ALBERT

Description: Abstract 1854 Poster Board I-880 Background: MLN4924 is a first-in-class small molecule inhibitor of NEDD8-activating enzyme (NAE), an essential component of the NEDD8 conjugation pathway in the ubiquitin-proteasome system. Inhibition of NAE with MLN4924 prevents conjugation of NEDD8 to the Cullin Ring Ligases (CRLs). This subsequently prevents ubiquitination and proteasomal degradation of CRL substrates, which include proteins involved in cell cycle regulation (p27), signal transduction (pIκBá), DNA replication (Cdt-1), stress response (Nrf-2), and other processes important to tumor cell growth and survival. MLN4924 has demonstrated potent antitumor activity in vitro against multiple myeloma (MM) and non-Hodgkin lymphoma (NHL) cell lines as well as in mouse xenograft models of NHL. This phase 1 dose-escalation study is the first investigation of MLN4924 in MM or NHL patients (pts). The primary objectives were to determine the maximum tolerated dose (MTD) and safety profile of MLN4924, describe the pharmacokinetics (PK) and pharmacodynamics (PD) in blood (inhibition of NEDD8-Cullin levels in peripheral blood mononuclear cells [PBMCs]; Nrf-2 target gene transcription in whole blood), and investigate PD effects in skin (Cdt-1, Nrf-2 accumulation). Methods: Pts aged ≥18 yrs with relapsed and/or refractory MM or NHL after ≥2 prior lines of therapy were eligible. Pts received escalating doses of MLN4924 by IV infusion on days 1, 2, 8, and 9 of 21-day cycles; once the MTD for this schedule was reached, MTDs for other schedules will be investigated. MTD determination was based on an adaptive approach using a Bayesian continual reassessment method, with the MTD defined as the dose level closest to that predicted to result in a dose-limiting toxicity (DLT) rate of 25%. DLT was defined as: grade 4 neutropenia or thrombocytopenia for 〉7 days; grade 3 neutropenia with fever/infection or thrombocytopenia with bleeding; grade ≥3 non-hematologic toxicity except arthralgia/myalgia, brief fatigue, or fever without neutropenia; and grade ≥2 MLN4924-related toxicities requiring dose reduction/discontinuation. For PD analysis, PBMCs and whole blood were isolated at screening, baseline, and following MLN4924 administration; skin biopsies for Cdt-1 and Nrf-2 assays were performed at baseline and after the second dose. Results: Among 22 pts enrolled to date, median age was 65 years, 13 were male, 14 had MM, and 8 had NHL (4 FL, 1 MCL, 1 DLBCL, 1 B-cell CLL/SLL, 1 transformed CLL). All MM pts had received prior autologous SCT, and 13, 9, and 9 had prior bortezomib, lenalidomide, and thalidomide, respectively. Seven NHL pts had received prior autologous SCT, 1 had a prior allogeneic SCT, and 8 had prior rituximab. Pts received MLN4924 at 6 dose levels: 25 (n=3), 50 (n=2), 65 (n=3), 83 (n=2), 110 (n=9), and 147 mg/m2 (n=3). Of the 15 (68% of the 22 enrolled) pts who received all 4 scheduled doses or had a DLT in cycle 1 (DLT-evaluable pts), 4 experienced a DLT: 1 grade 4 febrile neutropenia at 65 mg/m2; 1 grade 3 liver function tests at 110 mg/m2, and 1 grade 4 muscle cramps and 1 grade 2 myalgia that was considered dose limiting at 147 mg/m2. Thus, the MTD for this schedule was determined to be 110 mg/m2. The most common adverse events (AEs, NCI CTCAE v3.0) included fatigue, nausea, myalgia, and elevated liver enzymes. With the exception of the grade 4 neutropenia seen at 65 mg/m2, myelosuppression was limited. No infusion-related reactions were noted. Elevated CRP levels appeared transient in most cases. There have been no treatment-related deaths; 1 MM pt died due to progressive disease. MLN4924 displayed a multiexponential PK profile with a half life of 4–9 hours, relatively low PK variability, and approximately dose-proportional increases in total plasma exposure over the 25–147 mg/m2 dose range. NEDD8-Cullin levels in PBMCs were inhibited and Nrf-2 target gene transcripts in whole blood were higher vs baseline after MLN4924 administration, indicative of NAE inhibition. Cdt-1 and Nrf-2 levels in skin increased above baseline following the second dose of MLN4924, indicative of NAE inhibition in peripheral tissue. The 110 mg/m2 dose level is being expanded to more fully characterize safety, PK, and PD in MM and NHL. Subsequent pts will be enrolled to receive MLN4924 on 2 other schedules. Conclusions: This early analysis provides evidence of PD inhibition of NAE activity by MLN4924 in blood and skin, and supports continued investigation of MLN4924. Disclosures: Shah: Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Elan: Consultancy; Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Off Label Use: MLN4924 is not approved for the treatment of multiple myeloma or non-Hodgkin lymphoma.. Jakubowiak:Bristol-Myers-Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Centocor Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Exelixis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Berger:Millennium Pharmaceuticals, Inc.: Employment. Mulligan:Millennium Pharmaceuticals, Inc.: Employment. Petruzzelli:Pfizer: Equity Ownership; Millennium Pharmaceuticals, Inc.: Employment; Amgen: Equity Ownership. Pickard:Millennium Pharmaceuticals, Inc.: Employment. Smith:Millennium Pharmaceuticals, Inc.: Employment. Venkatakrishnan:Millennium Pharmaceuticals, Inc.: Employment. Lonial:Novartis: Consultancy; Gloucester: Research Funding; BMS: Consultancy; Millennium Pharmaceuticals, Inc.: Consultancy, Research Funding; Celgene: Consultancy.

Description: Introduction: B-cell malignancies may depend on the histone methyltransferase EZH2 to perpetuate a less differentiated state, with activating mutations of EZH2 being potential oncogenic drivers. Tazemetostat, a potent, selective EZH2 inhibitor, is in phase 2 clinical development in relapsed or refractory (R/R) non-Hodgkin lymphoma (NHL). Objective responses were observed in patients (pts) with EZH2 mutant or wild type tumors in the phase 1 part of the phase 1/2 study. The ongoing phase 2 study is enrolling pts with mutant or wild type EZH2 having R/R diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma (FL) to determine efficacy and safety. The primary endpoint is overall response rate. Here we report results of an updated molecular analysis of archived tumor and circulating tumor DNA (ctDNA) collected from pts plasma and associations with preliminary response data, including the discovery of novel candidate molecular predictors of tazemetostat response. Methods: Archived tumor and/or plasma-derived ctDNA samples were obtained during screening from R/R DLBCL and FL pts enrolled to the phase 2 trial of tazemetostat in NHL (NCT01897571). Next generation sequencing was performed retrospectively on archived tumor DNA (target coverage of 1,500X) and ctDNA (20,000X for somatic mutations and 5,000X for structural alterations). DNA was isolated from archived tumor and was tested across a panel of approximately 200 genes, including 62 genes commonly altered in NHL to identify somatic mutations, amplifications, and translocations. The ctDNA sequencing was restricted to the 62 gene NHL panel. Best objective overall response data (Cheson 2007) as of May 1, 2018 were used to generate two groups: responders (R = CR + PR), and non-responders (NR = progressive disease, stable disease, or unknown clinical response). Fisher's exact test (two tailed) was performed to identify DNA variants associated with either the R or NR groups for FL+DLBCL combined and for each NHL sub-type independently (Table 1). All gene associations met an unadjusted P-value threshold of ≤ 0.1. Results: In the combined analysis for all NHL pts, EZH2, BRCA2, ETV6, IKZF3 and TNFRSF14 mutations were associated with clinical response. Eleven genes, including BCL6, BCL2, TP53, PIM1, HIST1H1B and HIST1H1E, mutations were associated with a lack of response to tazemetostat. Association of detection of mutations in EZH2, BCL6 and HIST1H1E and response were detected in both ctDNA and archived tumor analysis, with all other genes achieving P ≤ 0.1 in either the ctDNA or the archived tumor analysis. Analysis of the NHL subcategories revealed in FL that mutations in EZH2 and STAT6 were associated with clinical response while mutations in BCL2, TNFAIP3, FOXO1 and MYD88 were identified as negative predictors. EZH2 was the only gene identified in both the ctDNA and archived tumor analyses in FL. In DLBCL, while EZH2 was not detected as a predictor of response to tazemetostat at P ≤ 0.1, the number of pts with mutant rather than wild type EZH2 was higher in responders than non-responders in both analyses. Positive predictors of tazemetostat response were detected in DLBCL: MYD88, MEF2B, ETV6, MLH1, RECQL4, and RNF43. Negative predictors of response were also identified in DLBCL: BCL2, PDL1, PDL2 and SOCS1. When the genes identified as associated with tazemetostat response in DLBCL were compared between the archived tumor and ctDNA analyses, no genes in common were identified. Conclusions: These analyses confirm genes previously described as potential predictors of tazemetostat response in analyses using smaller patient numbers and less mature clinical response data such as STAT6, EZH2, MYD88, TP53,BCL2, BCL6, PIM1 and HIST1H1E. In addition, identification of PDL1/PDL2 and RECQL4/MLH1/BRCA2 as novel predictors points to potential relevance of immune checkpoint and DNA damage repair pathways as additional pathways that may be of relevance to tazemetostat response in NHL. Subanalyses performed independently on each NHL subtype revealed disease specific biomarkers of response that may point to differences in the factors that can influence response to tazemetostat in DLBCL and FL. Disclosures McDonald: Epizyme: Employment, Equity Ownership. Thomas:Epizyme: Employment, Equity Ownership. Daigle:Epizyme: Employment, Equity Ownership. Morschhauser:Epizyme: Consultancy; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees. Salles:Morphosys: Honoraria; Servier: Honoraria; Janssen: Honoraria, Other: Advisory Board; Takeda: Honoraria; Pfizer: Honoraria; Servier: Honoraria, Other: Advisory Board; Merck: Honoraria; BMS: Honoraria, Other: Advisory Board; Gilead: Honoraria, Other: Advisory Board; Amgen: Honoraria; Epizyme: Honoraria; Acerta: Honoraria; Abbvie: Honoraria; Celgene: Honoraria, Other: Advisory Board, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria. Ribrag:Servier, Pharmamar, Nanostring, Gilead, Infinity, BMS, MSD, Epizyme: Consultancy; ESAI: Honoraria, Research Funding; Roche: Other: Travel, expenses, accommodation. McKay:Epizyme: Consultancy, Honoraria. Tilly:Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees. Johnson:Kite: Consultancy; Genmab: Consultancy; Incyte: Consultancy; Zenyaku Kogyo: Other: Travel, accommodations, expenses; Bristol-Myers Squibb: Honoraria; Eisai: Research Funding; Takeda: Honoraria, Travel, accommodations, expenses; Boeringher Ingelheim: Consultancy; Epizyme: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Research Funding; Celgene: Honoraria; Novartis: Honoraria. Dickinson:GSK: Consultancy. Opat:Roche, Celgene, Mundipharma, Janssen: Honoraria; Roche, Celgene, Mundipharma, Janssen: Consultancy. Jurczak:Gilead: Consultancy; Morphosys: Research Funding; Roche: Research Funding; Servier: Research Funding; TG Therapeutics: Research Funding; Merck: Research Funding; Pharmacyclics: Research Funding; European Medicines Agency: Consultancy; AstraZeneca/Acerta: Consultancy, Research Funding; Sandoz-Nowartis: Consultancy; Afimed: Research Funding; BeiGene: Research Funding; Celgene: Research Funding; Epizyme: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Nordic Nanovector: Research Funding; Janssen: Consultancy. Cartron:Gilead Sciences: Honoraria; Celgene: Consultancy, Honoraria; Janssen: Honoraria; Sanofi: Honoraria; Roche: Consultancy, Honoraria. Zinzani:Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; MSD: Honoraria, Speakers Bureau; Astra Zeneca: Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SERVIER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Assouline:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Novartis: Research Funding; Roche: Honoraria, Research Funding, Speakers Bureau. Radford:BMS: Consultancy, Speakers Bureau; Seattle Genetics: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Pfizer: Research Funding; Celgene: Research Funding; Takeda: Consultancy, Research Funding, Speakers Bureau; GlaxoSmithKline: Equity Ownership; AstraZeneca: Equity Ownership; ADC Therapeutics: Consultancy, Research Funding. Gribben:Novartis: Honoraria; Medical Research Council: Research Funding; Acerta Pharma: Honoraria, Research Funding; Roche: Honoraria; Abbvie: Honoraria; Cancer Research UK: Research Funding; Kite: Honoraria; NIH: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Research Funding; TG Therapeutics: Honoraria; Pharmacyclics: Honoraria; Unum: Equity Ownership; Wellcome Trust: Research Funding. Haioun:Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Sciences: Consultancy, Honoraria; Gilead Sciences: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Le Gouill:Roche: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria. Clawson:Epizyme: Employment, Equity Ownership. Larus:Epizyme: Employment, Equity Ownership. Blakemore:Epizyme: Employment, Equity Ownership.

Description: Background: Mantle cell lymphoma (MCL) is an aggressive NHL subtype that remains incurable in most cases with conventional chemotherapy. However, individual patient survival can vary considerably, and studies have identified potential markers of prognostic subgroups, including Ki-67 and the cyclin-dependent kinase (CDK) inhibitor p27. Genomic studies further highlight tumor proliferation status as the major driver of differential prognosis in MCL (Rosenwald et al, Cancer Cell 2003). Such studies were conducted prior to the introduction of bortezomib, a first-in-class proteasome inhibitor that was recently approved for the treatment of relapsed MCL. Plausible hypotheses for the activity of this novel agent in MCL include inhibition of NF-κB activity and disruption of the cell cycle via stabilization of CDK inhibitors such as p27. In order to test these hypotheses directly and also examine how this new agent impacts prognostic subgroups of MCL, the PINNACLE trial, the largest phase 2 study of bortezomib in MCL, specified the collection of archived tumor specimens for immunohistochemical (IHC) analyses. In this trial 155 relapsed/refractory MCL patients received single-agent bortezomib at a dose of 1.3 mg/m2 on days 1, 4, 8, and 11 of 21-day cycles. A 33% overall response rate was observed (8% CR/CRu), with a median time to progression (TTP) of 6.2 months and a responder TTP of 10.6 months (Fisher et al, J Clin Oncol 2006). Methods: A series of unstained tumor slides from the diagnostic tumor specimens were collected from a subset of 70 evaluable patients. This subset formed the basis of the current IHC analysis and the clinical parameters of this subset appeared similar to those of the study as a whole. Several proteins were assessed via IHC; these included p27, SKP2, cyclin D1, and Ki-67, as well as candidate markers correlated with bortezomib efficacy in a genomic study of multiple myeloma, including NF-κB and proteasome subunits (Mulligan et al, Blood 2007). Association with response was evaluated using Fisher’s Exact Test. A Cox proportional hazard linear model adjusted for clinical covariates was used to determine the effect of these parameters on TTP. Results: In this subset analysis no protein markers were significantly associated with response to therapy. However, patients with 〉50% Ki-67-positive tumor cells were more likely to progress than patients with

Description: Background: Proteasome inhibitors (PI) affect a variety of intracellular processes, including the cell cycle, NF-kB and other signal transduction pathways, and the balance of bcl-2-related proteins, yet it remains unknown which of these targets are most crucial in determining response to PI treatment in non-Hodgkin Lymphoma (NHL). Responses to bortezomib (btz) have been seen in different NHL subtypes in several phase 2 studies. The consistency of response suggests that there may be biological characteristics that determine individual tumor responses to PIs. We used tissue microarray (TMA) technology to determine if pre-treatment tumor characteristics can predict outcome of btz treatment in NHL. Methods: TMAs were constructed using paraffin-embedded blocks of pre-treatment tissue from patients enrolled in a phase 2, CTEP-NCI sponsored trial of btz in NHL. Microsections were stained for 18 proteins with reported relevance to proteasome function and/or lymphoma prognosis. All stains were evaluated and scored by a central pathologist. Staining was correlated with quality and duration of response (DOR) or time to treatment failure (TTF). The best response was treated as a binary outcome (PR/CR vs SD/POD). Correlation between each marker and response was assessed by univariate analysis. The exact Wilcoxon rank-sum test was used to compare DOR and TTF with respect to the binary marker scores. Results: This analysis includes 35 of 75 patients treated with btz between 11/6/01 and 2/23/07 for whom pre-treatment tissue blocks could be obtained. Response data were available for 32 of these patients. Diagnoses include: FL (N=10), SLL (N=4), MCL (N=14) and MZL (N=4). Expression of the cell-cycle inhibitor p27 was more common in responding vs non-responding pts (p=0.024), and was also significantly associated with longer DOR (p=0.020) and longer TTF (p=0.021; figure). Presence of Bcl-6 was significantly associated with shorter DOR (p=0.049) and shorter TTF (p=0.048; figure), but not response. Conclusions: Using TMA analysis, we have shown that NHL patients with pre-treatment p27 staining were more likely to respond to btz, and had a longer DOR and TTF than those without baseline staining. Conversely, patients with baseline Bcl-6 expression had a shorter DOR and TTF than those without. Although the number of patients in this analysis is small, the data will help us generate hypotheses about disease and treatment biology that can be tested in larger studies. We are currently collecting samples from a broader pool of patients treated with btz at MSKCC to verify these results. TTF with bortezomib by (A) p27 and (B) Bcl-6 status. Figure. Figure.

Description: Studies using genetically deficient mice have revealed that members of the NF-kB family play key roles in B-cell development. IKK2 which activates NF-kB by targeting degradation of IkB, is also required for B-cell development. We have studied the role of IKK2 in hematopoiesis using a chemical specific inhibitor (ML120B). Mice given daily oral dosing of ML120B for 4 days had severe B-cell depletion in spleen and bone marrow. B-cells at all stages (pro-B, pre-B, immature and mature B) were depleted 10 fold in the bone marrow while granulocyte numbers were largely unaffected. IKK2 inhibition in vivo showed selective sensitivity of B-cell progenitors (4 fold decrease) in the marrow compared to myeloid progenitors which were unaffected at an equivalent dose. Foci of cells with an apoptotic morphology were visible in bone marrow and spleen within 6 hours of a single oral dose. Apoptotic cells detected by labeling fragmented DNA were increased within splenic follicles (6 fold) and bone marrow. Also an increase in B220+ / annexin V+ cells and a decrease in pre-B (B220+/IgM−) cells in the marrow were observed. RNA expression studies in the marrow 6 hours after a single oral dose revealed a decrease in IL-7 and increased GM-CSF expression. Image analysis of B220 in spleens within 18 hours of a single dose of an IKK inhibitor revealed decreased follicle size. In order to evaluate hematopoietic progenitor sensitivity to NF-kB inhibition, dose responses to ML120B, panepoxydone (PPD) and proteasome inhibitor Lactacystin (Lcyst) were evaluated in B-cell and myeloid bone marrow colony assays. Inhibitors PPD and Lcyst were more effective at inhibiting B-cell colony growth than myeloid colony growth. In summary, pharmacologic inhibition of IKK2 results in a rapid induction of apoptosis with preferential depletion of B-cells and retention of myeloid cells and progenitors within the bone marrow.