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Introduction : CD47, a cell-surface ligand overexpressed in various malignancies, binds to SIRPα on effector macrophages to promote tumor cell evasion of phagocytosis. Blockade of the CD47-SIRPα interaction provides a pro-phagocytic signal which enhances phagocytosis mediated by tumor-targeting antibodies such as rituximab. Agents targeting CD47 in combination with rituximab have demonstrated promising clinical activity in R/R NHL; however, the broad expression of CD47 leads to frequent on-target, off-tumor toxicities, including treatment-emergent hemolytic anemia. CC-95251 is a novel, fully human immunoglobulin G1 antibody that binds to SIRPα on monocytes and macrophages to potently block the CD47-SIRPα interaction. Here we report interim results from a phase 1 study evaluating CC-95251 combined with rituximab in patients (pts) with R/R NHL. Methods: CC-95251-ST-001 (NCT03783403) is a multicenter, open-label, phase 1, dose-escalation and expansion study of CC-95251 in pts with advanced solid tumors and CD20+ R/R NHL. The primary objectives of the dose-escalation stage presented here are to evaluate the safety and tolerability of escalating doses of CC-95251 combined with rituximab and to define the maximum tolerated dose (MTD) and/or recommended phase 2 dose for the combination in pts with R/R NHL. Pts with CD20+ NHL who had progressed on standard anticancer therapy or for whom no approved conventional therapy was available were eligible for inclusion. Dose-escalation was conducted using an adaptive, 2-parameter Bayesian logistic regression model with overdose control. Pts were treated in 28-day cycles with CC-95251 administered intravenously at 3, 10, or 20 mg/kg every week (QW) and rituximab 375 mg/m 2 given on days 1, 8, 15, and 22 of cycle 1, on day 1 of cycles 2-5, and on day 1 of every other cycle from cycle 6 to 24 until disease progression or unacceptable toxicity. Results: As of April 30, 2021, 18 pts were enrolled and 17 had received ≥ 1 dose of CC-95251 and rituximab. Median age of the enrolled study population was 69 (range 30-84) years. Treated pts had received a median of 4 (range 1-7) prior systemic therapies, including 14/17 (82%) pts with confirmed prior rituximab exposure. Enrolled tumor types included R/R diffuse large B-cell lymphoma in 14 (78%) pts, follicular lymphoma in 2 (11%), and mantle cell lymphoma and marginal zone lymphoma in 1 (6%) pt each. Of the pts with available response data for prior lines of therapy (LOT), 7 (41%) had disease confirmed to be refractory to any prior LOT, including 5 (29%) refractory to rituximab-containing regimens, and 5 (29%) to their last LOT. Pts received a median of 3 (range 1-12) cycles of CC-95251, with a median duration of treatment of 14.1 (range 1.1-47.3) weeks. There were no CC-95251 dose reductions. Five (29%) pts experienced ≥ 1 treatment-emergent adverse events (TEAEs) leading to CC-95251 dose interruption. To date, the MTD has not been reached for the combination of CC-95251 and rituximab. The most common (≥ 30%) TEAEs of any grade were neutropenia (11/17 [65%]), infections (9/17 [53%]), hypokalemia (6/17 [35%]), and hypomagnesemia (6/17 [35%]; Table). Grade ≥ 3 TEAEs reported in ≥ 2 pts were neutropenia (9/17 [53%]) and infections (4/17 [24%]). No treatment-related anemia, a notable toxicity of some anti-CD47 therapies, was reported with CC-95251 and there were no treatment-related deaths. The overall response rate was 41% (7/17), with 2/17 (12%) pts achieving a complete response (Figure). The median time to response was 7.6 weeks. Median duration of response has not been reached as responses are ongoing. Pharmacokinetic (PK) analysis showed that CC-95251 exhibited dose-proportional increases in exposure at doses > 3 mg/kg QW indicating saturation of nonlinear clearance pathways or target saturation. Full receptor occupancy on peripheral monocytes was achieved at doses > 3 mg/kg QW. The estimated terminal half-life when in the linear range of clearance was approximately 12 days. No substantial differences in PK were observed at the given dose levels for CC-95251 monotherapy versus CC-95251 combined with rituximab. Conclusions: CC-95251, a novel anti-SIRPα antibody, demonstrated a manageable safety profile and promising efficacy in combination with rituximab in pts with heavily pretreated CD20+ R/R NHL. The study continues to enroll in the dose-expansion phase. Updated safety and efficacy data will be presented. Figure 1 Figure 1. Disclosures Strati: Astrazeneca-Acerta: Research Funding; Roche-Genentech: Consultancy. Hawkes: Regeneron: Speakers Bureau; Specialised Therapeutics: Consultancy; Bristol Myers Squib/Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck KgA: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel and accommodation expenses, Research Funding, Speakers Bureau; Janssen: Speakers Bureau; Antigene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Merck Sharpe Dohme: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees. Ghosh: Karyopharma: Consultancy, Honoraria; Genmab: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; Adaptive Biotech: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding, Speakers Bureau; TG Therapeutics: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria, Speakers Bureau; Genentech: Research Funding; AbbVie: Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; ADC Therapeutics: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Epizyme: Honoraria, Speakers Bureau. Tuscano: BMS, Seattle Genetics, Takeda, Achrotech, Genentech, Pharmacyclics, Abbvie: Research Funding. Chu: Abbvie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; BI: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria; Eli Lilly: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Merck KgaA: Other: DSMB. Patel: Bristol Myers Squibb: Current Employment. Burgess: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hege: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Chhagan: Bristol Myers Squibb: Current Employment. Boyanapalli: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Day: Tracey Day: Current Employment. Shen: Bristol Myers Squibb: Current Employment. Mehta: Affirmed; Kite/Gilead; Roche-Genetech; Celgene/BMS; Oncotartis; Innate Pharmaceuticals; Seattle Genetics; Incyte; Takeda; Fortyseven Inc/Gilead; TG Therapeutics; Merck; Juno Pharmaceuticals/Bristol Myers Squibb: Research Funding; Seattle Genetics; Incyte; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Seattle Genetics; Incyte; TG Therapeutics: Consultancy.

Introduction: BCMA is a tumor necrosis factor (TNF) receptor superfamily transmembrane glycoprotein essential for the maturation and survival of plasma cells. CC-93269 is an asymmetric 2-arm humanized IgG TCE that binds bivalently to BCMA and monovalently to CD3ε in a 2+1 format (Seckinger A, et al. Cancer Cell. 2017;31:396-410). The CC-93269-mediated interaction between T cells and BCMA-expressing myeloma cells induces T cell receptor/CD3 crosslinking leading to T cell activation, and release of proinflammatory cytokines and cytolytic enzymes, resulting in myeloma cell death. In preclinical studies with CC-93269 and related molecules, 2+1 BCMA TCEs induced tumor regression in animal models and promoted myeloma cell death in primary pt myeloma cells. Here we report interim results from a phase 1 dose-finding study (CC-93269-MM-001; NCT03486067) evaluating CC-93269 in pts with RRMM. Methods: Eligible pts had RRMM and had received ≥ 3 prior regimens without prior BCMA-directed therapy. In dose escalation, CC-93269 was administered intravenously over 2 hours on Days 1, 8, 15, and 22 for Cycles 1-3; Days 1 and 15 for Cycles 4-6; and on Day 1 for Cycle 7 and beyond, all in 28-day cycles. Dose escalation involved 2 stages: in stage 1, CC-93269 was given in fixed doses; in stage 2, pts received a fixed first dose on Cycle 1 Day 1, followed by intrapatient dose escalation on Cycle 1 Day 8. Primary objectives were to assess the safety and tolerability of CC-93269 and define the maximum tolerated dose (MTD), non-tolerated dose (NTD), and/or recommended phase 2 dose (RP2D). Minimal residual disease (MRD) was assessed after clinical response in pt bone marrow aspirate samples by Next Generation Flow using the EuroFlow panel. MRD negativity was reported only if a minimum sensitivity of < 1 tumor cell in 105 nucleated cells was achieved. Results: As of May 24, 2019, 19 pts had received CC-93269. Median age was 64 years (range 51-78), with a median of 6.2 years (range 1.4-13.9) since initial diagnosis. The median number of prior regimens was 6 (range 3-12) and included treatment with autologous stem cell transplantation (73.7%), allogenic stem cell transplantation (10.5%), lenalidomide (100%), pomalidomide (84.2%), bortezomib (100%), carfilzomib (84.2%), and daratumumab (DARA; 94.7%). All pts had MM refractory to their last line of therapy, with 16 (88.9%) refractory to DARA, 17 (89.5%) to their last proteasome inhibitor, and 16 (84.2%) to their last immunomodulatory agent. CC-93269 doses ranged from 0.15 to 10 mg; median duration of treatment was 14.6 weeks (range 1.6-32.0) with pts receiving a median of 4 cycles (range 1-8). Grade 3-4 treatment-emergent adverse events were reported in 15 (78.9%) pts and included 10 (52.6%) pts with neutropenia, 8 (42.1%) with anemia, 5 (26.3%) with infections, and 4 (21.1%) with thrombocytopenia. No pt required dose modifications. Cytokine release syndrome (CRS) was reported in 17 (89.5%) pts, the majority of whom reported a maximum grade 1 (n = 11 [57.9%]) or grade 2 (n = 5 [26.3%]), and occurred most frequently with the first or second dose (n = 22 of 27 events [81.5%]). CRS prophylaxis was implemented with dexamethasone for first dose and dose increases in pts receiving ≥ 6 mg. Of 27 CRS events, 8 (29.6%) were managed with dexamethasone and 10 (37.0%) with tocilizumab. One pt receiving 6 mg CC-93269 as first dose and 10 mg on Cycle 1 Day 8 died on study in the setting of CRS, with a potential infection as a contributing factor. Dose-related pharmacodynamic activity, including peripheral blood immune cell redistribution and transient release of pro- and anti-inflammatory cytokines, was observed in pts. Of the 12 pts treated with ≥ 6 mg CC-93269 in Cycle 1, 10 pts achieved a partial response (PR) or better (overall response rate; 83.3%), including 7 (58.3%) with a very good partial response (VGPR) or better and 4 (33.3%) with a stringent complete response (sCR) (Table); 9 (75.0%) pts achieved MRD negativity. The median time to response was 4.2 weeks (range 4.0-13.1), and 10 of 10 responses were ongoing with follow-up ranging from 2.1 to 4.7 months. The NTD, MTD, and RP2D have not yet been reached. Conclusions: CC-93269, a 2+1 BCMA TCE, shows a manageable safety profile and promising efficacy, including MRD-negative sCRs, in pts with heavily pretreated RRMM. The study continues to enroll in the dose escalation phase. Updated safety and efficacy data will be presented at the meeting. Disclosures Costa: Fujimoto Pharmaceutical Corporation Japan: Other: Advisor; Karyopharm: Consultancy; Abbvie: Consultancy; Sanofi: Consultancy, Honoraria, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Research Funding, Speakers Bureau. Wong:Genentech: Research Funding; Janssen: Research Funding; Celgene Corporation: Research Funding; Fortis: Research Funding; Juno: Research Funding. Bermúdez:MSD: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Fresenius: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. de la Rubia:AMGEN: Consultancy; Celgene Corporation: Consultancy; AbbVie: Consultancy; Takeda: Consultancy; Janssen: Consultancy. Mateos:Pharmamar: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria; EDO: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ocio:BMS: Honoraria; Sanofi: Research Funding; Mundipharma: Research Funding; Takeda: Consultancy, Honoraria; Seattle Genetics: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Array Pharmaceuticals: Research Funding; Pharmamar: Consultancy; Novartis: Consultancy, Honoraria; AbbVie: Consultancy; Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria. Rodríguez-Otero:Celgene Corporation: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria; Takeda: Consultancy; BMS: Honoraria; Kite Pharma: Consultancy. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria. Li:Celgene Corporation: Employment, Equity Ownership. Sarmiento:Celgene Corporation: Employment. Lardelli:Celgene Corporation: Employment, Equity Ownership. Gaudy:Celgene Corporation: Employment, Equity Ownership. Boss:Celgene Corporation: Employment, Equity Ownership. Kelly:Celgene Corporation: Employment. Burgess:University of California: Other: Volunteer clinical faculty, without salary, Patents & Royalties: Patent - T315A and F317I mutations of BCR-ABL kinase domain; Celgene Corporation: Employment, Equity Ownership, Patents & Royalties: Patent - CD47 antibodies and methods of use thereof. Hege:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties; Arcus Biosciences: Membership on an entity's Board of Directors or advisory committees; Society for Immunotherapy of Cancer: Membership on an entity's Board of Directors or advisory committees; Mersana Therapuetics: Membership on an entity's Board of Directors or advisory committees. Bensinger:Amgen, Celgene: Other: Personal Fees, Research Funding, Speakers Bureau; Takeda, Janssen: Speakers Bureau; Sanofi, Seattle Genetics, Merck, Karyopharm: Other: Grant.

Background : CD47 is a transmembrane protein ubiquitously expressed in human cells. CD47 is overexpressed in various malignancies and is correlated with negative prognosis in AML and MDS (Chao et al. Curr Opin Immunol.2012; Majeti et al. Cell.2009). Interaction of CD47 with signal-regulatory protein alpha (SIRPα) expressed on macrophages inhibits phagocytosis (Kim et al. Leukemia.2012). CC-90002, a humanized anti-CD47 monoclonal antibody, blocks CD47/SIRPα interactions, thereby enabling macrophage-mediated killing of tumor cells. In preclinical studies, CC-90002 demonstrated antibody-mediated phagocytosis of several hematologic cancer cell lines, including AML cells. CC-90002 also demonstrated a rapid and substantial reduction in tumor burden in AML xenograft models. Herein, we report results from CC-90002-AML-001 evaluating CC-90002 in patients (pts) with R/R AML and high-risk MDS. Methods : In this phase I multicenter study (NCT02641002), CC-90002 was administered intravenously once/week for 4 weeks of each 42-day cycle during cycles 1−4 then once every 4 weeks during a maintenance phase of 28-day cycles. Pts were enrolled in cohorts of escalating dose levels using a modified 3+3 design. The primary objectives were to determine preliminary safety and tolerability, non-tolerated dose (NTD), maximum tolerated dose (MTD), and/or recommended phase 2 dose. Secondary objectives were to measure preliminary efficacy, pharmacokinetics, and the presence and frequency of anti-drug antibodies (ADAs). Results: As of July 18, 2018, 24 pts with R/R AML and 4 pts with high-risk R/R MDS were enrolled. Pts received CC-90002 at 0.1 mg/kg (n=6), 0.3 mg/kg (n=6), 1 mg/kg (n=6), 2 mg/kg (n=4), and 4 mg/kg (n=6). Median age was 70 years (range, 28-85) and 16 (57%) were male. The most common AML subtypes were AML with myelodysplasia-related changes (n=9) and AML not otherwise specified (n=9). All 4 pts with MDS were classified as having refractory anemia with excess blasts-2 and high- or very high-risk disease per the Revised International Prognostic Index Scoring System. The median number of prior systemic anticancer regimens was 3 (range, 1-10), and 29% of pts had prior stem cell transplants. The median treatment duration was 6.9 weeks (range, 2-44). Four pts experienced a dose-limiting toxicity, consisting of grade 4 disseminated intravascular coagulation and grade 4 cerebral hemorrhage in 1 pt (0.1 mg/kg), grade 3 purpura in 1 pt (0.3 mg/kg), grade 4 congestive cardiac failure and grade 4 acute respiratory failure in 1 pt (1 mg/kg), and grade 4 sepsis in 1 pt (4 mg/kg). The most common (≥30%) any-grade treatment-emergent adverse events (TEAEs) were diarrhea (46%); thrombocytopenia (39%); febrile neutropenia and aspartate aminotransferase increased (36% each); and anemia, alanine aminotransferase increased, and cough (32% each). A total of 23 pts (82%) had serious TEAEs with febrile neutropenia (n=10), bacteremia (n=4), pneumonia (n=4), and general physical health deterioration (n=3) occurring in>2 pts. No TEAEs led to dose reductions; however, 7 pts (25%) discontinued due to TEAEs. Overall, 82% of pts were dependent on red blood cell (RBC) transfusions and CC-90002 treatment did not interfere with continued RBC transfusion in pts on study. No pts experienced hemolysis, tumor lysis syndrome, or macrophage activation/cytokine release syndrome. Sixteen pts died during the study. The best overall response observed was stable disease in 2 pts with MDS. CC-90002 serum exposures appeared to increase with doses above 0.3−4.0 mg/kg and the terminal half-life ranged from 4.6−17.0 hours. Development of ADAs targeting CC-90002 occurred at all dose levels tested and the proportion of pts testing positive for ADAs in cycle 1 increased over time (4/27 pts at day 8, 6/25 pts at day 15, and 8/22 pts at day 22). ADAs continued to be present across different doses with increases in median serum ADA titers after cycle 1. No apparent dose-ADA relationship was observed. Conclusion: CC-90002 showed a lack of objective responses in pts with R/R AML and high-risk MDS. The MTD and NTD were not established. The CC-90002-AML-001 study was discontinued in dose escalation for lack of preliminary monotherapy activity and evidence of ADAs in most pts. CC-90002 in combination with rituximab is being explored in CD20+ NHL to enhance efficacy of CD47 blockade while reducing ADAs (CC-90002-ST-001; NCT02367196). Disclosures Zeidan: BeyondSpring: Honoraria; Seattle Genetics: Honoraria; Acceleron Pharma: Consultancy, Honoraria, Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Otsuka: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Medimmune/AstraZeneca: Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Research Funding; Jazz: Honoraria; Ariad: Honoraria; Agios: Honoraria; Novartis: Honoraria; Astellas: Honoraria; Daiichi Sankyo: Honoraria; Cardinal Health: Honoraria. DeAngelo:Novartis: Consultancy, Research Funding; Jazz Pharmaceuticals Inc: Consultancy; Shire: Consultancy; Incyte: Consultancy; Blueprint: Consultancy, Research Funding; Pfizer: Consultancy; Amgen: Consultancy; GlycoMimetics: Research Funding; Celgene: Consultancy; Abbvie: Research Funding; Takeda Pharmaceuticals: Consultancy. Seet:University of California, Los Angeles: Employment. Tallman:Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Cellerant: Research Funding; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biosight: Research Funding; Biosight: Research Funding; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; UpToDate: Patents & Royalties; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding. Wei:Celgene Corp.: Employment, Equity Ownership. Li:Celgene Corp.: Employment, Equity Ownership. Hock:Celgene Corp.: Employment, Equity Ownership. Burgess:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties: Patent - CD47 antibodies and methods of use thereof; University of California: Other: Volunteer clinical faculty, without salary, Patents & Royalties: Patent - T315A and F317I mutations of BCR-ABL kinase domain. Hege:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties; Mersana Therapuetics: Membership on an entity's Board of Directors or advisory committees; Arcus Biosciences: Membership on an entity's Board of Directors or advisory committees; Society for Immunotherapy of Cancer: Membership on an entity's Board of Directors or advisory committees. Stock:Agios: Membership on an entity's Board of Directors or advisory committees; Research to Practice: Honoraria; UpToDate: Honoraria; Daiichi: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Kite, a Gilead Company: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Loss of all or part of chromosome 7 [-7/del(7q)] is a common, high-risk cytogenetic abnormality identified in a variety myeloid malignancies, including myelodysplastic syndrome (MDS), acute myeloid leukemia, therapy-related myeloid neoplasms, juvenile myelomonocytic leukemia (JMML) and chronic myelomonocytic leukemia (CMML). CUX1 is a non-clustered, homeodomain-containing transcription factor encoded on the commonly deleted region of 7q22. CUX1 inactivating mutations occur across solid tumors and myeloid neoplasms, and carry a poor prognosis in myeloid malignancies independently of 7q status. Previously, we reported that CUX1 is a conserved, haploinsufficient, myeloid tumor suppressor. To determine the mechanism by which CUX1 regulates leukemogenesis, we generated an inducible, shRNA-transgenic, Cux1 -knockdown mouse model. Insufficient Cux1 levels caused early mortality in mice, due to a spontaneous myeloproliferative disorder with features of CMML/JMML and MDS. Cux1 deficient mice had a significant increase in myelomonocytic cell numbers in the peripheral blood, bone marrow, and spleen. Cux1 deficiency led to extramedullary hematopoiesis, splenomegaly, and an increase in megakaryocyte numbers and megakaryocyte dysplasia. In addition, Cux1 is required for normal red cell development, as Cux1 knockdown led to a normocytic anemia due to an erythroblast maturation arrest. To identify CUX1 transcriptional targets, we performed gene expression profiling after CUX1 knockdown in CD34+ human hematopoietic stem and progenitor cells (HSPCs). Differentially expressed genes were enriched for genes deregulated in JMML and -7/del(7q) MDS, in addition to altered expression of quiescent, proliferative, and myeloid differentiation pathways. Consistent with these gene signatures, Cux1 maintains hematopoietic stem cell quiescence and represses HSPC proliferation, self-renewal, and granulocyte/monocyte differentiation. Thus Cux1 has pleotropic roles in multiple myeloid lineages. Deletion of CUX1, in the context of -7/del(7q), may facilitate myeloid transformation through aberrant transcriptional regulation of HSPC differentiation and proliferation. Disclosures No relevant conflicts of interest to declare.

Abnormal expression of the transcription factor EVI1 through chromosome 3q26 rearrangements has been implicated in the development of one of the most therapeutically challenging high-risk subtypes of acute myeloid leukemia (AML). Here we integrated genomic and metabolic screening of hematopoietic stem cells to reveal that EVI1 overexpression altered cellular metabolism. A pooled shRNA screen targeting metabolic enzymes identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as a druggable dependency in EVI1-positive AML. Of 18 screened AML cell lines harboring various genetic alterations, only the four EVI1-expressing lines exhibited markedly elevated CKMT1 protein expression and activity. Treatment of this cell line panel with either CKMT1-targeting shRNAs or cyclocreatine, an analog of the CKMT1 substrate creatine and inhibitor of the creatine biosynthesis pathway, showed that elevated CKMT1 protein expression correlated with sensitivity to CKMT1 pathway inhibition. Consistent with these data, flow cytometry analysis of a panel of 68 unselected primary AML patient specimens revealed that the four leukemias with the highest levels of EVI1 expression also had elevated CKMT1 protein levels and enhanced sensitivity to cyclocreatine treatment. We next established that enforced EVI1 expression increased CKMT1 protein and mRNA levels and that three independent shRNA molecules targeting EVI1 drastically reduced CKMT1 expression in two EVI1-positive AML cell lines. A luciferase-based reporter system established that RUNX1 represses CKMT1 expression through direct binding to its promoter. ChIP-qPCR approaches were then applied to dissect the sequential events involved in EVI1-induced CKMT1 upregulation and the possible role of RUNX1 as an intermediate. In both primary AML samples and cell lines, we determined that EVI1 represses RUNX1 expression by directly binding to its promoter. This, in turn, eliminates repressive RUNX1 binding at the CKMT1 promoter and thereby promotes CKMT1 expression. Based on these data, we explored the relationship between EVI1 and RUNX1 expression with CKMT1 mRNA levels in two AML transcriptional datasets (GSE14468 and GSE10358). We divided these cohorts into four subgroups with high versus low expression of EVI1 and RUNX1. Consistent with our mechanistic analysis, primary AML samples within the EVI1high/RUNX1low subgroup were significantly more likely to express high levels of CKMT1 than AML samples in the other three subgroups. CKMT1 promotes the metabolism of arginine to creatinine. To determine the effect of CKMT1 suppression on this pathway, we measured the metabolic flux of stable-isotope labeled L-arginine 13C6 through creatine synthesis using mass spectrometry. CKMT1-directed shRNAs or cyclocreatine selectively decreased intracellular phospho-creatine and blocked production of ATP by mitochondria. Salvage of the creatine pathway by exogenous phospho-creatine restored normal mitochondrial function, prevented the loss of viability of human EVI1-positive AML cells induced by cyclocreatine or CKMT1-directed shRNAs, and maintained the serial replating activity of Evi1-transformed bone marrow cells. Primary human EVI1-positive AML is frequently associated with somatic NRAS mutations. Thus, to investigate whether EVI1 over-expression sensitizes primary AMLs to CKMT1 inhibition in vivo, we transplanted primary NrasG12D mutant AMLs with and without elevated Evi1 expression into congenic recipient mice. In this system, Ckmt1 knockdown did not significantly alter the outgrowth of control Nras mutant AML cells compared to a shControl (63% versus 71%). By contrast, NrasG12D AML cells characterized by elevated Evi1 expression were profoundly depleted by Ckmt1 suppression to 2% versus 58% in shControl recipients. Consistent with these results, pharmacologic or genetic inhibition of the CKMT1-dependent pathway blocked disease progression and prolonged the survival of mice injected with human EVI1-positive cells but not with EVI1-negative cells, without noticeable cytotoxic effect on normal murine cells. In conclusion, we have integrated "omic" approaches to identify CKMT1 as a druggable liability in EVI-positive AML. This study supports a potential therapeutic avenue for targeting the creatine kinase pathway in EVI1-positive AML, which remains one of the worst outcome subtypes of AML. Disclosures DeAngelo: Incyte: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Baxter: Consultancy; Pfizer: Consultancy; Ariad: Consultancy. Stone:Pfizer: Consultancy; Agios: Consultancy; Jansen: Consultancy; Celator: Consultancy; Merck: Consultancy; Amgen: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy; Novartis: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Xenetic Biosciences: Consultancy; Sunesis Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy; Roche: Consultancy; Juno Therapeutics: Consultancy; ONO: Consultancy.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) raised the plasminogen activator (PA) activity of cultured human monocytes. This activity was characterized to be urokinase-PA (u-PA) by incubation with specific IgG and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography. Increased u-PA activity reflected GM-CSF- induction of u-PA mRNA levels. The stimulatory properties of GM-CSF for monocyte PA activity differed from those of interleukin-4, which induced monocyte tissue-type PA (t-PA) activity, and of interferon- gamma (IFN-gamma), which alone was not stimulatory but augmented lipopolysaccharide-induced t-PA activity. GM-CSF alone did not stimulate detectable monocyte t-PA activity but combined with IFN-gamma to promote this activity. Plasmin formation arising from GM-CSF-induced u-PA in monocytes may contribute to the matrix turnover involved in, eg, cell migration and inflammation, and may explain some of the pathology seen in GM-CSF transgenic mice.

Recent investigations into the relationship between cancer and cellular metabolism have revealed the strong dependency of different cancers on a diverse array of metabolic pathways. For instance, a variety of cancers depend on the PI3K/AKT pathway for a wide range of glucose-related mechanisms, including AKT-mediated membrane translocation of glucose transporters, activation of the glycolytic enzymes hexokinase and phosphofructokinase, and up-regulation of de novo fatty acid synthesis. A major area of therapeutic and biological interest in the study of cancer metabolism is the process by which these metabolic pathways become deregulated in the first place en route to, or as a result of, the development of cancer. The deregulation of metabolic pathways can occur through alterations in the cellular landscape brought about by mutations in metabolic enzymes (e.g., IDH1/IDH2 and SDH), aberrant expression of transcription factors, such as the proto-oncogene MYC, or the loss of tumor suppressors, such as p53. Through gene expression and metabolic profiling analyses, we found that the transcription factor EVI-1, whose overexpression in acute and chronic myeloid leukemia (AML and CML) is correlated with poor patient outcome, induced key metabolic perturbations in hematopoietic progenitor cells. These result in a decrease of the mitochondrial oxygen consumption rate, a blockade of the de novo purine and pyrimidine synthesis, and an increase in glycolysis. Using a library of pooled shRNAs targeting genes involved in each of these pathways, we then established a direct link between EVI-1 expression and the development of a heretofore undescribed cellular dependency on the overexpression of the ATP-buffering mitochondrial creatine kinase protein CKMT1B. We showed that EVI-1 directly promotes CKMT1B expression through repression of the master regulator of myeloid differentiation RUNX1. Alteration of the CKMT1B-dependent pathway, either with shRNA or with the small molecule cyclocreatine, impairs production of intracellular phospho-creatine, which in turn alters cell viability specifically in EVI-1-positive (n = 8) versus EVI-1-negative AML cell lines (n = 8), and in EVI-1-positive (n = 5) versus EVI-1-negative (n = 17) primary AML blasts. This decrease in cell viability is associated with the activation of an erythroid differentiation program concomitant with a downregulation of the immature lineage marker c-KIT, both induced by GSK3A/B inhibition. Overexpression of a constitutively activated form of GSK3B (S9A) or GSK3A (S21A) impairs the cell differentiation induced by CKMT1B inhibition. Finally, suppression of CKMT1B alleviates leukemic burden in vivo in two AML mouse models: i) an orthotopic model of transplanted human EVI1-positive AML cells and ii) a syngeneic model of transplanted murine NrasG12D + Evi1 AML cells. This new interplay between EVI-1 and the creatine pathway uncovers CKMT1B as a new target of interest in EVI-1-positive AML, a high-risk subtype of AML for which current treatment regimens remain inadequate. Disclosures DeAngelo: Novartis: Consultancy; Ariad: Consultancy; Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Incyte: Consultancy; Agios: Consultancy; Celgene: Consultancy. Stone:Celgene: Consultancy; Agios: Consultancy; Karyopharm: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Novartis: Research Funding; Celator: Consultancy; Merck: Consultancy; Roche/Genetech: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; AROG: Consultancy; Juno: Consultancy. Stegmaier:Novartis Pharmaceuticals: Consultancy.

The myelodysplastic syndromes (MDS) are clonal hematologic neoplasms characterized by normal, increased or decreased bone marrow cellularity, ineffective production of one or more blood cell lineages, morphologic dysplasia, and a variable risk of progression to acute myeloid leukemia. Somatic monosomy 7 (-7) and deletions affecting the long arm of chromosome 7 [del(7q)] are highly prevalent cytogenetic abnormalities in de novo and therapy-related MDS that are associated with advanced age, prior exposure to alkylating agents, a high risk of leukemic transformation, and intrinsic drug resistance. Although recent genome-wide and targeted DNA sequencing efforts have uncovered mutations in genes involved in chromatin modification, transcription, RNA splicing, and signal transduction in MDS, extensive sequencing and expression analysis have not revealed recurring “second hit” mutations of any gene located on chromosome band 7q22, which is commonly deleted in patients with myeloid malignancies. Therefore, answering the long-standing question of how recurrent deletions of the long arm of chromosome 7 contribute to initiation, maintenance, and clinical outcome of MDS remains a fundamental challenge. Given the importance of understanding the biology of -7/del(7q) and the urgent need to develop new mechanism-based treatments for hematologic cancers with these deletions, we generated mice with a heterozygous germline deletion of a 2 Mb interval of chromosome band 5A3 syntenic to an interval of human chromosome band 7q22 commonly deleted in human patients (Blood 88:1930,1996). We find that 5A3 haploinsufficiency perturbs hematopoietic stem cell (HSC) development and function in vivo. 5A3+/del mice exhibit a reduced bone marrow cellularity, an expanded proportion of long-term HSCs, and an altered distribution of lineage-committed progenitors. 5A3+/del HSCs display impaired competitive reconstitution of lymphoid hematopoietic cells and a myeloid output bias, but also paradoxically show enhanced stem cell reconstitution upon aging and after serial transplantation. These defects are cell autonomous. Like WT HSCs, 5A3+/del HSCs exhaust their self-renewal potential by the tertiary round of transplantation. Taqman quantitative real-time PCR confirmed that all seven genes within the deleted interval that are expressed at detectable levels in HSC and multi-potent progenitors (MPP)(Mll5, Armc10, Psmc2, Dnajc2, Orc5l, Pmpcb and Napepld) showed ~50% reduction in transcripts in 5A3+/delHSC and MPP. Transcriptome sequencing (RNA-seq) analysis identified broad changes in lineage signature gene expression, as well as down-regulation of genes and pathways involved in oxidative phosphorylation (OXPHOS) in 5A3+/del HSCs. Whereas reactive oxygen species (ROS) are increased in aged 5A3+/del MPP, treatment of 5A3+/delmice with antioxidant N-Acetyl Cysteine failed to correct the HSC defects, suggesting that increased ROS is not the primary cause of these hematopoietic defects. Together, the abnormal properties of 5A3+/del hematopoietic cells support a mechanistic role of 7q22 deletions as contributing to cardinal features of MDS, which include impaired differentiation, myeloid lineage output bias, and a pronounced age-associated increase in disease incidence. Disclosures No relevant conflicts of interest to declare.

Functional analysis of therapeutic outlier responses in cancer therapy can identify unexpected synthetic lethal interactions and uncover biomarkers that predict enhanced sensitivity to targeted agents. We performed preclinical trials of the MEK inhibitor PD0325901 (PD901) in primary murine acute myeloid leukemias (AMLs) generated using retroviral insertional mutagenesis in KrasG12D “knock-in” mice. Similar to previous observations Nf1 and Nras mutant leukemias, treatment with PD901 prolonged survival in six independent leukemias harboring diverse retroviral integrations. Intriguingly, one outlier leukemia, clone Kras.101, demonstrated a greater than 4-fold improvement in median overall survival when treated with the MEK inhibitor. Importantly, this AML demonstrated a novel retroviral integration at relapse, indicating clonal evolution during treatment. The relapsed clone displayed phenotypic resistance upon transplantation into secondary recipients and retreatment with PD901. We performed a detailed analysis of the paired PD901-sensitive/resistant Kras.101 clones to identify the mediators of mitogen-activated protein kinase pathway (MAPK) addiction. Functional studies demonstrated that the novel dominant retroviral integration in the PD901 resistant clone did not directly alter drug response. However, genome-wide analysis of copy number alterations revealed a gain of chromosome 6 involving the Kras locus in the resistant clone, which was confirmed by spectral karyotyping and fluorescence in situ hybridization. Surprisingly, the PD901-sensitive leukemia harbored two copies of oncogenic KrasG12D due to somatic uniparental disomy, while the resistant clone harbored two mutant and one wild-type Kras alleles. This observation and paired single nucleotide polymorphism analysis indicated that the PD901-resistant leukemia was evolutionarily ancestral to the drug sensitive clone. Consistent with this hypothesis, competitive repopulation experiments with fluorescently labeled leukemia cells demonstrated increased fitness of the drug sensitive leukemia in the absence of the MEK inhibitor and over-expression of wild type K-Ras in the drug-sensitive leukemia reduced fitness in vivo. Finally, utilizing a tetracycline-inducible system, we found that wild-type Kras expression enhances the fitness of the Kras.101 clone only in the presence of the MEK inhibitor. Together, these data suggest that loss of wild-type Kras imparts a fitness advantage at the expense of increased MAPK pathway addiction, which results in enhanced sensitivity to MEK inhibition in leukemias driven by oncogenic K-Ras. Disclosures No relevant conflicts of interest to declare.

Oncogenic NRAS mutations are highly prevalent in hematologic malignancies. In acute myeloid leukemia (AML), genetic analysis supports the hypothesis that NRAS mutations cooperate with antecedent molecular lesions in leukemogenesis. Furthermore, NRAS mutations identified at diagnosis may disappear at relapse, raising questions regarding the potential clinical benefits of inhibiting oncogenic N-Ras in AML. To directly investigate the consequences of Nras inactivation in normal hematopoiesis, we used the Mx1-Cre transgene to inactivate a conditional mutant Nras allele and analyzed hematopoiesis and hematopoietic stem and progenitor cells (HSPC) under normal and stressed conditions. We show that HSPCs lacking Nras expression are functionally equivalent to normal HSPCs in the adult mouse. Importantly, shRNA-mediated knockdown in human AML cell lines and primary mouse leukemias with oncogenic NRAS/Nras mutations revealed dependence on continued oncogene expression in vitro and in vivo. Next, we interrogated the functional consequences of pharmacologic inhibition of the canonical Ras effector pathways, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways,alone and in combination. Recipient mice transplanted with five independent primary mouse AMLs generated by infecting NrasG12D “knock in” mice with the MOL4070LTR retrovirus (Li et al, Blood 2011; 117:2022) were treated with the allosteric MEK inhibitors PD0325901 (PD901) or trametinib or the PI3K inhibitor GDC-0941. Both MEK inhibitors significantly prolonged survival and reduced proliferation and blast colony formation, but did not induce apoptosis, differentiation, or promote clonal evolution. PI3K inhibition alone was ineffective in vivo and combinations of MEK and PI3K inhibitors were no better than MEK inhibition alone. All mice ultimately succumbed from progressive leukemia. These data, along with observations that Nras is dispensable for normal hematopoiesis, validate oncogenic N-Ras signaling as a therapeutic target in AML and support testing combination regimens that include MEK inhibitors in leukemias harboring NRAS mutations. Disclosures No relevant conflicts of interest to declare.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) raised the plasminogen activator (PA) activity of cultured human monocytes. This activity was characterized to be urokinase-PA (u-PA) by incubation with specific IgG and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography. Increased u-PA activity reflected GM-CSF-induction of u-PA mRNA levels. The stimulatory properties of GM-CSF for monocyte PA activity differed from those of interleukin-4, which induced monocyte tissue-type PA (t-PA) activity, and of interferon-gamma (IFN-gamma), which alone was not stimulatory but augmented lipopolysaccharide-induced t-PA activity. GM-CSF alone did not stimulate detectable monocyte t-PA activity but combined with IFN-gamma to promote this activity. Plasmin formation arising from GM-CSF-induced u-PA in monocytes may contribute to the matrix turnover involved in, eg, cell migration and inflammation, and may explain some of the pathology seen in GM-CSF transgenic mice.

Point mutations in the kinase domain of BCR-ABL are the most common cause of resistance to imatinib therapy in CML. E255K and T315I are detectable before the initiation of imatinib therapy in a small population of CML patients, suggesting that certain imatinib-resistant BCR-ABL mutants display altered biological fitness relative to wild-type p210 BCR-ABL. To test this hypothesis, we performed primary murine bone marrow transformation assays using BCR-ABL-expressing retroviruses. In a survey of six clinically imatinib-resistant mutations (Y253F, Y253H, E255K, E255V, T315I, M351T) as well as five additional mutations that confer in vitro resistance to the dual SRC/ABL inhibitor dasatinib (L248R, V299L, T315A, F317L, F317V), we identified several that altered oncogenic fitness either positively or negatively relative to p210. To elucidate the molecular basis for altered transformation potential, we examined the kinase activity of p210 versus mutant full-length BCR-ABL. Enzyme kinetics studies with an optimized Abl peptide substrate as well as gel-based in vitro kinase assays illustrated that intrinsic kinase activity does explain some, but not all, of the mutant oncogenic potency phenotypes. The imatinib- and dasatinib-resistant mutant T315I, for instance, was a gain-of-fitness mutant in the bone marrow assay but exhibited a three-fold lower Vmax than p210. In contrast, T315A, a dasatinib-resistant mutation at the T315 gatekeeper residue, exhibits reduced transforming potential relative to wt p210 and is completely unable to phosphorylate Abl substrates in the kinase assays, although it does retain the ability to autophosphorylate. Subsequent kinase assays on immunoprecipitations from stably expressing IL-3-independent Ba/F3 cells illustrated that certain mutants bind and phosphorylate distinct substrates from p210. We further delineated signaling differences between p210 and the mutant panel by utilizing a mass spectrometry-based global phosphorylation profiling technique. Trypsinized proteins from stably expressing p210 and mutant Ba/F3 cells were immunoprecipitated using anti-phosphotyrosine antibodies, then identified and quantified by two dimensional mass spectrometry. Phosphorylation of specific substrates correlates with gain- or loss-of fitness observed in the bone marrow assay. We conclude that certain BCR-ABL kinase domain mutations, in addition to conferring drug resistance, also affect the oncogenic potency of BCR-ABL by altering intrinsic kinase activity as well as downstream targets. Elucidation of alternative signaling pathways activated by these mutants by the combination of biochemical and mass spectrometric methods may provide novel therapeutic targets for imatinib- and dasatinib-resistant CML.

A novel dual SRC/ABL kinase inhibitor, BMS-354825, is showing promise for the treatment of imatinib-resistant chronic myeloid leukemia not only in vitro (Shah NP, et al., Science 305:399), but also in a phase I clinical trial (ASH abstract: Sawyers CL, et al.) Resistance to imatinib is increasingly found in patients due to point mutations in the BCR-ABL kinase domain that do not impair kinase activity but prevent drug binding. BMS-354825 is more potent than imatinib and retains activity against 14 of 15 imatinib-resistant BCR-ABL mutants in vitro. The compound’s ability to inhibit imatinib-resistant forms of BCR-ABL is presumed to be due to its relaxed binding requirements, whereas imatinib requires the adoption of a closed conformation of the kinase to bind. We addressed the hypothesis that the relaxed binding requirements of BMS-354825 would limit the range of BCR-ABL mutations that confer drug resistance. To address this question, we employed a saturation mutagenesis experiment as described by others (Azam M, et al., Cell 112:831) and found that the spectrum of BMS-354825-resistant mutants was reduced compared to that of imatinib. In a series of such screens, mutations at only four amino acids have been isolated, two of which account for the vast majority of resistant clones. In contrast, Azam et al. isolated over 20 mutations in a screen for imatinib resistance, a finding which has been generally reproduced in our lab. All four BMS-354825-resistant mutations map to known BMS-354825 contact residues as shown by co-crystallographic studies (ASH abstract: Tokarski JS et al., Bristol-Myers Squibb). Mutations at L248, T315, and F317 show BMS-354825 resistance and have been previously reported to confer imatinib resistance. Mutation at V299 represents a novel mode of resistance. Interestingly, some point mutations conferring BMS-354825 resistance were at positions known to be mutated in cases of imatinib resistance, but the mutated residues differed. Furthermore, the identity of the mutated residue was crucial in conferring sensitivity or resistance to an individual drug as shown by comparison of cellular IC50’s (see table). For example, F317L was shown previously to confer imatinib resistance. F317V, on the other hand, demonstrates relative BMS-354825-resistance but is still exquisitely sensitive to imatinib. In a screen for mutants simultaneously resistant to both drugs, we consistently recover 30–50 fold fewer mutant clones compared to single drug treatment. All such clones isolated to date encode for T315I. Kinase domain point mutation is becoming an increasingly encountered clinical problem in diseases treated with small molecule inhibitors. Our findings suggest that combination therapy with imatinib and BMS-354825 may be of clinical utility in CML, particularly by delaying the development of resistance. IC50 for growth (nM) Baf3 Clone imatinib BMS-354825 p210 wt < 1,000 < 5 T315I > 10,000 > 500 T315A 1,000 100 F317L 2,000 10 F317V < 1,000 60 V299L 1,000 20 L248R > 10,000 20

Tissue-type plasminogen activator (t-PA) is involved in the lysis of blood clots (fibrinolysis) and is used clinically for this purpose. Endothelial cells are one source of the t-PA present in blood. We report here that interleukin-4 (IL-4) (0.1 to 0.25 U/mL; 1 to 3 x 10(- 11) mol/L), but not interferon-gamma (IFN-gamma), elevates t-PA messenger (m)RNA expression and secretion of t-PA activity by human monocytes, with the maximum response at 2.5 U/mL. Supernatant t-PA activity was detected within three hours of exposure to IL-4 and maximum activity within six hours. Thus, IL-4 may control fibrin deposition at sites of inflammation during cell-mediated immune responses, as well as having a therapeutic role in thrombolysis.

Tissue-type plasminogen activator (t-PA) is involved in the lysis of blood clots (fibrinolysis) and is used clinically for this purpose. Endothelial cells are one source of the t-PA present in blood. We report here that interleukin-4 (IL-4) (0.1 to 0.25 U/mL; 1 to 3 x 10(- 11) mol/L), but not interferon-gamma (IFN-gamma), elevates t-PA messenger (m)RNA expression and secretion of t-PA activity by human monocytes, with the maximum response at 2.5 U/mL. Supernatant t-PA activity was detected within three hours of exposure to IL-4 and maximum activity within six hours. Thus, IL-4 may control fibrin deposition at sites of inflammation during cell-mediated immune responses, as well as having a therapeutic role in thrombolysis.