Search

Rearrangements of the MLL gene define a genetically distinct subset of acute leukemias with poor prognosis. Current treatment options are of limited effectiveness; thus, there is a pressing need for new therapies for this disease. Genetic and small molecule inhibitor studies have demonstrated that the histone methyltransferase DOT1L is required for the development and maintenance of MLL-rearranged leukemia in model systems. Here we describe the characterization of EPZ-5676, a potent and selective aminonucleoside inhibitor of DOT1L histone methyltransferase activity. The compound has an inhibition constant value of 80 pM, and demonstrates 37 000-fold selectivity over all other methyltransferases tested. In cellular studies, EPZ-5676 inhibited H3K79 methylation and MLL-fusion target gene expression and demonstrated potent cell killing that was selective for acute leukemia lines bearing MLL translocations. Continuous IV infusion of EPZ-5676 in a rat xenograft model of MLL-rearranged leukemia caused complete tumor regressions that were sustained well beyond the compound infusion period with no significant weight loss or signs of toxicity. EPZ-5676 is therefore a potential treatment of MLL-rearranged leukemia and is under clinical investigation.

The activity and safety of the histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid, SAHA) were evaluated in patients with refractory cutaneous T-cell lymphoma (CTCL). Group 1 received vorinostat 400 mg daily, group 2 received vorinostat 300 mg twice daily for 3 days with 4 days rest, and group 3 received vorinostat 300 mg twice daily for 14 days with 7 days rest followed by 200 mg twice daily. Treatment continued until disease progression or intolerable toxicity. The primary objective was to determine the complete and partial response (PR) rate. Time to response (TTR), time to progressive disease (TTP), response duration (DOR), pruritus relief, and safety were determined. Thirty-three patients who had received a median of 5 prior therapies were enrolled. Eight patients achieved a PR, including 7 with advanced disease and 4 with Sézary syndrome. The median TTR, DOR, and TTP for responders were 11.9, 15.1, and 30.2 weeks, respectively. Fourteen of 31 evaluable patients had pruritus relief. The most common drug-related AEs were fatigue, thrombocytopenia, diarrhea, and nausea. The most common grade 3 or 4 drug-related AEs were thrombocytopenia and dehydration. Vorinostat demonstrated activity in heavily pretreated patients with CTCL. The 400 mg daily regimen had the most favorable safety profile and is being further evaluated.

The t(6;11)(q27;q23) is a recurrent chromosomal rearrangement that encodes the MLLAF6 fusion oncoprotein and is observed in patients with diverse hematologic malignancies. The presence of the t(6;11)(q27;q23) has been linked to poor overall survival in patients with AML. In this study, we demonstrate that MLL-AF6 requires continued activity of the histone-methyltransferase DOT1L to maintain expression of the MLL-AF6-driven oncogenic gene-expression program. Using gene-expression analysis and genome-wide chromatin immunoprecipitation studies followed by next generation sequencing, we found that MLL-fusion target genes display markedly high levels of histone 3 at lysine 79 (H3K79) dimethylation in murine MLL-AF6 leukemias as well as in ML2, a human myelomonocytic leukemia cell line bearing the t(6;11)(q27;q23) translocation. Targeted disruption of Dot1l using a conditional knockout mouse model inhibited leukemogenesis mediated by the MLL-AF6 fusion oncogene. Moreover, both murine MLL-AF6–transformed cells as well as the human MLL-AF6–positive ML2 leukemia cell line displayed specific sensitivity to EPZ0004777, a recently described, selective, small-molecule inhibitor of Dot1l. Dot1l inhibition resulted in significantly decreased proliferation, decreased expression of MLL-AF6 target genes, and cell cycle arrest of MLL-AF6–transformed cells. These results indicate that patients bearing the t(6;11)(q27;q23) translocation may benefit from therapeutic agents targeting aberrant H3K79 methylation.

Abstract 2379 The enzymatic activity of the protein methyltransferase (PMT) DOT1L has been shown to be a driver of cell proliferation in MLL-rearranged leukemia. Our group has previously reported the design of potent and selective aminonucleoside inhibitors of DOT1L [Daigle et al. (2011) Cancer Cell 20: 53–65; Basavapathruni et al. (2012) Chem. Biol. Drug Design, in press]. Structure-guided design, together with robust biochemical and biological assays, was used to optimize the potency, selectivity and pharmacological features of the aminonucleosides, resulting in the compound EPZ-5676. EPZ-5676 is an S-adenosyl methionine (SAM) competitive inhibitor of DOT11L that displays a Ki value of 80 pM and a drug-target residence time of > 24 hours. The compound is highly selective for DOT1L, demonstrating > 37,000-fold selectivity against all other PMTs tested. Crystallographic studies reveal that the high affinity, durable inhibition of DOT1L by EPZ-5676 has its origin in a conformational adaptation of the protein that attends inhibitor binding, extending the compound binding pocket to include novel recognition elements beyond the SAM binding active site. Treatment of leukemia cells with EPZ-5676 results in concentration- and time-dependent reduction of H3K79 methylation without effect on the methylation status of other histone sites. The reduction of H3K79 methylation leads to inhibition of key MLL target genes and selective, apoptotic cell killing in MLL-rearranged leukemia cells, but has minimal impact on non-rearranged cells. EPZ-5676 is highly soluble in aqueous solution and can thus be formulated for intravenous administration. The effective pharmacokinetic half-life of EPZ-5676 in systemic circulation has been measured to be 0.25 and 1.5 h in rats and dogs, respectively. A nude rat subcutaneous xenograft model of MLL-rearranged leukemia has been established. Continuous intravenous infusion of EPZ-5676 for 21 days in this model leads to dose-dependent anti-tumor activity. At the highest dose, complete tumor regressions are achieved with no regrowth for up to 32 days after the cessation of treatment (Figure 1). Figure 1. EPZ-5676 causes complete and sustained tumor regression in a MV4–11 nude rat xenograft model of MLL-rearranged leukemia. No significant weight loss or obvious toxicity was observed in rats treated with EPZ-5676 during this efficacy study. EPZ-5676 is thus a potent, selective inhibitor of DOT1L that demonstrates strong efficacy in a rat xenograft model of MLL-rearranged leukemia. Details of the preclinical characterization of this compound will be presented. Figure 1. EPZ-5676 causes complete and sustained tumor regression in a MV4–11 nude rat xenograft model of MLL-rearranged leukemia. . / No significant weight loss or obvious toxicity was observed in rats treated with EPZ-5676 during this efficacy study. EPZ-5676 is thus a potent, selective inhibitor of DOT1L that demonstrates strong efficacy in a rat xenograft model of MLL-rearranged leukemia. Details of the preclinical characterization of this compound will be presented. Disclosures: Pollock: Epizyme: Employment, Equity Ownership. Daigle:Epizyme: Employment, Equity Ownership. Therkelsen:Epizyme: Employment, Equity Ownership. Basavapathruni:Epizyme: Employment, Equity Ownership. Jin:Epizyme: Employment, Equity Ownership. Allain:Epizyme: Employment, Equity Ownership. Klaus:Epizyme, Inc.: Employment, Equity Ownership. Raimondi:Epizyme: Employment, Equity Ownership. Porter Scott:Epizyme: Employment, Equity Ownership. Chesworth:Epizyme: Employment, Equity Ownership. Moyer:Epizyme: Employment, Equity Ownership. Copeland:Epizyme Inc.: Employment, Equity Ownership. Richon:Epizyme, Inc.: Employment, Equity Ownership. Olhava:Epizyme: Employment.

Abstract 3502 The t(6;11)(q27;q23) produces a chimeric MLL-AF6 oncogene, and is a recurrent chromosomal rearrangement observed in patients with diverse hematologic malignancies such as acute myelogenous leukemia (AML), as well as both B-cell and T-cell acute lymphoblastic leukemias (ALL). The presence of an MLL-AF6 translocation predicts a particularly poor prognosis. Of particular biological interest, the MLL-AF6 translocation is the most common fusion event in which MLL fuses to a predominantly cytoplasmic protein. Very little is known about the molecular mechanisms of transformation mediated by the MLL-AF6 fusion oncogene, forestalling the development of specific therapeutic strategies for t(6;11)(q27;q23) positive leukemias. Recent studies suggest that the histone methyltransferase DOT1L could be an important therapeutic target in MLL-rearranged leukemias. We sought to assess whether MLL-AF6 mediated transformation is also dependent on aberrant H3K79 methylation using genomic, genetic and pharmacological approaches. First, we performed chromatin immuno-precipitation using H3K79me2 specific antibodies followed by next generation sequencing (ChIP-seq) on murine MLL-AF6 leukemias as well as on ML2, the human myelomonocytic leukemia cell line bearing the MLL-AF6 fusion gene. We observed that in both murine and human MLL-AF6 leukemia cells, MLL-fusion target genes display markedly high levels of H3K79 dimethylation as compared to other highly expressed genes. We then investigated whether MLL-AF6-induced transformation was dependent on aberrant H3K79 methylation through genetic or pharmacologic inhibition of the Dot1l histone methyltransferase. Lineage negative/Sca-1 positive/Kit positive (LSK) cells from mice bearing homozygous Dot1l floxed alleles were immortalized by retroviral expression of the MLL-AF6 fusion gene. Cre-recombinase mediated excision of Dot1l from MLL-AF6 transformed bone marrow cells resulted in a significant reduction in H3K79 dimethylation at the promoters of the MLL-target genes Hoxa9, Hoxa10 and Meis1, with a concomitant decrease in their expression. Dot1l excision significantly diminished the clonogenic capacity, abrogated blast colony formation in methylcellulose based medium, and enhanced differentiation of MLL-AF6 transformed cells. We then sought to assess whether EPZ004777, a recently described specific small molecule inhibitor of DOT1L could show efficacy against murine and human MLL-AF6 transformed cells. Dot1l inhibition using EPZ004777 significantly diminished H3K79 dimethylation globally (as assessed by immunoblotting) as well as on MLL-target genes (as assessed by ChIP-qPCR) using H3K79me2 specific antibodies. Importantly, EPZ004777 treatment significantly impaired the proliferation of both murine MLL-AF6 transformed cells as well as the ML2 cell line, whereas the proliferation rates of Hoxa9-Meis1 transformed cells as well as the human MLL-germline cell line HL60 were unaffected despite a similar decrease in H3K79me2 levels. EPZ004777 treatment induced cell cycle arrest as well as increased apoptosis in MLL-AF6 positive, but not control leukemia cells, demonstrating a selective activity of the DOT1L inhibitor EPZ004777 on MLL-AF6 transformed cells. In summary, we demonstrate that the MLL-AF6 oncoprotein requires continued activity of the histone methyltransferase DOT1L for aberrant epigenetic activation of downstream target oncogenes. More studies are needed to understand the mechanisms by which DOT1L is recruited to MLL-target genes by the MLL-AF6 fusion, since AF6 is not believed to normally associate with DOT1L. Nevertheless, the demonstration that H3K79 methylation is important for MLL-AF6 mediated transformation indicates that patients bearing the t(6;11)(q27;q23) translocation may benefit from therapeutic agents targeting aberrant H3K79 methylation. Disclosures: Olhava: Epizyme: Employment. Daigle:Epizyme, Inc.: Employment. Richon:Epizyme, Inc.: Employment, Equity Ownership. Pollock:Epizyme Inc.: Employment, Equity Ownership. Armstrong:Epizyme: Consultancy.

Abstract 2384 The t(10;11)(p12;q23) and the t(10;11)(p12;q14), which encode the MLL-AF10 and CALM-AF10 fusion oncoproteins respectively, are two recurrent chromosomal rearrangements observed in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Patients with AML harboring either MLL-AF10 or CALM-AF10 rearrangements have a particularly poor outcome compared to patients whose leukemia cells do not harbor these translocations. Thus new therapeutic approaches are clearly needed for patients with leukemias bearing rearrangements of the AF10 gene. Previous studies have suggested that the histone H3 lysine 79 (H3K79) methyltransferase DOT1L is recruited by a part of the AF10 protein retained in leukemic MLL-AF10 and CALM-AF10 fusions, suggesting a role of this methyltransferase in transformation mediated by these fusion oncoproteins. The general amenability of enzymes to pharmacological inhibition makes DOT1L an attractive therapeutic target in leukemias bearing AF10-fusions. We decided to test whether transformation mediated by the MLL-AF10 or CALM-AF10 fusions can be impaired by Dot1l inhibition. We transformed bone marrow cells with either the MLL-AF10 or the CALM (400–648)-AF10 (677–758) fusion proteins that have been previously shown to immortalize hematopoietic progenitors in vitro and in vivo. In contrast to the large, highly clonogenic compact colonies observed in methylcellulose culture of wildtype transformed cells, Dot1l excision from the MLL-AF10 or CALM-AF10 immortalized progenitors led to the formation of small, diffuse colonies that had lost their replating capability. In MLL-AF10 or CALM-AF10 transformed cells, Dot1l- inactivation significantly reduced global H3K79 dimethylation as assessed by Western blotting, and Wright Giemsa staining confirmed morphological features consistent with myeloid differentiation. Moreover, the expression of MLL-AF10 and CALM-AF10 targets, such as Hoxa cluster genes and Meis1, decreased significantly after Dot1l inactivation as assessed by quantitative PCR. We then assessed the impact of Dot1l deletion on the in vivo leukemogenic activity of MLL-AF10 and CALM-AF10 transformed bone marrow cells. Ongoing experiments clearly demonstrate that Dot1l excision strongly impairs the initiation and maintenance of both MLL-AF10 as well as CALM-AF10 mediated murine leukemias. Having established that genetic inactivation of Dot1l inhibits H3K79 methylation and consequently transforming potential of the AF10-fusions, we investigated the efficacy of Dot1l inhibitors against MLL-AF10 or CALM-AF10 transformed murine bone marrow cells. Treatment of MLL-AF10 or CALM-AF10 transformed cells with EPZ004777, the first specific small-molecule inhibitor of Dot1l, suppressed expression of downstream oncogenic targets such as Hoxa cluster genes and Meis1, and selectively impaired the proliferation of MLL-AF10 and CALM-AF10 transformed, but not Hoxa9/Meis1 transformed cells. MLL-AF10 and CALM-AF10 transformed cells underwent differentiation and cell cycle arrest after EPZ004777 treatment. Results from in vivo colony-forming units-spleen (CFU-S) assays showed that pre-treatment of MLL-AF10 or CALM-AF10 transformed cells with the EPZ004777 inhibitor profoundly impaired their spleen-colony forming ability, suggesting that EPZ004777 may show in vivo efficacy against AF10-fusion transformed cells. Taken together, our results demonstrate that Dot1l inhibition impairs the in vitro and in vivo oncogenic activity of the MLL-AF10 and CALM-AF10 fusion oncogenes. These results indicate that patients with leukemias bearing chromosomal rearrangements of the AF10 gene may benefit from small molecule inhibition of H3K79 methylation. Disclosures: Olhava: Epizyme: Employment. Daigle:Epizyme, Inc.: Employment. Richon:Epizyme, Inc.: Employment, Equity Ownership. Pollock:Epizyme Inc.: Employment, Equity Ownership. Armstrong:Epizyme: Consultancy.

Abstract 3712 The coupled enzymatic activity of wild-type and mutant EZH2 results in hyper-trimethylation of histone H3 lysine 27 (H3K27), which drives lymphomagenesis in heterozygous patients bearing the EZH2 mutations. Our group has previously reported that selective inhibition of EZH2 in cell culture results in selective killing of lymphoma cells bearing EZH2 mutations, with minimal effect on non-mutant lymphoma cells, suggesting that EZH2 enzymatic activity is a required driver of proliferation in the mutant-bearing cells [Knutson et al. (2012) Nature Chemical Biology, in press]. Through iterative medicinal chemistry we have developed a selective inhibitor of EZH2 with good pharmacological properties, E7438. E7438 binds to the enzyme in a manner competitive with S-adenosyl methionine (SAM) and a Ki for wild-type EZH2 of 2.5 ± 0.5 nM. The compound potently inhibits all known mutants of EZH2 that have been identified in non-Hodgkin lymphoma (NHL) patient samples. E7438 displays about 35-fold less activity against the closely related enzyme EZH1, and is >4500-fold selective with respect to all other protein methyltransferases tested. Lymphoma cells treated with E7438 display concentration- and time-dependent loss of H3K27 methylation with no effect on the methylation status of any other histone sites. The loss of H3K27 methylation results in selective killing of EZH2 mutant-bearing lymphoma cell lines. E7438 displays good oral bioavailability and pharmacokinetic properties. Various EZH2 mutant-bearing human lymphoma tumors were subcutaneously implanted in nude, SCID or NSG mice. Oral administration of E7438 to tumor bearing mice resulted in significant anti-tumor activity. The responses ranged from dose-dependent tumor growth inhibition to complete and sustained regressions. For example, KARPAS422 tumors in nude mice showed complete tumor elimination after 28 days of dosing, with mice remaining tumor free for up to 90 days after treatment cessation. Figure 1. E7438 causes complete and sustained tumor regression in a KARPAS422 nude mouse xenograft model of EZH2-mutated NHL. Dosing was on a BID schedule. * P< 0.05, Repeated measures ANOVA, Dunnett's post test vs. vehicle. Figure 1. E7438 causes complete and sustained tumor regression in a KARPAS422 nude mouse xenograft model of EZH2-mutated NHL. Dosing was on a BID schedule. * P< 0.05, Repeated measures ANOVA, Dunnett's post test vs. vehicle. Mice and rats tolerated E7438 administration well at doses representing high multiples of doses that show antitumor activity in mice. Activity against the EZH2 target in both species was demonstrated by dose-dependent diminution of H3K27me3 levels, assessed by ELISA, in samples of tumor, bone marrow, skin and peripheral blood mononuclear cells (PBMCs). Highly sensitive detection of existing H3K27me3 signal could also be observed with this ELISA assay in drug-naïve samples of human PBMCs. The ability to measure dose-dependent changes in H3K27me3 levels in skin and PBMCs portends the use of signal from these surrogate tissues as a non-invasive pharmacodynamics biomarker in human clinical trials. Disclosures: Keilhack: Epizyme Inc.: Employment, Equity Ownership. Yokoi:Eisai Co., Ltd.: Employment. Knutson:Epizyme Inc.: Employment, Equity Ownership. Wigle:Epizyme Inc.: Employment, Equity Ownership. Warholic:Epizyme Inc.: Employment, Equity Ownership. Kawano:Eisai Co., Ltd.: Employment. Minoshima:Eisai Co., Ltd.: Employment. Huang:Eisai Inc.: Employment. Kuznetsov:Eisai Inc.: Employment. Kumar:Eisai Inc.: Employment. Klaus:Epizyme, Inc.: Employment, Equity Ownership. Allain:Epizyme Inc.: Employment, Equity Ownership. Raimondi:Epizyme Inc.: Employment, Equity Ownership. Porter Scott:Epizyme: Employment, Equity Ownership. Chesworth:Epizyme: Employment, Equity Ownership. Moyer:Epizyme: Employment, Equity Ownership. Uenaka:Eisai Co., Ltd.: Employment. Copeland:Epizyme Inc.: Employment, Equity Ownership. Richon:Epizyme, Inc.: Employment, Equity Ownership. Pollock:Epizyme Inc.: Employment, Equity Ownership. Kuntz:Epizyme Inc.: Employment, Equity Ownership.

Abstract 62 Leukemias associated with translocations of the Mixed Lineage Leukemia (MLL) gene account for a significant percentage of both AML and ALL, and often carry a poor prognosis. The exact molecular mechanisms by which MLL-fusion proteins transform cells are incompletely understood. One proposed model involves the aberrant activation of transcriptional programs through epigenetic changes that ultimately lead to leukemogenesis. The histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been shown to be recruited by the most common MLL fusion proteins, and MLL fusion protein target loci are associated with H3K79 methylation (H3K79me2/3) in mouse models and MLL-rearranged human leukemia samples. However, it is currently unclear whether H3K79 methylation is indeed a necessary step in leukemogenesis. We sought to investigate in detail the importance of Dot1l in MLL-fusion mediated leukemia, using an shRNA approach and a newly developed conditional loss of function mouse model of Dot1l (Dot1lflox/flox). shRNA mediated suppression of Dot1l in a panel of MLL-rearranged human leukemia cell lines led to a decrease in growth rate and viability, induction of apopotosis and cell cycle arrest. Bioluminescent in vivo tracking of MLL-rearranged human leukemia cell lines in xenotransplant recipients revealed that the onset of leukemia was significantly delayed after Dot1l suppression. To complement and confirm the sh-RNA results, we developed a conditional loss of function mouse model for Dot1l. In this model, deletion of the active site of Dot1l severely impaired or abrogated serial replating of Mll-Af9 transduced lineage negative (lin-) cells, and fully developed Mll-Af9 leukemia cells. Immunoblot of total cellular H3K79 and chromatin immunoprecipitation of known MLL target loci showed loss of H3K79 methylation. In addition, we observed variable induction of additional silencing mechanism such as Polycomb Repressor Complex 2 (PRC2) mediated H3K27 methylation on selected loci such as HoxA10. These epigenetic changes correlated with a reduction in expression of known MLL-fusion downstream targets. When Dot1l was inactivated in Mll-Af9 leukemia cells, in vitro colony and cell morphology demonstrated loss of blast-like phenotype and induction of differentiation. Furthermore, mice transplanted with Mll-Af9 leukemia cells from primary recipients failed to induce leukemia in secondary recipients after cre-mediated deletion of Dot1l. The role of Dot1l in normal hematopoiesis is not defined, and it is possible that deletion of Dot1l induces catastrophic collapse of the entire hematopoietic system, including leukemia cell compartments. To exclude this possibility, we are analyzing the phenotype of hematopoiesis specific deletion of Dot1l in normal mice through the use of the Vav-cre system. Initial results show that Dot1lflox/flox-Vav-Cre mice are born at mendelian ratios and display neutrophil and lymphocyte counts at the lower limit of normal despite near complete absence of H3K79 methylation in peripheral blood leukocytes. A more detailed analysis of the hematopoietic phenotype of loss of Dot1l, including hematopoietic stem cell compartments, is currently ongoing in our laboratory. These data demonstrate that Dot1l is indeed a central player in MLL-fusion mediated leukemogenesis and required for leukemia cell survival. The low-normal Dot1l-/- neutrophil and lymphocyte counts observed in the Dot1lflox/flox-Vav-Cre mice suggest that Dot1l is more critical for leukemia cells than normal hematopoietic cells. This predicts a therapeutic window for pharmacologic inhibitors of Dot1l, and highlights their potential as targeted therapy for MLL-rearranged leukemias. Disclosures: Pollock: Epizyme, Inc: Employment. Richon:Epizyme, Inc: Employment. Armstrong:Epizyme, Inc: Consultancy.

Abstract 780 Rearrangements of the mixed lineage leukemia (MLL) gene on chromosome 11q23 are found in over 70 % of infant leukemias, and approximately 10% of adult acute myeloid leukemias (AML). Patients with MLL-rearranged leukemias have aggressive disease with a poor prognosis. Recent studies suggest that DOT1L, a histone methyltransferase that methylates lysine 79 of histone H3 (H3K79), plays a fundamental role in the development and maintenance of this genetically defined subset of leukemia. Rearrangements of the MLL gene result in the expression of MLL-fusion proteins that gain the ability to recruit DOT1L to chromatin. This leads to aberrantly high levels of H3K79 methylation and gene expression at specific genomic loci, including HOXA9 and MEIS1 that are thought to promote leukemogenesis. These findings, together with studies demonstrating a key role for DOT1L in propagating the transforming activity of MLL-fusion proteins in model systems, support the development of inhibitors of this enzyme as targeted therapeutics for patients bearing MLL-rearranged leukemias. To this end, we have used mechanism-guided design to identify EPZ01, the first small molecule DOT1L inhibitor. This compound is a potent and specific inhibitor of DOT1L methyltransferase activity with a Ki of ~ 400 pM in biochemical assays. EPZ01 acts as a competitive inhibitor with the co-factor S-adenosyl-methionine (SAM), and demonstrates greater than 500-fold selectivity for DOT1L over other lysine and arginine histone methyltransferases. Incubation of MLL-rearranged leukemic cell lines with EPZ01 leads to a dramatic decrease in cellular H3K79 methylation but does not affect the methylation of other histone residues, including H3K4, H3K27, H3K36 and H3K9. Analysis of the effects of EPZ01 on the proliferation of a panel of acute lymphoid leukemia (ALL) or AML-derived human MLL-rearranged cell lines including SEMK2, MV4-11, RS4;11, MOLM-13 and THP-1, and non-rearranged leukemia cell lines including HL-60, Jurkat and U937, reveals anti-proliferative activity that is remarkably selective for cell lines bearing the MLL-rearrangement. EC50 values for inhibition of proliferation by EPZ01 are in the nanomolar to low micromolar range for all MLL-rearranged lines tested. In contrast, EPZ01 shows little or no effect on the proliferation of cells lacking an MLL-rearrangement despite an equal decrease in cellular H3K79 methylation. A more detailed analysis of the cellular effects of EPZ01 in MLL-rearranged cell lines reveals that treatment with the inhibitor causes a decrease in mRNA expression of known MLL-fusion target genes including HOXA9 and MEIS1, cell cycle arrest in G0/G1, an increase in expression of differentiation markers in MLL-rearranged AML cells and death by apoptosis. We are currently evaluating the effects of EPZ01 and related compounds in in vivo models of MLL-rearranged leukemia where preliminary results indicate that we are able to achieve inhibition of DOT1L activity. EPZ01 therefore represents the first example of a histone methyltransferase inhibitor that selectively kills tumor cells bearing a defined genetic lesion. These data provide compelling validation for the development of DOT1L inhibitors as targeted therapeutics for MLL-rearranged leukemias and we are currently working towards this goal. Disclosures: Pollock: Epizyme, Inc: Employment. Daigle:Epizyme, Inc: Employment. Olhava:Epizyme, Inc: Employment. Therkelsen:Epizyme, Inc: Employment. Majer:Epizyme, Inc: Employment. Song:Epizyme, Inc: Employment. Allain:Epizyme, Inc: Employment. Sneeringer:Epizyme, Inc: Employment. Johnston:Epizyme, Inc: Employment. Porter Scott:Epizyme, Inc: Employment. Jin:Epizyme, Inc: Employment. Kuntz:Epizyme, Inc: Employment. Chesworth:Epizyme, Inc: Employment. Moyer:Epizyme, Inc: Employment. Armstrong:Epizyme, Inc: Consultancy. Copeland:Epizyme, Inc: Employment. Richon:Epizyme, Inc: Employment.

Abstract 707 EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2), catalyzes the mono- through tri-methylation of lysine 27 on histone H3 (H3K27). While overexpression of EZH2 and increased H3K27 methylation have generally been associated with both hematologic malignancies and solid tumors, inactivating somatic mutations of Tyr641 (Y641F, Y641N, Y641S and Y641H) of EZH2 were recently reported to be associated with follicular lymphoma (FL) and the GCB subtype of diffuse large B-cell lymphoma (DLBCL) (Morin, Nat Genet 2010; 42: 181). In all cases, occurrence of the mutant EZH2 gene was heterozygous, and expression of both wild type and mutant alleles was detected in the mutant samples profiled by transcriptome sequencing. Further, the mutant forms of EZH2 could be incorporated into the multi-protein PRC2 complex, but the resulting complexes lacked the ability to catalyze trimethylation of an unmethylated H3K27 peptide substrate. To explore further the role of EZH2 in lymphomagenesis, we have evaluated the catalytic activity of the mutant EZH2 proteins in greater detail. Recombinant PRC2 complexes were prepared with wild type and Tyr641 mutant EZH2 forms. As previously reported, the wild type enzyme demonstrated robust activity but none of the mutant enzymes displayed significant methyltransferase activity on an unmodified H3K27 peptide. We next evaluated the activity of the enzymes using native avian erythrocyte olignucleosomes as the substrate in the reaction. In contrast to the peptide result, we found that the wild type and all of the mutant enzymes were active methyltransferases against the native nucleosome substrate. Since native nucleosome represents an admixture of the unmodified and mono-, di- and tri-methylated H3K27 we next evaluated the activity of the wild type and mutant enzymes on unmodified, and mono- and di-methylated H3K27 peptide. We demonstrate that the wild type enzyme displays greatest catalytic efficiency (kcat/K) for the zero to mono-methylation reaction of H3K27, and diminished efficiency for subsequent (mono- to di- and di- to tri-methylation) reactions. In stark contrast, the disease-associated Y641 mutants display very limited ability to perform the first methylation reaction, but have enhanced catalytic efficiency for the subsequent reactions, relative to WT-enzyme. Catalytic coupling between the mutant EZH2 species and PRC2 complexes containing either wild type EZH2 or wild type EZH1 are predicted to augment H3K27 trimethylation and thus produce the malignant phenotype associated with mutant heterozygosity. To test this prediction, the level of H3K27 methylation was evaluated in lymphoma cell lines harboring only wild type EZH2 (OCI-LY-19) or heterozygous for EZH2 Y641N (DB, KARPAS and SU-DHL-6) or EZH2 Y641F (WSU-DLCL2) by immunoblotting. As predicted by simulations, the level of H3K27 trimethylation was elevated in all of the lymphoma cell lines harboring the mutant EZH2 relative the wild type. Additionally, we observe decreased H3K27 dimethylation and monomethylation in the cells harboring the mutated EZH2 relative to wild type enzyme; these reductions in di- and monomethylation are likewise consistent with expectations based on steady state kinetic simulations. The present results imply that the malignant phenotype of follicular lymphoma and diffuse large B cell lymphoma of the GCB subtype, associated with expression of mutant forms of EZH2, results from of an overall gain-of-function with respect to formation of the trimethylated form of H3K27. These data suggest that selective, small molecule inhibitors of EZH2 enzymatic function may form a rational underpinning for molecularly targeted therapeutics against mutant-harboring lymphomas and other malignancies in which EZH2 gain-of-function is pathogenic. Disclosures: Richon: Epizyme, Inc: Employment. Sneeringer:Epizyme: Employment. Porter Scott:Epizyme, Inc: Employment. Kuntz:Epizyme, Inc: Employment. Knutson:Epizyme, Inc.: Employment. Pollock:Epizyme, Inc: Employment. Copeland:Epizyme, Inc: Employment.

Bortezomib, a proteasome inhibitor, is extremely effective for the treatment of multiple myeloma (MM). However, nearly all patients will eventually become refractory to bortezomib. Vorinostat is a potent inhibitor of histone deacetylase enzymes, and has been shown to affect growth of various cell lines, including MM, in a variety of in vitro non-clinical studies. Furthermore, the combination of bortezomib and vorinostat has demonstrated synergy in several in vitro and murine models. Subsequently, 2 multicenter, open-label, Phase I clinical trials have been conducted to investigate the combination of vorinostat with bortezomib in patients with MM. In the first trial, 34 patients with relapsed/refractory MM were enrolled. Patients received escalating doses of vorinostat (200 mg bid or 300–400 mg daily for 14 days) and bortezomib (0.7, 0.9, 1.1, or 1.3 mg/m2 on days 1, 4, 8, and 11); cycles were repeated every 21 days for ≤8 cycles or until progressive disease (PD) or intolerable toxicity. In the event of PD, oral dexamethasone (20 mg on days 1–4 and 17–20) could be added to the bortezomib plus vorinostat combination. The highest dose level of vorinostat was 400 mg daily for 14 days and bortezomib 1.3 mg/m2. The maximum tolerated dose (MTD) was not determined because ≥2 dose-limiting toxicities (DLTs) did not occur at any dose level. The most common drug-related adverse events were nausea (61.8%), diarrhea (58.8%), thrombocytopenia (50%), and vomiting (50%). Two patients experienced a DLT; grade 3 transient AST elevation was experienced by 1 patient receiving 400 mg vorinostat daily and 0.9 mg/m2 bortezomib, and grade 4 thrombocytopenia was experienced by 1 patient receiving 400 mg vorinostat daily and 1.3 mg/m2 bortezomib. Among 34 evaluable patients, the best response to vorinostat plus bortezomib was a partial response (PR) in 9 (26%) patients, minimal response (MR) in 7 (21%) patients, and stable disease (SD) in 18 (53%) patients. Mean duration of SD was 89 days, range 9–369 days. Of the 13 evaluable patients who had previously been treated with bortezomib, 5 achieved a PR, 1 had a MR, and 7 had SD. The effect of adding dexamethasone will also be analyzed and presented. The second trial enrolled 23 patients who received vorinostat (100–500 mg on days 4–11) and bortezomib (1–1.3 mg/m2 on days 1, 4, 8, and 11). Dexamethasone was added at cycle 2 for 6 patients who achieved less than a PR, and at cycle 4 for 5 patients with PD and at cycle 6 for 2 patients with PD. There was no upgrade in response for any patients who received additional dexamethasone. Two patients in the vorinostat 500 mg group experienced DLT (fatigue and prolonged QTc); MTD was identified as vorinostat 400 mg plus bortezomib 1.3 mg/m2. The main toxicities in this trial were hematologic (anemia, neutropenia, and thrombocytopenia). Twenty-one patients were evaluable for response (2 achieved very good PR, 7 PR, 10 SD, 2 progressive PD). Of the 9 patients who were refractory to bortezomib, 3 had PR, 4 had SD, 1 had PD, and 1 was non-evaluable. Co-administration of bortezomib did not alter the pharmacokinetics of vorinostat. In conclusion, these data suggest that the combination of vorinostat plus bortezomib is active for treatment of MM, even among some patients with prior exposure to bortezomib.

Vorinostat is a histone deacetylase inhibitor currently under evaluation in numerous oncology clinical trials. In a Phase IIb trial, oral vorinostat resulted in a 29.7% overall objective response rate in patients (pts) with advanced cutaneous T-cell lymphoma (CTCL) and had an acceptable safety profile. These results prompted efforts to identify gene expression patterns that could elucidate the molecular mechanism of action (MOA), assess exposure to vorinostat and enrich for pts who are likely to respond. In the Phase IIb trial, gene expression profiles were obtained from 24 predose and 30 postdose (2 hr postdose on Day 15) PBMC samples. The gene expression associated with Sezary burden was easily identified in predose samples and consistent with published results. Although the power of this dataset was limited for development of a predose predictor of response, we identified three biologically-relevant pathways that correlated with response and deserve further validation. First, we found a coherent cluster of proliferation/cell cycle genes to be associated with resistance to therapy. This may imply that tumor aggressiveness is an important factor for clinical response. Second, a set of antioxidant genes was upregulated in non-responders. The generation of reactive oxygen species (ROS) is a component of the vorinostat MOA and increased ROS scavenging ability may confer resistance. Finally, cytotoxic cell markers were upregulated in responders and may represent another factor associated with contribution of T and NK cells to response. Each of these 3 patterns, if confirmed, would allow for 20–50% responder enrichment. We observed robust postdose gene expression changes in which ~942 genes exhibited significant regulation (fold-change>2, P

Purpose: To evaluate the activity and safety of the histone deacetylase inhibitor vorinostat in advanced cutaneous T-cell lymphoma (CTCL) patients (pts). Patients and Methods: Advanced CTCL pts received oral vorinostat 400 mg daily until disease progression (PD) or intolerable toxicity in this open label Phase IIb trial. Eligible pts must have received ≥ 2 prior systemic therapies which included bexarotene unless intolerable. The planned sample size was ≥ 50 evaluable pts with stage ≥ IIB. The primary endpoint was the objective response rate (ORR) as measured by a modified skin severity weighted assessment tool (SWAT). The study would be positive if the ORR in ≥ stage IIB pts was ≥ 20%. Time to response, time to progression (TTP), response duration (DOR, from time of first response), pruritus relief, safety and gene expression changes were also evaluated. Results: Seventy-four pts (median age, 60 y [range, 39–83]; median 3 prior systemic therapies) were enrolled (61 pts ≥ stage IIB) at 18 centers. Data cut-off was 5/06 with a median follow-up of 10 m. Efficacy data are shown (Table 1). The ORR was 29.5% in ≥ stage IIB pts. The median DOR and TTP were not reached but estimated to be at least 6.1 m and 9.8 m, respectively, for ≥ stage IIB responders. Median TTP was 4.9 m for all pts. Overall, 32% of pts had pruritus relief. The most common drug-related adverse experiences (AE) were diarrhea (49%), fatigue (46%), nausea (43%) and anorexia (26%), and were mostly ≤ grade 2. Seven pts discontinued and 10 had dose modification due to drug-related AE. Drug-related AE ≥ grade 3 included fatigue (5%), pulmonary embolism (5%), thrombocytopenia (5%) and nausea (4%). Fifteen pts, including 9 responders, were receiving treatment as of 11/05. Three of these pts discontinued due to PD (n = 2) or unacceptable toxicity (n = 1), and 12 have received vorinostat for > 1 y. A pharmacodynamic signature of vorinostat exposure based on gene expression changes was detected. Conclusion: These updated results with additional follow-up continue to demonstrate that oral vorinostat remains effective in advanced CTCL with an acceptable safety profile. Table 1. Efficacy of Vorinostat in Advanced CTCL Population n Objective Response* Median (range) n Pruritus Relief† *Objective response = confirmed complete or partial response. †Sustained pruritus reduction of ≥ 3 points or complete resolution for ≥ 4 wks; 2 pts with missing baseline scores were excluded. ‡Kaplan-Meier estimates were not reached but DOR was ≥ 165, 185 and 165 d, respectively, with a median follow-up of 10 m. §Stages IIB, III, IVA or IVB. CI = confidence interval n (%) (95% CI) TTR, d DOR, d n (%; 95% CI) All pts 74 22 (29.7) (19.7, 41.5) 55 (28–171) NR‡ (34+, 441+) 72 23 (31.9; 21.4, 44.0) Stage IB or IIA 13 4 (30.8) (9.1, 61.4) 42.5 (30–57) 80.5 (48+, 418+) 13 5 (38.5; 13.9, 68.4) ≥Stage IIB§ 61 18 (29.5) (18.5, 42.6) 56 (28–171) NR‡ (34+, 441+) 59 18 (30.5; 19.2, 43.9) Sezary syndrome 30 10 (33.3) (17.3, 52.8) 56 (28–171) NR‡ (34+, 244+) 30 9 (30.0; 14.7, 49.4)

The hydroxamic acid vorinostat (SAHA) is an HDAC inhibitor that induces differentiation, growth arrest and/or apoptosis of malignant cells both in vitro and in vivo, and is currently being tested in the clinic for a variety of indications. In particular, vorinostat has demonstrated an overall response rate of approximately 30% in advanced cutaneous T-cell lymphoma (CTCL). The purpose of this study was to identify biomarkers that predict response to vorinostat in CTCL using preclinical model systems based upon gene expression profiling and pathogenesis of lymphoma. Herein we report the results of our evaluation of the STAT (signal transducer and activator of transcription) signaling pathway. The relative level of mRNA and protein expression as well as activation status of several members of this family have been evaluated in lymphoid cell lines with diverse vorinostat sensitivity (proliferation/viability IC50 range: 0.3–14 mM). Among them, the more responsive CTCL cells lines HH and HUT102 exhibited an IC50 ≃ 0.8 mM. These cells underwent apoptosis in response to vorinostat as assessed by TUNEL assay, in contrast to Hut78 and MJ cells (IC50 > 2 mM) that did not show signs of DNA fragmentation upon incubation in vorinostat-containing media for 48 h. Our results indicate that in lymphoma cell lines elevated protein levels and persistent activation of STAT1, 3 and 5 correlate with resistance to vorinostat. Immunofluorescence microscopy studies revealed that STAT proteins preferentially localize to the nuclear compartment in cells with impaired vorinostat response, consistent with the expected distribution for this group of transcription factors in the active state. Simultaneous treatment with a pan-JAK inhibitor (JAK inhibitor I; Calbiochem) resulted in an additive antiproliferative effect consistent with a survival, antiapoptotic role for STAT proteins in the response to vorinostat treatment. Immunohistochemical analysis of STAT1 in skin biopsies isolated from CTCL patients (N = 49) enrolled in the vorinostat Phase IIb clinical trial showed that malignant T cells were positively stained in approximately half of the samples (21 positively stained; 25 negatively stained; 3 poor quality specimens), and in those cases a relationship between nuclear accumulation of STAT1 and lack of response to treatment exists (p=0.007

Vorinostat is a histone deacetylase inhibitor with potent in vitro and in vivo anti-leukemia activity. Several gene expression signatures were developed from preclinical models as biomarkers for clinical response to vorinostat. The signatures were evaluated in peripheral blood mononuclear cell (PBMC) samples from a Phase I study of oral vorinostat in patients with advanced leukemias or myelodysplastic syndromes (MDS). Of 41 registered patients, 31 had acute myeloid leukemia (AML), 4 had chronic lymphocytic leukemia, 3 had MDS, 2 had acute lymphoblastic leukemia, and 1 had chronic myeloid leukemia. Seven of 41 patients showed improvement with treatment, 2 complete responses, 2 complete responses that were not maintained for 28 days, and 3 with >50% decreases in blast counts without count recovery. All responses occurred in patients with AML at or below the MTD. The following three clinical gene expression signatures were shown to be predictive of clinical response in PBMC samples of AML patients. First, a positive prediction trend was observed when a preclinical 40-gene resistance prediction signature was evaluated in baseline (pre-dose and first day of dosing) AML PBMC samples (8 responder and 21 non-responder samples) or all PBMC samples (14 responder and 56 non-responder samples, including non-AML patients). If this trend is maintained in pretreatment samples of a larger patient population, it may allow for exclusion of up to 50% of non-responders from future clinical trials. Second, a similar prediction of resistance was obtained based on expression levels of known reactive oxygen species scavengers that were upregulated in non-responders relative to responders, indicating that cellular ability to withstand oxidative stress may be protective with respect to vorinostat exposure. This supports the hypotheses that oxidative stress pathways play an important role in the response to vorinostat. Finally, overexpression of p21 and several p53-responsive genes was also associated with resistance, consistent with preclinical observations. These results are being validated at the protein level in a tissue array platform. Post-dose downregulation of genes associated with cellular proliferation was also observed in responders but not in non-responder PBMC samples. This suggests that proliferation may be used as a post-dose efficacy biomarker. Further evaluation of vorinostat in patients with leukemias and MDS, including analysis of a potentially predictive gene expression signature for response to vorinostat, is warranted.

Vorinostat, formerly known as suberoyl anilide hydroxamic acid, (SAHA) is a histone deacetylase inhibitor with potent in vitro antileukemia activity. We conducted a phase I study of two dose schedules of vorinostat: oral three times a day (TID) x 14 days every 21 days; or oral twice a day (BID) x 14 days every 21 days. Patients with relapsed or refractory chronic and acute leukemia or myelodysplastic syndrome (MDS), with adequate renal, hepatic functions and performance status were eligible. Older patients with untreated AML/MDS were also eligible. Forty-one patients were registered and dosed. Median age was 54 years (range 18 to 90), 31 (76%) had AML, 4 (10%) CLL, 3 (7%) MDS, 2 (5%) ALL and 1 (2%) CML. The median number of prior therapies was 2 (range 0–7). The starting dose of the oral TID schedule was 100 mg po TID and it was increased in 50 mg po steps using a 3+3 design. As these patients were thrombocytopenic at baseline due to their underlying disease, thrombocytopenia was not considered to be a dose-limiting toxicity. A dose of 300 mg po TID was considered above the maximally tolerated dose (MTD) with 2 out of 3 patients developing grade 3 toxicity (nausea, vomiting and diarrhea). Subsequently, 7 patients were treated at a dose of 300 mg po BID x 14 days every 21 days. Two patients developed gastrointestinal toxicity (typhlitis) in the setting of profound neutropenia, and the dose was reduced in the next 6 patients to 200 mg po BID x 14 days. No excess toxicity was observed at that dose level. Subsequently, 6 more patients were treated at a dose of 200 mg po TID x 14 days (The MTD of 250 mg po TID X 14 days could not be further evaluated as the 50 mg capsule was no longer available). Only one out 6 patients developed grade 3 toxicity (fatigue). More frequently observed toxicities, regardless of causality, were nausea, vomiting, diarrhea, anorexia, headache, fatigue, typhlitis, and dyspepsia that resolved upon cessation of therapy. Laboratory abnormalities included pancytopenia, hyperglycemia, hypokalemia, hypocalcemia and hypophosphatemia. Overall 9 patients (21%) had objective evidence of response: 1 CR, 2 CRp (CR criteria but no recovery of platelet counts), 1 partial response and 5 complete marrow responses (blasts less than 5%). All responses were observed in patients with AML, and 5 (41%) of these responses were observed at a dose of 200 mg po tid. Histone acetylation was observed in all patients at all dose levels. In summary, the MTD of oral vorinostat is either 200 mg po TID or 200 mg po BID x 14 days every 21 days in patients with leukemia. Significant activity was observed at a dose of 200 mg po TID x 14 days in patients with AML. This single agent activity warrants additional investigation of the role of vorinostat in the therapy of AML and may be guided by the development of informative biomarkers.

Hsp90 is an ATP-dependent molecular chaperone, which helps in folding its client proteins, e.g., Bcr-Abl, FLT-3, c-Raf and Akt, into active conformation. Geldanamycin analogue, 17-AAG (Kosan Biosciences Inc., Hayward, CA) inhibits the chaperone function of hsp90, which promotes polyubiquitylation and proteasomal degradation of the misfolded hsp90 client proteins. We recently reported that, by inhibiting the activity of histone deacetylase 6, the hydroxamate HDIs such as vorinostat (Merck & Co., Inc.) induce acetylation and inhibition of hsp90, thus also causing the depletion of its client proteins. In the present studies, we determined the anti-leukemia effects of the novel, highly soluble, hsp90 antagonist IPI504 (Infinity Pharmaceuticals), which, in vitro and in vivo, interconverts with 17-AAG, ± vorinostat, against human cultured or primary, wild type or mutant Bcr-Abl or mutant FLT-3 containing acute leukemia cells. Treatment with IPI504 (0.5 to 2.0 μM) for 24 to 48 hours, in a dose dependent manner, induced apoptosis of WT Bcr-Abl-expressing K562 and LAMA-84 cells. This was associated with attenuation of the levels of Bcr-Abl, pCrkL, pSTAT5, c-Raf and pAkt. In a dose dependent manner (50 to 500 nM for 48 hours), IPI504 also induced apoptosis of FLT-3 internal tandem duplication (ITD)-containing human acute leukemia MV4-11 cells, which was associated with attenuation of the levels of FLT-3, pAkt, pSTAT5, pERK1/2. Notably, treatment with IPI504 induced similar level of apoptosis of mouse bone marrow BaF3 cells, which had been transformed and rendered IL-3 independent for growth by ectopic expression of WT Bcr-Abl, its P-loop (Bcr-Abl-E255K) or highly imatinib mesylate (IM) resistant, contact-inhibition (Bcr-Abl-T315I) point mutant. This was also associated with attenuation of the levels of WT and mutant Bcr-Abl-E255K or Bcr-Abl-T315I. In previous studies we had demonstrated that treatment with vorinostat depletes WT and mutant Bcr-Abl levels and induces apoptosis of expressing human leukemia cells. Therefore, we determined the effect of the co-treatment of IPI504 (1.0 μM) and vorinostat (1.0 μM) against cultured or primary human CML cells. Co-treatment with IPI504 and vorinostat induced significantly more apoptosis of K562 and MV4-11 cells, which was associated with more depletion of WT-Bcr-Abl and FLT-3-ITD levels in K562 and MV4-11 cells, respectively. Notably, co-treatment with IPI504 and vorinostat, versus treatment with either agent alone, also induced more apoptosis of primary CML cells (4 samples) derived from patients with IM-resistant CML, including a sample of cells documented to have Bcr-Abl-T315I mutation. Additionally, as compared to treatment with either agent alone, the combination of IPI504 and vorinostat also induced more apoptosis of primary AML cells (4 samples), including two samples that contained FLT-3-ITD. These findings demonstrate that the combination of IPI504 with vorinostat exerts a high level of in vitro activity against FLT-3-ITD-containing acute leukemia, as well as against highly IM-resistant mutant Bcr-Abl-expressing leukemia cells.

We have previously reported that agents that inhibit ATP binding and chaperone function of hsp90 are highly active against wild type and mutant Bcr-Abl and mutant FLT-3 containing human acute leukemia cells. In the present studies, we determined the effects of a more soluble and potent geldanamycin analogue, DMAG (Kosan Biosciences Inc.), and/or hydroxamate histone deacetylase inhibitor (HHDI), vorinostat (Merck & Co., Inc.), against human MCL Jeko1 and MO2058 cells. These cells contain the characteristic MCL-associated chromosomal translocation t(11; 14)(q13;q32), which results in the overexpression of cyclin D1. Recently, HHDIs, such as vorinostat, have been shown to inhibit HDAC6, which results in the acetylation of hsp90 and inhibition of its ATP binding and chaperone function. Treatment with vorinostat (0.5 to 2.0 μM) induced the accumulation of the cells in the G1 and DMAG (0.1 to 0.5 μM) in the G2/M phase of the cell cycle. Both agents induced apoptosis in a dose-dependent manner (up to 50%). While vorinostat induced both p21 and p27 levels, DMAG only increased the intracellular levels of p21. Treatment with either agent depleted the intracellular levels of c-Myc, c-Raf, Akt and cdk4 in a dose dependent manner. It is well established that the chaperone association with hsp90 maintains Akt, c-Raf, cyclin D1 and cdk4 in the native and active conformation, and inhibition of hsp90 promotes their polyubiquitylation and proteasomal degradation. Notably, co-treatment with DMAG (e.g., 0.25 μM) and vorinostat (e.g., 2.0 μM), more than either agent alone, markedly attenuated the levels of cyclin D1 and cdk4, as well as the levels of c-Myc, c-Raf and Akt. The combination of DMAG and vorinostat also induced significantly more apoptosis of Jeko1 and MO2058 cells, as compared to the treatment with either agent alone (p < 0.01). These findings demonstrate that the combined treatment with vorinostat and DMAG is highly active against human MCL cells, and support the rationale to determine the in vivo efficacy and safety of the combination against human MCL.

Introduction: Histone deacetylases (HDACs) affect cell growth at the transcriptional level by regulating the acetylation status of nucleosomal histones, and HDAC inhibition induces differentiation and/or apoptosis in transformed cells. We have recently shown that HDAC inhibitors, such as suberoylanilide hydroxamic acid (SAHA), induce apoptosis of human multiple myeloma (MM) cells via a constellation of antiproliferative and/or proapoptotic molecular events, including decreased expression of multiple signaling molecules and oncogenes implicated in MM pathophysiology. Based on these promising pre-clinical data, we embarked on a phase I clinical trial of oral SAHA in patients with advanced MM. Methods: An open-label phase I dose-escalation of oral SAHA (200, 250 and 300 mg po bid for 5 consecutive days followed by 2 days of rest) was administered in 4-week cycles in pts with relapsed/refractory MM. The primary objective was to determine the maximum tolerated dose (MTD), and secondary objectives included evaluation of tumor response, as well as assessment of markers of biologic activity in peripheral blood mononuclear cells and bone marrow plasma cells. Dose limiting toxicity (DLT) was defined as grade 4 or greater hematologic toxicity and/or grade 3 or greater non-hematologic toxicity within the first 28 days of treatment. Results: To date, 8 pts with advanced MM (5 relapsed and 3 with relapsed, refractory MM) have been enrolled at the first 2 dose levels, receiving a median of 3 cycles (range 2–9) of therapy. In 7 evaluable pts, one pt at the 2nd dose level (250 mg po bid) developed DLT with grade 3 fatigue, prompting dose reduction with the next cycle. Other side effects have included grade 2 fatigue (3 pts), grade 2 diarrhea (2 pts), grade 2 indigestion (2 pts) and grade 2 dehydration (2 pts), which have been manageable with appropriate supportive care. In one patient, during cycle 4 at dose level 2, grade 3 dehydration occurred with associated metabolic abnormalities that readily resolved with electrolyte supplementation and rehydration. The patient has continued on therapy at reduced dose (250 mg po bid, 4 days on, 3 days off) without recurrence of this toxicity. Importantly, no significant myelosuppression, neuropathy or sedation, which are associated with other anti-MM agents, has been seen. In 7 evaluable pts: minor responses (MR) were documented in 2 patients (25–50% reduction in serum paraprotein levels); stable disease (SD: less than 25% reduction in paraprotein levels) was observed in 2 pts; and progressive disease (PD) was documented in 3 pts. Conclusion: SAHA is an orally administered HDAC inhibitor with manageable toxicity and preliminary evidence of antitumor activity in advanced MM. Clinical evaluation of this agent continues, with enrolment at 250 mg b.i.d. ongoing, to further define the safety and tolerability at this dose level and provide insight into the future uses of SAHA, either alone or in combination with other agents, to treat pts with advanced MM.