ALBERT

Description: Abstract 3937 CHOP and CHOP-like chemotherapy programs remain the most commonly used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite often sub-optimal results. Histone deacetylase inhibitors (HDACIs) are epigenetic agents known to be active in T-cell lymphoma. Recently romidepsin (R) was approved for patients with relapsed or refractory CTCL. Both R and belinostat (B) are being investigated in patients with relapsed or refractory PTCL. We have previously shown that hypomethylating agents as decitabine (D) produce synergistic interactions with HDACIs in B-cell lymphomas. We investigated the in vitro and in vivo activity of D, R and B alone or in combination in different T-cell lymphoma and leukemia cell lines including CTCL (H9, HH), and T- acute lymphoblastic leukemia (T-ALL) lines resistant to gamma-secretase inhibitors (GSI) (P12, PF-382). For all cytotoxicity assays, luminescent cell viability was performed using CellTiter-Glo™ followed by acquisition on a Biotek Synergy HT. The IC50s for D, B and R were calculated using the Calcusyn software (Biosoft). Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR) based on the GraphPad software (RRR 20 uM. In the cytotoxicity assays, the combination of D and B or R at 72 hours showed synergism in all the cell lines studied. The most representative RRRs are showed in table 1. Table 1 D 0.5 uM 1uM B (nM) RRR H9 50 0.7 0.7 70 0.6 0.6 100 0.4 0.5 PF 382 150 0.8 0.7 0.5 uM 1 uM R (nM) RRR H9 0.5 0.9 0.9 1 0.8 0.8 2 0.3 0.3 PF 382 1 0.8 0.7 1.5 0.4 0.4 2 0.1 0.1 When H9, HH, P12 and PF382 cell lines were treated with D and B or R for 72 hours, all the combination groups showed significantly more apoptosis than the single drug exposures and controls. Table 2 displays the range of apoptosis induction for B, R and D or for them used in combination and the RRR value after the analysis for the most significant data. Table 2 B D B + D RRR (% Apoptotic + Dead Cells) H9 100nM (22.9%) 500nM (17.9%) 51.5% 0.7 HH 100nM (42.9%) 1uM (46.9%) 61.3% 0.8 P 12 150nM (16%) 1uM (42.7%) 80.1% 0.4 PF 382 100nM (8.3%) 1uM (27.9%) 40.1% 0.8 R D R + D H9 2nM (22.2%) 500nM (17.9%) 63.6% 0.5 HH 2nM (80%) 1uM (46.9%) 89.7% 0.6 P 12 2nM (9.9%) 10uM (58.7%) 98% 0.03 PF 382 2nM (54.5%) 500nM (17.9%) 88.7% 0.2 Increased acetylation of H3 was observed when H9 cells were treated with R alone and synergistically increased after exposing cells to the combination of D + B and D + R. The expression of phosphorylated Stat3 was decreased after exposure of H9 cells to the combination of D and R. Additional interrogation of the effects of this epigenetic therapy on the JAK-STAT signaling pathway are now underway. An in vivo xenograft study in six to eight weeks old female SCID beige mice injected subcutaneously with 2 × 107 HH cells has also begun and will be reported. Mice were separated into different cohorts and treated with intraperitoneal injections of D or B or their combination according to the following schedules: D alone at 1.5 mg/kg on days 1, 5; B alone at 35 mg/Kg/day for 7 days. Collectively, the data suggest that the combination of a hypomethylating agent like D and a HDACI (B and R) are synergistic in in vitro models of human T-cell lymphoma, and may lead to a new platform for the treatment of these diseases. Disclosures: O'Connor: Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Abstract 2727 CHOP and CHOP-like chemotherapy remain the most commonly used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite sub-optimal results. Histone deacetylase inhibitors (HDACIs) are presently approved for the treatment of relapsed or refractory cutaneous T- cell lymphomas (CTCL) and peripheral T-cell lymphomas (PTCL) given their marked single agent activity in these diseases. The interaction between the HDACIs (depsipeptide (R) and belinostat (B)) and a DNMT inhibitor (decitabine (D)) was investigated in vitro, in vivo and at the molecular level in different T-cell lymphoma and leukemia cell lines including CTCL (H9, HH), and T- acute lymphoblastic leukemia (T-ALL) lines resistant to gamma-secretase inhibitors (P12, PF-382). For all cytotoxicity assays, a luminescence based cell viability assay was used (CellTiter-Glo™) followed by acquisition on a Biotek Synergy HT. Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR

Description: Key Points Treatment of DLBCL with the combination of sirtuin and DAC inhibitors leads to synergistic cytotoxicity and acetylation of Bcl6 and p53. The overall response rate of relapsed lymphoma patients treated with vorinostat and niacinamide was 24%, and an additional 57% achieved stable disease.

Description: Abstract 435 Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoid malignancy, representing approximately 30–40% of all lymphomas. While significant progress has been made in treating this disease over the past decade, it is still regarded as a heterogeneous disease which, after being classified as relapsed or refractory, is fatal in about one-third of patients. Histone deacetylase inhibitors (HDACI) are presently approved for the treatment of relapsed or refractory cutaneous T- cell lymphomas (CTCL), and have marked activity in peripheral T-cell lymphomas (PTCL), though their effectiveness in DLBCL is less established. DNA methyltransferases (DNMTs) are known to recruit and cooperate with histone deacetylases to induce gene silencing. Combinations of drugs affecting these pathways have emerged as active and important, mostly in myeloid leukemias. We hypothesized that the combination of HDACI and DNMT inhibitors (DNMTI) in DLBCL may be active only in combination and not as single agents. We examined the interaction between a broad range of HDACI including vorinostat, depsipeptide, panobinostat and DNMTI using in vivo and in vitro models of DLBCLs, clearly confirming that these agents are in fact synergistic with decitabine. Synergy was measured by relative risk ratio (RRR) and the values obtained were as low as 0.01, representing very strong synergy. This combination of drugs, specifically panobinostat and decitabine, was also shown to be strongly synergistic in a murine xenograft model of DLBCL. In addition, we analyzed the molecular basis for this synergistic effect by evaluating the global gene expression and methylation using microarrays on the cells treated with the single agents and combination in DLBCL. Three DLBCL lines (OCI-Ly1, OCI-Ly10 and Su-DHL6) were treated with decitabine alone (2.5 μ M), panobinostat alone (2.5 nM) or their combination for 48h hours. DNA and RNA from untreated and treated cells were used for genome wide methylation analysis through Illumina Humanmethyation27 platform and gene expression profiling analysis with Illumina HumanHT-12 v3 Expression arrays. 3D principal component analysis clearly clustered the samples treated with panobinostat and combination therapy together and at greater distances from untreated samples and samples treated by decitabine alone. Therefore, the contribution to the gene expression phenotype of the combination was greater from the HDACI than with DNMTI. Consistent with this observation, the top network of genes differentially expressed (p

Description: When considering ASCT for high risk NHL patients with dose-related toxicity concerns, choosing an appropriate conditioning chemotherapy regimen that is both safe and effective presents a challenge. ASCT in elderly population is a feasible and safe procedure, but characterized by higher transplant related mortality compared to younger population. Reports on ASCT in patients with PTLD are rare and limited to case reports without clear understanding of the impact ASCT has on the function of the transplanted organ. Initial outcomes of patients with amyloidosis undergoing ASCT have been marred by significant toxicity, particularly driven by cardiac damage due to amyloid. As such we sought to improve the overall outcomes of our select patients by allowing them to proceed with ASCT, while attempting to reduce toxicity of the conditioning regimen. We used a modified mini-BEAM as conditioning regimen with full dose of melphalan (140mg/m2). This dose was also used due to its efficacy as conditioning regimen in patients with multiple myeloma undergoing ASCT who are elderly or have kidney impairment. 6 patients treated at our center received mini BEAM with full dose melphalan (beaM) as conditioning regimen before ASCT. Patients' characteristics are summarized in Table 1. Two had relapsed PTLD post cardiac transplant (including one with multiply relapsed PTCL), two had Waldenström macroglobulinemia and kidney amyloidosis, one had relapsed DLBCL and one had ALK- ALCL. Treatment prior to ASCT is also listed in Table 1. Conditioning regimen was as follows: carmustine 60mg/m2 on day -6, etoposide 75mg/m2 on days -5 to -2, cytarabine 100mg/m2 on days -5 to -2 and melphalan 140 mg/m2 on day -1. Stem cell infusion was performed on day 0. Stem cell mobilization was performed with filgrastim and plerixafor and was successful in all 6 patients with a median of 1.5 apheresis sessions (range 1-3). Median number of stem cells collected was 6.2 x106/kg (range 3.12 - 12.51 x106/kg). Post ASCT complications included one septic shock with lethal outcome for patient 6 due to MDR E. coli infection. Other side effects (≥grade 3) were: febrile neutropenia (n=2), non-infectious diarrhea (n=3), mucositis (n=1), cellulitis (n=1), seizure in a patient with previous seizure disorder (n=1), C. difficile infection (n=1), pneumonia (n=1), acute kidney injury (n=1), transaminitis (n=1), urinary tract infection (n=1) and HHV6 infection (n=1). Median time to neutrophil engraftment was 12 days (range 9-18) and median time to platelet engraftment was 20.5 days (18-23). Outcomes of patients before and after ASCT are summarized in Table 1. Both patients with PTLD achieved CR to pralatrexate and R-EPOCH respectively and are in remission 499 and 531 days following transplant. WM/amyloidosis patients achieved VGPR for their amyloidosis and one is in CR with respect to his lymphoma. Their last follow up was 399 and 308 days following transplant. Patient with DLBCL relapsed 364 days after transplantation and patient with ALCL died from MDR E. Coli sepsis 10 days following transplant. Mini-BEAM with full-dose of melphalan (beaM) is a safe and efficacious regimen that can be used in select lymphoma patients, particularly those with relapsed PTLD, and patients who are elderly or have impaired organ function. Disclosures Amengual: Bristol-Myers Squibb: Research Funding; Acetylon Pharmaceuticals: Research Funding. Lentzsch:BMS: Consultancy; Foundation One: Consultancy; Celgene: Consultancy, Honoraria. O'Connor:Seattle Genetics: Research Funding; Spectrum: Research Funding; Spectrum: Research Funding; Seattle Genetics: Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; TG Therapeutics: Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Celgene: Research Funding; Celgene: Research Funding.

Description: The first two authors contributed equally to this work Identifying pharmacologic strategies to inhibit the activation of NF-κB and its target genes has been a major research pursuit. To date, no direct inhibitors of the NF-κB subunits have been explored in the clinic. Based on the constitutive activation of NF-κB in diffuse large B-cell lymphoma (DLBCL), we used this disease model to develop drugs targeting NF-κB. Using a fluorescence-based high throughput screening (HTC) approach, a unique N-quinoline-benzenesulfonamide (NQBS) scaffold was identified as potential small molecule inhibitor of the NF-κB pathway. A confocal microscopy based HTC assay performed in human umbilical vein endothelial cells (HUVEC) identified hit compounds that contained a unique NQBS core structure. The assay screened for compounds that inhibited nuclear translocation of NF-κB subunits in TNFα-induced HUVEC cells. To date over 100 NQBS analogs have been synthesized with varying potency and cytotoxicity in inhibiting growth of DLBCL lines (OCI-Ly10, RIVA, HBL-1 and OCI-Ly3). Cytotoxicity assays demonstrated that the most potent compounds exhibit IC50s in the 0.5 to 1.5 µM range. These most potent NQBS analogs identified as CU-O42 CU-O47 and CU-O75 were also able to induce apoptosis and caspase activation. Apoptosis was preceded by exclusion of the NF-κB proteins from the nucleus. To analyze the localization of NF-κB proteins within the cell compartments before and after the treatment with CU-O42, CU-O47 and CU-O75, we used confocal microscopy, electromobility shift (EMSA) and ELISA assays. Control cells tested positive for p50/p65 both within the cytoplasm and the nucleus. Following treatment with CU-O42 NF-κB was sequestered within the cytoplasm of the cell which occurred as early as 3h after exposure. In addition, all three analogs reduced the nuclear levels of NF-κB in a concentration-dependent manner when measured by EMSA and ELISA. Furthermore, CU-O47 and CU-O75 were able to inhibit TNFα induced luciferase expression in a HEK293T cell model where luciferase is controlled by an NF-κB promoter. A KINOMEscan platform (examining the activity of over 450 different kinases) showed that no NQBS analog screened (CU-O42 and CU-O75) inhibited any of the kinases in the assay. In addition, a proteasome inhibition assay tested negative for trypsin-like and chromotrypsin-like protease activity (CU-O42, CU-O47 and CU-O75). Stabilization of the inactive trimer of p50, p65 and IκBα was hypothesized as a potential mechanism of action of CU-O42 and CU-O75 through Internal Coordinate Mechanics (ICM) software. This binding hypothesis was further corroborated by cellular thermal shift assays (CETSA) with an increase of the IκBα melting temperatures (2.5-3°C) in whole cell lysates following rapid (30min) exposure to CU-O42 and CU-O75. Using a genome-wide regulatory network perturbation analysis (DeMAND) based on the RNA-Seq data collected from OCI-Ly10 cells treated with CU-O75, we identified IκBα as one of the potential targets of the compounds. Gene set enrichment analysis demonstrated NF-κB target gene downregulation using IC20 of CU-O75 at 24h (p=0.045). In vivo experiments were conducted in two models: (1) xenografts with human DLBCL cell lines of both ABC and GC subtype; and (2) myc cherry luciferase mouse model where mice spontaneously develop aggressive lymphomas. In both models, CU-O42 was able to inhibit tumor growth. Interestingly, in the xenograft model, malignant cell growth was inhibited in both ABC (HBL-1) and GC (OCI-Ly1) cells when compared to controls (p=0.01 and p=0.02). However, overall survival of mice with ABC xenografts treated with CU-042 significantly exceeded the survival of mice with GC xenografts (p

Description: Interactions between histone deacetylase inhibitors (HDACIs) and decitabine were investigated in models of diffuse large B-cell lymphoma (DLBCL). A number of cell lines representing both germinal center B-like and activated B-cell like DLBCL, patient-derived tumor cells and a murine xenograft model were used to study the effects of HDACIs and decitabine in this system. All explored HDACIs in combination with decitabine produced a synergistic effect in growth inhibition and induction of apoptosis in DLBCL cells. This effect was time dependent, mediated via caspase-3 activation, and resulted in increased levels of acetylated histones. Synergy in inducing apoptosis was confirmed in patient-derived primary tumor cells treated with panobinostat and decitabine. Xenografting experiments confirmed the in vitro activity and tolerability of the combination. We analyzed the molecular basis for this synergistic effect by evaluating gene-expression and methylation patterns using microarrays, with validation by bisulfite sequencing. These analyses revealed differentially expressed genes and networks identified by each of the single treatment conditions and by the combination therapy to be unique with few overlapping genes. Among the genes uniquely altered by the combination of panobinostat and decitabine were VHL, TCEB1, WT1, and DIRAS3.

Description: Abstract 3733 Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin's Lymphoma. Despite advances in treatment, 1/3 of patients die from their disease. Gene expression profiling has delineated three subtypes with different genetic features known to be prognostic: the Activated B-cell (ABC), Germinal Center (GC), and grey zone types. For example, ABC DLBCL is addicted to NFkB over-expression. The oncogene, BCL6, encodes a transcription factor that functions as a transcriptional repressor within normal germinal center B-cells. Constitutive activation of Bcl-6 leads to GC-type DLBCL by turning off genes expressing cell cycle dependent kinase inhibitors, and essential tumor suppressor genes, like p53. There is a critical inverse relationship between Bcl-6 and p53, the functional status of which is linked to each transcription factor's degree of acetylation. Deacetylation of Bcl-6 is required for maintaining its effects as a transcriptional repressor. Conversely, acetylation of p53 is activating when class III histone deacetylases (HDAC), also known as sirtuins, are inhibited by drugs such as niacinamide. HDAC inhibitors are presently approved for T-cell lymphoma and may require the targeting of additional pathways to be effective in B-cell lymphomas. Trichostatin A and niacinamide modulate Bcl-6 in lymphoma cell lines. One therapeutic strategy that could favorably shift the relationship between oncogenes and tumor suppressors is the pharmacologic modification of Bcl-6 and p53 using HDAC inhibitors. Eight DLBCL cell lines were screened (4 ABC: Su-DHL2, HBL-1, OCI-Ly10, RIVA; 4 GC:OCI-Ly1, OCI-Ly7, Su-DHL6, Su-DHL4) with four class I/II HDAC inhibitors (romidepsin, vorinostat, panobinostat and belinostat) in combination with niacinamide (sirtuin inhibitor) at two dose levels each at three time points. Cell growth inhibition was measured by luminescence cell viability and apoptosis flow cytometry assays. Synergy was measured by the relative risk ratio (RRR) calculation where values

Description: Abstract 2725 There is a critical inverse relationship between Bcl6 and p53, the functional status of which is linked to each transcription factor's degree of acetylation. Deacetylation of Bcl6 is required for its transcriptional repressor effects allowing for the oncogene to drive lymphomagenesis. Conversely, acetylation of p53 is activating when class III HDACs, or sirtuins, are inhibited by agents such as niacinamide. One therapeutic strategy for lymphomas addicted to Bcl6 overexpression is the pharmacologic modification of Bcl6 and p53 using HDAC inhibitors. Here we report on the combination of sirtuin inhibitor, niacinamide (NIA), plus HDAC inhibitors in preclinical models, and a phase Iclinical trial in patients with relapsed or refractory lymphoma. Cytotoxicity was measured in 8 diffuse large B-cell lymphoma (DLBCL) cell lines (4 ABC and 4 GC) with 4 HDAC inhibitors (romidepsin, vorinostat, panobinostat and belinostat) in combination with NIA. Synergy was achieved predominantly in GC vs ABC cell lines, with romidepsin plus NIA having the greatest synergy. Acetylation of Bcl6 was observed in cells treated with HDAC inhibitors, or the combination, compared with control as measured by immunoprecipitation. Cells treated with the combination had increased acetylated-p53, p21 and BLIMP-1 content. In vivo effects of the drug combination were studied in a double transgenic mouse model of aggressive spontaneous B-cell lymphoma (l-myc overexpressing crossed with CD19-tagged mCherry luciferase). These mice express equal basal levels of Bcl6 and p53 as GC cell lines. Mice treated with NIA and romidepsin for 5 hrsachieved increased acetylation of Bcl6 and p53, and accumulation of p21 and BLIMP1. Mice treated with the combination exhibited decreased tumor burden achieving complete responses (CR=30%) compared to single agents and control (CR=0) after 3 weeks of treatment. The combination was well tolerated. Based on this rationale, a phase I clinical trial of vorinostat plus NIA was conducted in patients with relapsed or refractory lymphoid malignancies. Twenty-five patients enrolled between 3/2009 and 3/2011. Median age was 43 (range:25–75), 44% female, and 84% white. This was a group of heavily pretreated patients, median number of therapies was 4; with 16 auto- and 4 allo-transplants. Patients were treated on a 14/21 day cycle. Vorinostatwas given orally as a fixed dose of 400 mg daily. NIA was given orally, daily, in escalating doses from 20–100 mg/kg. A range of 1–18 cycles were given. The most common toxicities included fatigue (84%), nausea (80%), diarrhea (72%), and anorexia (56%). There was a DLT in cohort 4 (vorinostat 400 mg/NIA 80 mg/kg) of grade 3 infection. Two DLTs occurred at dose level 5 (vorinostat 400mg/NIA 100 mg/kg), a grade 4 transaminitis, not otherwise explained; and grade 4 hypotension. These events led to the determination of dose cohort 4 as the MTD. In total, there were 12 different grade 3–4 toxicities including neutropenia, infection, and transaminitis that occurred in 11 patients. There were two deaths that occurred within safety follow-up period in patients who had fulminant disease progression (POD); none of these events were related to the study drugs. Overall this was a very well tolerated treatment regimen and the responses seen were with the weakest HDAC and sirtuininhibitors available given to extremely heavily pretreated patients. The overall response rate was 24%, with 2 CRs and 3 partial responses (PR). CRs were maintained for 13 to 18 weeks. All PRs were in HL patients with 6–10 prior regimens including auto- and allo-transplants, and maintained for 4–13 weeks. Twelve patients achieved stable disease (SD) (57%), 2 of whom achieved near-PR. Of the 7 patients with germinal center derived lymphomas (4 DLBCL + 3 FL), there was 1 CR, 3 SD, and 3 POD. The preclinical and clinical data establish proof-of-principle that the Bcl6: p53 axis may be therapeutic targets, and that acetylation of these transcription factors could potentially serve as biomarkers for activity. Currently we are exploring new, more potent sirtuin inhibitors with second generation HDAC inhibitors, and working to further define mechanism of action. By targeting discrete molecular subtypes of DLBCL it may be possible to increase complete response rates in this challenging disease. Disclosures: Amengual: Acetylon Pharmaceuticals, Inc: Research Funding. Off Label Use: Vorinostat is not FDA approved for the treatment of B-cell and Hodgkins lymphoma. Niacinamide is not FDA approved for the treatment of lymphoma. Neylon:Seattle Genetics, Inc: Consultancy, Speakers Bureau; Allos Therapeutics, Inc: Speakers Bureau. Zain:Allos Therapeutics, Inc: Speakers Bureau; Seattle Genetics, Inc: Speakers Bureau. O'Connor:Allos Therapeutics, Inc: Consultancy; Seattle Genetics, Inc: Membership on an entity's Board of Directors or advisory committees; Millenium Pharaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics, Inc: Consultancy.