanine causes charge neutralization similar to acetylation of a lysine residue.
  • Hdac1,2 regulate FEN1-WRN interaction during replication stress
  • co-IPs to determine changes in their interaction in the presence of Hdac1,2-selective inhibitor upon hydroxyurea-induced replication stress in Pre-B-ALL cells.
  • WRN helicase activity at the Holliday junction intermediate will provide a free 5′ssDNA to be cleaved by FEN1 ( FIGS. 23A and 23B ).
  • Hdac1,2 activities regulate FEN1 activity to resolve recombination intermediates arising from fork stalling we will perform Holliday junction cleavage assays.
  • Hdac1,2 inhibition decreased homologous recombination (HR) in a BCR-ABL transformed cell line ( FIG. 23D ) containing a stably integrated DR-GFP reporter with an I-Scel site.
  • HR homologous recombination
  • FEN1 K375 mutants K75A/K375R/K375Q
  • FEN1 K375 mutations on the viability of SupB15 cells using FEN1 shRNA-mediated knockdown/complementation approach as described above.
  • Hdac1,2 inhibition affects the interaction of BCR-ABL with WRN and the ability of BCR-ABL in phosphorylating and nuclear-targeting of WRN using co-IP and cell fractionation, respectively.
  • Hdac1,2 inhibition does not affect the interaction or activity of BCR-ABL towards WRN, then it will support our possibility that Hdacs1, 2 work primarily by affecting the overall functions of WRN and FEN1. If however Hdac1,2 inhibition disrupts the interaction or activity of BCR-ABL on WRN, then we will test whether these two proteins are direct targets of Hdacs1,2. Overall, successful completion of studies described in Examples 23 and 24 would for show the direct functions for Hdac1,2 in the DNA repair steps after replication stress via targeting FEN1 acetylation and delineate the precise mechanism by which Hdac1,2 inhibition causes genotoxic stress in Pre-B-ALL cells.
  • BCR-ABL promotes DNA repair to prevent DNA damage accumulation in Philadelphia chromosome-positive (Ph+) leukemia cells.
  • SupB15 (Ph+PreB-ALL) cells were treated with 2 ⁇ M ACY1035 or 2 ⁇ M ACY1071 or 2 ⁇ M ACY957 for 48 hours.
  • a dramatic increase in DNA damage response was observed in SupB15 cells upon HDAC1,2 inhibitor treatment ( FIG. 24A ).
  • Quantitation of ⁇ H2AX foci a marker of double-strand breaks, showed a statistically significant increase in DNA damage following ACY957 or ACY1035 or ACY1071 treatments.
  • HDAC1,2 impairs of DNA repair via FEN1 acetylation.
  • Experiments were performed to determine whether inhibition of HDAC1,2 affects other DNA repair and replication proteins in Pre-B-ALL cells expressing BCR-ABL.
  • a large-scale quantitative LC-MS/MS (1D-LC/MS-MS) proteomic screen was performed in BAF3/BCR-ABL cells following treatment with DMSO; HDAC1,2 inhibitor; doxorubicin; or HDAC1,2 inhibitor plus doxorubicin ( FIG. 25A ).
  • a time point where only DNA damage but no cell death was activated was chosen for this analysis.
  • BAF180 is a subunit of PBAF SWI/SNF family chromatin remodeler complex and localizes to double-strand breaks, where it promotes transcriptional silencing during DNA repair via H2AK119 monoubiquitylation (H2AK119ub1, another transcriptional repression marker).
  • EPC2 is required for sustained oncogenic potential of leukemia stem cells, and a decrease in EPC2 can result in activation of DNA damage response in leukemia stem cells.
  • Treatment with HDAC1, 2 inhibitors led to cytoxic or cytostatic effects and impaired DNA repair in CD34+Ph+ALL stem/progenitor cells ( FIG. 28 ).
  • Doxorubicin can be used to treat leukemias in addition to lymphomas.
  • Doxorubicin is a DNA intercalating agent that impairs DNA repair.
  • Doxorubicin inhibits the process of DNA replication and activates the DNA damage response in cancer cells.
  • Experiments were conducted to determine what chromatin-bound proteins are affected upon treatment of BCR-ABL containing cells with ACY1035 in the presence of low doses of doxorubicin. For the mass spectrometry analysis, BAF3/BCR-ABL cells were treated with 2 ⁇ M ACY1035 for 48 hours. Doxorubicin at a final concentration of 0.1 ⁇ M was added during the last 10 hours of treatment.
  • HDAC1,2 Inhibition of HDAC1,2 triggers death in Ph+Pre-B-ALL cell lines.
  • MNC primary Ph+ALL mononuclear cells
  • Normal mononuclear cells served as a negative control.
  • FIGS. 30A and 30B Neutral comet assays revealed an increase in damaged DNA in these primary patient samples following HDAC1,2 inhibition.
  • chromatin-bound EPC2 the chromatin remodeler that promotes leukemia stem cell potential
  • HDAC1,2 inhibitors were effective against Ph+ALL stem/progenitor cells.
  • Stem/progenitor cells were FACS-sorted using human CD34 stem/progenitor cell surface marker and used for propidium-iodide cell cycle analysis as well as comet assays.
  • a G1 arrest or an increase in subG1 (dead) population was observed in Ph+ALL CD34+ cells following HDAC1,2 inhibition ( FIG. 28A ). Since the majority of CD34+ stem/progenitor cells are quiescent, no significant population of S-phase cells was detected ( FIG. 28A ).
  • HDAC1,2 inhibitors are very potent in primary patient samples.
  • HDAC1,2 selective inhibitors to treat the hyperactive repair addicted BCR-ABL expressing Ph+Pre-B-ALL cells in the clinic.
  • Pre-B-ALL cell lines as well as Ph+ALL primary patient samples were engrafted in NOD scid gamma (NSG) mice to examine the efficacy of Hdac1,2 inhibitor in killing cells in vivo.
  • NSG mice NOD scid gamma mice
  • SupB15 cells (1 ⁇ 10 6
  • Ph+Pre-B-ALL primary patient cells monitored engraftment by flow cytometry using anti-human CD45/CD19 antibodies every week for 4-5 weeks.
  • We saw successful engraftment with SupB15 and primary Ph+Pre-B-ALL cells FIG. 32 ).
  • FIG. 32A FACS analysis showed human CD45+ cells in bone marrow and spleen of xenografts. Similar results were obtained with human CD19 antibody in combination with CD45 or in isolation (data not shown).
  • IHC analysis To examine the extent of leukemia infiltration in spleen and bone marrow, we performed IHC analysis. Human TdT (a B-cell marker highly expressed in Pre-B-ALL patients) staining showed leukemia infiltration in bone marrow ( FIG. 32B ) and in spleen of xenograft mice (data not shown). We also observed human CD19 surface staining by IHC ( FIG. 32C ).
  • a mixture of bone marrow and spleen cells from the primary xenograft mice was then injected into a cohort of sub-lethally irradiated NOD/SCID mice.
  • Mice were divided into four treatment groups: solvent, ACY1035, doxorubicin, and ACY1035 plus doxorubicin.
  • Xenograft mice were injected according to one of the four treatment groups after 5 days post-xenograft induction, when human CD45 marker reached around 5-10% in the peripheral blood.
  • Mice were injected with 25 mg/kg ACY1035 every other day via i.p. and 0.25 mg/kg doxorubicin once a week via i.v., a dosing schedule that has minimal toxicity in immune compromised mice.
  • Pre-B-ALL will be induced by injecting BALB/c bone marrow cells (1 ⁇ 10 6 ) retrovirally transduced with a vector for co-expression of BCR-ABL and a GFP marker into lethally irradiated recipients. These mice develop leukemia in 1-2 weeks and start to die by 3-4 weeks. If needed, survival will be increased to perform analyses in a longer time window by injecting fewer cells.
  • GFP+B220+ cells will be measured in peripheral blood at 3-days interval after 10 days following retroviral transduction. Heska HemaTrue blood cell counter will be used to measure blood counts, and FACS will be used to measure GFP+ cells to diagnose the onset of leukemia.
  • Intraperitoneal (IP) injections with DMSO or Hdac1,2 inhibitor will occur when GFP+B220+ cells reach 1% of total white blood cells, and animals will be monitored to determine if leukemia is reversed over time. The percentage of GFP+B220+ cells in the peripheral blood will also be monitored to track leukemia regression following Hdac1,2 inhibitor treatment, for example, treatment with ACY1035.
  • mice will be subjected to histopathological analysis to examine spleen, liver, bone marrow and any other macroscopically involved organs. Immunohistochemistry (IHC) analysis with leukemia markers will also be performed.
  • IHC Immunohistochemistry
  • Karpas-422 is a doxorubicin resistant cell line, and Karpas-422 cells were far less sensitive to doxorubicin compared to the SUDHL5 cell line with wild type EZH2 in the neutral comet assays ( FIG. 34A ). However, the refractory Karpas-422 cells are sensitive to HDAC1,2 inhibition and HDAC1,2 inhibition makes these cells sensitive to doxorubicin treatment, because an increase in comet tail moment and a decrease in comet head intensity was observed with ACY957 alone and ACY957+doxorubicin treatments ( FIG. 34A ).
  • HDAC1,2 inhibitor causes replication fork collapse in S-phase to trigger DNA damage response, and transformed cancer cells have increased DNA replication when compared to normal B cells.
  • transformed cancer cells are extraordinarly sensitive to HDAC1,2 inhibition.
  • EZH2 GOF DLBCL cells also have increased DNA repair compared to EZH2 WT DLBCL cells ( FIG. 34A ), which can also be targeted by HDAC1,2 inhibitor as it impairs the HDAC1,2 regulated DNA repair processes used by EZH2 GOF DLBCL cells for their survival.
  • H3K27me3 recruits the PRC1 complex containing Bmi-1 E3 ligase to catalyze H2AK119 ubiquitination and facilitates further downstream EZH2 signaling.
  • a decrease in repair factor Bmi-1 and an increase in serine 2-phosphorylated form of RNA polymerase II (a transcriptional elongation mark) at laser break sites was observed following HDAC1,2 inhibition ( FIG. 34C ).
  • the disclosed results for the first time show the requirement of HDAC1,2 activities in EZH2-mediated DSB repair and the control of transcription at break sites.
  • Acetylation at H2AK119 is a Novel Target of HDAC1,2 in EZH2 GOF DLBCL Cells
  • H2AK119 monoubiquitination (H2AK119ubl) catalyzed by the PRC1 complex is downstream of the EZH2-containing PRC2 complex during DNA repair.
  • H2AK119ub1 is required for recruiting several repair proteins including RNF8 and subsequent recruitment of BRCA1 and 53BP1 to the break sites in order to propagate damage signaling.
  • HDAC1,2 regulate EZH2-mediated DSB repair signaling in the EZH2 GOF DLBCL cells experiments were performed to assess whether HDAC1,2 regulates H2AK119ub1 via targeting H2AK119 acetylation (H2AK119ac).
  • H2AK119ac Inhibition or knockdown of HDAC1,2 increases H2AK119ac ( FIG. 35A ). An increase in H2AK119ac is also accompanied by a decrease in global chromatin-associated H2AK119ub1 ( FIG. 35B ). HDAC1,2 inhibition thus decreases H2AK119ub1 indirectly via decreasing H3K27me3 ( FIG. 34C ) and directly by increasing H2AK119ac ( FIG. 35 ), which adds another layer to the regulation of EZH2-mediated repair by HDAC1,2.
  • HDAC1,2 Inhibition Increases H4K91ac to Alter the H4K91Acetyl-Ubiquityl Switch Following Doxorubicin Treatment
  • BBAP is overexpressed in the high risk, chemotherapy-resistant and aggressive form of DLBCL.
  • BBAP catalyzes H4K91 monoubiquitination (H4K91ub1), which is proposed to protect cells from death when exposed to DNA damaging agents.
  • H4K91ub1 H4K91 monoubiquitination
  • Inhibition of HDAC1,2 increased global H4K91ac levels in both SUDHL4 and Karpas-422 cells.
  • ACY957 treatment resulted in a decrease in the BBAP-mediated H4K91 monoubiquitination following addition of doxorubicinl.
  • HDAC1,2 selective inhibition of HDAC1,2 triggers apoptosis in chemoresistant/refractory EZH2 GOF DLBCL cells.
  • a first-of-its-kind study was carried out to test the therapeutic benefits of selective inhibition of HDAC1,2 as a treatment for chemoresistant EZH2 GOF DLBCL using a xenograft mouse model.
  • HDAC1,2 inhibition alone or in combination with doxorubicin can causes tumor regression using this mouse model.
  • the in vivo testing used a doxorubicin-resistant EZH2 GOF DLBCL (Karpas-422/NSG) mouse model and a doxorubicin-sensitive EZH2 WT DLBCL (SUDHL8/NSG) mouse model ( FIG. 36 ).
  • a doxorubicin-resistant EZH2 GOF DLBCL Kerpas-422/NSG
  • a doxorubicin-sensitive EZH2 WT DLBCL SUVHL8/NSG mouse model
  • FIG. 36 NOD-SCID-Gamma mice were injected intravenously (i.v.) with 1 ⁇ 10 6 Karpas-422 cells (chemoresistant and expressing the EZH2 gain-of-function mutant) or SUDHL8 cells (chemosensitive and expressing wild-type EZH2).
  • Glioma is a type of primary brain tumor that originates in the glial cells present in the brain or spinal cord.
  • the glioma cell lines U-87 and U-251 were treated with either DMSO or 2 ⁇ M ACY957 for 72 hours and then subjected to propidium iodide staining and FACS analysis to evaluate cell cycle progression. No effect on the cell cycle was observed in glioma cells following HDAC1,2 inhibition ( FIG. 37 ).
  • Clause 1 A method of treating a cancer characterized by BCR-ABL expression or BBAP overexpression in a subject in need thereof, the method comprising administering to the subject an agent that selectively inhibits HDAC1 and HDAC2.
  • Clause 6 The method of any one of clauses 1-5, wherein the cancer is further characterized as being dependent upon on a double-stranded break repair pathway.
  • Clause 8 The method of any one of clauses 1-7, wherein the cancer is further characterized by increased H3K27me3.
  • Clause 9 The method of any one of clauses 1-8, further comprising determining if the cancer is characterized by BCR-ABL expression or BBAP overexpression, wherein determining comprises detecting a level of BCR-ABL, a level of BBAP, or a level of BCR-ABL and a level of BBAP in a sample obtained from the subject.
  • Clause 10 The method of clause 9, further comprising comparing the detected levels of BCR-ABL, BBAP, or BCR-ABL and BBAP to levels of BCR-ABL, BBAP, or BCR-ABL and BBAP in a control sample, wherein if the detected level of BCR-ABL is increased relative to the control level of BCR-ABL, the cancer is characterized by BCR-ABL expression, and wherein if the detected level of BBAP is increased relative to the control level of BBAP, the cancer is characterized by BBAP overexpression.
  • Clause 11 The method of any one of clauses 1-10, further comprising sensitizing the cancer to a chemotherapeutic agent.
  • Clause 13 The method of any one of clauses 1-12, further comprising administering doxorubicin to the subject.
  • Clause 14 A method of sensitizing a cancer characterized by BCR-ABL expression or BBAP overexpression to a chemotherapeutic agent in a subject in need thereof, the method comprising administering an agent that selectively inhibits HDAC1 and HDAC2 to the subject.
  • Clause 15 The method of clause 14, wherein the cancer is a B cell malignancy.
  • Clause 19 The method of any one of clauses 14-18, wherein the cancer is further characterized as being dependent upon on a double-stranded break repair pathway.
  • Clause 21 The method of any one of clauses 14-20, wherein the cancer is further characterized by increased H3K27me3.
  • Clause 22 The method of any one of clauses 14-21, further comprising determining if the cancer is characterized by BCR-ABL expression or BBAP overexpression, wherein determining comprises detecting a level of BCR-ABL, a level of BBAP, or a level of BCR-ABL and a level of BBAP in a sample obtained from the subject.
  • Clause 23 The method of clause 22, further comprising comparing the detected levels of BCR-ABL, BBAP, or BCR-ABL and BBAP to levels of BCR-ABL, BBAP, or BCR-ABL and BBAP in a control sample, wherein if the detected level of BCR-ABL is increased relative to the control level of BCR-ABL, the cancer is characterized by BCR-ABL expression, and wherein if the detected level of BBAP is increased relative to the control level of BBAP, the cancer is characterized by BBAP overexpression.
  • a method for determining if a cancer is sensitive to an agent that selectively inhibits HDAC1 and HDAC2 comprising: (a) obtaining a sample from a subject suffering from the cancer; (b) measuring a level of one or more markers in the sample, wherein the one or more markers are selected from the group consisting of: BCR-ABL and BBAP; (c) comparing the measured level of the one or more markers in the sample to a level of the one or more markers in a control sample; and (d) determining that the cancer is sensitive to an agent that selectively inhibits HDAC1 and HDAC2 when the measured level of the one or more markers in the sample is increased relative to the level of the one or more markers in the control sample.
  • Clause 25 The method of clause 24, further comprising administering the agent that selectively inhibits HDAC1 and HDAC2 to the subject suffering from the cancer that is sensitive to the agent.
  • Clause 26 The method of clause 24 or 25, wherein measuring the level of the one or markers includes an immunoassay, fluorescence in situ hybridization, or polymerase chain reaction.
  • Clause 27 The method of any one of clauses 24-26, wherein the cancer is a B cell malignancy.
  • Clause 31 The method of any one of clauses 24-30, wherein the cancer is characterized as being dependent upon on a double-stranded break repair pathway.
  • Clause 32 The method of clause 31, wherein the double-stranded break repair pathway includes one or more of the group consisting of FEN1, EPC2, BAF180, and DNA Ligase I.
  • Clause 33 The method of any one of clauses 24-32, wherein the cancer is characterized by increased H3K27me3.
  • a method for monitoring the efficacy of a treatment for a cancer that includes administration of an agent that selectively inhibits HDAC1 and HDAC2, the method comprising: (a) obtaining a first sample from the subject before the treatment and a second sample from the subject during or after the treatment; (b) measuring a first level of one or more markers in the first sample and a second level of the one or more markers in the second sample, wherein the one or more markers are selected from the group consisting of: 53BP1, and ⁇ H2AX; (c) comparing the first level of the one or more markers and the second level of the one or more markers; and (d) determining that the treatment is effective when the second level of the one or more markers is higher than the first level of the one or more markers.
  • Clause 35 The method of clause 34, wherein measuring first and second levels of the one or more markers includes measuring foci formation of the one or more markers.
  • Clause 36 The method of clause 34 or 35, wherein the cancer is a B cell malignancy.
  • Clause 40 The method of any one of clauses 34-39, wherein the cancer is characterized as being dependent upon on a double-stranded break repair pathway.
  • Clause 42 The method of any one of clauses 34-41, wherein the cancer is characterized by increased H3K27me3.
  • Clause 43 The method of any one of clauses 34-42, further comprising administering a chemotherapeutic agent to the subject when the treatment is determined to be effective.
  • Clause 44 The method of clause 43, wherein the chemotherapeutic agent is doxorubicin.

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