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Effective therapies for non–small cell lung cancer (NSCLC) remain challenging despite an increasingly comprehensive understanding of somatically altered oncogenic pathways. It is now clear that therapeutic agents with potential to impact the tumor immune microenvironment potentiate immune-orchestrated therapeutic benefit. Herein, we evaluated the immunoregulatory properties of histone deacetylase (HDAC) and bromodomain inhibitors, two classes of drugs that modulate the epigenome, with a focus on key cell subsets that are engaged in an immune response. By evaluating human peripheral blood and NSCLC tumors, we show that the selective HDAC6 inhibitor ricolinostat promotes phenotypic changes that support enhanced T-cell activation and improved function of antigen-presenting cells. The bromodomain inhibitor JQ1 attenuated CD4+FOXP3+ T regulatory cell suppressive function and synergized with ricolinostat to facilitate immune-mediated tumor growth arrest, leading to prolonged survival of mice with lung adenocarcinomas. Collectively, our findings highlight the immunomodulatory effects of two epigenetic modifiers that, together, promote T cell–mediated antitumor immunity and demonstrate their therapeutic potential for treatment of NSCLC.Significance: Selective inhibition of HDACs and bromodomain proteins modulates tumor-associated immune cells in a manner that favors improved T-cell function and reduced inhibitory cellular mechanisms. These effects facilitated robust antitumor responses in tumor-bearing mice, demonstrating the therapeutic potential of combining these epigenetic modulators for the treatment of NSCLC. Cancer Discov; 7(8); 852–67. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 783

Supplementary Table 1. HDAC inhibitors tested with healthy donor PBMCs and their biochemical potency (nM) across HDACs 1, 2, 3, and 6. Supplementary Table 2. Information for consented Non-small cell lung cancer (NSCLC) patients that underwent surgical resectioning as part of their treatment plan and whose tumor specimen and blood samples obtained after surgery were analyzed. Supplementary Figure 1. Reduced CD4+FOXP3+ Treg cells in healthy donor and NSCLC patient PBMC in the presence of ricolinostat. Supplementary Figure 2. Up-regulation of CD69 on T cells in healthy donor PBMC cultures in the presence of ricolinostat. Supplementary Figure 3. Viability of immune cells within dissociated tumor specimens cultured in the presence of ricolinostat or entinostat. Supplementary Figure 4. Effector function of T cells within 2-D cultures of disaggregated tumor specimens from NSCLC patients. Supplementary Figure 5. Increased expression of MHC class II and CD86 on monocytes in healthy donor PBMC in the presence of ricolinostat. Supplementary Figure 6. Phenotype of T cells infiltrating lung tumors of genetically engineered mice treated with ricolinostat. Supplementary Figure 7. Gene expression profile of Tumor-infiltrating T cells. Supplementary Figure 8. Ricolinostat promotes up-regulation of MHC class II and CD86 on tumor-associated macrophages. Supplementary figure 9. Immunohistochemical and flow cytometric analyses of acetylated α-tubulin in lung tumors of KP mice. Supplementary Figure 10. Kinetics of tumor growth in KP mice treated with ricolinostat. Supplementary Figure 11. Phenotype of tumor-infiltrating T cell subsets in lung tumors of KP mice treated with JQ1. Supplementary Figure 12. Immunohistochemical and flow cytometric analyses of phospho-STAT5 levels in lung tumors of KP mice. Supplementary Figure 13. Suppressive function of Tregs isolated from the spleen of lung tumor-bearing KP mice treated with JQ1. Supplementary Figure 14. Phenotype of Tregs in the spleen or lung tumors of genetically engineered mouse models (GEMM) of NSCLC. Supplementary Figure 15. Phenotype of Tregs within cultures of dissociated tumor specimen from NSCLC patients. Supplementary Figure 17. Histology and immunohistochemical staining of lung tumor sections from treated KP mice. Supplementary Figure 18. Kinetics of tumor growth in TL or wild-type mice. Supplementary Figure 19. Quantification of T cell subsets and TAMs in tumors of KP mice. Supplementary Figure 20. Phenotype of CD8+T cells infiltrating lung tumors of treated KP mice. Supplementary Figure 21. Gene expression profile of tumor-infiltrating macrophages (TAMs) in treated KP mice. Supplementary Figure 22. Gene expression profile of tumor-infiltrating macrophages T cells in treated KP mice. Supplementary Figure 23. Proportion of CD4+Foxp3+ Tregs present within tumor-infiltrating T cells utilized in gene expression studies. Supplementary Figure 24. Gating strategy. Supplementary Figure 25. Correlation plot for the expression of indicated genes in CD45+ leukocytes in KP tumors as evaluated by single cell RNA-Sequencing.

Interactions between the HDAC6 inhibitor ricolinostat (ACY1215) and the irreversible proteasome inhibitor carfilzomib were examined in non-Hodgkin lymphoma (NHL) models, including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and double-hit lymphoma cells. Marked in vitro synergism was observed in multiple cell types associated with activation of cellular stress pathways (e.g., JNK1/2, ERK1/2, and p38) accompanied by increases in DNA damage (γH2A.X), G2–M arrest, and the pronounced induction of mitochondrial injury and apoptosis. Combination treatment with carfilzomib and ricolinostat increased reactive oxygen species (ROS), whereas the antioxidant TBAP attenuated DNA damage, JNK activation, and cell death. Similar interactions occurred in bortezomib-resistant and double-hit DLBCL, MCL, and primary DLBCL cells, but not in normal CD34+ cells. However, ricolinostat did not potentiate inhibition of chymotryptic activity by carfilzomib. shRNA knockdown of JNK1 (but not MEK1/2), or pharmacologic inhibition of p38, significantly reduced carfilzomib–ricolinostat lethality, indicating a functional contribution of these stress pathways to apoptosis. Combined exposure to carfilzomib and ricolinostat also markedly downregulated the cargo-loading protein HR23B. Moreover, HR23B knockdown significantly increased carfilzomib- and ricolinostat-mediated lethality, suggesting a role for this event in cell death. Finally, combined in vivo treatment with carfilzomib and ricolinostat was well tolerated and significantly suppressed tumor growth and increased survival in an MCL xenograft model. Collectively, these findings indicate that carfilzomib and ricolinostat interact synergistically in NHL cells through multiple stress-related mechanisms, and suggest that this strategy warrants further consideration in NHL. Mol Cancer Ther; 13(12); 2886–97. ©2014 AACR.

Supplementary Figures and Tables. Fig 1: Synergistic interactions between CFZ and ACY1215 lead to induction of apoptosis in a time-dependent manner. Fig 2: Knocking down HDAC6 expression by shRNA or pharmacologic inhibition by ACY1215 or Tubastatin A potentiates proteasome inhibitor lethality. Fig 3: Combined CFZ/ACY1215 exposure activates stress pathways and increases DNA damage in SUDHL16 cell. Fig 4: Pre-treatment of cells with pan-caspase inhibitor BOC does not protect JNK activation, induction of DNA damage and inactivation of ERK. Fig 5: Knocking down HDAC6 expression by shRNA recapitulate signaling events when treated with ACY1215 alone similar to combined treatment of CFZ+ ACY1215 to untransfected cells. Fig 6: CFZ and ACY1215 interact synergistically in bortezomib-resistant DLBCL and MCL cells. Fig 7: Constitutive MEK/ERK phosphorylation does not protect SUDHL4 cells from the ACY1215+CFZ regimen. Fig 8: Knocking down of Histone1.2 circumvent the lethality of ACY1215+CFZ drug regimen in U2932 cells. Sup Table 1: Addition of ACY1215 to CFZ does not enhance inhibition of chymotryptic activity in DLBCL cells. Sup Table 2: Combined ACY1215t/CFZ exposure leads to G2M arrest of DLBCL cells.

Purpose: Histone deacetylase (HDAC) inhibition improves the efficacy of proteasome inhibition for multiple myeloma but adds substantial toxicity. Preclinical models suggest that the observed synergy is due to the role of HDAC6 in mediating resistance to proteasome inhibition via the aggresome/autophagy pathway of protein degradation. Experimental Design: We conducted a phase I/II trial of the HDAC6-selective inhibitor ricolinostat to define the safety, preliminary efficacy, and recommended phase II dose in combination with standard proteasome inhibitor therapy. Patients with relapsed or refractory multiple myeloma received oral ricolinostat on days 1–5 and 8–12 of each 21-day cycle. Results: Single-agent ricolinostat therapy resulted in neither significant toxicity nor clinical responses. Combination therapy with bortezomib and dexamethasone was well-tolerated during dose escalation but led to dose-limiting diarrhea in an expansion cohort at a ricolinostat dose of 160 mg twice daily. Combination therapy at a ricolinostat dose of 160 mg daily in a second expansion cohort was well tolerated, with less severe hematologic, gastrointestinal, and constitutional toxicities compared with published data on nonselective HDAC inhibitors. The overall response rate in combination with daily ricolinostat at ≥160 mg was 37%. The response rate to combination therapy among bortezomib-refractory patients was 14%. Samples taken during therapy showed dose-dependent increases of acetylated tubulin in peripheral blood lymphocytes. Conclusions: At the recommended phase II dose of ricolinostat of 160 mg daily, the combination with bortezomib and dexamethasone is safe, well-tolerated, and active, suggesting that selective inhibition of HDAC6 is a promising approach to multiple myeloma therapy. Clin Cancer Res; 23(13); 3307–15. ©2017 AACR.

Introduction: The Philadelphia (Ph) chromosome was the first reported chromosomal rearrangement linked to a human malignancy. Ph-positive precursor B-cell acute lymphoblastic leukemic (Ph+Pre-B-ALL) expressing the BCR-ABL1 oncoprotein is a major subclass of ALL with poor prognosis and high relapse rates in adults. BCR-ABL1 drives an aberrantly hyperactive DNA repair that confers survival advantages to leukemic cells and serves as its ‘Achilles' heel'. We used HDAC1,2 selective inhibitor in isolation or in combination with doxorubicin (a chemotherapy regimen component used for treating pre-B-ALL) to combat the aberrant DSB repair processes. Experimental design: We used a combination of molecular and cell biology, proteomics and functional genomics-based approaches and primary patient-derived xenograft mouse models to understand the genome stability mechanisms targeted by HDAC1,2 inhibition to overcome oncogenic signals promoted by the BCR-ABL1 fusion protein. Results: Mass-spectrometry analysis and DNA repair assays showed that HDAC1,2 inhibition and doxorubicin treatments impact common as well as distinct DNA repair and genome maintenance networks including decreased chromatin association of Mre11-Rad51-DNA ligase 1, the central hub of DNA repair. Translating this mechanistic knowledge, we then showed that the HDAC1,2 inhibitor either alone or when combined with a low dose doxorubicin caused cytotoxicity in primary patient Ph+ Pre-B-ALL cells, and decreased leukemia burden without adverse side-effects in vivo in refractory Ph+ Pre-B-ALL PDX models that we created. We recently found that HDAC1,2 targets H4K91ac mark in Ph+Pre-B-ALL cells. H4K91ac occurs within the body of nucleosome core and thus directly controls nucleosome or chromatin integrity. Increased H4K91ac following HDAC1,2 inhibition occurs around DSB sites genome-wide could cause a delay in the rate at which DSBs are repaired in Ph+Pre-B-ALL cells as a result of defective nucleosome structure around DSB sites. Conclusion: Our comprehensive study using a multipronged approach showed that HDAC1,2 inhibition as a monotherapy or in combination with a low-dose doxorubicin can override DSB repair ‘addiction' in Ph+ Pre-B-ALL. We are currently using the genome-wide ChIP-seq approach and an homing endonuclease DSB reporter system to examine how HDAC1,2 inhibition changes the chromatin landscape in Ph+Pre-B-ALL cells. Citation Format: Danielle Johnson, Shwetha Tharkar, Simon Jones, Matt Jarpe, Mahesh Chandrasekharan, Srividya Bhaskara. Targeting DNA repair in Philadelphia positive B-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5844.

Effective therapies for non-small cell lung cancer (NSCLC) remain challenging despite an increasingly comprehensive understanding of somatically altered oncogenic pathways. It is now clear that therapeutic agents with potential to impact the tumor immune microenvironment potentiate immune-orchestrated therapeutic benefit. This study evaluated the immunoregulatory properties of two classes of drugs that modulate the epigenome, histone deacetylase (HDAC) and bromodomain inhibitors with a focus on key cell subsets that are engaged in an immune response. By evaluating human peripheral blood and NSCLC tumors, we show that the selective HDAC6 inhibitor ricolinostat promotes phenotypic changes associated with enhanced T-cell priming and function of antigen presenting cells. The bromodomain inhibitor JQ1 attenuated CD4+Foxp3+ T regulatory cell suppressive function and synergized with ricolinostat to facilitate immune-mediated tumor growth arrest, leading to prolonged survival of mice with lung adenocarcinomas. Collectively, our findings highlight immunomodulatory effects of two epigenetic modifiers that together promote T-cell-mediated anti-tumor immunity and demonstrate their therapeutic potential for NSCLC treatment. Citation Format: Dennis O. Adeegbe, Yan Liu, Patrick Lizotte, Yusuke Kamihara, Mark Awad, David Barbie, Jerome Ritz, Simon Jones, Steven Quayle, Peter Hammerman, Kwok-Kin Wong. Synergistic immunostimulatory effects and therapeutic benefit of combined histone deacetylase and bromodomain inhibition in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3682. doi:10.1158/1538-7445.AM2017-3682

Alteration of the epigenetic landscape of immune cells can modify the pattern of gene expression, thus resulting in phenotypic and functional changes. Small molecule inhibitors targeting epigenetic modifiers, such as histone deacetylases (HDACs), have been shown to reduce tumor growth. Besides promoting direct anti-tumor effects, HDAC inhibitors also target immune cells and alter their gene regulation. Here, we demonstrate that the HDAC6 selective inhibitors ACY-241 and ACY-1215 (ricolinostat) decrease the function of myeloid derived suppressor cells (MDSC) and T regulatory (Treg) cells, maintain an effector phenotype by CD8+ T cells, and do not reduce viability of immune cells. First, peripheral blood mononuclear cells derived from melanoma patients were treated with ACY-241, and the phenotype of MDSCs was assessed. Expression of the suppressive molecules ARG1 (p Citation Format: Andressa L. Sodre, David M. Woods, Amod Sarnaik, Brian C. Betts, Steven Quayle, Simon Jones, Jeffrey Weber. Epigenetic reprograming of immune cells through selective inhibition of HDAC6 reduces suppressive phenotypes and augments anti-tumor properties of T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 638. doi:10.1158/1538-7445.AM2017-638

ACY 241 is a new, orally available and selective histone deacetylase (HDAC) 6 inhibitor in clinical development in combination with pomalidomide and dexamethasone in multiple myeloma. Like the structurally related drug ricolinostat (ACY-1215), ACY-241 has the potential for a substantially reduced side effect profile versus current nonselective HDAC inhibitor candidates due to reduced potency against Class I HDACs while retaining the potential for anticancer effectiveness. One of the major effects of HDAC6 inhibition is hyperacetylation of á-tubulin, which is suggestive of potential combination activity with taxanes in cancer treatment. Paclitaxel is a chemotherapeutic agent approved for use in the treatment of multiple solid tumor types, including breast cancer, ovarian cancer, non-small cell lung cancer and Kaposi's sarcoma (Weaver, 2014). Recent studies indicate that clinically relevant low concentrations of paclitaxel caused multipolar spindle formation resulting in aberrant mitosis and cell death, which further contributes to the anti-cancer efficacy of paclitaxel (Zasadil et al., 2014). In cell lines, combination treatment with ACY-241 and paclitaxel resulted in enhanced inhibition of proliferation and increased cell death. The combination of ACY-241 plus paclitaxel also demonstrated enhanced efficacy in xenograft models of pancreatic and ovarian cancer. Cell-cycle analysis indicated that ACY-241 treatment in combination with paclitaxel caused synergistic inhibition of tumor cell proliferation through enhanced reduction of DNA replication in S-phase and increased induction of cell death and aneuploidy. ACY-241 treatment in combination with paclitaxel did not increase mitotic arrest but did increase the frequency of multipolar spindle formation during mitosis, leading to aberrant mitoses, abnormal nuclei, and cell death in agreement with a previous report (Zasadil et al). At the molecular level, the enhanced anti-cancer efficacy resulting from combination treatment with ACY-241 and paclitaxel in cell lines was associated with further increased hyperacetylation of á-tubulin, suggesting these agents synergistically impact the regulation of tubulin biology. Interestingly,gamma-tubulin was not always detectable at each pole of these multipolar spindles, suggesting that multipolar spindle formation may be independent of centrosome amplification and/or splitting. Additional studies investigating the association between localization of spindle proteins and multipolar spindle formation are in progress. The enhanced efficacy of paclitaxel plus ACY-241 observed here, in addition to the anticipated superior safety profile of a selective HDAC6 inhibitor, provides a strong rationale for clinical development of this combination in patients with advanced solid tumors. Citation Format: Pengyu Huang, Ingrid Almeciga-Pinto, Min Yang, Simon S. Jones, Steven N. Quayle. Combination efficacy of the selective HDAC inhibitor ACY-241 and paclitaxel in solid tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4822.

INTRODUCTION: HDACs are a family of enzymes that remove acetyl groups from proteins and are involved in key cellular processes. Nonselective HDAC inhibitors have shown promise in treating MM in combination with standard therapies such as proteasome inhibitors (PIs) and immunomodulatory agents, but are limited by adverse effects. The selectivity profile of clinical HDAC inhibitors is critically important as the same drug may exert its effects via alternative mechanisms depending on the partner therapeutic. An example is HDAC6 inhibitors in combination with bortezomib, a proteasome inhibitor (PI). Delivery of protein-bearing vesicles to the aggresome involves HDAC6 through its role in vesicle trafficking, thus supporting a secondary survival pathway. In the case of lenalidomide or pomalidomide, inhibiting class I HDACs can suppress MYC protein levels, which potentially enhances activity in a clinical setting. Ricolinostat (ACY-1215) and ACY-241 that are currently in clinical trials have a selectivity profile tuned toward HDAC6 over HDACs 1, 2, and 3, which is hypothesized to enhance anti-cancer activity with multiple standard of care regimes while minimizing toxicity. METHODS: Peripheral blood from patients that received escalating oral doses of ricolinostat or ACY-241 that range from 40 mg to 360 mg was collected for matched PK and PD assessment. Lymphocytes were examined for tubulin and histone hyper-acetylation by flow cytometry. RESULTS & CONCLUSION: Plasma concentration for both ricolinostat and ACY-241 reach a maximal level (Cmax) 1-2 hr after administration. An exposure plateau was reached at dose levels ≥160 mg of ricolinostat, with Cmax observed to be ∼2 μM. ACY-241 exposures reached higher levels, Cmax of ∼3 μM and ∼7 μM at 180 and 360 mg, respectively, and no evidence of an exposure plateau. Correlating to the PK levels, acetyl-tubulin increases to a maximal level by 1-2 h after dosing compared to predose, and declines to basal level by >4 hr. As plasma levels increased with dose from 40 mg to 160 mg for ricolinostat so do the number of patients that have an increase in acetyl-tubulin such that at dose levels ≥80 mg all patients have a measurable increase in acetylated tubulin. Levels of acetyl-tubulin were similar at dose levels of 180 and 360 mg of ACY-241. Acetyl-histone levels were low- moderate at the 160 and 180 mg dose level for ricolinosat and ACY-241, respectively, and for ACY-241 increased with increasing exposure at doses > 180 mg similar to results observed in vitro by immunoblot analysis of MM cells treated with ACY-241. In conclusion, the PK and PD results suggest that both ricolinostat and ACY-241 have reached a pharmacologically relevant level of HDAC inhibition as determined by acetylated tubulin and histone at the clinical dose levels examined. Citation Format: David L. Tamang, Jeffery Supko, Kailash Bhol, Catherine Wheeler, Simon S. Jones, Min Yang. Tubulin hyper-acetylation in blood lymphocytes: pharmacodynamic (PD) biomarker for the selective histone deacetylase (HDAC) 6 inhibitors ricolinostat and ACY-241 in multiple myeloma (MM) patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3958.

INTRODUCTION: Neuroblastoma (NB) is an extra-cranial solid cancer and is among the most common cancers in infants less than 1 year of age, with 650 new cases each year in the United States. Half of the children with NB have high risk disease and 20-50% of those will fail to respond adequately to current therapies, illustrating an urgent unmet medical need. Current treatment for high-risk disease is aggressive, including chemotherapy, surgery, radiation with stem cell transplant, anti-GD2/cytokine immunotherapy and retinoic acid (RA) treatment. RA is a pro-differentiation agent that slows growth and promotes cell death. A gene expression pattern associated with RA-induced NB differentiation was identified, and chemical inhibition of HDAC1/2 was shown to induce a similar expression pattern. METHODS: In this work, we examine the activity of an orally bioavailable HDAC1/2 inhibitor (HDAC1/2i) on NB cell differentiation, proliferation and apoptosis. RA combined with HDAC1/2i enhances gene expression patterns associated with differentiation, slows cellular proliferation and more rapidly induces dendrite formation than RA can achieve alone. The mechanisms leading to the differentiated phenotype were examined by RT-PCR, gene expression microarray and retinoic acid receptor (RAR) chromatin immunoprecipitation followed by high-througput sequening (ChIP-Seq). RESULTS: HDAC1/2i and RA together caused increased localization of the RAR to its own RARα and RARβ promoter regions, and increased in RAR mRNA and protein relative to the RA treatment condition alone. Additionally, expression of Cyp26, an enzyme responsible for clearing intercellular RA, was reduced in the combination setting. Gene set enrichment analysis of the microarray data comparing the combination setting against RA as a single agent suggested that the addition of HDAC1/2i enhanced apoptotic pathways and decreased E2F driven cell cycle signaling. We confirmed enhanced apoptosis in the combination setting by measuring caspase 3 and PARP cleavage. Consistent with this finding, we observed reduced proliferation, increased sub-G1 cell frequency in cell cycle assays and ablation of emergent RA-resistant NB colonies after combination treatment. Further, combination treatment reduced the E2F-activators CDK4 and CDK6 at the protein level while the CDK inhibitor, p21, was dramatically increased. Hypo-phosphorylation of retinoblastoma protein, directly linked to E2F complex inactivation, was also observed and consistent with reduced proliferation and the decreased frequency of S-phase cells observed in EDU incorporation assays. Xenograft models of NB with RA and HDAC1/2i are in progress, as are HDAC1, HDAC2, acetylated histone H3K9 ChIP-seq experiments, and will be discussed. Taken together, these findings support a role for selective HDAC1/2i in combination with RA for the treatment of patients with high risk NB. Citation Format: David L. Tamang, Pengyu Huang, Olga Golonzhka, Jeffrey R. Shearstone, Steven N. Quayle, Simon S. Jones, Min Yang. An orally bioavailable and selective histone deacetylase (HDAC) 1 & 2 inhibitor enhances retinoic acid mediated differentiation of neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2477.

Class I family enzymes, histone deacetylases 1 and 2 (HDAC1,2), are targets of FDA-approved small molecule inhibitors currently used in cancer therapy. HDAC inhibitors impair DNA repair and replication in cancer cells to trigger genotoxic stress and cause cell death. How HDAC1,2 function in genome maintenance processes, DNA replication and repair, in mammalian cells is far from fully understood. Using small molecules to selectively inhibit HDAC1,2 activities, we have systematically defined their direct functions in DNA replication and repair. Our studies show that HDAC1,2 inhibition reduces replication fork velocity and activates the replication stress associated DNA damage independent of its effect on transcription. HDAC1,2 inhibition increases histone H4K16 acetylation (H4K16ac) and nuclease sensitivity of nucleosomes associated with newly replicated DNA, suggesting that HDAC1,2 are required for chromatin compaction during DNA replication. Thus, mechanistically, HDAC1,2 activities are required for maintaining nascent chromatin structure and dynamics required for efficient progression of the replication fork. In addition to H4K16ac, we have found that HDAC1,2 inhibition activities increases H3K27ac and H4K91ac present on the histone tail and core regions, respectively. Increase in these histone acetyl marks perturbs the acetyl-methyl or the acetyl-ubiquityl switch during DNA repair leading to impaired EZH2-mediated H3K27me3-dependent DNA repair signaling and the H4K91ubiquityl-dependent 53BP1-mediated DNA repair signaling in chemoresistant EZH2 gain-of-function diffuse large B-cell lymphoma cells. We are currently investigating the therapeutic benefits of HDAC1,2 inhibition in mouse models of aggressive chemoresistant diffuse large B-cell lymphoma and in primary patient samples. Overall, our studies have uncovered novel functions for HDAC1,2 in genome maintenance processes and provide insight into the mode-of-action of HDAC1,2 inhibitors in overcoming chemoresistance in cancer cells (via targeting the DNA repair process) and in inducing DNA damage and death in the rapidly cycling cancer cells (via targeting DNA replication). Note: This abstract was not presented at the conference. Citation Format: Danielle Johnson, Simon Jones, Steven Quayle, Jeff Shearstone, Srividya Bhaskara. Inhibition of histone deacetylases 1 and 2 (HDAC1,2) perturbs DNA replication and DNA repair in cancer cells: Implications in mechanism-based therapeutic strategies. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B29.

Neuroblastoma (NB) is an extra-cranial solid cancer arising from the neural crest and is among the most common cancers in infants less than 1 year of age (Park, JR et al., 2008). Approximately one child per 100,000 is diagnosed with NB, resulting in 650 new cases each year in the United States. Half of the children with NB have high risk disease and 20-50% of those will fail to respond adequately to current therapies, illustrating a clear unmet medical need. Current treatment for high-risk disease is aggressive, including chemotherapy, surgery, radiation with stem cell transplant, anti-GD2/cytokine immunotherapy and retinoic acid (RA) (Yang, RK et. al., 2010; Cheung, NK et. al., 2012). RA is a pro-differentiation agent that slows growth and promotes cell death. A gene expression pattern associated with RA-induced NB differentiation was identified (Hahn, CK et. al., 2008; Frumm, SM et. al., 2013), and inhibition of HDAC1/2 was shown to induce a similar expression pattern. In this work, we examine the activity of an orally bioavailable HDAC1/2 inhibitor (HDAC1/2i) on NB cell differentiation, proliferation and apoptosis. RA combined with HDAC1/2i enhances gene expression patterns associated with differentiation, slows cellular proliferation and more rapidly induces dendrite formation than RA can achieve alone. The mechanisms leading to the differentiated phenotype were examined by microarray and retinoic acid receptor (RAR) ChIP-seq. HDAC1/2i and RA together caused increased localization of the RAR to its own RARa and RARß promoter regions, and an increase in mRNA and protein relative to the RA treatment condition alone. Additionally, expression of Cyp26a1/b1, enzymes responsible for clearing intercellular RA, were reduced in the combination setting. Gene set enrichment analysis of the microarray data comparing the combination setting against RA as a single agent suggested that the addition of HDAC1/2i was enhancing apoptotic pathways and decreasing E2F driven cell cycle signaling. In further experiments, we confirmed enhanced apoptosis in the combination setting by measuring caspase 3 and PARP cleavage, which is consistent with reduced proliferation, increased sub-G1 cell frequency in cell cycle assays and ablation of emergent RA-resistant NB colonies. Further, the E2F-activators, CDK4 and CDK6, were reduced at the protein level in the combination setting while the CDK inhibitor, p21, was dramatically increased. Hypo-phosphorylation of retinoblastoma protein, directly linked to E2F complex inactivation, was also observed and consistent with reduced proliferation and the decreased frequency of S-phase cells observed in EDU incorporation assays. Xenograft models of NB with RA and HDAC1/2i are in progress and will be discussed. Taken together, these findings support a role for selective HDAC1/2i in combination with RA for the treatment of patients with high risk NB. Citation Format: David L. Tamang, Pengyu Haung, Olga Golonzhka, Jeff Shearstone, Steven N. Quayle, Simon S. Jones, Min Yang. A novel, orally bioavailable and selective histone deacetylase (HDAC) 1 & 2 inhibitor enhances retinoic acid mediated differentiation of neuroblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A136.

AML is a heterogeneous group of hematopoietic stem cell disorders characterized by defects in myeloid differentiation and increased proliferation of neoplastic hematopoietic precursor cells. There is a great need for novel approaches to treat AML due to limited improvements in the treatment of patients over the past several decades. Aberrant epigenetic regulation plays an important role in the pathogenesis of AML, and the DNA methyltransferase inhibitor azacitidine is approved for the treatment of myelodysplastic (MDS), which frequently progresses to AML, and is commonly used in AML patients. Numerous clinical studies are ongoing to investigate the benefit of combining azacitidine with other investigational agents in AML. HDAC inhibitors are promising agents for the treatment of AML. Selective HDAC inhibitors are predicted to reduce combination drug toxicity and other side effects observed with pan-HDAC inhibitors, while still enabling therapeutic benefits. In this work, we demonstrate that potent and selective inhibitors of HDAC1/2 are highly efficacious both as single agents and in combination with azacitidine in preclinical models of AML, including established AML cell lines and bone marrow samples freshly derived from AML patients. Combination treatment in vivo demonstrated good tolerability, and ongoing efficacy studies in AML xenograft models will be discussed. At the molecular level, gene expression profiling and DNA methylation mapping were performed on MV4-11 cells treated with an HDAC1/2 inhibitor, azacitidine, or the combination. Molecular signatures were analyzed by GSEA. Significantly more genes and signatures were upregulated than downregulated by the single agent and combination treatments, consistent with the transcription activation mechanism of these compounds. Signatures and genes involved in myeloid cell differentiation, apoptosis and cell cycle arrest were identified as potential mediators of the combinatorial effects of HDAC1/2 inhibition with azacitidine. Together, these findings support the clinical evaluation of selective HDAC1/2 inhibitors in combination with azacitidine in AML and potentially MDS patients. Citation Format: Chengyin Min, Steven N. Quayle, Pengyu Huang, Jeffrey R. Shearstone, Simon S. Jones, Min Yang. Novel and selective inhibitors of histone deacetylases (HDAC) 1 and 2 significantly enhance the activity of the DNA methyltransferase inhibitor azacitidine in acute myeloid leukemia (AML). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B84.

Combinations of HDAC inhibitors with chemotherapeutic agents have demonstrated evidence of clinical benefit, for example in ovarian cancer combining belinostat with carboplatin and paclitaxel (Dizon, Int J Gynecol Cancer, 2012), and disease stabilization with vorinostat and tamoxifen in hormone receptor therapy-refractory breast cancer patients (Munster, Br J Cancer, 2011). However, the toxicity profile of pan-HDAC inhibitors frequently limits their use in combination with other drugs commonly used in hematologic and solid tumors. The search for combination therapies that are better tolerated has motivated the development of more selective HDAC inhibitors with improved safety profiles. ACY-241 is a new, orally available and selective histone deacetylase (HDAC) 6 inhibitor under clinical development in combination with pomalidomide and dexamethasone in multiple myeloma (NCT02400242). ACY-241 has the potential for a substantially reduced side effect profile versus current nonselective HDAC inhibitor drug candidates due to reduced potency against Class I HDACs while retaining the potential for anticancer effectiveness. In this study the potential activity of ACY-241 in combination with paclitaxel was explored in preclinical models of solid tumors. In cell lines generated from multiple solid tumor lineages, combination treatment with ACY-241 and paclitaxel resulted in enhanced inhibition of proliferation and increased cell death relative to either single agent alone. The combination of ACY-241 plus paclitaxel also demonstrated enhanced efficacy in xenograft models of pancreatic and ovarian cancer relative to either single agent. Combination dosing was well tolerated as treated animals continued to gain weight throughout the dosing period, and similar results were obtained when ricolinostat, an alternative HDAC6 inhibitor, was tested in combination with paclitaxel. Beyond cell cycle arrest and apoptosis, treatment with paclitaxel was recently shown to induce multipolar spindle formation during mitosis, subsequently leading to aberrant cell division and death (Zasadil, Sci Transl Med, 2014). While ACY-241 treatment alone had minimal effect on mitotic spindle formation, combination treatment with ACY-241 and paclitaxel resulted in more frequent occurrence of mitotic cells with multipolar spindles. Thus, in addition to potentially blocking entry into S phase, combination treatment also leads to increased frequency of aberrant mitoses. At the molecular level, treatment with paclitaxel results in enhanced stability of microtubules, which in turn leads to increased acetylation of α-tubulin. HDAC6 directly regulates acetylation of α-tubulin, and ACY-241 treatment results in a dose-dependent increase in α-tubulin hyperacetylation. The enhanced anti-cancer efficacy resulting from combination treatment with ACY-241 and paclitaxel in cell lines was associated with further increased hyperacetylation of α-tubulin, suggesting these agents synergistically impact the regulation of tubulin biology. The enhanced efficacy of ACY-241 plus paclitaxel observed here, in addition to the anticipated superior safety profile of a selective HDAC6 inhibitor, provides a strong rationale for clinical development of this combination in patients with advanced solid tumors. Citation Format: Pengyu Huang, Ingrid Almeciga-Pinto, Morgan Jordan, Min Yang, Simon S. Jones, Steven N. Quayle. Selective HDAC inhibition by ACY-241 enhances the activity of paclitaxel in solid tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A187.

Neuroblastoma is an extra-cranial solid cancer arising from the neural crest and is among the most common cancers in infants less than 1 year of age (Park, JR et al., 2008). Approximately one child per 100,000 is diagnosed with neuroblastoma, resulting in 650 new cases each year in the United States. Half of the children with neuroblastoma have high risk disease and 20% - 50% of those children will fail to respond adequately to current therapies, illustrating a clear unmet medical need. Current treatment for high-risk disease is aggressive, including chemotherapy, surgery, radiation with stem cell transplant, anti-GD2/cytokine immunotherapy and retinoic acid (Yang, RK et. al., 2010; Cheung, NK et. al., 2012). Retinoic acid is a pro-differentiation agent that acts on neuroblastoma cells to slow growth and promote cell death. A gene expression pattern associated with retinoic acid induced neuroblastoma differentiation was recently identified (Hahn, CK et. al., 2008; Frumm, SM et. al., 2013), and it was further shown that inhibition of HDAC1/2 was able to induce a similar expression pattern. In this work, we demonstrate that next generation selective and orally bioavailable HDAC1/2 inhibitors can induce gene expression changes in neuroblastoma cells consistent with differentiation. The action of HDAC1/2 inhibitors potently enhances the retinoic acid differentiation effect at sub-optimal concentrations of retinoic acid or HDAC inhibitor, as well as with intermittent (pulse) HDAC1/2 inhibition. Retinoic acid alone and in combination with HDAC1/2 inhibitors is able to slow cell proliferation in long term growth assays and alter morphology in a manner consistent with differentiation. The observed enhancement of differentiation by selective HDAC1/2 inhibitors occurs at concentrations below that required for cell death as evidenced by viability assays and caspase 3/7 activation. Acute toxicity is induced by elevated concentrations of HDAC1/2 inhibitors, and synergy is observed in combination with retinoic acid. Ongoing studies exploring global gene expression changes, ChIP-seq examining retinoic acid receptor and HDAC1/2 chromatin binding, and activity of the selective HDAC1/2 inhibitor in combination with retinoic acid in animal models of neuroblastoma will be discussed. Taken together, these findings support a role for selective HDAC1/2 inhibitors in combination with retinoic acid for the treatment of patients with high risk neuroblastoma. Citation Format: David L. Tamang, Emily Lurier, Olga Golonzhka, Steven N. Quayle, Simon S. Jones, Min Yang. Novel and selective histone deacetylase (HDAC) 1 & 2 inhibitors enhance differentiation of neuroblastoma cells in combination with retinoic acid. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2196. doi:10.1158/1538-7445.AM2015-2196

AML is a heterogeneous group of hematopoietic stem cell disorders characterized by defects in myeloid differentiation and increased proliferation of neoplastic hematopoietic precursor cells. There is a great need for novel approaches to treat AML due to limited improvements in the treatment of patients over the past several decades since the development of cytarabine and daunorubicin combination therapy. Aberrant epigenetic regulation plays an important role in the pathogenesis of AML, and the DNA methyltransferase inhibitor azacitidine is approved for the treatment of myelodysplastic (MDS) which frequently progresses to AML. Numerous clinical studies are ongoing to investigate the benefit of combining azacitidine with other investigational agents in AML. HDAC inhibitors are emerging as promising agents for the treatment of AML. Selective HDAC inhibitors are predicted to reduce the combination drug toxicity and other side effects observed with non-selective HDAC inhibitors, while also realizing beneficial therapeutic effects. One example is ricolinostat (ACY-1215), a first-in-class orally available HDAC inhibitor that is 11-fold selective for HDAC6, synergizes with proteasome inhibitors (Santo, et al, Blood, 2012) and immunomodulatory agents (Quayle, et al, ASH, 2013) in preclinical models of B-cell malignancies, which has thus far demonstrated an improved safety and tolerability profile in Phase I trials in multiple myeloma (Raje, Haematological, 2014, Suppl 1). In this work, we evaluated the efficacy of azacitidine in combination with selective inhibitors of either HDAC1/2, HDAC3 or HDAC6 on AML cells. Comparison of the panel of selective HDAC inhibitors demonstrated that HDAC1/2 inhibition is sufficient to reduce cell viability, induce differentiation, arrest cell cycle progression and initiate apoptosis in multiple AML cell lines. Selective inhibition of HDAC3 induced differentiation yet had no effect on cell cycle progression or apoptosis, while selective inhibition of HDAC6 had no effect on differentiation, cell cycle arrest or apoptosis in these cells. Furthermore, combining HDAC1/2 inhibition with azacitidine led to significantly enhanced induction of differentiation and apoptosis, and decreases in AML cell viability compared to either agent alone. Ongoing studies exploring the molecular mechanism underlying the activity of selective HDAC1/2 inhibitors, as well as the activity of combination treatment with azacitidine in primary AML cells and animal models of AML, will be discussed. Together, these findings provide support for the clinical evaluation of selective HDAC1/2 inhibitors in combination with azacitidine in AML and potentially MDS patients. Citation Format: Chengyin Min, Steven N. Quayle, David Tamang, Simon S. Jones, Min Yang. Novel and selective inhibitors of histone deacetylases (HDAC) 1 and 2 significantly enhance the activity of the DNA methyltransferase inhibitor azacitidine in acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5372. doi:10.1158/1538-7445.AM2015-5372

Histone deacetylase (HDAC) enzymes are attractive therapeutic targets in oncology, but non-selective HDAC inhibitors have led to dose-limiting toxicities in patients, particularly in combination with other therapeutic agents. Ricolinostat (ACY-1215), a first-in-class orally available HDAC inhibitor that is 11-fold selective for HDAC6, synergizes in vitro and in vivo with bortezomib in models of MM (Santo, Blood, 2012) and with carfilzomib in models of lymphoma (Dasmahapatra, Mol Cancer Ther, 2014). Furthermore, ricolinostat has demonstrated an excellent safety and tolerability profile in Phase I trials (Raje, Haematologica, 2014, Suppl 1). In addition to ongoing clinical studies with ricolinostat, we have identified ACY-241 as a next generation, orally available, selective inhibitor of HDAC6 for clinical evaluation. The IMiD® immunomodulatory class of drugs, including lenalidomide (len) and pomalidomide (pom), exhibit striking anti-myeloma properties in a variety of MM models, and have demonstrated significant clinical activity in MM patients. Beyond MM, len is also approved for the treatment of MCL patients. In support of our ongoing clinical development programs for ricolinostat and ACY-241, we show here that combinations with either len or pom leads to synergistic decreases in the viability of MM and MCL cells in vitro. Time course studies demonstrated accumulation of cell cycle arrest in cells after prolonged exposure to len or pom, as well as progressive induction of apoptosis in these cells. Notably, though, the addition of ricolinostat or ACY-241 to either len or pom resulted in synergistic increases in the percentage of MM and MCL cells undergoing apoptosis. At the molecular level, treatment with IMiD® immunomodulatory compounds reduced expression of the critical transcription factors MYC and IRF4, which were reduced even further by treatment with ricolinostat or ACY-241 in combination with either len or pom. The molecular mechanism underlying this effect is currently being explored, though retention of low level inhibition of HDAC1, 2, and 3 by these compounds may contribute to the enhanced effects on gene expression reported here in combination with len or pom. Finally, prolonged treatment with ACY-241 plus pomalidomide was well tolerated in vivo with no overt evidence of toxicity, and combination treatment resulted in a significant extension of survival in a xenograft model of MM. Given the comparable tolerability profiles of ricolinostat and ACY-241, and their preclinical activity in combination with IMiD® immunomodulatory drugs, these findings support clinical evaluation of these agents in MM and MCL patients. Citation Format: Steven N. Quayle, Ingrid Almeciga-Pinto, David Tamang, Min Yang, Simon S. Jones. Selective HDAC inhibition by ricolinostat (ACY-1215) or ACY-241 synergizes with IMiD® immunomodulatory drugs in Multiple Myeloma (MM) and Mantle Cell Lymphoma (MCL) cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5380. doi:10.1158/1538-7445.AM2015-5380

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Histone deacetylase (HDAC) enzymes represent attractive therapeutic targets in MM, but non-selective HDAC inhibitors have led to dose-limiting toxicities in patients, particularly in combination with other therapeutic agents. ACY-1215 is a first-in-class, orally available HDAC inhibitor that is 11-fold selective for HDAC6, and synergizes in vitro and in vivo with bortezomib in preclinical models of MM without inducing unfavorable toxicities (Blood, 20[210]: 4061). In support of its potential tolerability benefits, ongoing Phase Ib clinical trials with ACY-1215 have thus far demonstrated an excellent safety and tolerability profile (Yee, et al, ASH, 2013). The IMiD class of drugs, including lenalidomide and pomalidomide, exhibit striking anti-myeloma properties in a variety of MM models, and have demonstrated significant clinical activity in MM patients. Prior clinical studies have demonstrated activity of the non-selective HDAC inhibitor vorinostat in combination with lenalidomide and dexamethasone in MM patients (Richter, et al, ASH, 2011). However, many patients experienced considerable toxicities with this regimen that significantly limited clinical utility. In support of our ongoing clinical development program for ACY-1215 in MM, we show here that combining ACY-1215 with either IMiD leads to synergistic decreases in the viability of MM cells in vitro. The relevance of inhibition of HDAC6 to this effect was validated by demonstrating synergistic interactions of either IMiD molecule with ACY-775, which is more than 300-fold selective for HDAC6 over class I HDAC's. Time course studies demonstrated accumulation of cell cycle arrest in cells after prolonged exposure to either IMiD, as well as progressive induction of apoptosis in these cells. Notably, though, the addition of ACY-1215 to either IMiD resulted in synergistic increases in the percentage of MM cells undergoing apoptosis. At the molecular level, MM cells are known to be dependent on expression of the MYC and IRF4 transcription factors. Both ACY-1215 and IMiDs as single agents reduced expression of the critical genes MYC and IRF4, which were reduced even further by combination treatment. The molecular mechanism underlying this effect is currently being explored. Finally, the combination of ACY-1215, lenalidomide, and dexamethasone was well tolerated in vivo with no overt evidence of toxicity, and combination efficacy studies with this combination are now being explored in models of MM. By demonstrating that a selective inhibitor of HDAC6 synergizes with IMiD's while maintaining an improved safety profile, these results provided a rational basis for the clinical development of the orally available combination of ACY-1215 and lenalidomide plus dexamethasone in the ongoing Phase Ib clinical trial ([NCT01583283][1]) for the treatment of MM. Citation Format: Steven N. Quayle, David Tamang, Min Yang, Simon S. Jones. ACY-1215, a selective inhibitor of HDAC6, synergizes with immunomodulatory drugs (IMiDs) to induce apoptosis of multiple myeloma (MM) cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5456. doi:10.1158/1538-7445.AM2014-5456 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01583283&atom=%2Fcanres%2F74%2F19_Supplement%2F5456.atom

Histone deacetylase inhibitors (HDACi) are being evaluated as novel chemotherapeutics in combination with approved and investigational drugs for multiple myeloma (MM) therapy. Despite the rapid advancement in the use of HDACi in MM therapy, it is still not clear which HDAC needs to be inhibited to attain maximal MM cell death. HDAC6, a mediator of the proteasome pathway through its ability to deacetylate tubulin, is considered to be a critical target to promote MM cell apoptosis. However the contribution of Class I HDACs is still unclear. To determine the HDAC that needs to be inhibited in MM, cell death induced by pan-HDACi (LBH589) was compared to Class I HDACi (FK228), selective Class I and HDAC6 inhibitors (ACY-1215, ACY-738) and a specific HDAC6 inhibitor (ACY-738) utilising human myeloma cell lines (HMCL). The specificity of these HDACi was determined by characterizing the acetylation induced in histones (H2A, H2B, H3 and H4) and tubulin through flow cytometry. The histone acetylation pattern of LBH589 and FK228 were similar, ACY-1215 and ACY-738 have comparatively lesser but discernible histone acetylation, while ACY-775 did not acetylate histones. ACY-1215, ACY-738 and ACY-775 acetylate tubulin at amounts significantly higher than LBH589, while FK228 did not acetylate tubulin. To determine if there was a correlation between the pattern of acetylation and MM cell death, eight HMCLs were treated with each HDACi and proportion of cell death measured through flow cytometric enumeration of propidium iodide staining. LBH589 (50 nM) or FK228 (50 nM) induced the same amount of cell death irrespective of the HMCL used whereas ACY-1215 and ACY-738 were able to induce cell death comparable to the LBH589 and FK228 only at the highest concentration used (10 μM). Conversely, ACY-775, which induces negligible histone acetylation, was not able to induce cell death comparable to the other inhibitors except against RPMI-8226 where ACY-775 (10 μM) alone was able to cause a significant amount of cell death. Assessment of apoptosis following exposure of primary MM cells (n=8) to HDACi recapitulated the findings with the HMCL wherein ACY-775 caused the least amount of cell death. ACY-1215 and ACY-738 at higher concentrations (10 μM) were comparable to LBH589 and FK228 in all samples tested and as in the case with the HMCLs, a minority of patients (2/8) showed some cell death with ACY-775 treatment alone. These data suggest that activity against Class I HDACs is more effective than HDAC6 alone in inducing MM cell death. Specific HDAC6 inhibition does induce comparable cell death of some MM cells suggesting that in a subset of patients, inhibiting HDAC6 alone may be as efficient as Class I HDAC inhibition and identifying these patients is important. However, it is clear that in several instances histone acetylation mediated by inhibition of Class I HDAC is sufficient to induce significant MM cell death. Citation Format: Sridurga Mithraprabhu, Tiffany Khong, Simon S. Jones, Andrew Spencer. Histone acetylation mediated by inhibition of Class I histone deacetylases is critical for induction of cell death in multiple myeloma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1007. doi:10.1158/1538-7445.AM2013-1007