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Phosphatidylinositol 3 kinase (PI3K) inhibitors such as idelalisib have been associated with potentially severe autoimmune toxicity. In the present study, we demonstrate that relapsed refractory patients with chronic lymphocytic leukaemia treated with idelalisib rituximab on the phase III registration trial show uniform decrease in regulatory T cells (Tregs) and increase in CD8 T cells with treatment. Patients who do not develop toxicity show enrichment for T cells expressing multiple chemokine receptors, while those who do develop toxicity have an activated CD8 T cell population with T helper 17 cell differentiation at baseline, which then increases, leading to an increased CD8:Treg ratio that likely triggers autoimmune toxicity.

ispartof: LEUKEMIA vol:36 issue:4 pages:1171-1175 ispartof: location:England status: published

Background: Acalabrutinib (A) is a next-generation, highly selective, covalent Bruton tyrosine kinase (BTK) inhibitor approved for marketing by the US FDA in patients (pts) with chronic lymphocytic leukemia (CLL). While responses to A monotherapy are durable in CLL, response depth may be enhanced with combination therapy and potentially allow treatment discontinuation. The safety and efficacy of A plus a CD20 antibody (obinutuzumab [O] or rituximab [R]) and the BCL-2 inhibitor venetoclax (V) were examined in a phase 1b study (CL-003; NCT02296918) in relapsed/refractory (R/R) or treatment-naïve (TN) CLL pts. Methods: Pts were aged ≥18 years with intermediate or high-risk CLL (R/R or TN) and an Eastern Cooperative Oncology Group performance status (ECOG PS) ≤2. Prior BTK inhibitor treatment was allowed for R/R pts if discontinuation was not due to CLL progression. R/R CLL pts received oral (PO) A 100 mg twice daily (BID) until progression, plus intravenous (IV) R 375 mg/m2 for 9 infusions (cycles 2-7), plus V PO daily per standard dosing (cycles 3-15). TN CLL pts received PO A and V (as above) plus IV O per standard dosing (cycles 2-7). The primary endpoint was safety. Secondary endpoints included investigator-assessed overall response rate (ORR, partial response [PR] or better) at cycle 16, complete response (CR) rate, undetectable minimal residual disease (uMRD) rate, duration of response (DOR), progression-free survival (PFS), overall survival (OS), and pharmacokinetics (PK). Results: Twelve pts were included in each cohort (R/R: male, n=9 [75%], median [range] age: 66 [55-72] y; TN: male, n=9 [75%], median [range] age: 61 [42-73] y). All pts had ECOG PS ≤1; 6/8 (75%) R/R and 8/10 (80%) TN pts had unmutated IGHV ( At a median follow-up of 23.2 mo (range: 19.8-25.3) in R/R pts and 22.0 mo (range: 19.5-24.4) in TN pts, 11 (92%) and 10 (83%) pts remained on treatment, respectively. Reasons for treatment discontinuation were adverse events (AEs) in 1 pt (8%) in each cohort (R/R: grade 1 purpura; TN: grade 2 head discomfort), and withdrawal by 1 pt (8%) in the TN cohort. The median number of treatment cycles in R/R pts was 23 (range: 14.9-27.5) for A, 6 (6-6) for R, and 13 (10.0-13.7) for V, and in TN pts was 23 (18.0-24.8) for A, 6 (6-6) for O, and 13 (13.0-13.5) for V. Reported AEs were similar to the individual agents' toxicity profiles (Table 1). Among AEs of interest, 7 R/R pts (58%) and 4 TN pts (33%) had cardiac AEs; grade ≥3 cardiac AEs were reported in 1 pt (8%). Atrial fibrillation was reported in 1 pt (grade 3; R/R pt with prior atrial fibrillation history). No ventricular arrhythmias were reported. Three R/R pts (25%) and 7 TN pts (58%) had hypertension AEs; none were grade ≥3. The most frequent infections (≥40%) were upper respiratory tract infection (URTI; 50%) in R/R pts and URTI (67%), sinusitis (42%), and nasopharyngitis (42%) in TN pts. Grade ≥3 infections occurred in 3 TN pts (lung infection, n=2 [17%]; prostate infection, staphylococcal bacteremia, staphylococcal sepsis, n=1 [8%] each) and no R/R pts. Six R/R pts (50%) and 3 TN pts (25%) had infusion-related reactions; none were grade ≥3. One R/R pt (8%) and 6 TN pts (50%) had decreased neutrophil counts; all were grade ≥3. No tumor lysis syndrome AEs, Richter transformations, or deaths were reported. After 16 cycles, ORR was 92% (95% CI: 62-100) in R/R pts and 100% (95% CI: 74-100) in TN pts. At the time of data cutoff, 50% of pts in each cohort had achieved CR or CR with incomplete marrow recovery (CRi). All pts with CR or CRi achieved uMRD (10-4) in peripheral blood (PB) at the time of CR/CRi or earlier. Overall, 8 (67%) R/R and 9 (75%) TN pts achieved uMRD (10-4) in PB at cycle 10 (Table 2). Median DOR, PFS, and OS were not reached in either group. Estimated 18-mo PFS and OS rates were 100% (95% CI: not estimable) in both cohorts. The PK of A and its active metabolite, ACP-5862, were consistent when given as monotherapy or with V, R, or O. The PK of V remained within the range observed with monotherapy (Salem et al. J Clin Pharmacol. 2017;57:484-492) when given with A. Conclusions: Combination therapy with A plus an anti-CD20 antibody and BCL-2 inhibitor leads to a tolerable safety profile with high CR and uMRD rates in both R/R and TN CLL pts, with minimal to no drug-drug interactions. Disclosures Woyach: Pharmacyclics, Janssen, Morphosys, Karyopharm, Verastem, Abbvie, Lox: Research Funding; Pharmacyclics LLC, an AbbVie Company, AbbVie, Janssen, AstraZeneca, ArQule: Honoraria; Janssen, Pharmacyclics, AstraZeneca, Abbvie, Arqule: Consultancy. Blachly:AbbVie, AstraZeneca, KITE Pharma: Consultancy. Rogers:Janssen: Research Funding; AstraZeneca: Consultancy, Other: Travel Funding; Pharmacyclics: Consultancy; AbbVie: Consultancy, Research Funding; Genentech: Research Funding; Acerta Pharma: Consultancy. Lozanski:Genentech, Novartis, Beckman Coulter: Research Funding. Andersen:Ohio State University: Current Employment, Research Funding. Patel:AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Munugalavadla:Acerta Pharma/AstraZeneca: Current Employment, Current equity holder in publicly-traded company; Gilead Sciences: Current equity holder in publicly-traded company, Other: Family Association. Butturini:Puma Biotechnology: Divested equity in a private or publicly-traded company in the past 24 months; Dynavax, Puma Biotechnology: Ended employment in the past 24 months; Roche/Genentech, Pfeizer, AstraZeneca, Amgen: Current equity holder in publicly-traded company; Acerta Pharma: Current Employment. Xu:Acerta Pharma: Current Employment, Research Funding; AstraZeneca: Research Funding. Wang:Acerta Pharma: Current Employment. Byrd:Acerta Pharma: Research Funding; Syndax: Research Funding; Novartis: Research Funding; Kartos Therapeutics: Research Funding; Trillium: Research Funding; Leukemia and Lymphoma Society: Other; Janssen: Consultancy; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, BeiGene: Research Funding; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, Novartis, Janssen: Speakers Bureau; Pharmacyclics LLC, an AbbVie Company, Janssen, Novartis, Gilead, TG Therapeutics: Other; Vincera: Research Funding.

Context: Results from ELEVATE-TN (NCT02475681) at a median follow-up of 28.3 months demonstrated superior efficacy of acalabrutinib (A) ± obinutuzumab (O) compared with O + chlorambucil (Clb) in TN CLL (Sharman et al. Lancet 2020;395:1278-91). Objective: To report the results from a 4-year update of ELEVATE-TN. Design: Randomized, multicenter, open-label, 3-arm, Phase 3 study. Patients: TN CLL. Interventions: Patients received A±O or O+Clb. Crossover to A monotherapy was permitted in patients who progressed on O+Clb. Main Outcome Measures: Investigator-assessed (INV) progression-free survival PFS, INV ORR, OS, and safety were evaluated. Results: 535 patients (A+O, n=179; A, n=179; O+Clb, n=177) were randomized with a median age of 70 y; 14% had del(17p) and/or mutated TP53. At a median follow-up of 46.9 months (range, 0.0–59.4; data cutoff: Sept 11, 2020), median PFS was not reached (NR) for A+O and A patients vs 27.8 months for O+Clb patients (both P Conclusions: With a median follow-up of 46.9 months (~4y), the efficacy and safety of A+O and A monotherapy was maintained, with an increase in CR since the interim analysis (from 21% to 27% [A+O] and from 7% to 11% [A]) and low rates of discontinuation.

Background: First-line conventional chemoimmunotherapy in MCL can be improved. Promising results have been seen with Bruton tyrosine kinase inhibitors (BTKis) in combination with venetoclax (V) and an anti-CD20 monoclonal antibody in patients (pts) with relapsed/refractory or TN MCL. Acalabrutinib (A) is a next-generation, highly selective, covalent BTKi currently approved for relapsed/refractory MCL. We report initial safety and efficacy results of the ongoing, multicenter, open-label phase 1b study of A, V, and rituximab (R) (AVR) in TN MCL. Methods: TN MCL pts aged ≥18 y with ECOG PS ≤2 were eligible. Starting on cycle 1 day 1, A was administered at 100 mg BID until disease progression or discontinuation for other reasons. R was administered at 375 mg/m 2 on day 1 of each 28-day cycle for 6 cycles, followed by maintenance every other cycle for pts achieving complete response (CR) or partial response (PR), through cycle 24. Starting on cycle 2 day 1, V was administered via an initial 5-wk ramp-up schedule (20, 50, 100, 200, and 400 mg/d) to 400 mg/d, through cycle 25. Dose-limiting toxicity (DLT) was assessed from cycle 2 day 1 to cycle 3 day 28. Primary endpoint was AVR safety. Secondary endpoints were overall response rate (ORR), duration of response (DOR), and progression-free survival (PFS) per Lugano criteria. Positron-emission tomography (PET)/computed tomography (CT) scans were performed after 3 and 6 cycles and to confirm CR at any time. CT scans were performed after 3, 6, 9, and 12 cycles, and then every 6 cycles. Longitudinal minimal residual disease (MRD) was assessed using the clonoSEQ assay in peripheral blood at PR, CR, every 6 cycles post-CR, and treatment end. Results: 21 pts were enrolled (median age 66 y [range 51-85]; ECOG PS ≤1 20 [95%]; Ann Arbor stage IV disease 19 [90%]; bulky disease >5 cm 7 [33%]; intermediate- and high-risk simplified MCL International Prognostic Index scores 11 [52%] and 4 [19%], respectively; blastoid variant 1 [5%]; and Ki-67 proliferation index ≥50% 3 [14%]). Fifteen (71%) pts had bone marrow (BM) involvement at baseline. As of March 19, 2021, median time on study was 16 mo (range 8-26.2). Median (range) number of cycles administered was 15 (7-27) for A, 13.5 (5-23) for 400 mg daily V, and 12 (6-15) for R. Seventeen (81%) pts remain on study treatment and 4 (19%) have discontinued (progressive disease: n=1; COVID-19 infection: n=3). No DLTs were observed; V 400 mg daily after ramp-up was the dose chosen for triple therapy. Most common any-grade AEs in ≥20% of pts were diarrhea (13 [62%]), headache (11 [52%]), fatigue (10 [48%]), neutropenia (6 [29%]), paresthesia (6 [29%]), cough (6 [29%]), dyspnea (6 [29%]), myalgia (5 [24%]), dizziness (5 [24%]), and hypoesthesia (5 [24%]). Grade 3/4 AEs in ≥2 pts were neutropenia (5 [24%]) and pneumonia (2 [10%]). Serious any-grade AEs in ≥2 pts were COVID-19 infection (4 [19%]) and pneumonia (2 [10%]). In the 4 pts with COVID-19 infection, the events led to triple-drug discontinuation and death in 3 pts and to dose holds of A and V and event resolution in 1 pt (all considered unrelated to study treatment). Diarrhea led to V dose reduction in 1 pt. AEs led to dose holds in 12 (57%) pts and were associated with A, V, and R in 52%, 48%, and 14%, respectively. Events of clinical interest are shown in Table 1. At the end of cycle 6, ORR was 100%, with CR/PR in 90%/10% by PET/CT alone (11 of the 13 CRs by PET/CT lacked BM confirmation); the CR/PR rate by Lugano criteria with BM confirmation was 38%/62% (Table 2). Median DOR was 19 mo (95% CI 17-not estimable [NE]) overall, and not reached when the 3 pts with COVID-19 deaths were censored. Median PFS and OS were not reached. The 1-y PFS and OS rates were 89% (95% CI 62-97) and 95% (95% CI 71-99), respectively. Treating the 3 COVID-19 deaths as censored, the 1-y PFS rate was 93.8% (95% CI 63.2-99.1). Median time to initial response and best response was 2.8 mo. Twelve of 16 (75%) pts with available MRD results at cycle 6 achieved MRD negativity (10 -6), including 6 pts with CR who attained MRD negativity at the time of CR or earlier and continued to be MRD negative at cycle 24. Six pts with PR also achieved MRD negativity, suggesting deeper molecular responses. One pt with PR became MRD positive before having clinical disease progression. Conclusions: The triple combination of acalabrutinib, venetoclax, and rituximab is well tolerated and provides a 100% clinical response rate and high rate of complete molecular response in TN MCL. Figure 1 Figure 1. Disclosures Wang: Loxo Oncology: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Honoraria, Research Funding; Physicians Education Resources (PER): Honoraria; Scripps: Honoraria; InnoCare: Consultancy, Research Funding; Molecular Templates: Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; Oncternal: Consultancy, Research Funding; BGICS: Honoraria; DTRM Biopharma (Cayman) Limited: Consultancy; Mumbai Hematology Group: Honoraria; Anticancer Association: Honoraria; Genentech: Consultancy; OMI: Honoraria; Dava Oncology: Honoraria; Epizyme: Consultancy, Honoraria; CAHON: Honoraria; CStone: Consultancy; Bayer Healthcare: Consultancy; Chinese Medical Association: Honoraria; Imedex: Honoraria; Newbridge Pharmaceuticals: Honoraria; Moffit Cancer Center: Honoraria; Clinical Care Options: Honoraria; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Kite Pharma: Consultancy, Honoraria, Research Funding; Hebei Cancer Prevention Federation: Honoraria; Lilly: Research Funding; Celgene: Research Funding; BioInvent: Research Funding; VelosBio: Consultancy, Research Funding; Juno: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; The First Afflicted Hospital of Zhejiang University: Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding. Robak: Biogen, Abbvie, Octapharma, Janssen: Honoraria, Other: Advisory board; AstraZeneca, Abbvie, Janssen, Octapharma, Gilead,Oncopeptides AB, Pharmacyclics, Pfizer, GlaxoSmithKline, Biogen: Research Funding; Medical University of Lodz: Current Employment. Maddocks: Karyopharm: Divested equity in a private or publicly-traded company in the past 24 months; KITE: Divested equity in a private or publicly-traded company in the past 24 months; Celgene: Divested equity in a private or publicly-traded company in the past 24 months; ADC Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months; Beigene: Divested equity in a private or publicly-traded company in the past 24 months; Merck: Divested equity in a private or publicly-traded company in the past 24 months; Seattle Genetics: Divested equity in a private or publicly-traded company in the past 24 months; Janssen: Divested equity in a private or publicly-traded company in the past 24 months; Novatis: Divested equity in a private or publicly-traded company in the past 24 months; BMS: Divested equity in a private or publicly-traded company in the past 24 months; Morphosys: Divested equity in a private or publicly-traded company in the past 24 months; Pharmacyclics: Divested equity in a private or publicly-traded company in the past 24 months. Phillips: Abbvie: Consultancy, Research Funding; ADCT: Consultancy; AstraZeneca: Consultancy; Bayer: Research Funding; BMS: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, 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; KITE: Membership on an entity's Board of Directors or advisory committees; Lymphoma Connect: Honoraria; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Smith: Beigene: Consultancy, Research Funding; Merck Sharp & Dohme Corp: Research Funding; Millenium/Takeda: Consultancy; Incyte: Consultancy; Acerta Pharma BV: Research Funding; Ignyta (spouse): Research Funding; Bayer: Research Funding; AstraZeneca: Consultancy, Research Funding; Karyopharm: Consultancy; ADC Therapeutics: Consultancy; Genentech: Research Funding; Portola Pharmaceuticals: Research Funding; Ayala (spouse): Research Funding; Incyte Corporation: Research Funding; De Novo Biopharma: Research Funding; Bristol Myers Squibb (spouse): Research Funding; KITE pharm: Consultancy. Calvo: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Wun: AstraZeneca: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months. Munugalavadla: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Jurczak: AstraZeneca, BeiGene, Janssen, Loxo Oncology, Sandoz, Roche: Membership on an entity's Board of Directors or advisory committees; Abbvie, AstraZeneca, BeiGene, Celtrion, Celgene, Debbiopharm, Epizyme, Incyte, Janssen, Loxo Oncology, Merck, Mei Pharma, Morphosys, Novo Nordisk, Roche, Sandoz, Takeda, TG Therapeutics: Research Funding.

Background: Bruton's tyrosine kinase (BTK) inhibition is a key component in B-cell receptor signaling and is one of the most prominent therapeutic targets in hematologic malignancies. Acalabrutinib is a highly selective, covalent, potent next-generation inhibitor of BTK (Barf et al, 2017) and is approved for the treatment of mantle cell lymphoma and chronic lymphocytic leukemia (CLL). However, a small subset of patients develop resistance to acalabrutinib over time. While the major mechanism of resistance to covalent BTK inhibitors is explained by acquired mutations in BTK-Cys481 and its downstream target PLCg2, additional mechanisms are under investigation. Previously, we presented that high surface expression of CD49d (VLA-4) and CD79B correlates with acalabrutinib resistance in CLL patients (Bibikova et al, 2019). In the present study, we performed optimized whole genome methylation sequencing on 42 clinical samples from 22 patients in the ACE-CL-001 clinical trial (NCT02029443) to better understand novel mechanisms of acalabrutinib resistance at an epigenetic level in CLL patients. Methods: DNA was extracted from peripheral blood mononuclear cells of patients with available samples at baseline (n=20), following acalabrutinib treatment at the time of progressive disease (n=8), or at 6 months post treatment start (n=14, used as non-progressor controls). While bisulfite conversion has been the gold standard for DNA methylation analysis for decades, it damages DNA and performs suboptimally with clinical FFPE and cfDNA samples that are often in limited quantity. Enzymatic methods have recently been reported that do not induce DNA damage, making them more suitable to routine clinical samples that are already DNA-damaged (eg, by formalin fixation) and often have limited DNA amounts. Before performing methylome analysis, we conducted a comprehensive comparison of bisulfite and enzymatic DNA methylation assays in reference cell lines and clinically relevant samples . This revealed that enzymatic methods outperformed bisulfite in several library quality metrics and resulted in increased library yield with the same DNA input and PCR amplification cycles. Enzymatic methods also captured a larger fraction of GC-rich regions, including CpG islands, in all comparisons. Prior to interrogating the full CLL 22 patient sample set, the enzymatic methods were validated in a subset of 6 CLL samples. We conducted a comprehensive analysis of methylation data sets with previously established RNA-seq analysis from paired samples. Results: Analysis of the acalabrutinib posttreatment samples revealed that the non-progressed patients had significantly higher methylation of 440 genes, whereas progressed samples had higher methylation of only 2 genes. CDKN1C, IL15, and AXL were significantly more methylated in non-progressed samples (Figure 1A), and these genes have been shown to be related to proliferation, survival, or response to BTK inhibition in CLL (De Totero et al, 2008; Sinha et al, 2018; Gupta et al, 2019; Lu et al, 2021; Kagiyama et al, 2021). Using RNA-seq data from paired samples, we showed that, as expected, IL15 methylation negatively correlated with IL15 expression (Figure 1B; R=−0.73, p=7.1e-09). IL15 is a proinflammatory cytokine that has downstream signaling targets in the NF-kB pathway, similar to BTK, indicating that alternative methods of NF-kB signaling may affect response. To support this hypothesis, we found that IL15 methylation significantly correlated with BIRC2 (R=0.59, p=1.4e-5) and RELA (R=0.42, p=0.0034) expression. Both genes play a role in NF-kB signaling, where BIRC2 encodes for the protein cIAP1 that regulates NF-kB activation and RELA encodes for p65, a member of the NF-kB transcription factor family. To our knowledge, it is not known if DNA methylation of IL15-induced expression plays a role in CLL resistance to BTK inhibition, and this finding may have important implications for future combination treatments. To note, CDKN1C, IL15, and AXL methylation were also significantly different between the progressed and non-progressed patients before acalabrutinib treatment, indicating they may be playing a role in innate resistance to acalabrutinib treatment. Conclusions: Using EM-seq to analyze DNA methylation, we found that reduced IL15 methylation and increased IL15 expression correlates with acalabrutinib innate resistance in CLL patients. Figure 1 Figure 1. Disclosures Burke: AstraZeneca: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company, Divested equity in a private or publicly-traded company in the past 24 months. Nuttall: AstraZeneca: Current Employment, Current holder of individual stocks in a privately-held company. Karl: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Callahan: AstraZeneca: Current Employment; Veritas Genetics: Current holder of individual stocks in a privately-held company. Mendler: AstraZeneca: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months; BISC Global, Inc.: Ended employment in the past 24 months. Criscione: AstraZeneca: Current Employment, Current holder of individual stocks in a privately-held company, Divested equity in a private or publicly-traded company in the past 24 months. Naumenko: AstraZeneca: Consultancy. Bibikova: AstraZeneca: Current Employment, Current equity holder in publicly-traded company, Ended employment in the past 24 months; Acerta Pharma: Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Bloecher: AstraZeneca: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company. Dougherty: AstraZeneca: Current Employment, Current equity holder in publicly-traded company, Research Funding; Pfizer: Current equity holder in publicly-traded company. Barrett: AstraZeneca: Current Employment, Current holder of individual stocks in a privately-held company, Research Funding. Scaltriti: AstraZeneca: Current Employment, Current holder of stock options in a privately-held company; MSKCC: Research Funding. Cingolani: AstraZeneca: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months. Byrd: Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Newave: Membership on an entity's Board of Directors or advisory committees. Furman: Oncotracker: Consultancy; Sunesis: Consultancy; Janssen: Consultancy, Honoraria; Acerta/AstraZeneca: Consultancy; Beigene: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Loxo Oncology: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy; Incyte: Consultancy; Abbvie: Consultancy, Honoraria, Other: Expert testimony; AstraZeneca: Honoraria; Morphosys: Consultancy; Sanofi: Consultancy; X4 Pharmaceuticals: Consultancy. Brown: Invectys: Other: Data Safety Monitoring Committee Service; TG Therapeutics: Research Funding; Beigene: Consultancy; Genentech/Roche: Consultancy; MEI Pharma: Consultancy; Morphosys AG: Consultancy; Bristol-Myers Squib/Juno/Celegene: Consultancy; Rigel: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Catapult: Consultancy; Acerta/Astra-Zeneca: Consultancy; Eli Lilly and Company: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Nextcea: Consultancy; Loxo/Lilly: Research Funding; Sun: Research Funding; SecuraBio: Research Funding; Gilead: Research Funding. Mortlock: ARTICA Therapeutics B.V.: Membership on an entity's Board of Directors or advisory committees; Anavo Therapeutics: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Hadfield: AstraZeneca: Current Employment, Current holder of individual stocks in a privately-held company, Divested equity in a private or publicly-traded company in the past 24 months. Munugalavadla: AstraZeneca: Current Employment, Current equity holder in publicly-traded company.

Background: Patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) after prior autologous stem cell transplant (ASCT) or chimeric antigen receptor T-cell (CAR-T) therapy have poor outcomes with limited treatment options. Bruton's tyrosine kinase (BTK) inhibitors are safe and effective agents in subsets of DLBCL with chronic active B-cell receptor (BCR) signaling, but durations of remission are short. Acalabrutinib, a highly selective, covalent, potent next-generation inhibitor of BTK (Calquence ® prescribing information [USPI]), is approved for the treatment of mantle cell lymphoma and chronic lymphocytic leukemia and is being explored in combination with other rational targeted agents in DLBCL. High levels of signal transducer and activator of transcription 3 (STAT3) expression and activation have been preferentially detected in activated B-cell DLBCL (Ding et al, 2008), and inhibition of STAT3 has suppressed DLBCL in preclinical models (Scuto et al, 2011). A phase 1b study demonstrated the safety and tolerability of AZD9150 in DLBCL with some evidence of clinical activity including 2 complete responses (CRs) and 2 partial responses (PRs) in 27 patients (Reilley et al, 2018). We report results from one of the arms of a phase 1 master protocol PRISM study (NCT03527147: A Platform Protocol for the Treatment of Relapsed/Refractory Aggressive Non-Hodgkin's Lymphoma), which evaluated combination therapy of acalabrutinib with the anti-STAT3 allele-specific oligonucleotide AZD9150 in patients with R/R DLBCL. Methods: Study participants were patients with R/R DLBCL aged ≥18 years with Eastern Cooperative Oncology Group performance status ≤2 and after ≥1 line of prior chemo-immunotherapy (including patients failing or ineligible for ASCT or CAR-T therapy). Starting on cycle 1 day 1 (C1D1), acalabrutinib was administered at 100 mg twice daily until disease progression or discontinuation. AZD9150 200 mg was administered as a 1-hour intravenous (IV) infusion on D1, D3, and D5 of C1, followed by weekly infusions (starting D8C1 and beyond). The primary endpoint was safety, and there was a dose-limiting toxicity (DLT) analysis after 6 subjects completed D28. Disease response (secondary endpoint) was assessed using Response Evaluation Criteria in Lymphoma 2017. Exploratory analyses included longitudinal peripheral blood and tumor tissue samples including immunophenotyping by flow cytometry and gene expression from total blood RNA (using the Nanostring - PanCancer IO 360™ Panel). Molecular classification at baseline was explored using available tumor tissue samples by LymphGen classification (Wright et al, 2020). Circulating tumor DNA (ctDNA) analysis was conducted using whole gene sequencing and a custom next-generation sequencing panel (AZHeme 600; Collins GP et al, 2021) and correlated with clinical response. Results: A total of 17 patients were enrolled; median age was 72 (range, 34-88) years and 47% (8/17) of patients were ≥75 years. Median number of prior lines of therapy was 2 (range, 1-6). Overall response rate was 24% with a CR rate of 12%. The most commonly reported adverse events (≥25%) of any grade independent of study drug attribution were anemia, AST/ALT elevation, thrombocytopenia, neutropenia, and fatigue. One DLT of grade 3 AST/ALT increase was observed, and there were no treatment-related deaths. Total cell-free DNA (cfDNA) levels and the allele frequency of mutations within ctDNA correlated with treatment response. One patient achieved an early CR to therapy after C2 (Figure 1A), which correlated with dynamic changes in ctDNA (Figure 1B) including the disappearance of copy number changes (Figure 1C). Patients with persistent copy number changes detected (≥1 alteration) as early as C1D8 were less likely to respond to acalabrutinib in combination with AZD9150. Cell of origin phenotype of DLBCL or genetic subtype did not correlate with response. The pharmacokinetics of acalabrutinib, ACP-5862 (active metabolite of acalabrutinib), and AZD9150 were generally consistent with historical monotherapy profiles. Conclusions : Targeting BTK and STAT3 is safe and tolerable in relapsed DLBCL but has limited efficacy. Early clearance of copy number variations and decreased cfDNA levels were associated with clinical responses and may be a useful biomarker with targeted agents. Figure 1 Figure 1. Disclosures Munugalavadla: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Nuttall: AstraZeneca: Current Employment, Current holder of individual stocks in a privately-held company. Burke: AstraZeneca: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company, Divested equity in a private or publicly-traded company in the past 24 months. Acar: AstraZeneca: Current Employment; Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months. White: AstraZeneca: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months. Udriste: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Sharma: AstraZeneca: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Dougherty: Pfizer: Current equity holder in publicly-traded company; AstraZeneca: Current Employment, Current equity holder in publicly-traded company, Research Funding. Flinn: Incyte: Research Funding; MorphoSys: Consultancy, Research Funding; AbbVie: Consultancy; Great Point Partners: Consultancy; Iksuda Therapeutics: Consultancy; IGM Biosciences: Research Funding; Karyopharm Therapeutics: Research Funding; Juno Therapeutics: Consultancy, Research Funding; Janssen: Research Funding; Merck: Research Funding; Loxo: Research Funding; KITE Pharma: Consultancy, Research Funding; Acerta: Research Funding; Gilead Sciences: Consultancy, Research Funding; Hutchison MediPharma: Consultancy; Nurix Therapeutics: Consultancy; Agios: Research Funding; Calithera: Research Funding; Infinity Pharmaceuticals: Research Funding; Rhizen Pharmaceuticals: Research Funding; Century Therapeutics: Consultancy; Beigene: Consultancy, Research Funding; ArQule: Research Funding; AstraZeneca: Consultancy, Research Funding; Celgene: Research Funding; Constellation Pharmaceuticals: Research Funding; Curis: Research Funding; Forma Therapeutics: Research Funding; Forty Seven: Research Funding; Genentech: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; Portola Pharmaceuticals: Research Funding; Pharmacyclics: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Servier Pharmaceuticals: Consultancy; Takeda: Consultancy, Research Funding; Teva: Research Funding; TG Therapeutics: Consultancy, Research Funding; Trillium Therapeutics: Research Funding; Triphase Research & Development Corp.: Research Funding; Unum Therapeutics: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Vincerx Pharma: Consultancy; Yingli Pharmaceuticals: Consultancy. Saba: AbbVie (venetoclax): Consultancy, Honoraria, Speakers Bureau; AbbVie, PCYC, Janssen (ibrutinib): Consultancy, Honoraria, Speakers Bureau; Kyowa Kirin (mogamulizumab-kpkc): Honoraria, Other: advisory board ; Kite: Honoraria, Other: advisory board ; TG Therapeutics: Honoraria, Other: advisory board ; Epizyme: Honoraria, Other: advisory board ; Karyopharm: Honoraria, Other: advisory board . Reagan: Genentech: Research Funding; Kite, a Gilead Company: Consultancy; Curis: Consultancy; Seagen: Research Funding. Collins: AstraZeneca: Honoraria, Research Funding; Amgen: Research Funding; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celleron: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck Sharp & Dohme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Pfizer: Honoraria; Novartis: Honoraria, Speakers Bureau; Beigene: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Speakers Bureau. Mortlock: ARTICA Therapeutics B.V.: Membership on an entity's Board of Directors or advisory committees; Anavo Therapeutics: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Patel: Portola Pharmaceuticals: Research Funding; Vigeo: Research Funding; Vedanta: Research Funding; Verastem: Research Funding; Alexion, AstraZeneca Rare Disease: Other: Study investigator; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Exelixis: Membership on an entity's Board of Directors or advisory committees; Xencor: Research Funding; TopAlliance: Research Funding; Tesaro: Research Funding; Taiho: Research Funding; Stemline Therapeutics: Research Funding; Synthorx: Research Funding; Syndax: Research Funding; Ribon Therapeutics: Research Funding; Revolution Medicines: Research Funding; Qilu Puget Sound Biotherapeutics: Research Funding; Prelude Therapeutics: Research Funding; Placon Therapeutics: Research Funding; Phoenix Molecular Designs: Research Funding; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; GlaxoSmithKline: Research Funding; H3 Biomedicine: Research Funding; Hengrui: Research Funding; Hutchinson MediPharma: Research Funding; Ignyta: Research Funding; Incyte: Research Funding; Jacobio: Research Funding; Jounce Therapeutics: Research Funding; Klus Pharma: Research Funding; Kymab: Research Funding; Loxo Oncology: Research Funding; Lycera: Research Funding; Mabspace: Research Funding; Macrogenics: Research Funding; Millennium Pharmaceuticals: Research Funding; Mirati Therapeutics: Research Funding; ModernaTX: Research Funding; Forma Therapeutics: Research Funding; Evelo Biosciences: Research Funding; Eli Lilly: Research Funding; EMD Serono: Membership on an entity's Board of Directors or advisory committees, Research Funding; Effector Therapeutics: Research Funding; Daiichi Sankyo: Research Funding; Cyteir Therapeutics: Research Funding; Curis: Research Funding; Ciclomed: Research Funding; Clovis: Research Funding; Checkpoint Therapeutics: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Calithera: Research Funding; Boehringer Ingelheim: Research Funding; BioNTech: Research Funding; Takeda: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; ORIC Pharmaceuticals: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; LSK Biopartners: Research Funding; Seven and Eight Biopharmaceuticals: Research Funding; Merck: Research Funding; Bicycle Therapeutics: Research Funding; AstraZeneca: Research Funding; Artios Pharma: Research Funding; Aileron Therapeutics: Research Funding; Agenus: Research Funding; ADC Therapeutics: Research Funding; Acerta Pharma: Research Funding; Florida Cancer Specialists: Research Funding.

Introduction: Acalabrutinib (Calquence ®), a selective Bruton tyrosine kinase (BTK) inhibitor, is approved for the treatment of mantle cell lymphoma (relapsed/refractory) and chronic lymphocytic leukemia. Patients with hematologic malignancies may require acid-reducing agents (including proton pump inhibitors [PPIs]) for the treatment of gastroesophageal reflux or peptic ulcer disease. The solubility of acalabrutinib is reduced with increasing pH; concomitant administration of acalabrutinib capsules with PPIs reduces acalabrutinib exposure and is currently not recommended. Additionally, many cancer patients are unable to swallow capsules and require alternative methods to deliver acalabrutinib, such as a suspension administered orally or via a nasogastric (NG) tube. To enable the use of acalabrutinib in patients who require co-treatment with PPIs and/or are unable to swallow capsules, a new maleate salt of acalabrutinib, formulated as an immediate-release film-coated tablet (acalabrutinib maleate tablet [AMT]), has been developed which shows fast and complete in vitro release at all physiologic pH. We evaluated the pharmacokinetics (PK), pharmacodynamics (PD), safety, and tolerability of AMT administered orally or via NG tube in the presence or absence of a PPI. In addition, the effect of food on AMT was evaluated to confirm the absence of a clinically relevant impact, consistent with acalabrutinib capsules. Methods: Three Phase 1, open-label, single-dose, cross-over studies were conducted in healthy subjects to establish PK similarity (bioequivalence) between 100-mg AMT and 100-mg acalabrutinib capsules (N=66); evaluate PPI effect by comparing PK of 100-mg AMT administered in the presence vs absence of rabeprazole (PPI; N=14); evaluate food effect by comparing PK of 100-mg AMT administered with a high-fat diet vs fasted (N=16); and assess PK following administration of 100-mg acalabrutinib maleate suspension (in 15 mL water) delivered via NG tube, in the presence vs absence of rabeprazole (N=20). PD was assessed by measuring BTK target occupancy (BTK-TO) in peripheral blood mononuclear cells across all treatment arms and studies. Results: Exposure geometric mean ratios and 90% confidence intervals (CIs) are shown in Table 1 with the PK profiles shown in Figure 1. Systemic exposures (C max and AUC) of acalabrutinib and its major pharmacologically active metabolite, ACP-5862, between AMT and acalabrutinib capsules were bioequivalent ( Conclusions: Acalabrutinib maleate, administered as a tablet or suspension, is safe and well tolerated. Based on the PK (and associated variability), BTK-TO, and established exposure-efficacy/safety relationship, AMT clinical effect is expected to be comparable to acalabrutinib capsules at the approved 100-mg BID dosing, regardless of use of PPIs and ingestion of food. Additionally, AMT improves swallowing ability given the film coating and a 50% reduced volume compared with the capsule, and can be easily suspended in a small amount of water to allow dosing in patients unable to swallow tablets. Figure 1 Figure 1. Disclosures Sharma: AstraZeneca: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Pepin: AstraZeneca: Current Employment. Burri: AstraZeneca: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months. Zheng: AstraZeneca: Current Employment; Kite Pharma, a Group of Gilead: Ended employment in the past 24 months; Gilead Science Inc., AstraZeneca: Current equity holder in publicly-traded company; Gilead Science Inc.: Divested equity in a private or publicly-traded company in the past 24 months. Kuptsova-Clarkson: AstraZeneca: Current Employment, Current equity holder in publicly-traded company; AbbVie: Current holder of individual stocks in a privately-held company. de Jong: Acerta Pharma B.V. (A Member of the AstraZeneca Group): Current Employment. Yu: AstraZeneca: Current Employment; EMD Serono Research Institute: Ended employment in the past 24 months; AstraZeneca, Johnson and Johnson, AbbVie, Abbott: Current equity holder in publicly-traded company; Merck KGaA: Divested equity in a private or publicly-traded company in the past 24 months. MacArthur: AstraZeneca: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Majewski: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Ware: AstraZeneca: Current equity holder in publicly-traded company; Denali (DNLI) Therapeutics: Current equity holder in publicly-traded company. Mann: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Ramies: AstraZeneca: Consultancy. Munugalavadla: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Sheridan: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Tomkinson: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. OffLabel Disclosure: New Formulation

Background: Acalabrutinib is a highly selective, covalent irreversible Bruton tyrosine kinase inhibitor with minimal activity against other kinases. Acalabrutinib has demonstrated durable responses as a single agent or combined with O in treatment-naïve (TN) CLL. Here, interim results are presented for the multicenter, open-label Phase 3 ELEVATE-TN study (NCT02475681), which evaluated the efficacy and safety of acalabrutinib + O vs acalabrutinib alone vs O + Clb in pts with TN CLL. Methods: Eligible pts had TN CLL requiring treatment per iwCLL criteria and were aged ≥65 y or 6, creatinine clearance Results: From 9/14/2015-2/8/2017, 535 pts were randomized to the acalabrutinib + O (n=179), acalabrutinib (n= 179), or O + Clb (n=177) arms. The median age was 70 y (range, 41-91); 69% had high- and 12% had very high-risk CLL IPI scores. At a median follow-up of 28 mo, acalabrutinib + O significantly prolonged PFS vs O + Clb (median not reached [NR] vs 22.6 mo; HR 0.10, 95% CI 0.06-0.18, P IRC-assessed ORR was higher with acalabrutinib + O (94%; 95% CI, 89.3%-96.5%) vs O + Clb (79%; 95% CI, 71.9%-83.9%; P The median treatment duration was 27.7 mo for acalabrutinib + O (range, 2.3-40.3) and acalabrutinib (range, 0.3-40.2) and 5.6 mo (range, 0.9-7.4) for O + Clb. Common adverse events (AEs) are shown in the Table. AEs were similar between the acalabrutinib-containing arms. Infusion reactions were less frequent with acalabrutinib + O (13%) than with O + Clb (40%). AEs led to treatment discontinuation in 20 pts (11%) on acalabrutinib + O, 16 pts (9%) on acalabrutinib, and 25 pts (14%) on O + Clb. With >2 y of follow-up, 79.3% of pts in both the acalabrutinib-containing arms remain on single-agent acalabrutinib. AEs of interest (acalabrutinib + O or acalabrutinib vs O + Clb) were atrial fibrillation (any grade: 3% or 4% vs 1%), bleeding (any grade/Grade ≥3: 43%/2% or 39%/2% vs 12%/0%), and hypertension (Grade ≥3: 3% or 2% vs 3%). Conclusions: Acalabrutinib + O and acalabrutinib monotherapy significantly improved PFS vs O + Clb, with tolerable safety in pts with TN CLL. Despite cross over for disease progression in the O + Clb arm, a trend toward improved OS was observed in both acalabrutinib arms, though longer follow-up is needed. Disclosures Sharman: AbbVie: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; TG Therapeutics: Consultancy, Honoraria, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding; Acerta: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Research Funding. Banerji:CIHR: Research Funding; LLSC: Research Funding; Research Manitoba: Research Funding; CCMF: Research Funding; CancerCare Manitoba/University of Manitoba: Employment; CAPhO: Honoraria; BIOGEN: Other: Licensing fee; Dana-Farber Cancer Institute: Other: Licencing fee; Gilead: Consultancy, Honoraria, Research Funding; Astra-Zeneca: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Roche: Honoraria, Licensing fee, Research Funding; Abbvie: Consultancy, Honoraria. Herishanu:Janssen: Honoraria; AbbVie: Honoraria; Roche: Honoraria. Munir:Gilead: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Roche: Honoraria; Morphosys: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sunesis: Consultancy; Pharmacyclics: Other: TBC; Acerta: Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria; AbbVie: Honoraria. Walewska:Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Speakers Bureau; Takeda: Other: Travel grant; Novartis: Other: travel grant. Follows:Abbvie: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau. Karlsson:Skane University Hospital: Employment. Ghia:AbbVie: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria; Juno/Celgene: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy; ArQule: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Dynamo: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Corbett:Celgene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Tauranga Hospital: Employment; Pathlab Waikato: Equity Ownership. Walker:Peninsula Health (public hospital): Employment; Alfred health (public hospital): Employment; Roche: Other: Travel grant. Jurczak:Incyte: Research Funding; Takeda: Research Funding; Sandoz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Research Funding; Roche: Research Funding; Gilead: Research Funding; Celgene: Research Funding; MorphoSys: Research Funding; Celtrion: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Research Funding. Salles:Epizyme: Consultancy, Honoraria; BMS: Honoraria; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis, Servier, AbbVie, Karyopharm, Kite, MorphoSys: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Roche, Janssen, Gilead, Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Autolus: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Other: Educational events. Janssens:Novartis: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Celgene: Speakers Bureau; abbvie: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; idem consultancy: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau. Cymbalista:AstraZeneca: Honoraria; Janssen: Honoraria; Sunesis: Research Funding; Roche: Research Funding; Gilead: Honoraria; Abbvie: Honoraria. Wierda:Juno Therapeutics: Research Funding; Janssen: Research Funding; Cyclcel: Research Funding; KITE pharma: Research Funding; Loxo Oncology Inc.: Research Funding; Genentech: Research Funding; Xencor: Research Funding; Acerta Pharma Inc: Research Funding; Pharmacyclics LLC: Research Funding; Sunesis: Research Funding; AbbVie: Research Funding; Miragen: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Gilead Sciences: Research Funding; GSK/Novartis: Research Funding. Coutre:Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Astra Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Acerta: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; BeiGene: Other: Travel, Accommodations, Expenses & Data Safety Monitoring Committee; Genentech: Consultancy. Pagel:AstraZeneca: Consultancy; Gilead Sciences: Consultancy; Pharmacyclics: Consultancy. Skarbnik:Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Verastem Oncology: Honoraria, Research Funding, Speakers Bureau; Kite Pharma: Honoraria, Speakers Bureau; Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Acerta: Research Funding; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Honoraria, Speakers Bureau; CLL Society: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Speakers Bureau; Novartis: Speakers Bureau. Kamdar:AstraZeneca: Consultancy; University of Colorado: Employment; Celgene: Consultancy; Seattle Genetics: Speakers Bureau; Pharmacyclics: Consultancy. Woyach:Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Verastem: Research Funding. Izumi:AstraZeneca: Equity Ownership; Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acalabrutinib patents. Munugalavadla:Acerta Pharma: Employment; Gilead Sciences: Equity Ownership; AstraZeneca: Equity Ownership. Patel:Acerta Pharma: Employment, Equity Ownership; AstraZeneca: Equity Ownership. Wang:Acerta Pharma: Employment; AstraZeneca: Equity Ownership. Wong:Acerta Pharma: Employment. Byrd:Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Acerta: Research Funding; Acerta: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; BeiGene: Research Funding; BeiGene: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau.

Background Combinations of different targeted therapies, including Bruton tyrosine kinase (BTK) inhibitors and anti-CD20 monoclonal antibodies (mAbs) could improve treatment for CLL. Unexpectedly, the combination of ibrutinib (IBR) with rituximab did not show additional clinical benefit. However, IBR inhibits many off-target molecules that may limit therapeutic mAb clinical effectiveness and a more selective BTK inhibitor, such as acalabrutinib (ACALA), could be more effective in combination with mAb therapy. Initial data from the ELEVATE TN trial support this possibility. IBR off-target effects on antibody-dependent cellular phagocytosis (ADCP), the major mechanism of therapeutic mAb activity could explain this difference. Additionally, IBR induces a higher and longer duration increase in circulating lymphocytes than ACALA. IBR off-target effects on efferocytosis, another phagocytic process involved in apoptotic cell removal, might explain this difference. Methods Using state-of-the-art direct kinetic measurements of phagocytosis by time-lapse video, (Chu et al. J Cell Sci 2020;133:jcs237883) we investigated the effects of IBR and ACALA on phagocytosis (ADCP or efferocytosis) by human monocyte-derived macrophages (hMDM) in vitro. Live cell time-lapse video of 10 μg/ml rituximab (Genentech) mediated ADCP of CLL cells by CellTracker Deep Red (CTDR, Thermofisher) labeled hMDM (20:1 CLL:hMDM cell ratio) either untreated or treated with IBR or ACALA (3-fold serial dilutions from 100 to 0.41 μM) was imaged in a stage-top environmental chamber (37°C and 5% CO2) mounted onto a Nikon Ti-Eclipse inverted microscope with an ELWD 20x/0.45NA S Plan Fluor Ph1 objective and an Andor Zyla 5.5 sCMOS camera. Images were captured sequentially every 4 min over 2.8 h. For each experiment (n = 18), duplicate or triplicate wells for each drug concentration were imaged. For efferocytosis, live cell time-lapse video imaging of phagocytosis of pHrodo iFL Red STP ester (pHrodo Red, Thermo Fisher Scientific) labeled apoptotic CLL cells by CTDR-labeled hMDM (20:1 CLL:hMDM cell ratio) either untreated or treated with IBR or ACALA (2-fold serial dilutions from 10 to 1.25 μM) was collected every 4 min over 2.8 h. For each experiment (n = 7), duplicate or triplicate wells for each drug concentration was imaged and analyzed. Finally, for efferocytosis, the intensity of pHrodo Red dye, a pH-sensitive dye that increases in intensity with acidic pH, as found in the endolysosomes, was measured in the pHrodo Red color channel and analyzed. Results IBR significantly inhibited ADCP at all measured drug concentrations (0.41 μM, p < 0.05; 1.2 μM, p < 0.01; 3.7 - 100 μM, p < 0.001). The mean peak free drug concentration (Cmax) achieved clinically by standard doses for IBR is ~0.5 μM. ACALA only significantly inhibited ADCP at the highest concentration (100 μM, p < 0.001). The Cmax achieved clinically by standard doses for ACALA is ~1.2 μM. ACALA did not inhibit efferocytosis or subsequent transition to endolysosomal compartment at all tested concentrations (p > 0.05). IBR did not inhibit efferocytosis (p > 0.05) and only inhibited transition to endolysosomal compartment at highest concentration tested (10 μM, p < 0.01) Conclusion Our study shows that BTK inhibition does not block ADCP and a more selective BTK inhibitor may prove effective in combination with therapeutic anti-CD20 mAbs. IBR off-target inhibition specifically blocks ADCP and not efferocytosis. Thus, IBR off-target inhibition of ADCP should be via proximal signaling by antibody Fc receptors and not subsequent downstream phagocytic mechanisms in common with efferocytosis. These results also imply the lack of BTK and IBR off-target molecules involvement in efferocytosis. Finally, the increased lymphocytosis seen with IBR compared to ACALA treatment in CLL cannot be explained by IBR off-target effects on efferocytosis. These findings provide a critical understanding of macrophage phagocytosis reduction by BTK inhibitor selectivity that will have important consequences for the development of combination targeted therapies with mAbs. Disclosures Chu: Acerta Pharma/AstraZeneca: Research Funding; Pfizer: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months; TG Therapeutics: Research Funding. Izumi:AstraZeneca: Current equity holder in publicly-traded company; Acerta Pharma: Current equity holder in private company, Ended employment in the past 24 months, Patents & Royalties: Acalabrutinib patents (no royalties). Munugalavadla:Gilead Sciences: Current equity holder in publicly-traded company; AstraZeneca: Current equity holder in publicly-traded company; Acerta Pharma: Current Employment. Barr:Gilead: Consultancy; Morphosys: Consultancy; TG therapeutics: Consultancy, Research Funding; Seattle Genetics: Consultancy; Celgene: Consultancy; AstraZeneca: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Genentech: Consultancy; Abbvie/Pharmacyclics: Consultancy, Research Funding; Verastem: Consultancy. VanDerMeid:Acerta Pharma / AstraZeneca: Research Funding. Elliott:Acerta Pharma / AstraZeneca: Research Funding. Zent:Mentrik Biotech: Research Funding; TG Therapeutics, Inc: Research Funding; Acerta / Astra Zeneca: Research Funding.

Background: Acalabrutinib (ACP-196) is a highly selective, potent Bruton tyrosine kinase (BTK) inhibitor developed to minimize off-target activity. Acalabrutinib monotherapy shows promising safety and efficacy in CLL (Ghia et al 2019). However, a few patients (pts) develop resistance to acalabrutinib. A known mechanism of covalent BTK inhibitor resistance is acquired mutations in BTK (particularly Cys481) and its downstream target PLCg2. Alternate mechanisms and the contribution of the CLL microenvironment to acquired resistance remain to be elucidated. In this study, we performed cell surface phenotyping, intracellular signaling, and RNA-seq analyses on samples from 39 pts with relapsed/refractory or treatment-naive CLL from the ACE-CL-001 clinical trial (NCT02029443) to identify novel mechanisms of acalabrutinib resistance with focus on the CLL microenvironment. Methods : Pts were divided into 2 groups: those who continued to respond to treatment (non-progressed, NP, n=23) and those who developed progressive disease (progressed, PD, n=16) within 36 months of starting acalabrutinib. Blood mononuclear cells (PBMCs) were analyzed after 6 months of acalabrutinib therapy (100 mg twice a day) for the first (NP) group of patients, or at the time of progression for the second (PD) group of patients and compared with pre-treatment baseline. Expression of cell surface markers, including CD49d, CD38, and CD79b, was evaluated by flow cytometry. A 30% positive cut-off was used to identify pts that express high levels of CD49d, an α-chain of the VLA-4 integrin (Tissino, et al 2018), CD38, and CD79b. Intracellular flow cytometry was used to measure cell proliferation via Ki-67 staining. RNA-seq was used to measure changes in gene expression. Results: Cell surface phenotyping showed higher expression of CD49d (p Conclusions: High surface expression of CD49d (VLA-4) and CD79b prior to and after therapy correlates with acalabrutinib resistance in pts with CLL. Targeting CD49d may prove an effective strategy to overcome acalabrutinib resistance in CD49d high pts. Disclosures Bibikova: Acerta Pharma: Employment, Equity Ownership; AstraZeneca: Equity Ownership. Law:Acerta Pharma: Employment; AstraZeneca: Equity Ownership. Clevenger:Acerta Pharma: Employment; AstraZeneca: Equity Ownership. Cheung:AstraZeneca: Equity Ownership; Acerta Pharma: Employment, Equity Ownership. De Jesus:Acerta Pharma: Employment, Equity Ownership; AstraZeneca: Equity Ownership. Gulrajani:Acerta Pharma: Employment, Equity Ownership; AstraZeneca: Equity Ownership. Yamaguchi:AstraZeneca: Equity Ownership; Acerta Pharma: Employment. Do:Acerta Pharma: Employment, Equity Ownership; AstraZeneca: Equity Ownership. Burke:AstraZeneca: Employment, Equity Ownership. Brock:AstraZeneca: Equity Ownership; Acerta Pharma: Employment. Munugalavadla:Gilead Sciences: Equity Ownership; AstraZeneca: Equity Ownership; Acerta Pharma: Employment. Frigault:Acerta Pharma: Employment; AstraZeneca: Employment, Equity Ownership. Byrd:Acerta: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; BeiGene: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Novartis: Other: Travel Expenses, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S. Furman:Acerta Pharma: Consultancy; AstraZeneca: Consultancy; Incyte: Consultancy; Janssen: Consultancy; Beigene: Consultancy; Oncotracker: Consultancy; Pharmacyclics: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy; Genentech: Consultancy; Abbvie: Consultancy. Brown:Catapult Therapeutics: Consultancy; AbbVie: Consultancy; Genentech/Roche: Consultancy; Acerta Pharma: Consultancy; Pharmacyclics: Consultancy; Pfizer: Consultancy; Janssen: Honoraria; Teva: Honoraria; AstraZeneca: Consultancy; Octapharma: Consultancy; Invectys: Other: Data safety monitoring board; Morphosys: Other: Data safety monitoring board; Dynamo Therapeutics: Consultancy; BeiGene: Consultancy; TG Therapeutics: Consultancy; Sunesis: Consultancy; Loxo: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Juno/Celgene: Consultancy; Gilead: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Sun Pharmaceuticals: Research Funding; Novartis: Consultancy. Covey:AstraZeneca: Equity Ownership; Acerta Pharma: Employment, Equity Ownership.

The efficacy of many therapeutic unconjugated monoclonal antibodies (mAbs), including those targeting CD20 in CLL, requires immune cell-mediated cytotoxicity. mAbs have often been optimized for natural killer (NK) cell antibody-dependent cellular cytotoxicity (ADCC) activity. However, in vivo mouse studies have shown that antibody-dependent cellular phagocytosis (ADCP) by macrophages is the major mechanism of clearance of circulating B cells by anti-CD20 mAbs. To directly compare ADCC versus ADCP, we previously used a panel of anti-CD20 mAbs (rituximab, ofatumumab, obinutuzimab, ocaratuzumab) to test cytotoxicity of paired human NK cells and monocyte-derived macrophages (hMDM) against CLL cells in vitro. All mAbs demonstrated ADCP activity at least 10-fold greater than ADCC as measured by CLL cell depletion per effector cell. Moreover, ADCC and ADCP activity levels did not correlate, meaning that ADCC cannot be used as a surrogate measure of ADCP for these mAb. This could explain why mAb optimization for ADCC activity has often failed to translate into more efficacious treatment. Thus, ADCP may be an effective translational measurement of anti-CD20 mAb performance. Because of the clinical interest in combining anti-CD20 mAb with targeted small molecule inhibitors, we began studying the effects of the Bruton tyrosine kinase (BTK) inhibitors on anti-CD20 mAb-mediated ADCP. Our initial studies showed that ibrutinib (IBR), but not acalabrutinib (Acala), significantly decreased anti-CD20 ADCP as measured by a flow cytometry-based assay that measures single timepoint cell collections. These types of assays cannot easily determine the kinetics and individual effector cell activity of ADCP. Thus, to more fully study the BTK inhibitor effects on ADCP, we developed a live cell time-lapse imaging method for measuring ADCP, utilizing recent advances in microscopy, cellular dye labeling, digital imaging, imaging software and computing. Whole-cell labeling of macrophages enabled visualization of internalized CLL cells as regions of dye exclusion or "voids". Because of the vast number of images acquired during live cell time-lapse imaging, we utilized computer software-aided image recognition and enumeration to measure the number of macrophages and voids inside each macrophage. As a measure of phagocytic engulfment, we developed a void index, which provides a relative measure of phagocytic engulfment per macrophage. Measuring ADCP in this manner replicates clinical observation of mAb therapeutic activity. Clinically, intravenous anti-CD20 mAb therapy typically induces a rapid decrease in circulating CLL cells (within hours), followed by a long period (days) of stable to increased levels of circulating cells. Similarly, our live cell time-lapse video assay shows initial rapid ADCP over the first 2 hours followed by a prolonged period of "hypophagia" with little ADCP for the remainder of the assay (imaged every 2 minutes for 8 hours). This "hypophagia" phenomenon may explain the resistance to therapeutic mAb observed clinically. With these new tools for quantitation of ADCP, we compared the effects of serial dilutions of IBR or Acala on ADCP. Overall, as measured by Area Under the Curve (AUC) analysis, IBR decreased phagocytic capacity of anti-CD20 mediated CLL cell ADCP at concentrations of 0.41 μM and above. By contrast, Acala did not begin to decrease AUC measurements until 3.7 μM, and subsequent AUC values were higher in Acala versus IBR-treated ADCP assays up to the highest tested drug concentration (100 μM). Similarly, the initial ADCP kinetics (void index / min over the first hour) reflected a decrease with IBR treatment at 0.41 μM that continued until a nadir was reached at 33 μM. In contrast, Acala did not induce a decrease in this kinetic measurement until 3.7 μM and a nadir was not reached (up to 100 μM). Thus, IBR significantly decreases ADCP by hMDM at concentrations much lower than a more specific BTK inhibitor, Acala. This result suggests that BTK inhibition has little to no effect on ADCP and furthermore suggests that IBR off-target effects decrease ADCP. IBR off-target candidates include other tyrosine kinases in the TEC (tyrosine kinase expressed in hepatocellular carcinoma) family. These data suggest that a a highly selective BTK inhibitor with little effect on ADCP could be a more suitable drug to combine with therapeutic mAb(s). Disclosures Chu: Pfizer: Equity Ownership; Acerta Pharma: Research Funding. VanDerMeid:AstraZeneca: Research Funding. Izumi:Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acalabrutinib patents; AstraZeneca: Equity Ownership. Munugalavadla:Acerta Pharma: Employment; AstraZeneca, Gilead Sciences: Equity Ownership. Barr:TG Therapeutics: Consultancy, Research Funding; Celgene: Consultancy; Pharmacyclics LLC, an AbbVie company: Consultancy, Research Funding; Seattle Genetics: Consultancy; Merck: Consultancy; Genentech: Consultancy; Verastem: Consultancy; Gilead: Consultancy; Astra Zeneca: Consultancy, Research Funding; Janssen: Consultancy; AbbVie: Consultancy. Elliott:Astra Zeneca: Research Funding. Zent:Mentrik Biotech: Research Funding; Astra Zeneca: Research Funding.

Intracellular mechanism(s) that contribute to promiscuous signaling via oncogenic KIT in systemic mastocytosis and acute myelogenous leukemia are poorly understood. We show that SHP2 phosphatase is essential for oncogenic KIT-induced growth and survival in vitro and myeloproliferative disease (MPD) in vivo. Genetic disruption of SHP2 or treatment of oncogene-bearing cells with a novel SHP2 inhibitor alone or in combination with the PI3K inhibitor corrects MPD by disrupting a protein complex involving p85α, SHP2, and Gab2. Importantly, a single tyrosine at position 719 in oncogenic KIT is sufficient to develop MPD by recruiting p85α, SHP2, and Gab2 complex to oncogenic KIT. Our results demonstrate that SHP2 phosphatase is a druggable target that cooperates with lipid kinases in inducing MPD.

Mast cell maturation is poorly understood. We show that enhanced PI3K activation results in accelerated maturation of mast cells by inducing the expression of microphthalmia transcription factor (Mitf). Conversely, loss of PI3K activation reduces the maturation of mast cells by inhibiting the activation of AKT, leading to reduced Mitf but enhanced Gata-2 expression and accumulation of Gr1+Mac1+ myeloid cells as opposed to mast cells. Consistently, overexpression of Mitf accelerates the maturation of mast cells, whereas Gata-2 overexpression mimics the loss of the PI3K phenotype. Expressing the full-length or the src homology 3– or BCR homology domain–deleted or shorter splice variant of the p85α regulatory subunit of PI3K or activated AKT or Mitf in p85α-deficient cells restores the maturation but not growth. Although deficiency of both SHIP and p85α rescues the maturation of SHIP−/− and p85α−/− mast cells and expression of Mitf; in vivo, mast cells are rescued in some, but not all tissues, due in part to defective KIT signaling, which is dependent on an intact src homology 3 and BCR homology domain of p85α. Thus, p85α-induced maturation, and growth and survival signals, in mast cells can be uncoupled.

Objective Src family kinases (SFK) have been implicated in regulating growth factor and integrin-induced proliferation, migration, and gene expression in multiple cell types. However, little is known about the role of these kinases in the growth, homing, and engraftment potential of hematopoietic stem and progenitor cells. Results Here we show that loss of hematopoietic-specific SFKs Hck, Fgr, and Lyn results in increased number of Sca-1 + Lin − cells in the bone marrow, which respond differentially to cytokine-induced growth in vitro and manifest a significant defect in the long-term repopulating potential in vivo. Interestingly, a significant increase in expression of adhesion molecules, known to coincide with the homing potential of wild-type bone marrow cells is also observed on the surface of SFK −/− cells, although, this increase did not affect the homing potential of more primitive Lin − Sca-1 + SFK −/− cells. The stem cell−repopulating defect observed in mice transplanted with SFK −/− bone marrow cells is due to the loss of Lyn Src kinase, because deficiency of Lyn, but not Hck or Fgr, recapitulated the long-term stem cell defect observed in mice transplanted with SFK −/− bone marrow cells. Conclusions Taken together, our results demonstrate an essential role for Lyn kinase in positively regulating the long-term and multilineage engraftment of stem cells, which is distinct from its role in mature B cells and myeloid cells.

Objective Oncogenic activation loop mutations of KIT are observed in acute myeloid leukemia (AML) and in myeloproliferative disorders (MPD); however, the signaling pathways that contribute to transformation via these mutations in vivo are not known. Previous studies have demonstrated hyperactivation of p85α regulatory subunit of class IA phosphatidylinositol-3-kinase (PI3K) in cell lines expressing the activation loop mutant of KIT (KITD816V [human] and KITD814V [murine]). Although p85α is hyperphosphorylated and constitutively bound to KITD814V in cell-line models; the physiologic significance of this biochemical phenomenon in KITD814V-induced transformation is not known. Materials and Methods Here, we describe the generation of a new mouse model to study KITD814V-induced transformation in myeloid cells as opposed to previously described models that primarily result in the generation of disease resembling acute lymphocytic leukemia. Results Our results show that transplantation of KITD814V expressing bone marrow cells from C57/BL6 strain of mice into syngeneic recipients results in a fatal MPD. Importantly, in this model, transplantation of KITD814V expressing p85α-deficient bone marrow cells rescues the MPD phenotype. Conclusions Our results describe the generation of a new murine transplant model to study KITD814V-induced transformation and identify p85α as potential therapeutic target for the treatment of KITD814V-bearing diseases.

Oncogenic activation loop KIT mutations are observed in acute myeloid leukemia (AML) and systemic mastocytosis (SM); however, unlike the KIT juxtamembrane mutants, the activation loop mutants are insensitive to imatinib mesylate. Furthermore, as prior studies primarily used heterologous cell lines, the molecular mechanism(s) underlying oncogenic KIT-induced transformation in primary cells is poorly understood. We demonstrate that expression of KITD814V in primary hematopoietic stem/progenitor cells (HSC/Ps) and mast cell progenitors (MCps) induces constitutive KIT autophosphorylation, supports ligand-independent hyperproliferation, and promotes promiscuous cooperation with multiple cytokines. Genetic disruption of p85α, the regulatory subunit of class IA lipid kinase phosphoinositol-3-kinase (PI3K), but not of p85β, or genetic disruption of the hematopoietic cell-specific Rho GTPase, Rac2, normalizes KITD814V-induced ligand-independent hyperproliferation. Additionally, deficiency of p85α or Rac2 corrects the promiscuous hyperproliferation observed in response to multiple cytokines in both KITD814V-expressing HSC/Ps and MCps. Treatment of KITD814V-expressing HSC/Ps with a Rac inhibitor (NC23766) or with rapamycin showed a dose-dependent suppression in ligand-independent growth. Taken together, our results identify p85α and Rac2 as potential novel therapeutic targets for the treatment of KITD814V-bearing AML and SM.

Objective The intracellular signals that contribute to granulocyte colony–stimulating factor (G-CSF) receptor induced stem cell mobilization are poorly characterized. Methods We show enhanced G-CSF induced mobilization of stem cells in mice deficient in expression of Src family kinases ( SFK −/− ), which is associated with hypersensitivity of SFK −/− bone marrow cells to G-CSF as well as sustained activation of signal transducer and activator of transcription-3. Results A proteome map of the bone marrow fluid derived from wild-type and SFK −/− mice revealed a significant global reduction in the number of proteins in SFK −/− mice compared to controls, which was associated with elevated matrix metalloproteinase-9 levels, reduced stromal-derived factor-1 expression, and enhanced breakdown of vascular cell adhesion molecule-1. Transplantation of wild-type or SFK −/− stem cells into wild-type mice and treatment with G-CSF recapitulated the G-CSF–induced increase in stem cell mobilization noted in SFK −/− nontransplanted mice; however, the increase was significantly less. G-CSF treatment of SFK −/− mice engrafted with wild-type stem cells also demonstrated a modest increase in stem cell mobilization compared to controls, however, the observed increase was greatest in mice completely devoid of SFKs. Conclusions These data suggest an involvement of both hematopoietic intrinsic and microenvironmental factors in Src kinase–mediated mobilization of stem cells and identify Src kinases as potential targets for modulating stem cell mobilization.

Introduction: Idelalisib (IDELA) is a targeted PI3Kd inhibitor approved for the treatment of patients (pts) with relapsed CLL/FL. The established toxicity profile of IDELA includes diarrhea/colitis with a grade ≥3 incidence of ~15%. Here, we present the histopathological-as well as immune profiling and viral testing-results of gastrointestinal tissue specimens from pts who received diagnostic biopsies for abdominal pain or diarrhea during treatment with IDELA. Methods: Intestinal biopsy specimens obtained from 31 pts during treatment with IDELA across 10 trials (monotherapy n = 3 or in combination with: rituximab n = 9; ofatumumab n = 14; and bendamustine/rituximab n = 5) were reviewed separately by 2 independent pathologists with expertise in the area of gastrointestinal and transplant pathology. If more than 1 biopsy time point per pt was available (n = 3), all time points would be evaluated and, if discordant, would be reported separately. Evaluation included hematoxylin and eosin stain; and immunohistochemistry (IHC) for CD3, CD8, FOXP3, CD79a, CMV, and adenovirus (as adequate tissue allowed). Additionally, droplet digital PCR (ddPCR) was performed for cytomegalovirus (CMV), adenovirus, and human herpesvirus 6 (HHV-6). Colon/small bowel tissues from 18 normal individuals who underwent biopsy during routine screening colonoscopy and had normal pathology results were included as controls. Results: Baseline characteristics of pts included CLL n = 24 (77.4%) and iNHL n = 7 (22.6%), age range 50 to 82 years with median 65.6 years, male n = 19 (61.3%). IDELA median dose = 150 mg BID. Diarrhea/colitis (Grade 1-3) was reported in 24 cases (75%) including 1 case of hemorrhagic colitis. One patient each had concurrent laboratory-confirmed Mycobacterium avium-intracellulare and norovirus and 2 patients were excluded from histomorphological analysis, 1 due to celiac disease and 1 due to total loss of glands secondary to CMV. Four distinct morphological patterns were identified: 1) predominantly normal, 2) apoptotic, 3) inflammatory/ischemic, and 4) mixed apoptotic/inflammatory/ischemic. CMV IHC was performed in all 31 cases; 2 showed strong/moderate positivity, and 2 were weakly positive. Adenovirus IHC was performed on 29 specimens, and 1 was weakly positive. ddPCR performed on 27 formalin-fixed paraffin-embedded tissues identified 6 positive CMV cases (including all 4 IHC+ cases), 0 adenovirus cases, and 1 HHV-6-positive case. Among the CMV cases, the most common pattern was mixed, seen in 4 of 6 cases, whereas the most common morphologic feature was apoptosis, seen in 5 of 6 cases. A summary of findings is shown in Table 1. Conclusion: In this subset of pts who underwent intestinal biopsy during IDELA clinical trials, most biopsies (87%) were performed in the context of ongoing diarrhea/colitis and the majority of tissue samples (90%) revealed some component of inflammation. Immune profiling by IHC showed increased Tregs in patterns associated with glandular destruction. Immunohistochemistry and ddPCR for virus in conjunction with microbiology studies revealed that in 28% of these cases, a pathogen was identified. This dataset suggests that in a significant number of cases, idelalisib-associated diarrhea/colitis may be due to an infectious etiology secondary to immune dysfunction that results in either an inflammatory/ischemic or a mixed histologic pattern; however, the majority of these cases have no identifiable infectious etiology. Based on these findings, we recommend that a workup of pts with idelalisib-associated diarrhea/colitis should include a complete evaluation for infectious causes including intestinal biopsy when a pathogen is elusive with standard testing. Disclosures Yeung: Gilead Sciences: Research Funding. Hockenbery:Gilead Sciences: Research Funding. Coutre:Gilead Sciences: Consultancy, Research Funding. Dubowy:Gilead Sciences: Employment, Equity Ownership. Marcondes:Gilead Sciences: Employment, Equity Ownership. Munugalavadla:Gilead Sciences: Employment, Equity Ownership. Bosch:Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Research Funding.

Introduction: Idelalisib (IDELA) is a targeted PI3Kd inhibitor approved as monotherapy in relapsed follicular lymphoma and in combination with rituximab in relapsed chronic lymphocytic leukemia (CLL). Increased rates of adverse events (AEs) were recently observed in the IDELA vs placebo arms of randomized controlled trials (RCT) evaluating IDELA added to standard therapies in front-line CLL and early-line indolent non-Hodgkin lymphoma (iNHL). AEs leading to death were mainly infectious and included pneumocystis jirovecii pneumonia (PJP) and cytomegalovirus (CMV). This analysis across trials in the relapsed population evaluated whether quantitative changes in lymphocyte subsets may have contributed to these AEs. Methods: Peripheral blood immunophenotypic data available for analysis from patients (pts) (n = 1,480) treated in 5 IDELA RCTs were analyzed. Three studies (n = 787) included pts with relapsed CLL (NCT01569295: IDELA + bendamustine-rituximab [BR] vs placebo + BR; NCT01539512: IDELA + R vs placebo + R and NCT0165902: IDELA + ofatumumab [O] vs placebo + O) and 2 studies (n = 693) included R/R iNHL pts (NCT01732913: IDELA + R vs placebo + R and NCT01732926: IDELA +BR vs placebo + BR). Absolute numbers of T (CD4+ and CD8+), B (CD19+) and NK (CD16+/CD56+) cells were analyzed longitudinally in both IDELA and placebo pts across the 5 studies. Lymphocyte subsets were analyzed separately in those who died and then correlated with specific grade ≥3 AEs including infections, febrile neutropenia, and respiratory (acute respiratory failure, pneumonitis) events. Analysis was conducted within individual study and for all studies combined. Of note, samples were collected more frequently during the first 6 months (during combination therapy) and collection times varied among the 5 studies. Results: There was no specific trend noted with the CD8+ T-cells between treatment groups across the studies. Generally, NK-cells were decreased to a similar degree in both IDELA and placebo pts at weeks 10 to 12 with recovery starting around week 24. There were no differences in median NK- and CD8+ T-cell counts between pts with grade ≥3 AEs and no AEs within either group. In both BR trials, CD4+ T-cells nadir to 900 cells/µL. Conclusion: Within 5 RCTs evaluating IDELA vs placebo in combination with an anti-CD20 mAb or BR in R/R CLL or iNHL, there was no correlation between grade ≥3 AEs and NK- or CD8+ T-cell counts. Median CD4+ T-cells in pts on the BR trials were lower in both groups in those with and without AEs, compared with non-BR trials. In addition, pts with PJP and CMV infections were noted to have CD4+ T-cells Figure 1 Figure 1. Incidence of PJP and CMV Infections and Correlation with CD4 Count. Disclosures Sharman: Gilead Sciences, Inc.: Honoraria, Research Funding. Salles:Mundipharma: Honoraria; Amgen: Consultancy, Honoraria; Gilead: Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding. Jurczak:Celltrion, Inc: Research Funding; Janssen: Research Funding; Gilead Sciences: Research Funding; Acerta: Research Funding; Bayer: Research Funding. Jones:AbbVie: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Gilead Sciences: Consultancy, Research Funding; PCYC: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Owen:Janssen: Honoraria; Gilead: Honoraria, Research Funding; Pharmacyclics: Research Funding; Celgene: Honoraria, Research Funding; Abbvie: Honoraria; Lundbeck: Honoraria, Research Funding; Novartis: Honoraria; Roche: Honoraria, Research Funding. Munugalavadla:Gilead Sciences: Employment, Equity Ownership. Dreiling:Gilead Sciences: Employment, Equity Ownership. Xiao:Gilead Sciences: Employment, Equity Ownership. Rao:Gilead Sciences: Employment, Equity Ownership. Flinn:Janssen: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Gilead Sciences: Research Funding; ARIAD: Research Funding; RainTree Oncology Services: Equity Ownership. O'Brien:Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria.

Introduction: Idelalisib (IDELA) is a selective inhibitor of PI3Kδ that has demonstrated efficacy in indolent lymphoma. In the NCT01659021 (101-09) study in patients (pts) with previously treated iNHL, the ORR was 57%. However, in this heavily-treated and refractory population, 27.2% (34/125 pts) discontinued the study due to reported ≥ grade 3 adverse events (AEs). This analysis aims to investigate an underlying genetic molecular mechanism(s) that may contribute to IDELA associated AEs. Methods: We performed a genome-wide association study (GWAS) to determine if common genetic factors influence the risk of AEs. We utilized available DNA samples from 66 subjects from the 101-09 clinical study to conduct genotyping at 2,088,393 single nucleotide polymorphisms (SNPs) using the Illumina HumanOmni2.5 high-density BeadArray chip. In parallel, we also conducted Illumina whole exome sequencing (WES) to identify causal functional variation, as well as to examine the role of rare functional variations that may influence the development of AEs. The phenotypic endpoints assessed in this GWAS analysis were: grade ≥3 any AE, diarrhea/colitis (DC), abnormal liver function test (LFT) and pneumonitis. True controls were defined as subjects with no reported AEs of any grade. Results: Logistic regression analysis of the combined AE groups, DC, and pneumonitis status, identified no common SNPs among Caucasians, or any ethnic subgroups that exceeded the genome-wide threshold for statistical significance. For the abnormal LFT phenotype, a total of seven SNPs (four in the all ethnicity and three in the Caucasian subgroup analysis) were found to exceed the genome-wide threshold for statistical significance. Six of these SNPs were considered false positives resulting from the small sample size. The remaining SNP (rs1351623), located in intron 1 of the tachykinin receptor 3 (TACR3) gene, was present in 12 out of 23 controls (minor allele frequency of 28.3% vs. 17.6% in the general population), but was completely absent from the 9 pts in the abnormal LFT case group, suggesting that the minor allele may be protective against altered liver function. However, TACR3 does not have an obvious biological role in mediating liver function and we were unable to identify any potentially causal functional variants in TACR3 or any other genes in close proximity which could sufficiently explain the observed association. Follow-up analysis of this association using statistical tests more robust for rare data indicated that this association may much more likely be due to chance (p=0.0187; Firth logistic regression). Analyses of the WES dataset using single variant and gene-based collapsing tests showed no single nucleotide variants or genes that exceeded the threshold for statistical significance after correcting for multiple testing. Conclusion: With the current dataset, no evidence was observed to support a role for human genetic variation of large effect size in influencing the risk of AEs during IDELA therapy. In pts with abnormal LFTs, we identified 7 SNPs that exceeded the genome-wide threshold for statistical significance. All but one of these associations were found to be false positives and there was no compelling evidence to support the remaining association with the rs1351623 SNP in TACR3. In both the GWAS and WES, significance scores from virtually all association analyses showed evidence of moderate p-value deflation, suggesting that the analyses were underpowered due to the limited sample size of the study. Therefore, further genetic studies using larger cohorts of patients treated with IDELA are needed to determine if genetic factors influence the risk of adverse events. Disclosures Goldstein: Astra Zeneca: Consultancy; Janssen: Consultancy; Pairnomix: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Clarus: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; LabCorp: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Lilly: Consultancy. Munugalavadla:Gilead Sciences: Employment, Equity Ownership. Dreiling:Gilead Sciences: Employment, Equity Ownership. Jurczak:Acerta: Research Funding; Janssen: Research Funding; Bayer: Research Funding; Gilead Sciences: Research Funding; Celltrion, Inc: Research Funding. Flinn:Janssen: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Gilead Sciences: Research Funding; ARIAD: Research Funding; RainTree Oncology Services: Equity Ownership. Salles:Mundipharma: Honoraria; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Gilead: Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding.

Introduction: Complex karyotype (CKT), defined as the presence of ≥3 chromosomal aberrations, is emerging as an adverse prognostic factor in chronic lymphocytic leukemia (CLL) (Thompson et. al., Cancer 2015; Herling et. al., Blood, 2016). Idelalisib (IDELA) is a selective PI3Kd inhibitor approved for use in patients (pts) with relapsed CLL. A phase 3 randomized study of rituximab (R) plus IDELA or placebo (PBO) in pts with relapsed CLL and comorbidities (NCT1539512) demonstrated superior progression-free survival (PFS) and overall survival (OS) in the IDELA arm (Furman et. al. NEJM 2014). This report examines the effect of CKT on long-term outcome in that study. Methods: Key eligibility criteria included CLL requiring therapy after progression within 24 months of last therapy, and being unfit to receive cytotoxic therapy. Pts were randomized to receive IDELA at 150 BID (n = 110) or PBO (n = 110) in combination with R at 375 mg/m2 (first dose) and then 500 mg/m2 q2 weeks x 4, followed by q4 weeks x 3 (8 doses total). After progression, prior PBO+R pts could enroll into an extension study to receive IDELA at 150 mg BID. The primary endpoint of PFS was assessed by an IRC using standard criteria (Hallek et. al. Blood 2008; Blood 2012; Cheson et. al. J Clin Oncol 2012). After the first prespecified interim analysis, with median IDELA exposure time of 3.8 months, the study was stopped due to overwhelming difference in efficacy and pts on PBO arm were offered open-label IDELA on the extension study. Baseline peripheral blood samples for karyotype analysis, TP53 mutation (mut), and FISH for del(17p) were analyzed by central labs. Metaphase spreads were produced after IL-2/CpG-stimulated cell cultivation and karyotypes were analyzed per ISCN 2013 nomenclature. Results: Based on geography, two labs received a total of 283 screening blood samples and prepared metaphase spreads, with one lab completing the karyotypic analyses. The overall karyotypic success rate was 65%, but was 98% for samples initially processed in one of the labs. Karyotypes were successfully performed in 127 of 220 randomized pts. The median follow-up for karyotyped IDELA and PBO pts was 21.4 months (range 0.3-39.4) and 12.0 months (range 0.2-35.8), respectively. CKT and non-CKT were found in 26 and 39 IDELA pts and 24 and 38 PBO pts, respectively. Overall, 16 IDELA pts and 15 PBO pts had del(17p)/TP53mut along with CKT. Within the IDELA group: the overall response rate (ORR) was 80.8% (95% CI 60.6-93.4) with CKT and 89.7% (95% CI 75.8-97.1) with non-CKT; the median PFS was 20.9 months in CKT vs 19.4 in non-CKT, HR = 1.18 (p = 0.63); the med OS was NR vs NR in CKT vs non-CKT, HR = 1.78 (95%CI 0.69-4.64; p = 0.23). The presence or absence of del(17p)/TP53mut and CKT status did not significantly affect PFS or OS in pts randomized to IDELA (Figure 1). Prior to crossover, the median PFS for IDELA vs PBO pts with CKT was NR vs 3.7 months, HR = 0.16 (p Conclusion:In this first systematic evaluation of CKT in relapsed CLL, CKT was found in 44% of 185 successfully karyotyped screened patients, including 37% of those randomized on study. This post hoc exploratory analysis demonstrates the lack of any adverse prognostic effect of CKT on IDELA-treated patients with respect to PFS or OS, and there was no significant difference among subsets of these pts with and without del(17p)/TP53mut. As previously reported in the entire study intention-to-treat population, a survival and PFS advantage was imparted by the addition of IDELA to R in pts with CKT. Detailed analyses including other clinical and prognostic parameters will be presented. Figure Progression-Free Survival and Overall Survival in Patients Randomized to Idelalisib Figure. Progression-Free Survival and Overall Survival in Patients Randomized to Idelalisib Figure Figure. Disclosures Kreuzer: Gilead Sciences: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche Pharma GmbH and Mundipharma GmbH: Consultancy, Honoraria, Research Funding, Speakers Bureau. Furman:Abbvie: Consultancy, Honoraria; Gilead Sciences: Consultancy; Janssen: Consultancy; Pharmacyclics: Consultancy, Speakers Bureau; Genentech: Consultancy. Stilgenbauer:Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding. Dubowy:Gilead Sciences: Employment, Equity Ownership. Kim:Gilead Sciences: Employment, Equity Ownership. Munugalavadla:Gilead Sciences: Employment, Equity Ownership. Pettitt:Celgene: Speakers Bureau; Infinity: Research Funding; Gilead: Research Funding, Speakers Bureau; Roche: Research Funding, Speakers Bureau. Hallek:Gilead Sciences: Research Funding, Speakers Bureau.

Inhibitors of B-cell receptor (BCR) signaling have proven effective in the treatment of chronic lymphocytic leukemia (CLL). An important downstream mediator of BCR signaling is phosphoinositide 3-kinase delta (PI3Kd), which through activation of AKT, controls cell survival, growth and proliferation. Since it is expressed predominantly in cells of hematopoietic origin, PI3Kd is a promising target in CLL, and the novel PI3Kd inhibitor idelalisib is effective in treating relapsed/refractory CLL. However, a subset of patients relapse under therapy, and the mechanisms leading to resistance are not understood. To generate and study resistance mechanisms in vivo, a tool compound GS-649443 with favorable murine pharmacokinetic properties in vivo as compared to Idelalisib was used. GS-649443 is a highly specific and potent small molecule inhibitor of the PI3K isoform d, with an IC50 of 0.3nM and 86/420/4120 fold selectivity over PI3K isoforms g/b/a respectively. We performed an in vivo serial transfer and treatment scheme to generate specific resistance against GS-649443 using the transferable murine CLL clone TCL1-192(Chen SS et al., 2013). 5 million TCL1-192 cells were transferred by intravenous injection into the tail vein of CB17.SCID mice, followed by treatment with vehicle (N=12) or 5mg/kg GS-649443 (N=12) twice daily by oral gavage, starting from day 10. Analysis of the animals (N=6 vs. 6) at the death of the vehicle treated mice (11 days) showed a drastic reduction in spleen weight (median 0.650g vs. 0.345g, P=0.005), liver weight (median2.025g vs. 1.145g, P=0.0022) and WBC count (median220.4 cells/nl vs. 1.7 cells/nl, P=0.0078) as compared to vehicle treated mice. Also, GS-649443 treatment almost doubled the survival of the mice in comparison to vehicle treatment (Fig. 1, P=0.0011). To generate resistance against GS-649443 in vivo, TCL1-192 cells were kept under selection pressure using serial transfers and subsequent treatment. After three rounds of transfer and treatment, the mice treated with GS-649443 failed to respond and showed a survival identical to vehicle controls (Fig.1). Mutations acquired over time during the serial transfers in TCL1-192 cells from resistant mice were identified using whole exome sequencing. 64 treatment specific mutations were identified with a tumor variant allele frequency of ≥10%. Intriguingly, no single recurrent mutation that would likely contribute to drug resistance was identified. This is similar to the observation in patients refractory to idelalisib (abstract Ghia et al., submitted to ASH 2016). However, a subset of the genes such as Grb2, Mylk, Srfbp1, Ptk2, Cd44 and Prkd1 harboring mutations could be functionally grouped into integrin and extracellular matrix signaling. Consistent with this, all the resistant tumors showed a significant upregulation of genes involved in the integrin receptor complex such as talin 1 (P=0.004), talin 2 (P=0.004) and vinculin (P=0.009). RNAseq analysis for detecting changes in gene expression identified an upregulation of Igf1r expression in the resistant samples compared to vehicle treated samples (P The synergy between the receptor tyrosine kinases such as IGF1R and integrin mediated signaling is established in different cancers and a similar mechanism may play a role in compensating for the inhibition of BCR mediated PI3K/AKT signaling. In conclusion, resistance to a tool PI3Kd inhibitor is not mediated by unique recurrent mutations but by alterations in survival signaling with mutations playing a probable cooperative role. Treatment scheme for generation of resistance in vivo. Treatment scheme for generation of resistance in vivo. Disclosures Tausch: Celgene: Other: Travel support; Amgen: Other: Travel support; Gilead: Other: Travel support, Speakers Bureau. Yahiaoui:Gilead Sciences: Employment. Munugalavadla:Gilead Sciences: Employment, Equity Ownership. Tannheimer:Gilead Sciences: Employment. Stilgenbauer:Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding.

Introduction: Idelalisib (IDELA) is an ATP-competitive, reversible, and selective small molecule inhibitor of the delta isoform of phosphatidylinositol 3-kinase (PI3Kδ) approved for the treatment, in combination with rituximab, of patients with relapsed chronic lymphocytic leukemia (CLL). In the relapsed CLL randomized, controlled trials, IDELA + rituximab showed high response rates with improved progression-free and overall survival as compared with placebo + rituximab. While IDELA therapy has significant efficacy, disease progression after response occurs, indicating that escape mechanisms may develop. However, the molecular basis for relapse or progressive disease (PD) in CLL patients treated with IDELA has not yet been characterized. Methods: Peripheral blood mononuclear cells (PBMCs) were collected from 13 CLL patients enrolled in the phase 3 studies; NCT01539512 (study 116; IDELA + rituximab vs placebo + rituximab), 116 extension study NCT01539291 (study 117) and NCT01659021 (study 119; IDELA + ofatumumab). Sample selection criteria included treatment period of at least 180 days (range: 243-703 days), achieving at least partial nodal response followed by PD, PD did not occur within a drug interruption window, PD was not associated with Richter's transformation, and PBMC samples were available from both baseline and time of PD. Whole-exome sequencing (WES) was conducted on the matched samples from 13 subjects fitting the above criteria. In 6/13 cases, DNA was available from CD19+/CD5+ enriched tumor cells, and neutrophils or T-lymphocytes served as a source of germline DNA. These 6 patients were considered as a discovery set for mutational analysis. Established bioinformatics tools were used for detection of somatic mutations and for the comparison of baseline and PD samples. Results: Baseline clinical patient profiles indicated that 12 of 13 patients with PD had unmutated IGHV genes and 8 patients carried TP53 aberrations (ie, 17p deletion and/or TP53 mutation). WES resulted in a mean read depth of 106X within the targeted coding region across samples. In the discovery set, on average 25 somatic mutations (range: 4-44) at baseline and 32 mutations (range: 15-81) at progression were identified. By comparing baseline and PD samples, we identified 88 PD-associated mutations. These specific mutations were tested for in a complete set of 13 patient samples; however, no recurrent progression-associated mutations were identified in more than 1 patient. In particular, no progression-associated mutations were identified in the PI3K signaling pathway or in any other related pathway. Conclusion: Across 3 phase 3 studies in relapsed CLL, WES on 13 samples from patients with PD while on IDELA treatment were evaluated. This analysis detected no relapse-associated mutations in common across this patient set; in particular, no mutations were identified in the drug-binding site (ie, "gateway mutation") or in any other related signaling pathway. Based on these results, we conclude there is no common mutational mechanism of IDELA resistance in this patient group. Disclosures Ghia: Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Honoraria, Research Funding; Adaptive: Consultancy; Abbvie: Consultancy, Honoraria. Tausch:Gilead: Other: Travel support, Speakers Bureau; Celgene: Other: Travel support; Amgen: Other: Travel support. Owen:Roche: Honoraria, Research Funding; Pharmacyclics: Research Funding; Celgene: Honoraria, Research Funding; Abbvie: Honoraria; Lundbeck: Honoraria, Research Funding; Novartis: Honoraria; Janssen: Honoraria; Gilead: Honoraria, Research Funding. Barrientos:Gilead: Consultancy, Research Funding; Janssen: Consultancy; AbbVie: Consultancy, Research Funding. Munugalavadla:Gilead Sciences: Employment, Equity Ownership. Degenhardt:Gilead Sciences: Employment, Equity Ownership. Kim:Gilead Sciences: Employment, Equity Ownership. Dubowy:Gilead Sciences: Employment, Equity Ownership. Dreiling:Gilead Sciences: Employment, Equity Ownership. Rosenquist:Gilead Sciences: Speakers Bureau. Stilgenbauer:Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding.

We show that loss of p85α inhibits the growth and maturation of mast cells, whereas loss of p85β enhances this process. Whereas restoring the expression of p85α in P85α−/− cells restores these functions, overexpression of p85β has the opposite effect. Consistently, overexpression of p85β in WT mast cells represses KIT-induced proliferation and IL-3–mediated maturation by inhibiting the expression of Microphthalmia transcription factor. Because p85α and p85β differ in their N-terminal sequences, chimeric proteins consisting of amino or carboxy-terminal of p85α and/or p85β do not rescue the growth defects of p85α−/− cells, suggesting cooperation between these domains for normal mast cell function. Loss of p85β impaired ligand induced KIT receptor internalization and its overexpression enhanced this process, partly because of increased binding of c-Cbl to p85β relative to p85α. In vivo, loss of p85β resulted in increased mast cells, and bone marrow transplantation of cells overexpressing p85β resulted in significant reduction in some tissue mast cells. Overexpression of p85β suppressed the growth of oncogenic KIT-expressing cells in vitro and prolonged the survival of leukemic mice in vivo. Thus, p85α and p85β differentially regulate SCF and oncogenic KIT-induced signals in myeloid lineage-derived mast cells.

The microenvironment (ME) critically promotes progression of chronic lymphocytic leukemia (CLL), favoring leukemic cell survival and proliferation as well as inducing drug resistance. We aimed at reproducing the effects of ME stimuli for the development and optimization of an ex vivo assay that would enable to predict in vivo drug efficacy for agents interfering with ME protective effects e.g. the novel BCR inhibitors. To this purpose, we exploited the following 2 new approaches: 1) the Exvitech® proprietary automated flow cytometry-based platform by Vivia Biotech that enables evaluation of up to 20.000 wells and conditions per sample, and 2) Viviaxs Precision Medicine Native Environment approach that utilizes the whole blood sample rather than isolated leukocytes. We present this assay optimization using primary CLL samples and its validation when exposing CLL cells to the registered PI3Kd inhibitor, Idelalisib, for which only a weak pro-apoptotic effect has been reported, besides the known tissue mobilization activity in vivo. Cryopreserved peripheral blood (PB) mononuclear cells were provided from CLL patients in need of treatment. In order to more closely reproduce the complexity of the in vivo ME, the following elements were evaluated in different combinations: (i) 3 backbone stimulations, previously reported to improve, to different extent, CLL viability and proliferation: CD40L+CpG, CD40L+IL21, CpG+IL2; (ii) "Native Environment", defined as the plasma & erythrocyte/granulocyte fraction of a Ficoll gradient, already shown to improve ex vivo drug testing (Bennet et al. Clin Lymphoma Myeloma Leuk. 2014;14:305-18): the two fractions were added to thawed CLL samples and were obtained from fresh samples of normal donor PB or bone marrow as well as from CLL patients at different stages of disease; (iii) the stroma cell line H5S, added at different ratios (1:10 or 1:100); (iv) both human and bovine fetal serum (at 10 or 20% total volume); (v) stimulatory B cell factors, including IL-21, soluble CD40L, BAFF, and B cell receptor stimulation (anti-IG). CLL cell viability and proliferation was then tested and, although CLL cells from PB are notorious for a low proliferative index and tend to die quickly ex vivo by apoptosis, we achieved a median of 30±3% proliferation (assessed by the CFDA dye) and 60±5% viability (assessed by Annexin V staining) in cryopreserved progressive CLL samples. These results were obtained with the combination of the following assay conditions: CpG+IL2, HS5 (at 1:100 ratio), human serum 10%, and "native environment" from PB of CLL samples (pooled samples to prevent interpatient variability). We then tested the dose responses of Idelalisib in 16 cryopreserved progressive CLL samples and found little effect on the non-proliferative CLL fraction (Fig, 1A), suggesting a limited direct pro-apoptotic activity of the drug. In contrast, potent inhibition of proliferation with median potency (EC50) of 14 nM was observed (Fig 1B). The efficacy was nearly complete leaving a median of 5% resistant CLL cells that proliferated at the highest doses of Idelalisib. In conclusion, we report a novel ex vivo assay that enables high-throughput pharmacological characterization of compounds and combinations, optimized for CLL cells by incorporating ME stimuli and thereby more accurately simulating in vivo interactions. The increased cell viability and proliferation achieved with this innovative assay offers improved opportunities for ex vivo pharmacology, in particular unraveling a hitherto unknown anti-proliferative mode of action for Idelalisib, a drug interfering with the interaction of CLL cells with the ME. Figure 1. Dose response curves of Idelalisib incubated for 96 h with 16 CLL samples in the new Microenvironment Native Environment assay. The effect on non-proliferative (A) and proliferative (B) CLL cells identified using flow cytometry as subpopulations with different CFDA staining is shown. Figure 1. Dose response curves of Idelalisib incubated for 96 h with 16 CLL samples in the new Microenvironment Native Environment assay. The effect on non-proliferative (A) and proliferative (B) CLL cells identified using flow cytometry as subpopulations with different CFDA staining is shown. Disclosures Ballesteros: Vivia Biotech: Employment. Primo:Vivia Biotech: Employment. Robles:Vivia Biotech: Employment. Gorrochategui:Vivia Biotech: Employment. Munugalavadla:Gilead Sciences: Employment. Stamatopoulos:Gilead Sciences: Research Funding; Janssen Pharmaceuticals: Research Funding. Quéva:Gilead Sciences: Employment, Equity Ownership. Ghia:AbbVie: Consultancy; Pharmacyclics: Consultancy; Gilead: Consultancy, Research Funding, Speakers Bureau; Adaptive: Consultancy; Acerta Pharma BV: Research Funding; GSK: Research Funding; Roche: Consultancy, Research Funding; Janssen: Consultancy.

Erythropoiesis is regulated by a number of growth factors, among which stem cell factor (SCF) and erythropoietin (Epo) play a non-redundant function. Viable mice with mutations in the SCF gene (encoded by the Steel (Sl) locus), or its receptor gene c-Kit (encoded by the White spotting (W) locus) develop a hypoplastic macrocytic anemia. Mutants of W or Sl that are completely devoid of c-Kit or SCF expression die in utero of anemia between days 14 and 16 of gestation and contain reduced numbers of erythroid progenitors in the fetal liver. Likewise, Epo and Epo receptor (Epo-R)-deficient mice die in utero due to a marked reduction in the number of committed fetal liver derived erythroid progenitors. Thus, committed erythroid progenitors require both c-Kit and Epo-R signal transduction pathways for their survival, proliferation and differentiation. In vitro, Epo alone is capable of generating mature erythroid progenitors; however, a combined treatment of Epo and SCF results in synergistic proliferation and expansion of developing erythroid progenitors. This review summarizes recent advances made towards understanding the signaling mechanisms by which Epo-R and c-Kit regulate growth, survival, and differentiation of erythroid progenitors alone and cooperatively.

Abstract 1839 The PIM kinases are a family of 3 growth factor- & cytokine-induced proteins hypothesized to have redundant survival and growth functions. Although PIM-1, -2 have been noted as highly expressed in multiple myeloma (MM) (Claudio JO et al., 2002), there are few data to support potential therapeutic utility of PIM inhibition in this indication. Here we show that the myeloma cell lines express all PIM protein isoforms to varying extents, and we describe the properties of a novel pan-PIM inhibitor GNE-652 with picomolar biochemical potency, an excellent selectivity profile, and favorable ADME properties. Myeloma cell lines and patient samples exhibit a striking prevalence of response to GNE-652 (23 of 25 lines with IC50 < 1 micromolar, median < 0.1 micromolar) and synergy in combination with the PI3K inhibitor GDC-0941 (mean combination index values ∼0.2 (n=25)). MM cells respond to this combination with cell cycle arrest and marked apoptosis in vitro. Conversely, a PIM-1, -3 selective inhibitor, GNE-568, failed to suppress MM cell growth and also failed to provide synergy in combination with PI3K inhibition, suggesting PIM-2 is a critical driver of MM cell growth & survival. Additional results suggest that PIM signaling converges on both TORC1 and AKT to generate differential synergies with PI3K/AKT/mTOR pathway inhibitors. PIM has been shown to potentially inactivate PRAS40, a negative regulator of TORC1 (Zhang et al., 2009). We demonstrate that PIM or PI3K inhibition caused a loss of phosphorylation on PRAS40 and resulted in a physical association of PRAS40 and TORC1 and a decrease in phosphorylated p70S6K and S6RP. These reductions were apparent in 7 of 7 cell lines assayed and enhanced by the combination of PI3K and PIM inhibition. Consistent with prior reports (Hammerman et al., 2005), we show that a second node of convergence between PIM and TORC1 is 4E-BP1. Both GDC-0941 and GNE-652 treatments reduced phosphorylation of 4E-BP1 in all the myeloma cell lines tested. Since dephosphorylated 4E-BP1 competes with eIF4G for the mRNA cap binding factor eIF4E, we assayed immunoprecipitates of eIF4E for the presence of eIF4G and 4E-BP1 and observed increased BP1 and decreased 4G. The combination treatment significantly enhanced the loss of 4G relative to either single agent, and importantly, even at 5 × IC50 concentrations for single agents, combination drug treatment achieved greater extent of effect than single agent treatment. It has been hypothesized that a subset of mRNAs are particularly sensitive to inhibition of cap-dependent translation, including a number of oncogenes such as cyclin D1. We noted across 7 different myeloma cell lines, strong decreases in levels of cyclin D1, and D3 that were further decreased by combination treatment of PIM and PI3K inhibition. In summary, we have identified several points at which PIM and PI3K/AKT/mTOR converge to provide synergy in multiple myeloma cell lines. As PIM isoforms are highly expressed in MM cells, we hypothesized that this could be due to proteosomal-mediated stability, and interestingly, MG132 and velcade each stabilized all PIM isoforms. It is commonly known that the JAK/STAT pathway regulates PIM transcription, but we show JAK inhibitors failed to abolish the expression of PIM in myeloma cells, suggesting a role for additional regulators. Recent genome sequencing studies from human myeloma samples (Chapman MA et al., 2011) confirmed the prevalence of NF-kB pathway activation, consistent with prior observations made in MM cell lines (Demchenko YN et al., 2010). The relationship of PIM and NF-kB is controversial in the literature (Hammerman PS et al., 2004 & Zhu N et al., 2002), with some groups placing PIM upstream of NF-kB and others the converse. Using an IκBα inhibitor, BMS-345541, we have examined the role for NF-kB in the regulation of PIM kinases. Here, we show that the BMS-345541 could preferentially suppress PIM2 expression in a dose dependent manner while PIM 1, 3 levels are modestly affected, suggesting that the high levels of PIM2 expression observed are partly driven by deregulation of the NF-kB pathway in MM. In conclusion, we provide pharmacological and biochemical evidence to suggest that PIM2 differentially regulate growth and survival of myeloma cells. Our results provide the rationale for further preclinical development of PIM inhibitors and the basis for a possible clinical development plan in multiple myeloma. Disclosures: Munugalavadla: Genentech: Employment. Berry:Genentech: Employment. Chang:Genentech: Employment. Rosario:Genentech: Employment. Drummond:Genentech: Employment. Du:Genentech: Employment. Fitzgerald:Genentech: Employment. Friedman:Genentech: Employment. Gould:Genentech: Employment. Maecker:Genentech: Employment. Moffat:Genentech: Employment. Slaga:Genentech: Employment. Xiaojing:Genentech: Employment. West:Genentech: Employment. Yu:Genentech: Employment. Ebens:Genentech: Employment.

Abstract 868 Gain-of-function mutations in KIT receptor in humans are associated with gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), and acute myelogenous leukemia (AML). An activating KIT receptor mutation of aspartic acid to valine at codon 814 in mice (KITD814V) or codon 816 in humans (KITD816V) results in altered substrate recognition and constitutive tyrosine autophosphorylation leading to promiscuous signaling. Consequently, cells bearing oncogenic form of KIT (KITD814V) demonstrate ligand independent proliferation in vitro and MPD in vivo. However, the intracellular signals that contribute to KITD814V induced MPD are not known. Here, we show the constitutive phosphorylation of SHP2 in cells bearing KITD814V, but not WT KIT, which was inhibited by treatment with a novel SHP2 inhibitor, II-B08 (*p < 0.05). In addition, treatment with II-B08 suppressed the growth of cells bearing KITD814V, but not WT KIT (*p < 0.05), Human mast cell line HMC1.2 (*p < 0.05), and Human CD34+ cells bearing KITD816V (*p < 0.05). Likewise, deficiency of SHP2 in primary bone marrow cells resulted in a significant repression in constitutive growth of cells bearing KITD814V (*p < 0.05). To determine the mechanism behind the repression in ligand independent growth of cells bearing KITD814V by II-B08, we examined the role of SHP2 in cell survival. We observed a dose dependent increase in apoptosis of cells bearing KITD814V compared to WT KIT in the presence of II-B08 (*p < 0.05). Similarly, deficiency of SHP2 resulted in increased apoptosis of cells bearing KITD814V (*p < 0.05). In an effort to identify the mechanism behind reduced growth and increased apoptosis of cells bearing KITD814V in the presence of II-B08, we examined whether SHP2 regulates the activation of AKT in KITD814V bearing cells. We found constitutive activation of AKT in cells bearing KITD814V, but not WT KIT, which was significantly inhibited upon II-B08 treatment suggesting that SHP2 regulates ligand independent growth and survival of KITD814V bearing cells in part by regulating the activation of AKT. To further determine the signaling molecules that co-operate with SHP2 in KITD814V induced MPD, we examined proteins that potentially interact with SHP2 in KITD814V bearing cells and whether inhibition of SHP2 activity by II-B08 suppresses these co-operating protein interactions. SHP2 constitutively bound to p85α and Gab2 in cells bearing KITD814V, but not in WT KIT bearing cells, which was inhibited upon II-B08 treatment. To further assess whether the binding of SHP2 to p85α, Gab2 and KITD814V is sufficient to induce MPD, we generated a KIT mutant receptor, KITD814V-F7, in which seven tyrosine residues in KITD814V (known to bind SH2 containing proteins at tyrosine 567, 569, 702, 719, 728, 745, and 934) were converted to phenylalanine, and KITD814V-Y719, in which only tyrosine residue 719 (binding site for p85α) was added back to the KITD814V-F7 receptor. We observed constitutive binding of SHP2 and Gab2 to p85α in cells bearing KITD814V and KITD814V-Y719, but not in WT KIT or KITD814V-F7 bearing cells. In addition, conversion of all the seven intracellular tyrosine residues in KITD814V to phenylalanine (KITD814V-F7) resulted in complete loss of ligand independent growth in vitro (*p < 0.05) and significantly delayed the progression of MPD in vivo (*p < 0.05). Importantly, restoration of tyrosine residue at position 719 (KITD814V-Y719) was sufficient to induce ligand independent growth in vitro and MPD in vivo to KITD814V levels. Furthermore, deficiency of Gab2 resulted in significant repression in constitutive growth of cells bearing KITD814V (*p < 0.05). These results demonstrate that p85α recruits SHP2 and Gab2 to KITD814V at Y719, which might contribute to KITD814V induced MPD. Moreover, II-B08 enhances the efficacy of PI3Kinase inhibitor LY294002 in suppressing KITD814V induced ligand independent growth in vitro (*p < 0.05) and MPD in vivo (*p < 0.05). Taken together our results demonstrate that SHP2 is a druggable target which cooperates with PI3Kinase in inducing MPD. Disclosures: No relevant conflicts of interest to declare.

Abstract 4084 Related work from our group has shown the therapeutic utility of PIM inhibition in multiple myeloma cell lines, xenografts, and primary patient samples (Ebens A. et al., ASH 2010 submitted abstr.). In this study we provide detailed mechanistic findings to show that PIM kinase inhibition co-regulates several important elements of the PI3K/AKT/mTOR pathway, resulting in significant synergy for combination drug treatments. The PIM kinases are a family of 3 ser/thr growth factor- & cytokine-induced proteins hypothesized to have redundant survival and growth functions. GNE-652 is a pan-PIM kinase inhibitor with picomolar biochemical potencies and an excellent kinase selectivity profile. Myeloma cell lines exhibit sensitivity to single agent PIM inhibition and a striking synergy in combination with the PI3K inhibitor GDC-0941. Cells respond to this combination with cell cycle arrest and marked apoptosis in vitro. We tested a panel of selective PI3K/AKT/mTOR inhibitors and found PI3K and AKT inhibitors showed the greatest extent of synergy with GNE-652, whereas mTOR inhibitors were synergistic to a lesser extent. These results suggest that PIM signaling converges on both TORC1 and AKT to generate these differential synergies. BAD is a negative regulator of both Bcl-2 and Bcl-XL, and we were able to confirm previous reports that AKT and PIM cooperate to inactivate BAD (Datt et al., 1997; Yan et al., 2003). Pim has been shown to potentially inactivate PRAS40, a negative regulator of TORC1 (Zhang et al., 2009). We demonstrate that PIM or PI3K inhibition caused a loss of phosphorylation on PRAS40 and results in a physical association of PRAS40 and TORC1 and a decrease in phosphorylated p70S6K and S6RP. These reductions were apparent in 7 of 7 cell lines assayed and enhanced by the combination of PI3K and PIM inhibition in these cell lines. Consistent with prior reports (Hammerman et al., 2005), we show that a second node of convergence between PIM and TORC1 is 4E-BP1. Both GDC-0941 and GNE-652 treatments reduced phosphorylation of 4E-BP1 in 7 of 7 myeloma cell lines. Since dephosphorylated 4E-BP1 competes with eIF4G for the mRNA cap binding factor eIF4E, we assayed immunoprecipitates of eIF4E for the presence of eIF4G and 4E-BP1 and observed increased BP1 and decreased 4G. The combination treatment significantly enhanced the loss of 4G relative to either single agent, and importantly, even at 5× the IC50 concentrations for single agents, combination drug treatment achieved greater extent of effect than single agent treatment. Thus PI3K and PIM pathways are redundant at the level of cap-dependent translational initiation mediated by eIF4E. It has been hypothesized a subset of mRNAs are particularly sensitive to inhibition of cap-dependent translation, and that this includes a number of oncogenes such as cyclin D1. We assayed global protein synthesis in MM1.s cells using 35S-methionine and as expected we observed only a modest ≂∼f20% decrease caused by either GNE-652 or GDC-0941 and this decrease was not enhanced by combination treatment. However, we noted across 7 different myeloma cell lines, strong decreases in levels of cyclin D1 that were enhanced by combination treatment. In summary, we have identified several points at which PIM and PI3K/AKT/mTOR converge to provide synergistic apoptosis in multiple myeloma cell lines. These results provide the rationale for further preclinical development of PIM inhibitors and provide the basis for a possible clinical development plan in multiple myeloma. Disclosures: Munugalavadla: Genentech: Employment, Equity Ownership. Berry:Genentech: Employment, Equity Ownership. Chen:Genentech: Employment, Equity Ownership. Deshmukh:Genentech: Employment, Equity Ownership. Drummond:Genentech: Employment, Equity Ownership. Du:Genentech: Employment, Equity Ownership. Eby:Genentech: Employment, Equity Ownership. Fitzgerald:Genentech: Employment, Equity Ownership. S.Friedman:Genentech: Employment, Equity Ownership. E.Gould:Genentech: Employment, Equity Ownership. Kenny:Genentech: Employment, Equity Ownership. Maecker:Genentech: Employment, Equity Ownership. Moffat:Genentech: Employment, Equity Ownership. Moskalenko:Genentech: Employment, Equity Ownership. Pacheco:Genentech: Employment, Equity Ownership. Saadat:Genentech: Employment, Equity Ownership. Slaga:Genentech: Employment, Equity Ownership. Sun:Genentech: Employment, Equity Ownership. Wang:Genentech: Employment, Equity Ownership. Yang:Genentech: Employment, Equity Ownership. Ebens:Genentech Inc: Employment, Equity Ownership.

Abstract 3001 PIM kinases co-regulate several important elements of the PI3K/AKT/mTOR pathway in myeloma cells (Munugalavadla V. et al., ASH 2010 submitted abstr.). In this study we show that pan-PIM inhibition suppresses growth in myeloma cell lines, xenografts, and primary patient samples, both as a single-agent as well acting synergistically in combination with PI3K, AKT, and mTOR inhibition. The PIM kinases are a family of 3 ser/thr growth factor- & cytokine-induced proteins hypothesized to have redundant survival and growth functions. Although PIM-1,2 have been noted as highly expressed in myeloma (Claudio et al., 2002), there are few data to support potential therapeutic utility of PIM inhibition in this indication. We show myeloma cell lines express all 6 PIM protein isoforms to varying extents, and we describe the properties of a novel pan-PIM inhibitor GNE-652 with picomolar biochemical potency, an excellent selectivity profile, and favorable ADME properties. Myeloma cell lines exhibit a striking prevalence of response to GNE-652 (23 of 25 lines with IC50 < 1 micromolar, median < 0.1 micromolar) and synergy in combination with the PI3K inhibitor GDC-0941 (mean combination index values ~0.2 (n=25)). We used an unrelated compound GNE-568 which has a PIM-1,3 selective profile to test the hypothesis that PIM-2 may have a non-redundant role in myeloma cells. GNE-568 while having cellular potency against PIM-1 and PIM-3, did not have single agent activity in myeloma cell lines nor did it act synergistically with GDC-0941 (n=10 cell lines). Interestingly, PI3K and AKT inhibitors showed the greatest extent of synergy with GNE-652, whereas mTOR-selective inhibitors were synergistic to a lesser extent. Standard of care agents dexamethasone, revlimid, velcade, and melphalan also combined well with GNE-652, but to a lesser extent and not as broadly. The synergistic anti-tumor activity of GNE-652 and PI3K inhibitor GDC-0941 on cell lines or on primary myeloma bone marrow aspirates in vitro was associated with cell-cycle arrest and marked apoptosis. In addition, we found 4 of 4 myeloma xenograft mouse models tested with GNE-562 and GDC-0941 showed excellent combination efficacy that correlated with modulation of the expected pharmacodynamic markers. These results provide the rationale for further preclinical development of PIM inhibitors and provide the basis for a possible clinical development plan in multiple myeloma. Disclosures: Ebens: Genentech: Employment, Equity Ownership. Berry:Genentech: Employment, Equity Ownership. Chen:Genentech: Employment, Equity Ownership. Deshmukh:Genentech: Employment, Equity Ownership. Drummond:Genentech: Employment, Equity Ownership. Du:Genentech: Employment, Equity Ownership. Eby:Genentech: Employment, Equity Ownership. Fitzgerald:Genentech: Employment, Equity Ownership. S. Friedman:Genentech: Employment, Equity Ownership. E. Gould:Genentech: Employment, Equity Ownership. Kenny:Genentech: Employment, Equity Ownership. Maecker:Genentech: Employment, Equity Ownership. Moffat:Genentech: Employment, Equity Ownership. Moskalenko:Genentech: Employment, Equity Ownership. Pacheco:Genentech: Employment, Equity Ownership. Saadat:Genentech: Employment, Equity Ownership. Slaga:Genentech: Employment, Equity Ownership. Sun:Genentech: Employment, Equity Ownership. Wang:Genentech: Employment, Equity Ownership. Yang:Genentech: Employment, Equity Ownership. Munugalavadla:Genentech: Employment, Equity Ownership.

Abstract 77 Stem cell factor (SCF) mediated c-Kit receptor activation plays a pivotal role in mast cell growth, maturation and survival. However, the signaling events downstream from c-Kit are poorly understood. Mast cells express multiple regulatory subunits of class 1A PI 3-kinase including p85α, p85β, p50α, and p55α. While it is known that PI 3-kinase plays an essential role in mast cells; the precise mechanism by which these regulatory subunits impact specific mast cell functions including maturation, growth, and survival are not known. Using mice deficient in the expression of p85α or p85β or combination of both p85α/p55α/p50α as well as all four subunits we have examined the role of these subunits in mast cell functions. We show that loss of p85α subunit alone results in impaired bone marrow derived mast cell (BMMC) maturation, growth, and survival compared to wild-type (WT) controls, in spite of the continuous expression of p85β, p55α, and p50α subunits in these cells. Restoring the expression of p85α in p85α deficient mast cells restores the maturation and growth defects. To assess the contribution of p50α and p55α subunits, we generated mice using the Cre lox system that were deficient in the expression of all three subunits (i.e. p85α/p55α/p50α). Deficiency of p85α/p55α/p50α subunits in bone marrow cells completely blocked mast cell maturation and growth, suggesting an essential role for the smaller subunits p50 and p55 in addition to the full length form of p85. Curiously, over-expression of p50α in p85α deficient BMMCs only marginally rescued mast cell maturation and growth, suggesting that the full length form of p85α functions with specificity in regulating mast cell functions. Since the major difference between the shorter isoforms and the full length form of p85α is the absence of the amino terminal SH3 and BH domains, we generated two mutants of p85α lacking either the SH3 or the BH domain and expressed them in p85α−/− BMMCs. While both these mutants completely restored the maturation defect associated with p85α deficiency and showed normal binding to the c-Kit receptor upon SCF stimulation as well as to the p110 catalytic subunits; none of these mutants completely rescued SCF induced proliferation (50% and 70% respectively, n=3, p Disclosures: Munugalavadla: Genentech: Employment, Patents & Royalties.

Abstract 3788 Poster Board III-724 Multiple myeloma (MM) is a malignancy characterized by clonal expansion and accumulation of long-lived plasma cells within the bone marrow. Phosphatidylinositol 3' kinase (PI3K) -mediated signaling is frequently dysregulated in cancer and controls fundamental cellular functions such as cell migration, growth, survival and development of drug resistance in many cancers, including MM, and therefore represents an attractive therapeutic target. Here, we demonstrate in vitro, that a potent and selective pan-isoform PI3Kinhibitor, GDC-0941, modulates the expected pharmacodynamic markers, inhibits cell-cycle progression and induces apoptosis; overcomes resistance to apoptosis in MM cells conferred by IGF-1 and IL-6; and is additive or synergistic with current standard of care drugs including dexamethasone, melphalan, lenolidamide and bortezomib. In cell lines we find sensitivity to GDC-0941 is positively correlated with pathway activation as determined by phospho-AKT-specific flow-cytometry and Western-blot analysis. Preliminary results indicate apoptosis of MM cells is correlated with increased expression of the proapoptotic BH3-only protein BIM; mechanisms of increased apoptosis in MM will be further explored and an update presented. We further extend these in vitro findings to show that GDC-0941 has activity as a single agent in vivo and combines well with standard of care agents in several murine xenograft models to delay tumor progression and prolong survival. Our results suggest that GDC-0941 may combine well with existing therapies, providing a framework for the clinical use of this agent, and a rational approach to improving the efficacy of myeloma treatment. Disclosures: Munugalavadla: Genentech: Employment, Patents & Royalties. Berry:Genentech: Employment, Patents & Royalties. Du:Genentech, Inc.: Employment, Equity Ownership. Mariathasan:Genentech: Employment, Patents & Royalties. Slaga:Genentech: Employment, Patents & Royalties. Sun:Genentech Inc.: Employment. Chesi:Genentech, Inc.: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Merck: Research Funding. Bergsagel:Genentech: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Merck: Research Funding. Ebens:Genentech, Inc.: Employment, Equity Ownership, Patents & Royalties.

Abstract 3909 Poster Board III-845 Myeloproliferative disorder (MPD) and acute myeloid leukemia (AML) are heterogeneous hematologic malignancies which share the common characteristics of myeloid cell overproduction and aberrant differentiation. With the exception of chronic myelogenous leukemia (CML), therapeutic intervention for these diseases is largely ineffective. There are increasing examples of mutations in tyrosine kinases (TKs) that contribute to MPD as well as AML. Key examples are the well characterized BCR-ABL transgene causing constitutively activated Abl tyrosine kinase function in CML. FLT3 internal tandem duplications (ITDs) induce ligand-independent autophosphorylation in AML, and activating KIT mutations seen in AML and in over 90% cases of systemic mastocytosis (SM). Although hyperactivation of several signaling molecules downstream from these tyrosine kinases have been reported; little is known about the identity of signaling molecules that are shared among these mutations. Rho kinases or Rho-associated coiled coil-containing protein kinases (ROCK1 and ROCK2) are protein serine/threonine kinases that control a wide variety of cellular functions, including cell growth, survival and motility. Rho kinases have been shown to be hyperactive in several cancers. Importantly, in preclinical models, ROCK inhibitors have demonstrated significant efficacy in repressing aspects of tumorigenesis. However, the role of ROCK kinases in MPDs/Leukemogenesis has not been investigated. We show that expression of wild-type (WT) KIT receptor or vector alone in 32D murine myeloid cells or WT FLT3 receptor in BaF3 murine lymphoid cells does not result in ligand independent activation of ROCK kinases as evident by lack of phosphorylation of its substrate MYPT1. In contrast, expression of oncogenic KITD816V, FLT3N51, and BCR-ABL in these cells results in constitutive activation of Rho kinases, which is completely inhibited in the presence of Rho kinase inhibitor dimethyl-fasudil. Importantly, treatment of oncogene bearing cells with dimethy-fasudil did not affect the activation of PKA, PKC, AKT, STAT3 or ERK MAPkinase. Furthermore, treatment of oncogenic KITD816V, FLT3N51 and BCR-ABL bearing cells with additional Rho kinase inhibitors (Y27632 and fasudil) also resulted in a dose dependent repression in ligand independent growth in a thymidine incorporation assay. In contrast, WT KIT, FLT3, or vector alone containing cells demonstrated little to no repression of growth in the presence of cytokines and Rho kinase inhibitors. A human mast cell line (HMC1.2) derived from a patient with mastocytosis bearing the activation loop mutation D816V in the KIT receptor, also showed a dose dependent suppression in ligand independent growth upon treatment with Rho kinase inhibitors. Similar inhibition of growth was observed when primary hematopoietic stem and progenitor cells (HSC/Ps) bearing the oncogenic KITD814V, FLT3N51 and BCR-ABL mutations were treated with Rho kinase inhibitors. In an effort to identify the mechanism behind reduced growth, we examined opoptosis in oncogene bearing cells treated with Rho kinase inhibitors. We found increased apoptosis in these cells compared to WT KIT receptor, FLT3 receptor, or vector alone expressing cells. Apoptosis was observed as early as two hours after Rho kinase inhibitor treatment in oncogene bearing cells. To determine the mechanism of such rapid apoptosis in oncogene bearing cells, we examined the activation of several known downstream substrates of ROCK kinases. We found that cells bearing the oncogenic KITD814V, FLT3N51 and BCR-ABL receptors demonstrated enhanced and constitutive phosphorylation of myosin light chain (MLC). Since inhibition of MLC phosphorylation in breast cancer lines has been associated with apoptosis, we next measured the activation of MLC in the presence of ROCK inhibitors. We found that MLC was dephosphorylated within minutes of treating oncogene bearing cells with Rho kinase inhibitors but not in wildtype receptor bearing cells. Taken together, these results suggest that dephosphorylation of a major downstream Rho kinase substrate (i.e. MLC) plays an essential role in inducing cell death in oncogene bearing cells but not in normal cells. These studies indicate Rho kinases as possible therapeutic targets for treating diseases such as AML and MPD. Disclosures: No relevant conflicts of interest to declare.

Oncogenic activation-loop mutants of KIT, the receptor for stem cell factor (SCF), are commonly observed in acute myeloid leukemia (AML) and systemic mastocytosis (SM); however, unlike the KIT juxtamembrane mutants (found in patients with gastrointestinal stromal tumors [GISTs]), the activation-loop mutants are commonly insensitive to inhibition by tyrosine kinase inhibitors. Furthermore, little is known about the signaling pathways that contribute to oncogenic KIT-induced transformation in SM or AML. We demonstrate that expression of KITD814V (KIT activation-loop mutant) in primary hematopoietic stem and progenitor cells induces constitutive KIT autophosphorylation, promotes ligand-independent hyperproliferation, skews myeloid differentiation towards the granulocytic lineage, and promotes promiscuous cooperation with multiple cytokines, including G-CSF, M-CSF and IL-3. KITD814V expressing primary mast cells also demonstrated hyperproliferation in response to SCF, IL-3, IL-4 and IL-10. Biochemical analyses of KITD814V expressing cells revealed constitutively elevated levels of phosphatidylinositol-3-kinase (PI3K) and its downstream substrate, the Rho family GTPase Rac. Genetic disruption of p85a, the regulatory subunit of class IA PI-3Kinase, but not of p85β, or genetic disruption of the hematopoietic cell-specific Rho GTPase, Rac2, normalized KITD814V-induced ligand independent hyperproliferation in vitro. Additionally, deficiency of p85α or Rac2 corrected the promiscuous hyperproliferation observed in response to multiple cytokines in both KITD814V expressing stem/progenitor cells as well as mast cells in vitro. Although p85α is hyperphosphorylated and constitutively bound to KITD814V in bone marrow cells in vitro; its physiologic role in transformation in vivo is not known. To address this, we generated a new mouse model to study KITD814V induced transformation in myeloid cells as opposed to previously described models that primarily result in the generation of phenotypes resembling acute lymphocytic leukemia via this mutation. Our results show that transplantation of KITD814V expressing bone marrow cells from C57/BL6 strain of mice into syngeneic recipients results in a fatal myeloproliferative disease (MPD) characterized by leukocytosis, splenomegaly, disruption of the splenic architecture as well as myeloid cell infiltration in the lung and liver. Importantly, in this model, transplantation of KITD814V expressing p85α deficient bone marrow cells rescued the MPD phenotype, including splenomegaly, peripheral blood leukocytosis and the reduced life span associated with the transplantation of KITD814V expressing wildtype bone marrow cells. Treatment of KITD814V-expressing hematopoietic progenitors with either a Rac inhibitor (NC23766) or rapamycin showed a dose-dependent suppression in KITD814V induced growth. Taken together, our results describe the generation of a new murine transplant model to study KITD814V induced transformation and identify p85a and Rac2 as potential novel therapeutic target for the treatment of KITD814V-bearing diseases including SM and AML.

Mast cell activation plays a critical pathophysiologic role in asthma and allergy. A role for mast cell activation has also been described in multiple sclerosis, rheumatoid arthritis and coronary artery disease. In addition, these cells also play a prominent role in early phases of innate immunity to pathogenic bacteria. While several cytokines influence the growth, survival and maturation of mast cells; stem cell factor (SCF) and its interaction via the tyrosine kinase receptor, KIT is essential for normal mast cell development and function. However the intracellular signals that control mast cell growth, migration and maturation are not completely understood. In non-hematopoietic cells, Rho family GTPases are key regulators of many different biological processes including cell motility, growth, and differentiation. Cdc42, Rac and Rho are the most extensively studied members of this family. Although the role of Rac GTPases is becoming increasingly clear in mast cells and in hematopoietic cells in general, virtually nothing is known about the role of downstream effectors of Rho GTPases in these cells. RhoA and RhoC activate the serine/threonine protein kinases ROCKI and ROCKII. We show that both ROCKI and ROCKII are expressed in hematopoietic cells, including in bone marrow cells, splenocytes as well as in thymocytes. To determine the role of ROCK kinases in mast cells, we generated mice deficient in the expression of ROCKI. Here, we demonstrate that Rho-kinase ROCKI plays an essential role in regulating mast cell growth and maturation. We show that deficiency of ROCKI results in impaired maturation of bone marrow derived mast cells in response to IL-3 stimulation. Furthermore, the reduced maturation of ROCKI−/− mast cells is associated with reduced expression of KIT as well as reduced expression of the high affinity receptor for IgE at different stages of maturation (13% vs 7% at week1, 80% vs 52% at week2, and 93% vs 67% KIT/IgE receptor double positive cells at week3, n=3). KIT induced proliferation in response to SCF was also significantly reduced in ROCKI deficient mast cells, which was associated with reduced activation of MAPKinase Erk1 and Erk2. To test if the decreased growth in response to SCF was simply due to reduced KIT expression or due to cell intrinsic defects in ROCKI signaling, we isolated KIT positive WT and ROCKI−/− mast cells and measured growth in response to SCF and/or IL-3 stimulation by thymidine incorporation over a period of 24 and 48 hours. KIT positive ROCKI−/− mast cells showed reduced growth in response to SCF as well as in response to a combination of SCF and IL-3, suggesting a critical role of ROCKI in normal growth and maturation of mast cells. Since ROCK kinases also regulate migration in non-hematopoietic cells, we next examined the role of ROCKI in integrin (haptotactic) as well as in cytokine induced (chemotaxis) migration of mast cells. Mast cells deficient in ROCKI showed a 68% reduction in directional migration on fibronectin alone (64±7 [WT] vs 20±4, p

Osteoclasts (OCs) play an indispensable role in regulating bone remodeling. In adults, a significant number of skeletal diseases have been linked to abnormal osteoclast function(s), including rheumatoid arthritis, periodontal disease, multiple myeloma, and metastatic cancers. Although, a clear picture of the critical players that regulate osteoclastogenesis and bone resorption has begun to emerge; further studies detailing the intracellular signaling pathways is necessary for the rationale development of new drugs for the treatment of bone disorders involving OCs. While recent studies utilizing pharmacologic inhibitors of PI-3Kinase have suggested a role for this pathway in osteoclastogenesis, these inhibitors interfere with the function of all classes of PI-3Kinase and result in extensive in vivo toxicity. Therefore, to therapeutically manipulate PI-3Kinase signaling cascade in osteoclasts, additional data evaluating the specific role of individual PI-3Kinase isoforms is necessary. Class IA PI-3Kinase are heterodimeric kinases consisting of a regulatory subunit and a catalytic subunit. Five different proteins, namely p85α, p55α, p50α, p85β, and p55γ, have been identified to date as the regulatory subunits. The p85α, p55α, and p50α proteins are derived from the same gene locus by alternative splicing mechanism. In contrast, distinct genes encode the p85β and p55γ subunits. Utilizing mice deficient in the expression of p85α subunit, we have recently shown that p85α subunit of PI-3Kinase plays an important role in regulating growth and actin based functions in bone marrow (BM) derived macrophages. Here, we demonstrate that OCs express multiple regulatory subunits of class IA PI-3Kinase, including p85α, p85β, p50α and p55α. Deficiency of p85α in OCs alone results in a significant increase in bone mass and bone density (% bone volume [BV]/trabecular volume [TV]: WT 6.7±0.01 vs p85α−/− 14±0.01*, *p

Gain of function mutations in c-Kit are associated with a number of cancers in humans, including gastrointestinal stromal tumors (GIST), mastocytosis and acute myeloid leukemia (AML). Although, gleevec is a potent inhibitor of c-Kit juxtamembrane mutants that are associated with GISTs, little is known about the drugs that inhibit the activity of c-Kit with mutations in the catalytic domain (i.e. substitution of valine for aspartic acid in codon 816: c-Kit D816V), which are associated with mastocytosis and/or AML. Although, in vitro expression of c-Kit D816V in transformed cell lines results in ligand independent survival due to constitutive phosphorylation, the functional manifestation of this mutation in primary stem/progenitor cells (HSC/Ps) or in mast cells is poorly understood. In addition, little is known about the signaling pathways that contribute to c-Kit D816V induced transformation in HSC/Ps or mast cells. Here, we assessed the role of c-Kit D814V in primary bone marrow derived HSC/Ps and mast cells. Consistent with prior reports, expression of c-Kit D814V in HSC/Ps resulted in constitutive autophosphorylation of the c-Kit receptor. To determine the affect of this mutation on HSC/Ps, we first assessed alteration in the differentiation of HSC/Ps by assessing the cell surface expression of Gr-1, Mac-1 and F4/80. Compared to HSC/Ps transduced with the empty vector, no significant perturbation in the expression of Gr-1, Mac-1 or F4/80 was observed in c-Kit D814V expressing HSC/Ps (n=3, p

In adults, hematopoietic stem/progenitor (SC/P) cell expansion and lineage commitment is regulated by coordinated signals from cytokine receptors and transcription factors (TFs). Studies performed in cell lines using cytokine receptors lacking the binding sites for Src and PI-3Kinase family members have suggested a role for these signaling molecules in regulating growth and survival. However, the physiologic role(s) for these signaling molecules in primary SC/P cell growth and lineage commitment are poorly understood. Furthermore, the mechanism(s) via which these kinases regulate the expression of TFs in primary SC/Ps are not known. We demonstrate that Lyn Src family kinase and p85α subunit of class IA PI-3Kinase play opposing roles in regulating growth and lineage commitment in primary SC/Ps, in part by regulating the threshold of PI-3Kinase signaling, including the levels of phosphatidylinositol-3,4,5,-trisphosphate (PIP3) and Akt. Loss of Lyn in SC/P cells results in elevated PIP3 levels and hyperactivation of Akt. In contrast, loss of p85α subunit of class IA PI-3Kinase results in reduced levels of PIP3 and Akt activation. Functionally, Lyn−/− or Lyn+/− SC/P cells show enhanced growth and commitment towards myeloid lineage, including granulocytes, monocytes and mast cells, which is associated with enhanced and accelerated induction of myeloid lineage markers including c-Kit and the high affinity receptor for IgE. The enhanced myeloid lineage commitment in Lyn−/− SC/P cells is also associated with hyper induction of PU.1, CEB/Pα as well as members of the GATA family. In spite of high degree of homology between various Src family kinases, reconstitution of Lyn−/− SC/Ps with Lyn, but not with other related family members such as Hck or Fgr resulted in the rescue of Lyn−/− SC/P cell phenotype, suggesting that Lyn functions with specificity in regulating SC/P cell growth and survival, including lineage commitment. Enhanced lineage commitment, growth and survival as well as PIP3 levels seen in Lyn−/− SC/Ps were in part due to impaired activation of SHIP in these cells. Remarkably, in spite of continued reduction in the activation of SHIP and hyperactivation of Stat5 and Erk MAP kinase in Lyn−/− SC/P cells, haplo-insufficiency or complete deficiency of p85α subunit of class IA PI-3Kinase in the setting of Lyn deficiency or Lyn-haplo-insufficiency not only corrected the elevated PIP3 levels, cytokine induced hypersensitivity and lineage commitment to near wildtype levels, but in some cases made it worst than that seen in wildtype controls. Taken together, utilizing a genetic and a biochemical approach, our results illustrate a complex network of interactions between cytokine receptors and TFs involving Lyn, p85α and SHIP in positive and negative regulation of growth and lineage commitment in primary SC/P cells. Furthermore, our results demonstrate that the level of PIP3 in SC/P cells plays a critical role in determining the fate and lineage commitment in these cells. In vivo consequences on the long-term repopulating ability of stem cells lacking both Lyn and p85α subunit of class IA PI-3Kinase will be discussed.

Although it is clear that chemokines, cytokines and adhesion molecules play an essential role in regulating hematopoietic stem and progenitor cell (HSC/P) self-renewal, lineage commitment, apoptosis and mobilization, the intracellular signals that regulate these processes are poorly defined. Here, we demonstrate that the deficiency of hematopoietic specific Src family kinases (SFKs) in Lin- HSC/Ps results in reduced chemotaxis and adhesion via CXCR4 and β1 integrins, respectively (n=3, p

While it has been established that c-Kit/stem cell factor (SCF) interactions are indispensable for normal mast cell development and functions, the intracellular signals that regulate the development and function of these cells downstream from c-Kit are not fully understood. Here, we demonstrate that p85 regulatory subunits of class IA PI-3Kinase (p85α and p85β) play an essential role in regulating mast cell growth and maturation. We show that deficiency of p85α in mast cells results in reduced growth and survival in response to SCF stimulation, which is associated with impaired activation of Akt, Rac and JNK MAP kinase. Furthermore, we demonstrate that in addition to regulating growth and survival, loss of p85α also impairs the maturation of mast cells as seen by significantly reduced expression of c-Kit and the IgE receptor on p85α−/− mast cells compared to wildtype controls. To determine the extent to which Akt contributes to the growth, survival and maturation of p85α−/− mast cells, we reconstituted p85α−/− mast cells with a retrovirus encoding an activated form of Akt. Remarkably, p85α−/− mast cells expressing an activated Akt completely rescued the proliferative defect associated with p85α deficient mast cells in response to IL-3 as well as IL-3 plus SCF, and a 70% correction in response to SCF alone. The phenotypic abnormalities seen in p85α−/− mast cells were surprising in light of the sequence homology and continued expression in these cells of p85β subunit of class IA PI-3Kinase, and suggested that p85α might function with specificity in mast cells in regulating c-Kit functions. To examine the relationship between p85α and p85β in regulating mast cell functions, we generated mast cells from p85β−/− mice and compared them with p85α−/− mast cells. We found that p85α and p85β perform qualitatively distinct functions in regulating mast cell growth and differentiation. We found that although deficiency of p85α in mast cells consistently results in reduced growth and impaired maturation, deficiency of p85β in these same cells results in enhanced growth and no significant defect in maturation. To further demonstrate that p85α and p85β play qualitatively distinct functions in regulating mast cell growth and differentiation, a genetic intercross between p85α+/− and p85β+/− mice was performed. Since mice completely deficient in both the isoforms of p85 (α & β) die early in embryonic development, we analyzed mast cell growth and differentiation in mice that were deficient in the expression of p85α(p85α−/−) and heterozygous at the p85β (p85β+/−) locus, along with the appropriate controls. We found that heterozygosity of p85β (p85β+/−) in the setting of p85α deficiency (p85α−/−) significantly corrected the mast cell defect associated with p85α deficiency (p85α−/−) alone. Finally, we reconstituted p85α−/− mast cells with a bicistronic retrovirus encoding EGFP and a cDNA for p85β, and found that p85β could not rescue the mast cell defect associated with p85α deficiency. In contrast, reconstituting p85α−/− mast cells with a retrovirus encoding the p85α cDNA completely rescued the mast cell growth defect associated with p85α deficiency. The level of expression of p85α and p85β in p85α−/− reconstituted mast cells was similar as determined by western blot analysis. Taken together, our results demonstrate qualitatively distinct functions for two closely related regulatory subunits of class IA PI-3Kinase in controlling mast cell growth and maturation.