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Acalabrutinib is a selective irreversible Bruton tyrosine kinase (BTK) inhibitor that does not affect IL2-associated tyrosine kinase or antibody-dependent cellular cytotoxicity, making it an attractive candidate for combination therapy with anti-CD20 antibodies. We investigated acalabrutinib plus obinutuzumab in a phase Ib/II study (NCT02296918) of patients with treatment-naïve or relapsed/refractory chronic lymphocytic leukemia (CLL). Nineteen treatment-naïve and 26 relapsed/refractory patients were treated with acalabrutinib (100 mg twice daily) until progression and obinutuzumab (cycle 1: 100 mg day 1, 900 mg day 2, 1000 mg days 8 and 15; cycles 2–6: 1,000 mg day 1). Grade 3/4 adverse events occurred in 71% of patients. Overall response rates were 95% (treatment-naïve) and 92% (relapsed/refractory). Thirty-two percent of treatment-naïve and 8% of relapsed/refractory patients achieved complete remission. At 36 months, 94% (treatment-naïve) and 88% (relapsed/refractory) were progression free. Acalabrutinib plus obinutuzumab was well tolerated, producing high and durable responses in treatment-naïve and relapsed/refractory CLL.Significance:Rituximab plus the less selective BTK inhibitor ibrutinib has not shown benefit in CLL; however, the selective BTK inhibitor acalabrutinib plus the antibody-dependent cellular cytotoxicity–enhanced antibody obinutuzumab yielded durable responses that deepened over time in treatment-naïve and relapsed/refractory CLL, supporting the evaluation of this approach in larger, comparative studies in CLL.This article is highlighted in the In This Issue feature, p. 327

Figure S1 shows immunoglobulin A, B, and M levels in treated patients over time Figure S2 shows investigator-assessed progression-free survival by CLL-IPI score and presence or absence of complex karyotype

Acalabrutinib, a selective Bruton tyrosine kinase inhibitor, is approved for the treatment of mantle cell lymphoma and chronic lymphocytic leukaemia. Many critically ill patients are unable to swallow and need oral medications to be delivered via a nasogastric (NG) tube. Furthermore, critically ill patients are typically administered proton-pump inhibitors (PPIs) to prevent stress ulcers. Concomitant administration with PPIs reduces acalabrutinib exposure and is not currently recommended. To evaluate acalabrutinib in subjects co-administered with PPIs who require NG delivery, a phase 1, open-label, randomized, crossover, single-dose study was conducted in healthy subjects.The study assessed the relative bioavailability of an acalabrutinib suspension-in regular, degassed Coca-Cola-administered via NG tube (Acala-NG) versus the pharmacokinetics (PK) of an acalabrutinib capsule administered orally with water. In addition, the PPI effect was evaluated by comparing the PK following Acala-NG in the presence or absence of rabeprazole.Exposure of acalabrutinib and its active metabolite (ACP-5862) were comparable following administration of Acala-NG versus the oral capsule (Geo mean ratio, % ref [90% confidence interval, CI]: acalabrutinib AUCAcala-NG with or without a PPI is safe and well-tolerated without impeding bioavailability.

Supplementary Figure from Safety and Efficacy of Pembrolizumab in Combination with Acalabrutinib in Advanced Head and Neck Squamous Cell Carcinoma: Phase 2 Proof-of-Concept Study

Suppl Figures 1-3, Suppl Table 1 Supplemental Figure 1. Study schema. Supplemental Figure 2. Overall survival in patients treated with ENTO + 7+3. OS is censored on the date the patient was last known to be alive (if it was not known the patient had died by the end of study follow-up). Supplemental Figure 3. Heat map showing single patients (each column) by HOXA9 and MEIS1 categorization, cytogenetic or molecular risk and outcomes.

Supplementary Data from Safety and Efficacy of Pembrolizumab in Combination with Acalabrutinib in Advanced Head and Neck Squamous Cell Carcinoma: Phase 2 Proof-of-Concept Study

Purpose:Programmed cell death-1 (PD-1) receptor inhibitors have shown efficacy in head and neck squamous cell carcinoma (HNSCC), but treatment failure or secondary resistance occurs in most patients. In preclinical murine carcinoma models, inhibition of Bruton's tyrosine kinase (BTK) induces myeloid cell reprogramming that subsequently bolsters CD8+ T cell responses, resulting in enhanced antitumor activity. This phase 2, multicenter, open-label, randomized study evaluated pembrolizumab (anti–PD-1 monoclonal antibody) plus acalabrutinib (BTK inhibitor) in recurrent or metastatic HNSCC.Patients and Methods:Patients received pembrolizumab 200 mg intravenously every 3 weeks, alone or in combination with acalabrutinib 100 mg orally twice daily. Safety and overall response rate (ORR) were co-primary objectives. The secondary objectives were progression-free survival (PFS) and overall survival.Results:Seventy-six patients were evaluated (pembrolizumab, n = 39; pembrolizumab + acalabrutinib, n = 37). Higher frequencies of grade 3–4 treatment-emergent adverse events (AE; 65% vs. 39%) and serious AEs (68% vs. 31%) were observed with combination therapy versus monotherapy. ORR was 18% with monotherapy versus 14% with combination therapy. Median PFS was 2.7 [95% confidence interval (CI), 1.4–6.8] months in the combination arm and 1.7 (95% CI, 1.4–4.0) months in the monotherapy arm. The study was terminated due to lack of clinical benefit with combination treatment. To assess how tumor immune contexture was affected by therapy in patients with pre- and post-treatment biopsies, spatial proteomic analyses were conducted that revealed a trend toward increased CD45+ leukocyte infiltration of tumors from baseline at day 43 with pembrolizumab (monotherapy, n = 5; combination, n = 2), which appeared to be higher in combination-treated patients; however, definitive conclusions could not be drawn due to limited sample size.Conclusions:Despite lack of clinical efficacy, immune subset analyses suggest that there are additive effects of this combination; however, the associated toxicity limits the feasibility of combination treatment with pembrolizumab and acalabrutinib in patients with recurrent or metastatic HNSCC.

Purpose:Spleen tyrosine kinase (SYK) signaling is a proposed target in acute myeloid leukemia (AML). Sensitivity to SYK inhibition has been linked to HOXA9 and MEIS1 overexpression in preclinical studies. This trial evaluated the safety and efficacy of entospletinib, a selective inhibitor of SYK, in combination with chemotherapy in untreated AML.Patients and Methods:This was an international multicenter phase Ib/II study, entospletinib dose escalation (standard 3+3 design between 200 and 400 mg twice daily) + 7+3 (cytarabine + daunorubicin) in phase Ib and entospletinib dose expansion (400 mg twice daily) + 7+3 in phase II.Results:Fifty-three patients (n = 12, phase Ib and n = 41, phase II) with previously untreated de novo (n = 39) or secondary (n = 14) AML were enrolled (58% male; median age, 60 years) in this study. The composite complete response with entospletinib + 7+3 was 70%. Patients with baseline HOXA9 and MEIS1 expression higher than the median had improved overall survival compared with patients with below median HOXA9 and MEIS1 expression. Common adverse events were cytopenias, febrile neutropenia, and infection. There were no dose-limiting toxicities. Entospletinib-related skin rash and hyperbilirubinemia were also observed.Conclusions:Entospletinib with intensive chemotherapy was well-tolerated in patients with AML. Improved survival was observed in patients with HOXA9/MEIS1 overexpression, contrasting published data demonstrating poor survival in such patients. A randomized study will be necessary to determine whether entospletinib was a mediator this observation.

Introduction: Minimal or measurable residual disease (MRD) is an emerging independent predictor of progression-free and overall survival in several hematologic diseases. Despite many different methods used in clinical trials, few assays have clinical utility as a surrogate endpoint in a limited number of hematologic malignancies. MRD assays for lymphomas are mostly performed in research settings due to lack of harmonized methods, heterogeneity of diseases, and need for a primary tumor sample. We explored the feasibility of a pan hematologic malignancy classifier (“pan heme classifier”) based on GRAIL’s methylation platform as a potential tumor-agnostic plasma-based MRD assay for hematologic malignancies. Methods: First, 428 plasma samples from various hematologic malignancies including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and multiple myeloma (MM) were blindly tested retrospectively using the pan heme classifier. The majority of samples (375/428; 88%) were from relapsed/refractory (R/R) disease. Cancer signal detection and signal origin prediction were explored at various detection thresholds. Next, sensitivity of the pan heme classifier was evaluated using 74 (22 and 14 unique patients in CLL and MCL, respectively) post-treatment samples with orthogonal methods (flow cytometry in CLL and clonoSEQ in MCL). Finally, dilution experiments were carried out in 4 DLBCL and 4 CLL patient samples. Results: Of 428 samples, 408 (95%) passed quality control. At a prespecified detection threshold, we observed high (91%) cancer detection rate with high cancer signal origin accuracies: 100% in CLL, >98% in MM, >95% in non-Hodgkin lymphoma (DLBCL, FL, MCL), and 87% in AML samples. As expected, targeting lower detection thresholds resulted in increased cancer-positive calls. Cancer was reproducibly detected in 48 of 54 (89%) cases where paired samples were taken prior to treatment (screening and cycle 1, day 1), demonstrating high biologic reproducibility. Analysis of posttreatment CLL and MCL samples with cell-based orthogonal MRD assays suggests the current assay limit of detection (LOD) is ~10-3-10-4 MVAF (a methylation-based tumor fraction estimate). These data suggest that cancer signal score is strongly correlated to the observed MVAF. Furthermore, serially diluted DLBCL and CLL patient plasma samples were spiked into healthy volunteer plasma samples, suggesting an LOD of 10-4 MVAF. Conclusions: These data demonstrate that the pan heme classifier can identify cancer signal from R/R patients across multiple hematologic malignancies. We are currently working to further optimize the assay’s specificity and sensitivity. These results support the development of a blood-based tumor agnostic methylated ctDNA MRD assay with potential utility in several hematologic indications. Citation Format: Veerendra Munugalavadla, Gary de Jesus, Aleksandra Markovets, Qinwen Liu, Oliver Venn, Rafael White, Giulia Fabbri, Paul Labrousse, Dan Stetson, Brian Dougherty, Darren Hodgson, Jill Walker, Anas Younes, Daniel Auclair. Utility of ctDNA-based targeted methylation MRD assay for hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3369.

Purpose: Spleen tyrosine kinase (SYK) signaling is a proposed target in acute myeloid leukemia (AML). Sensitivity to SYK inhibition has been linked to HOXA9 and MEIS1 overexpression in preclinical studies. This trial evaluated the safety and efficacy of entospletinib, a selective inhibitor of SYK, in combination with chemotherapy in untreated AML. Patients and Methods: This was an international multicenter phase Ib/II study, entospletinib dose escalation (standard 3+3 design between 200 and 400 mg twice daily) + 7+3 (cytarabine + daunorubicin) in phase Ib and entospletinib dose expansion (400 mg twice daily) + 7+3 in phase II. Results: Fifty-three patients (n = 12, phase Ib and n = 41, phase II) with previously untreated de novo (n = 39) or secondary (n = 14) AML were enrolled (58% male; median age, 60 years) in this study. The composite complete response with entospletinib + 7+3 was 70%. Patients with baseline HOXA9 and MEIS1 expression higher than the median had improved overall survival compared with patients with below median HOXA9 and MEIS1 expression. Common adverse events were cytopenias, febrile neutropenia, and infection. There were no dose-limiting toxicities. Entospletinib-related skin rash and hyperbilirubinemia were also observed. Conclusions: Entospletinib with intensive chemotherapy was well-tolerated in patients with AML. Improved survival was observed in patients with HOXA9/MEIS1 overexpression, contrasting published data demonstrating poor survival in such patients. A randomized study will be necessary to determine whether entospletinib was a mediator this observation.

Acalabrutinib is a Bruton tyrosine kinase (BTK) inhibitor approved to treat adults with chronic lymphocytic leukemia, small lymphocytic lymphoma, or previously treated mantle cell lymphoma. As the bioavailability of the acalabrutinib capsule (AC) depends on gastric pH for solubility and is impaired by acid-suppressing therapies, coadministration with proton-pump inhibitors (PPIs) is not recommended. Three studies in healthy subjects (N = 30, N = 66, N = 20) evaluated the pharmacokinetics (PKs), pharmacodynamics (PDs), safety, and tolerability of acalabrutinib maleate tablet (AT) formulated with pH-independent release. Subjects were administered AT or AC (orally, fasted state), AT in a fed state, or AT in the presence of a PPI, and AT or AC via nasogastric (NG) route. Acalabrutinib exposures (geometric mean [% coefficient of variation, CV]) were comparable for AT versus AC (AUC

Purpose: Programmed cell death-1 (PD-1) receptor inhibitors have shown efficacy in head and neck squamous cell carcinoma (HNSCC), but treatment failure or secondary resistance occurs in most patients. In preclinical murine carcinoma models, inhibition of Bruton's tyrosine kinase (BTK) induces myeloid cell reprogramming that subsequently bolsters CD8+ T cell responses, resulting in enhanced antitumor activity. This phase 2, multicenter, open-label, randomized study evaluated pembrolizumab (anti–PD-1 monoclonal antibody) plus acalabrutinib (BTK inhibitor) in recurrent or metastatic HNSCC. Patients and Methods: Patients received pembrolizumab 200 mg intravenously every 3 weeks, alone or in combination with acalabrutinib 100 mg orally twice daily. Safety and overall response rate (ORR) were co-primary objectives. The secondary objectives were progression-free survival (PFS) and overall survival. Results: Seventy-six patients were evaluated (pembrolizumab, n = 39; pembrolizumab + acalabrutinib, n = 37). Higher frequencies of grade 3–4 treatment-emergent adverse events (AE; 65% vs. 39%) and serious AEs (68% vs. 31%) were observed with combination therapy versus monotherapy. ORR was 18% with monotherapy versus 14% with combination therapy. Median PFS was 2.7 [95% confidence interval (CI), 1.4–6.8] months in the combination arm and 1.7 (95% CI, 1.4–4.0) months in the monotherapy arm. The study was terminated due to lack of clinical benefit with combination treatment. To assess how tumor immune contexture was affected by therapy in patients with pre- and post-treatment biopsies, spatial proteomic analyses were conducted that revealed a trend toward increased CD45+ leukocyte infiltration of tumors from baseline at day 43 with pembrolizumab (monotherapy, n = 5; combination, n = 2), which appeared to be higher in combination-treated patients; however, definitive conclusions could not be drawn due to limited sample size. Conclusions: Despite lack of clinical efficacy, immune subset analyses suggest that there are additive effects of this combination; however, the associated toxicity limits the feasibility of combination treatment with pembrolizumab and acalabrutinib in patients with recurrent or metastatic HNSCC.

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.

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of treatment, and help guide dose and schedule setting of therapeutics. From a single blood draw, peripheral blood mononuclear cells can be isolated and processed to analyze and quantify protein markers, and plasma samples can be used for the analysis of circulating tumor DNA, cytokines, and plasma metabolomics. Longitudinal samples from a treatment provide information on the evolution of a given protein marker, the mutational status and immunological landscape of the patient. This can only be achieved if the processing of the peripheral blood is carried out effectively in clinical sites and samples are properly preserved from the bedside to bench. Here, we present an optimized general-purpose protocol that can be implemented at clinical sites for obtaining PBMC pellets and plasma samples in multi-center clinical trials, that will enable clinical professionals in hospital laboratories to successfully provide high quality samples, regardless of their level of technical expertise. Alternative protocol variations are also presented that are optimized for more specific downstream analytical methods. We apply this protocol for studying protein biomarkers against DNA damage response (DDR) on X-ray irradiated blood to demonstrate the suitability of the approach in oncology settings where DDR drugs and/or radiotherapy have been practiced as well as in preclinical stages where mechanistic hypothesis testing is required.

Introduction: Patients (pts) with CLL who relapse on therapies targeting B-cell receptor signaling pathways have limited treatment options. Cerala, a selective oral ATR inhibitor, has shown synergistic preclinical activity with a Bruton tyrosine kinase inhibitor (BTKi) in TP53- and ATM-defective CLL cells (Kwok et al. Blood. 2016;127:582-95). Acala is a selective, irreversible BTKi approved for CLL. This phase 1/2 proof-of-concept study investigated cerala alone and in combination with acala in high-risk R/R CLL. Methods: This multicenter, nonrandomized, open-label study (NCT03328273) enrolled adults with high-risk R/R CLL per iwCLL criteria and ECOG performance status ≤2. In Arm A, pts with TP53 mutation, del(17p), or del(11q) having exhausted available treatment options received cerala monotherapy (cohort 1: 160 mg BID continuously; cohort 2 [after ≥2 DLTs in cohort 1]: 160 mg BID 2 wk on/2 wk off). In Arm B, pts with del(11q) suitable for BTKi and cerala treatment per investigator judgment received acala 100 mg BID continuously alone for cycle 1 and acala 100 mg BID continuously + cerala 160 mg BID 1 wk on/3 wk off from cycle 2 onward. Primary objectives: safety and pharmacokinetics (PK). Secondary objective: activity—overall response rate (ORR), complete response rate, duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Pharmacodynamic changes were explored using novel multiple reaction monitoring-targeted mass spectrometry. Results: Eleven pts were treated (median age 64 y; median prior therapy lines 3). Pts in Arm A (n=8 [7 received prior BTKi]: cohort 1 n=5; cohort 2 n=3) received cerala for a median of 3.5 mo (range 0.5-7.7). Grade (G) ≥3 AEs in >1 pt were anemia (75%), thrombocytopenia (63%), and neutropenia (25%); 4 DLTs of G4 thrombocytopenia were reported (3 in 2 pts in cohort 1; 1 in 1 pt in cohort 2). No responses were observed in Arm A. At a median follow-up of 15.1 mo, median PFS was 3.8 mo (95% confidence interval [CI] 0.7-4.6); median OS was 16.9 mo (95% CI 6.6-not reached [NR]). All pts had baseline telomeres within a “fusogenic” range (negative prognostic indicator).In Arm B (n=3; all BTKi-naive), 2 pts received acala + cerala and 1 received acala only. Median (range) treatment duration was 7.2 mo (4.9-9.5) for cerala and 15.9 mo (9.7-18.4) for acala. No G≥3 AEs or DLTs were reported. ORR was 100% (all partial responses); median DOR was NR. At a median follow-up of 15.9 mo, median PFS and OS were NR. PK of acala and cerala were consistent with historical data, with no drug-drug interactions when combined; cerala did not affect BTK occupancy. Conclusions: Cerala monotherapy in BTKi-exposed, high-risk CLL pts showed limited clinical benefit. Acala + cerala was tolerable with preliminary clinical activity in pts with CLL and del(11q). The study terminated due to the evolving treatment landscape. Citation Format: Wojciech Jurczak, Nagah Elmusharaf, Christopher P. Fox, William Townsend, Amanda G. Paulovich, Shringi Sharma, Graeme Parr, Veerendra Munugalavadla, Fanny Krantz, Helen Yang, Emma Dean, Richa Manwani, Peter Hillmen. Phase 1/2 results of ceralasertib (Cerala) as monotherapy or in combination with acalabrutinib (Acala) in high-risk relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT532.

Background: The PI3K/AKT/mTOR pathway is an established therapeutic target in indolent B-cell non-Hodgkin lymphoma (B-NHL). Several PI3K inhibitors are approved to treat relapsed/refractory (R/R) follicular lymphoma (FL) and, more recently, marginal zone lymphoma (MZL). Nonetheless, potential class-specific and PI3K-isoform-related toxicities may limit their clinical utility. Capivasertib is an oral, potent, selective inhibitor of the 3 AKT isoforms that demonstrated a manageable safety profile (n>1500) and survival benefit in combination with other agents in patients (pts) with solid tumors. Unlike PI3K inhibitors, colitis or pneumonitis have not been a concern with monotherapy. Preclinically, capivasertib was active both in vitro in a large panel of B-NHL cell lines and in vivo in PTEN-null GCB-DLBCL PDX models, where it downregulated the oncogenic factor MYC. In the JVM2 mantle cell lymphoma (MCL) cell line, capivasertib showed superior activity vs PI3Ka/d (AZD8835) and PI3Kb/d (AZD8186) inhibitors. Of note, AKT activation following dysregulation of PTEN has been frequently described in FL (where >85% of tFL or FL/DLBCL exhibited a GCB phenotype). Therefore, capivasertib may represent an effective treatment option for pts with R/R B-NHL. Trial Design: This modular phase II study will enroll pts in 3 cohorts according to B-NHL type. Core inclusion criteria include: age ≥18 years and ECOG PS ≤2. Module-specific criteria include: cohort 1A: R/R FL after ≥2 prior systemic lines (max 5), including an anti-CD20 monoclonal antibody (mAb) and an alkylating agent; cohort 1B: R/R MZL of splenic, nodal, and extranodal subtypes after ≥2 prior systemic lines (max 5) including ≥1 anti-CD20 mAb; cohort 1C: R/R MCL after ≥2 prior systemic lines (max 4), previously exposed to anti-CD20 mAb and a BTK inhibitor. Pts with FL grade 3B, transformed disease, blastoid or TP53 mutation MCL, active CNS disease, diabetes requiring insulin, or being Funding: AstraZeneca Clinical Trial Registration: NCT050080552 Citation Format: Michael Wang, Ho-Jin Shin, Alex F. Herrera, Moshe Levy, Wong Seog Kim, Tae Min Kim, Jin Seok Kim, Dok Hyun Yoon, Vincent Ribrag, Miguel Canales, John Radford, Dima El-Sharkawi, Brandy Hemmer, Richa Manwani, Veerendra Munugalavadla, Robert Chen, Andrew Mortlock, Alessandra Forcina, Franck Morschhauser. Trial in progress: A phase II, open-label, multicenter study of capivasertib, a potent, oral pan-AKT inhibitor in patients with relapsed or refractory B-cell non-hodgkin lymphoma (CAPITAL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT127.

7539 Background: For ELEVATE-TN (NCT02475681), we previously reported superior efficacy of acalabrutinib (A) ± obinutuzumab (O) vs O + chlorambucil (Clb) in patients (pts) with treatment-naive (TN) chronic lymphocytic leukemia (CLL) at 28.3 and 46.9 months (mo) median follow-up. Now, we report results from a 5-y update. Methods: Pts were randomized to A+O, A, or O+Clb. Pts who progressed on O+Clb could cross over to A monotherapy. Investigator-assessed (INV) progression-free survival (PFS), INV overall response rate (ORR), overall survival (OS), and safety were evaluated. Results: A total of 535 pts (A+O, n=179; A, n=179; O+Clb, n=177) had a median age of 70 y. At a median follow-up of 58.2 mo (range, 0.0–72.0; data cutoff Oct 1, 2021), median PFS was not reached (NR) (hazard ratio [HR]: 0.11) for A+O and A (HR: 0.21) vs 27.8 mo for O+Clb (both P

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.

Spleen tyrosine kinase (SYK) signaling is a proposed target in acute myeloid leukemia (AML). Sensitivity to SYK inhibition has been linked toThis was an international multicenter phase Ib/II study, entospletinib dose escalation (standard 3+3 design between 200 and 400 mg twice daily) + 7+3 (cytarabine + daunorubicin) in phase Ib and entospletinib dose expansion (400 mg twice daily) + 7+3 in phase II.Fifty-three patients (Entospletinib with intensive chemotherapy was well-tolerated in patients with AML. Improved survival was observed in patients with

Acalabrutinib is a selective irreversible Bruton tyrosine kinase (BTK) inhibitor that does not affect IL2-associated tyrosine kinase or antibody-dependent cellular cytotoxicity, making it an attractive candidate for combination therapy with anti-CD20 antibodies. We investigated acalabrutinib plus obinutuzumab in a phase Ib/II study (NCT02296918) of patients with treatment-naïve or relapsed/refractory chronic lymphocytic leukemia (CLL). Nineteen treatment-naïve and 26 relapsed/refractory patients were treated with acalabrutinib (100 mg twice daily) until progression and obinutuzumab (cycle 1: 100 mg day 1, 900 mg day 2, 1000 mg days 8 and 15; cycles 2–6: 1,000 mg day 1). Grade 3/4 adverse events occurred in 71% of patients. Overall response rates were 95% (treatment-naïve) and 92% (relapsed/refractory). Thirty-two percent of treatment-naïve and 8% of relapsed/refractory patients achieved complete remission. At 36 months, 94% (treatment-naïve) and 88% (relapsed/refractory) were progression free. Acalabrutinib plus obinutuzumab was well tolerated, producing high and durable responses in treatment-naïve and relapsed/refractory CLL. Significance: Rituximab plus the less selective BTK inhibitor ibrutinib has not shown benefit in CLL; however, the selective BTK inhibitor acalabrutinib plus the antibody-dependent cellular cytotoxicity–enhanced antibody obinutuzumab yielded durable responses that deepened over time in treatment-naïve and relapsed/refractory CLL, supporting the evaluation of this approach in larger, comparative studies in CLL. This article is highlighted in the In This Issue feature, p. 327

Background: Acalabrutinib is a selective, covalent Bruton tyrosine-kinase inhibitor with activity in chronic lymphocytic leukaemia. We compare the efficacy of acalabrutinib with or without obinutuzumab against chlorambucil with obinutuzumab in patients with treatment-naive chronic lymphocytic leukaemia. Methods: ELEVATE TN is a global, phase 3, multicentre, open-label study in patients with treatment-naive chronic lymphocytic leukaemia done at 142 academic and community hospitals in 18 countries. Eligible patients had untreated chronic lymphocytic leukaemia and were aged 65 years or older, or older than 18 years and younger than 65 years with creatinine clearance of 30–69 mL/min (calculated by use of the Cockcroft-Gault equation) or Cumulative Illness Rating Scale for Geriatrics score greater than 6. Additional criteria included an Eastern Cooperative Oncology Group performance status score of 2 or less and adequate haematologic, hepatic, and renal function. Patients with significant cardiovascular disease were excluded, and concomitant treatment with warfarin or equivalent vitamin K antagonists was prohibited. Patients were randomly assigned (1:1:1) centrally via an interactive voice or web response system to receive acalabrutinib and obinutuzumab, acalabrutinib monotherapy, or obinutuzumab and oral chlorambucil. Treatments were administered in 28-day cycles. To reduce infusion-related reactions, acalabrutinib was administered for one cycle before obinutuzumab administration. Oral acalabrutinib was administered (100 mg) twice a day until progressive disease or unacceptable toxic effects occurred. In the acalabrutinib-obinutuzumab group, intravenous obinutuzumab was given on days 1 (100 mg), 2 (900 mg), 8 (1000 mg), and 15 (1000 mg) of cycle 2 and on day 1 (1000 mg) of cycles 3–7. In the obinutuzumab-chlorambucil group, intravenous obinutuzumab was given on days 1 (100 mg), 2 (900 mg), 8 (1000 mg), and 15 (1000 mg) of cycle 1 and on day 1 (1000 mg) of cycles 2–6. Oral chlorambucil was given (0·5 mg/kg) on days 1 and 15 of each cycle, for six cycles. The primary endpoint was progression-free survival between the two combination-therapy groups, assessed by independent review committee. Crossover to acalabrutinib was allowed in patients who progressed on obinutuzumab-chlorambucil. Safety was assessed in all patients who received at least one dose of treatment. Enrolment for this trial is complete, and the study is registered at ClinicalTrials.gov, NCT02475681. Findings: Between Sept 14, 2015, and Feb 8, 2017, we recruited 675 patients for assessment. 140 patients did not meet eligibility criteria, and 535 patients were randomly assigned to treatment. 179 patients were assigned to receive acalabrutinib-obinutuzumab, 179 patients were assigned to receive acalabrutinib monotherapy, and 177 patients were assigned to receive obinutuzumab-chlorambucil. At median follow-up of 28·3 months (IQR 25·6–33·1), median progression-free survival was longer with acalabrutinib-obinutuzumab and acalabrutinib monotherapy, compared with obinutuzumab-chlorambucil (median not reached with acalabrutinib and obinutuzumab vs 22·6 months with obinutuzumab, hazard ratio [HR] 0·1; 95% CI 0·06–0·17, p

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

Pim kinases have been targets of interest for a number of therapeutic areas. Evidence of durable single-agent efficacy in human clinical trials validated Pim kinase inhibition as a promising therapeutic approach for multiple myeloma patients. Here, we report the compound optimization leading to GDC-0339 (16), a potent, orally bioavailable, and well tolerated pan-Pim kinase inhibitor that proved efficacious in RPMI8226 and MM.1S human multiple myeloma xenograft mouse models and has been evaluated as an early development candidate.

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.

// Reuben Kapur 1, 2 , Jianjian Shi 1 , Joydeep Ghosh 2 , Veerendra Munugalavadla 3 , Emily Sims 1 , Holly Martin 1 , Lei Wei 1 , Raghuveer Singh Mali 1 1 Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA 2 Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA 3 Gilead Sciences, Inc., Foster City, CA, USA Correspondence to: Raghuveer Singh Mali, e-mail: rsmali@iupui.edu Keywords: mast cells, Rho kinase, LIM kinase, cell growth, anaphylaxis Received: October 21, 2015 Accepted: January 29, 2016 Published: March 02, 2016 ABSTRACT Understanding mast cell development is essential due to their critical role in regulating immunity and autoimmune diseases. Here, we show how Rho kinases (ROCK) regulate mast cell development and can function as therapeutic targets for treating allergic diseases. Rock1 deficiency results in delayed maturation of bone marrow derived mast cells (BMMCs) in response to IL-3 stimulation and reduced growth in response to stem cell factor (SCF) stimulation. Further, integrin-mediated adhesion and migration, and IgE-mediated degranulation are all impaired in Rock1-deficient BMMCs. To understand the mechanism behind altered mast cell development in Rock1−/− BMMCs, we analyzed the activation of ROCK and its downstream targets including LIM kinase (LIMK). We observed reduced activation of ROCK, LIMK, AKT and ERK1/2 in Rock1-deficient BMMCs in response to SCF stimulation. Further, loss of either Limk1 or Limk2 also demonstrated altered BMMC maturation and growth; combined deletion of both Limk1 and Limk2 resulted in further reduction in BMMC maturation and growth. In passive cutaneous anaphylaxis model, deficiency of Rock1 or treatment with ROCK inhibitor Fasudil protected mice against IgE-mediated challenge. Our results identify ROCK/LIMK pathway as a novel therapeutic target for treating allergic diseases involving mast cells.

BackgroundThe immunosuppressive desmoplastic stroma of pancreatic cancer represents a major hurdle to developing an effective immune response. Preclinical studies in pancreatic cancer have demonstrated promising anti-tumor activity with Bruton tyrosine kinase (BTK) inhibition combined with programmed cell death receptor-1 (PD-1) blockade.MethodsThis was a phase II, multicenter, open-label, randomized (1:1) clinical trial evaluating the BTK inhibitor acalabrutinib, alone (monotherapy) or in combination with the anti-PD-1 antibody pembrolizumab (combination therapy). Eligible patients were adults with histologically confirmed metastatic or locally advanced unresectable pancreatic ductal adenocarcinoma with an Eastern Cooperative Oncology Group Performance Status (ECOG PS) ≤1 who had received at least one prior systemic therapy. Oral acalabrutinib 100 mg twice daily was administered with or without intravenous pembrolizumab 200 mg on day 1 of each 3-week cycle. Peripheral blood was analyzed for changes in immune markers, and tumors from exceptional responders were molecularly analyzed.ResultsA total of 77 patients were enrolled (37 monotherapy; 40 combination therapy) with a median age of 64 years; 77% had an ECOG PS of 1. The median number of prior therapies was 3 (range 1–6). Grade 3–4 treatment-related adverse events were seen in 14.3% of patients in the monotherapy arm and 15.8% of those in the combination therapy arm. The overall response rate and disease control rate were 0% and 14.3% with monotherapy and 7.9% and 21.1% with combination therapy, respectively. Median progression-free survival was 1.4 months in both arms. Peripheral blood flow analysis demonstrated consistent reductions in granulocytic (CD15+) myeloid-derived suppressor cells (MDSCs) over time. Two exceptional responders were found to be microsatellite stable with low tumor mutation burden, low neoantigen load and no defects in the homologous DNA repair pathway.ConclusionsThe combination of acalabrutinib and pembrolizumab was well tolerated, but limited clinical activity was seen with either acalabrutinib monotherapy or combination therapy. Peripheral reductions in MDSCs were seen. Efforts to understand and target the pancreatic tumor microenvironment should continue.Trial registration numberNCT02362048.

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.

Aim: Idelalisib (IDELA) treatment is associated with diarrhea/colitis (incidence of ∼15% grade ≥3). We performed a retrospective analysis of gastrointestinal biopsies from 29 patients treated with IDELA across nine clinical trials. Methods: A central core laboratory performed histopathologic review, immunohistochemistry, and droplet digital PCR viral studies. These results were correlated with tissue immune profiling data and morphologic features per modified Geboes score. Results: Out of 29 eligible patients with abdominal pain or diarrhea, 24 (82.8%) had reported adverse event terms of diarrhea and/or colitis. Infectious pathogens were detected in 9/29 samples. Most biopsies presented with mixed/inflammatory infiltrates and contained increased numbers of FOXP3+ cells versus normal controls. Conclusion: This study revealed evidence of T-cell dysregulation and a substantial infectious component in association with IDELA-related diarrhea/colitis.

PI3Kδ (idelalisib) and BTK (ibrutinib) inhibitors have demonstrated significant clinical activity in chronic lymphocytic leukemia (CLL) interfering with the cross-talk between CLL cells and the lymph node microenviroment, yet their mechanism of action remains to be fully elucidated. Here, we developed an ex vivo model with the aim of reproducing the effects of the microenvironment that would help shed light on the in vivo mechanism of action of idelalisib and ibrutinib and predict their clinical efficacy in individual patients. First we explored the effects of various cell-extrinsic elements on CLL apoptosis and proliferation and found that the combination of CpG+IL2+HS5 stromal cell line + human serum +CLL plasma and erythrocyte fractions represented the best co-culture conditions to test the effects of the novel inhibitors. Then, using this assay, we investigated the impact of idelalisib and ibrutinib on both survival and proliferation in 30 CLL patients. While both drugs had a limited direct pro-apoptotic activity, a potent inhibition of proliferation was achieved at clinically achievable concentrations. Notably, up to 10% of CLL cells still proliferated even at the highest concentrations, likely mirroring the known difficulty to achieve complete responses in vivo. Altogether, this novel assay represents an appropriate ex vivo drug testing system to potentially predict the clinical response to novel inhibitors in particular by quantifying the antiproliferative effect., This work was supported in part by H2020 “MEDGENET, Medical Genomics and Epigenomics Network” (Grant 692298), funded by the European Union; the Swedish Cancer Society, the Swedish Research Council, and Lion’s Cancer Research Foundation; Associazione Italiana per la Ricerca sul Cancro AIRC (Investigator Grant #20246 to PG and Special Program Molecular Clinical Oncology - 5 per mille #9965), Milano, Italy, Ricerca; ERA-NET TRANSCAN-2 JTC 2016, GCH-CLL. Vivia has received unrestricted research support from Gilead.

Pim kinases have been identified as promising therapeutic targets for hematologic–oncology indications, including multiple myeloma and certain leukemia. Here, we describe our continued efforts in optimizing a lead series by improving bioavailability while maintaining high inhibitory potency against all three Pim kinase isoforms. The discovery of extensive intestinal metabolism and major metabolites helped refine our design strategy, and we observed that optimizing the pharmacokinetic properties first and potency second was a more successful approach than the reverse. In the resulting work, novel analogs such as 20 (GNE-955) were discovered bearing 5-azaindazole core with noncanonical hydrogen bonding to the hinge.

357 Background: Immunotherapy checkpoint inhibitors (ICIs) are now standard treatments (txs) in mUC, with response rates ranging 15-25%. A phase 2 randomized study (NCT02351739) treated 75 pts with mUC with either P or PA. We profiled peripheral blood mononuclear cells (PBMCs) to understand changes on tx. Methods: Comprehensive immune profiling of PBMCs was performed at baseline, weeks (wk) 4, 7, and 10. The Wilcoxon rank sum (WRS) test was used to compare tx arms, as well as pts with complete and partial response (CR/PR) vs disease progression (PD). Using the Benjamini-Hochberg (BH) method, results were deemed significant if they had false discovery rate (FDR) of 0.1 or less. Results: Clinical responses did not differ between tx arms (Zhang T et al, ESMO, 2017). The majority of patients showed largest immune changes from baseline at wk7. Comparing P vs PA pts, the PA group had statistically significant increases in PD-L1+ monocytes; CD8 T cells expressing: CD39, CTLA-4, ICOS, PD-1, TIM3, HLA-DR, Ki67, CD39/HLA-DR, CD39/Ki67, and PD-1/TIM-3; and exhausted (CD28-) CD8 T cells expressing ICOS and PD-L1 (Table). Comparing CR/PR vs PD pts, Treg subsets expressing CCR4/HLA-DR and CD39/HLA-DR were significant by WRS but not FDR criteria. Conclusions: From this extensive, exploratory, non-functional flow analysis of PBMCs, PA may potentiate immune changes of PD-L1+ monocytes and CD8+ T cell subsets. Increasing activated peripheral Tregs in CR/PR pts in either arm may indicate marginalization of Tregs from the tumor microenvironment. [Table: see text]

The phosphatidylinositol 3'-kinase (PI3K) pathway is dysregulated in multiple myeloma (MM); we therefore tested a highly selective class I PI3K inhibitor, GDC-0941, for anti-myeloma activity. Functional and mechanistic studies were first performed in MM cell lines, then extended to primary MM patient samples cultured in vitro. GDC-0941 was then assessed as a single agent and in various combinations in myeloma tumor xenograft models. We show p110 α and β are the predominant PI3K catalytic subunits in MM and that a highly selective class I PI3K inhibitor, GDC-0941, has robust activity as a single agent to induce cell cycle arrest and apoptosis of both MM cell lines and patient myeloma cells. Mechanistic studies revealed an induction of cell cycle arrest at G0/G1, with decreased phospho-FoxO1/3a levels, decreased cyclin D1 and c-myc expression, and an increase in the cell cycle inhibitor, p27kip. Induction of apoptosis correlated with increased expression of the pro-apoptotic BH3-only protein BIM, cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP). In vitro, GDC-0941 synergized with dexamethasone (Dex) and lenalidomide (combination index values of 0.3-0.4 and 0.4-0.8, respectively); in vivo GDC-0941 has anti-myeloma activity and significantly increases the activity of the standard of care agents in several murine xenograft tumor models (additional tumor growth inhibition of 37-53% (Dex) and 22-72% (lenalidomide)). These data provide a clear therapeutic hypothesis for the inhibition of PI3K and provide a rationale for clinical development of GDC-0941 in myeloma.