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.

Description: Introduction: Rearrangements of the MLL (mixed lineage leukemia) gene on chromosome 11q23 are present in 5-10% of either acute myeloid (AML) or acute lymphoblastic leukemia (ALL), and are associated with a poor prognosis. Fusion proteins involving the MLL histone methyltransferase (HMT) result in recruitment of another HMT, DOT1L, to a multi-protein complex leading to aberrant methylation of Histone H3 lysine 79 (H3K79) at MLL target genes and enhanced expression of leukemogenic genes such as HOXA9 and MEIS1. EPZ-5676 is a small molecule inhibitor of DOT1L with sub-nanomolar affinity and 〉37,000 fold selectivity against additional HMTs. EPZ-5676 treatment of cultured cells reduced histone H3K79 methylation, decreased MLL target gene expression and resulted in selective killing of MLL-rearranged (MLL-r) leukemia cells by inducing apoptosis and differentiation. EPZ-5676 administration led to tumor regression in an MLL-r leukemia xenograft model. We report preliminary results of an ongoing Phase 1 trial of EPZ-5676 in patients with advanced hematologic malignancies, including acute leukemia with MLL-r. Methods: This is a 2-part phase 1 open label dose escalation study investigating the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD) and anti-leukemic activity of EPZ-5676 in adult patients (pts) with relapsed/refractory leukemia (CT.gov: NCT01684150). In the dose-escalation phase, patients with AML, ALL, acute mixed lineage leukemia, myelodysplastic syndrome, myeloproliferative neoplasia (MPN) or chronic myeloid leukemia were eligible. Eligibility in the expansion phase of the study was restricted to pts with MLL-r or partial tandem duplication of MLL (MLL-PTD). EPZ-5676 was administered via continuous intravenous infusion (CIV) for 21 days of a 28 day cycle in the dose escalation phase and CIV for all 28 days of a 28 day cycle in the expansion phase, until disease progression or unacceptable toxicity. Results: Between October 2012 and July 2014, 37 pts have enrolled; 36 are evaluable for safety (received at least 1 dose of EPZ-5676) and 28 pts (19 MLL-r, 4 MLL-PTD) are evaluable for anti-leukemia activity (completed at least one post baseline marrow evaluation). The median age at time of enrollment was 53 years (range: 20 to 81 years); the median number of prior systemic therapies was 2 (range: 1 to 6) and 14 pts had a prior allogeneic stem cell transplant. Of the treated pts, disease characteristics were: 31 pts with AML (21 MLL-r, 5 MLL-PTD), 4 pts with ALL (3 MLL-r) and 1 pt with MPN (MLL-r). Table 1: Number of evaluable pts per cohort Dose (mg/m2/day) 12 24 36 54 80 90 21 day CIV 1 5 4 6 3 - 28 day CIV - - - - - 17 The median time on study was 29 days (range: 5-196 days). Adverse events (AEs) reported in 〉15% of pts were: anemia, fever/neutropenia, thrombocytopenia, constipation, diarrhea, nausea, chills, fatigue, mucosal inflammation, peripheral edema, hypomagnesemia, dyspnea and sepsis; of which leukocytosis, nausea and hypomagnesemia were assessed by the investigator as drug related. Grade I PR interval prolongation, without associated QTc changes, was observed in 2 pts. There were no deaths attributed to study drug treatment, and there were 2 pts with treatment discontinuation for potentially drug-related AEs. Dose proportional PK was observed with rapid attainment of steady-state plasma concentrations on Day 1 of treatment. Preliminary PD data demonstrated inhibition of H3K79 methylation from baseline in marrow (median: 52%, range 0-81%) and peripheral blood mononuclear cells (median: 43%, range 25-67%) at doses above 36 mg/m2/day. PD effects were observed by day 8. The 21 day CIV pts recovered H3K79 methylation towards baseline by day 28, and the 28 day CIV pts maintained methyl-mark inhibition throughout cycle 1. To date, best responses for pts with MLL-r are morphologic CR (1 pt.), cytogenetic CR (1 pt), and resolution of leukemia cutis (2 pts). In addition, a treatment-related increase in neutrophils (PMN) and/or monocytes was observed in 6 pts with a median day of onset of 15 days (range 8-28 days). The identification of the MLL-r by split signal FISH in mature PMN suggests a differentiation effect on the leukemic clone. Conclusions: EPZ-5676 is well tolerated and exhibits anti-leukemic activity in MLL-rearranged acute leukemia. These data provide rationale for continued clinical investigation of potential utility of EPZ-5676 in patients with rearrangements of the MLL gene. Disclosures Stein: Janssen Pharmaceuticals: Consultancy. Garcia-Manero:Epizyme, Inc: Research Funding. Rizzieri:Novartis, Inc: Consultancy, Speakers Bureau; Ariad, Inc: Speakers Bureau. Savona:Karyopharm: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Altman:Astellas: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Membership on an entity's Board of Directors or advisory committees. Armstrong:Epizyme, Inc: Consultancy. Pollock:Epizyme: Employment, Equity Ownership. Waters:Epizyme, Inc: Employment, Equity Ownership. Legler:Epizyme, Inc: Employment. Thomson:Epizyme, Inc: Employment. Daigle:Epizyme, Inc: Employment, Equity Ownership. McDonald:Epizyme, Inc: Employment. Campbell:Epizyme, Inc: Employment, Equity Ownership. Olhava:Epizyme, Inc: Employment. Hedrick:Epizyme, Inc: Employment; Pharmacyclics, Inc: Equity Ownership. Copeland:New Enterprise Associates: Ad hoc consultant, Ad hoc consultant Other; Mersana: Membership on an entity's Board of Directors or advisory committees; Epizyme, Inc: Employment, Equity Ownership.

Description: Iron and copper are essential for all organisms, assuming critical roles as cofactors in many enzymes. In eukaryotes, the transmembrane transport of these elements is a highly regulated process facilitated by the single electron reduction of each metal. Previously, we identified a mammalian ferrireductase, Steap3, critical for erythroid iron homeostasis. Now, through homology, expression, and functional studies, we characterize all 4 members of this protein family and demonstrate that 3 of them, Steap2, Steap3, and Steap4, are not only ferrireductases but also cupric reductases that stimulate cellular uptake of both iron and copper in vitro. Finally, the pattern of tissue expression and subcellular localization of these proteins suggest they are physiologically relevant cupric reductases and ferrireductases in vivo.

Description: Introduction: The histone methyl transferase EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2) and responsible for methylation of lysine 27 of histone H3 (H3K27), a modification of DNA associated with repressed transcription when trimethylated (H3K27me3). Aberrant EZH2 activity has been implicated as an oncogenic driver in non-Hodgkin's lymphoma (NHL). Here we report the phase 1 first-in-human experience to date with tazemetostat in patients (pts) with NHL. Methods: Tazemetostat was administered orally twice daily (BID) to subjects in five dose cohorts (100 mg [n=6], 200 mg [n= 3], 400 mg [n=3], 800 mg [n=14], 1600 mg [n=12], and one food effect cohort (400 mg [n=7]). Tumor response assessments were performed every 8 weeks. Archival tumor tissue from NHL pts was analysed for EZH2 hot spot mutations Y646X, A682G and A692V (by either amplicon-based next generation sequencing [NGS] or cobas ® EZH2 Mutation Test [in development]), and for additional somatic mutations by NGS focusing on a panel of 39 genes commonly mutated in NHL. In addition, cell-of-origin in Diffuse Large B-cell Lymphoma (DLBCL) patients was determined by immunohistochemistry on archival tumor tissue using the Hans algorithm (Blood, 2004). Results: As of 9-July 2015, 45 pts were enrolled to this trial (CT.gov: NCT01897571). To date 19 NHL pts: 13 DLBCL, 5 follicular lymphoma (FL) and 1 marginal zone lymphoma (MZL) were enrolled. Results to date on the 26 solid tumor pts have been reported separately (ECC, 2015). Adverse events (AE) occurring in 〉10% of the 45 pts regardless of attribution were: asthenia, anorexia, constipation, nausea, dysgeusia, vomiting and muscle spasms with 5 grade 3 or greater related AE's: thrombocytopenia, neutropenia, hypertension, anorexia and transaminase elevation. The median age of the NHL patients enrolled was 62 yrs (range 23-82) and 74% of pts were male. Of the fifteen evaluable NHL pts, objective responses were seen in: 5/9 DLBCL, 3/5 FL and 1/1 MZL. The majority of objective responses occurred at the Recommended Phase 2 Dose of 800 mg BID. EZH2 status in patient tumors was determined for 14/19 NHL patients (n=3 data pending, n=2 tissue unavailable) with 13/14 found to be wild-type (WT) and one patient, who experienced an ongoing PR at week 16, expressing an Y646H mutation. Updated data including duration of response will be presented. In addition, 10/13 patients had evidence of somatic mutations in 〉1 non-EZH2 genes among the 39 genes tested (allelic frequency 〉10% with coverage 〉1000X) known to be commonly mutated in NHL, e.g. MYD88 & CARD11, or involved in epigenetic signalling, e.g. EP300 & CREBBP. Table.Relapsed or refractory NHLTotal(n=19)Evaluable(n=15)Best Response CR+PRaBest Response SDaDLBCLGCB4220Non-GCB6520undetermined3210FL5531MZL1110aper IWG (Cheson, 2007), complete response (CR), partial response (PR), stable disease (SD) Conclusions: Tazemetostat demonstrates a safety profile favorable for chronic dosing and objective responses in pts with either EZH2 WT or mutant B-cell lymphoma, including both GCB and non-GCB sub-types of DLBCL. EZH2 pathway genes and genes commonly somatically mutated in lymphoma are under investigation to elucidate the mechanisms and biomarkers of clinical response to tazemetostat. Based upon the observed safety and efficacy profile of tazemetostat, a phase 2 trial in relapsed or refractory DLBCL and FL pts, stratified by cell-of-origin and EZH2 mutation status, is enrolling. Disclosures Ribrag: Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Pharmamar: Honoraria, Membership on an entity's Board of Directors or advisory committees. Thomson:Epizyme, Inc: Employment. Keilhack:Epizyme: Employment, Equity Ownership. Blakemore:Epizyme: Employment. Reyderman:Eisai: Employment. Kumar:Eisai: Employment. Fine:Epizyme: Employment. McDonald:Epizyme: Employment. Ho:Epizyme, Inc: Employment.

Description: An important goal of cancer therapy is to improve patient outcomes by driving to deep and durable tumor responses. The activity of single-agent targeted therapies, such as BTK inhibitors or IMiDs alone, has been modest in relapsed and refractory DLBCL, necessitating the use of combination therapy. Targeted protein degraders are heterobifunctional small molecules that co-opt the endogenous ubiquitin-proteasome system to drive the selective degradation of target proteins. Degraders of IRAK4, a key component of the myddosome complex, show potent and selective IRAK4 degradation and preferential activity in MYD88-mutant (MYD88MT) DLBCL models. Notably, the activity of IRAK4 degradation is superior to IRAK4 kinase inhibition, supporting the essential scaffolding role of IRAK4 in myddosome signaling. We have previously described IRAKIMiDs, novel IRAK4 degraders that utilize an IMiD as a pharmacologically active cereblon binder. These degraders simultaneously degrade both IRAK4 and IMiD substrates and show synergistic antitumor activity over either IRAK4 degraders or IMiDs alone, enabling a therapeutically relevant biological combination within a single small molecule. Here we describe KTX-120, a novel IRAKIMiD development candidate. KTX-120 is an equipotent degrader of both IRAK4 and the IMiD substrates Ikaros and Aiolos in lymphoma model systems with low single-digit nM DC50 for degradation of all substrates. The cell activity of KTX-120 has shown a high dependence on MYD88MT status: across a panel of MYD88MT cell lines, KTX-120 showed consistent and potent cell activity, with IC50 ranging from 7-29nM, whereas in MYD88WT lines, cell activity was poor ranging from 1800-3400nM. In the OCI-Ly10 ABC DLBCL cell line that harbors a MYD88L265P mutation, the cell activity of KTX-120 was associated with degradation of both IRAK4 and Ikaros, supporting the combined IRAK4 and IMiD targeting of this molecule as contributing to cell activity. Notably, the onset of cell death in OCI-Ly10 cells with KTX-120 treatment was rapid, with cells becoming committed to cell death within 72h of exposure, suggesting that continuous exposure to KTX-120 may not be necessary for antitumor activity. We explored the pharmacological activity of KTX-120 in several in vivo model systems. KTX-120 is orally bioavailable and shows dose-proportional exposure in several species. A single oral dose of KTX-120 (10 mg/Kg or 30 mg/Kg) showed significant degradation of both IRAK4 and Ikaros in a dose and time-dependent manner, with degradation of both substrates being sustained for 〉96h, further supporting the potential for intermittent dosing. To assess this, we have explored the antitumor efficacy of KTX-120 in intermittent dosing schedules. KTX-120 was well tolerated and showed potent antitumor activity in several CDX models of MYD88MT DLBCL, including OCI-Ly10, TMD8 and SUDHL2, achieving regressions in all models in as little as once every 2 weeks. As an example, in OCI-Ly10, a 30mpk dose Q2W drove 〉80% degradation of both IRAK4 and Ikaros and showed regressions (including CR) by D28. Similar activity and tolerability were seen with both PO and IV dosing at doses that achieve active exposure, enabling the potential for both oral and parenteral dosing. We have further explored the activity of KTX-120 in a collection of DLBCL patient derived xenograft models. KTX-120 shows robust activity (〉85% TGI) in 4/5 models of MYD88MT DLBCL and shows no or modest activity in 2/2 models of MYD88WT DLBCL, supporting the preferential activity of this mechanism in patient samples harboring MYD88 activating mutations. Importantly, activity was observed in models with a variety of co-mutations that activate the NFkB pathway, including alterations in, CD79B and TNFAIP3, suggesting that KTX-120 has the potential for activity in MYD88MT lymphoma regardless of other mutations. Collectively, these data support the combined IRAK4 degradation and IMiD activity of KTX-120 has the potential to achieve robust and durable regressions in MYD88MT lymphomas with the increased convenience of an intermittently administered single agent and decreased potential for drug combination challenges. Disclosures Walker: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mayo:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Klaus:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Chen:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bhaduri:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rusin:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. McDonald:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gollob:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mainolfi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Weiss:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Description: STAT3 (signal transducers and activators of transcription 3) is a transcription factor and a member of the STAT protein family that is activated through a variety of different cytokine and growth factor receptors via JAKs, as well as through oncogenic fusion proteins and gain-of-function (GoF) mutations in STAT3 itself. STAT3 hyperactivation and GoF mutations are found in numerous cancers, including clinically aggressive hematologic malignancies with high unmet medical need, such as peripheral T cell lymphomas (PTCLs) (Andersson et al., 2020). We have previously shown that a potent and selective STAT3 heterobifunctional degrader, KTX-201, strongly represses cell growth in models of STAT3-dependent heme malignancies (Csibi et al., 2019). Herein, we report on the cellular mechanisms underlying the anti-tumor effect of STAT3 degradation in PTCL and provide a model for the relationship between pharmacokinetics/ pharmacodynamics (PK/PD) and activity of KTX-201 in vivo. The relationship between STAT3 degradation by KTX-201, anti-tumor mechanism of action and in vivo activity were investigated in anaplastic large T cell lymphoma (ALCL) models, a subset of PTCLs. In vitro, a decrease of STAT3 by 90% for 48hr was required for ALCL cells to commit to death. To identify anti-tumor mechanism(s) of KTX-201 at the systems level, we performed a time-resolved analysis of the proteomic changes of SU-DHL-1 cells undergoing growth inhibition mediated by KTX-201 at GI95. We measured the abundance of 10,000 proteins and confirmed selective degradation of STAT3 by KTX-201 after 8h of treatment. Significant changes in several marker proteins known to be involved in STAT3-mediated proximal signaling in ALCL including SOCS3, Myc and Granzyme B were observed after 16h. Functional annotation analysis of proteins identified pathways that were significantly enriched in at least one time point. Using unsupervised hierarchical clustering of annotations, we found that proteins that increased in abundance over 48h of exposure to KTX-201 were associated with markers of apoptosis and those that decreased in abundance by 24h and 48h were associated with cytokine signaling and cell cycle, respectively. Based on these data, this study identifies inhibition of cytokine signaling, G1 cell cycle arrest and induction of apoptosis as key anti-tumor mechanisms associated with KTX-201 consistent with observed cell phenotypes. STAT3 degradation in tumor was characterized in mice bearing SU-DHL-1 tumors following single dose IV administration. The STAT3 PD response in tumor was correlated with exposures in tumor. At the dose of 25 mg/kg weekly where complete tumor regression was achieved, KTX-201 achieves 〉90% STAT3 degradation at 24h post dosing in SUDHL1 xenografts. STAT3 degradation was maintained at 90% at 4 days post dosing. The results from the PK/PD study suggests that STAT3 degradation in tumor of 〉90% is necessary for anti-tumor efficacy in vivo of KTX-201, but only for a limited duration, such as 4 days out of a weekly dosing cycle. Collectively, our data demonstrate that significant STAT3 degradation for a limited time during dosing interval with KTX-201 in ALCL promotes early changes in key signaling nodes involved with proliferation and cytokine stimulation, followed by profound changes in apoptotic proteins. By integrating mechanistic biology with a deep understanding of PK/PD and efficacy, this study provides a foundation for the clinical development of STAT3 degraders using intermittent dosing regimen for treatment of PTCL and other STAT3-dependent heme malignancies. Disclosures Rong: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Csibi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company, Ended employment in the past 24 months. Yang:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rusin:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Shi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dey:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karnik:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mayo:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yuan:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Chutake:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. McDonald:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zhu:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ji:Kymera Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Liu:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Li:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Walker:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gollob:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mainolfi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Desavi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.