Simultaneous Kinase Inhibition with Ibrutinib and BCL2 Inhibition with Venetoclax As a Therapeutic Strategy for Acute Lymphoblastic Leukemia
Authors: Eide Christopher A. , Kurtz Stephen E , Kaempf Andy , Long Nicola , Leonard Jessica , Chang Bill H. , Mori Motomi , Druker Brian J. , Tyner Jeffrey W. 
Published: November 2019

Background: In patients with acute lymphoblastic leukemia (ALL), patient outcomes vary considerably by patient age group, specific genetic subtypes, and treatment regimen. Large-scale sequencing efforts have uncovered a spectrum of mutations and gene fusions in ALL, suggesting that combinations of agents will be required to treat these diseases effectively. Previous preclinical studies have shown efficacy of the BCL2 inhibitor venetoclax alone or in combination in ALL cells (Chonghaile et al., Can Disc 2014; Leonard et al, STM 2018), and the multi-kinase inhibitor ibrutinib (approved for patients with chonic lymphoblastic leukemia (CLL)) has also shown potent activity in subsets of ALL (Kim et al., Blood 2017). However, the combination of ibrutinib and venetoclax has not been evaluated to date in patients with ALL.
Methods: We used a functional ex vivo screening assay to evaluate the potential efficacy of the combination of ibrutinib and venetoclax (IBR+VEN) across a large cohort (n=808) of patient specimens representing a broad range of hematologic malignancies. Primary mononuclear cells isolated from leukemia patients were plated in the presence of graded concentrations of venetoclax, ibrutinib, or the combination of both FDA-approved drugs. IC50 and AUC values were derived from probit-based regression for each response curve. A panel of clinical labs, treatment information, and genetic features for tested ALL patient specimens was collated from chart review. Single and combination drug treatment sensitivity were compared within groups by Friedman test, across groups by Mann-Whitney test, and with continuous variables by Spearman rank correlation.
Results: Consistent with clinical data and previous literature, IBR+VEN was highly effective in CLL specimens ex vivo (median IC50=0.015 µM). Intriguingly, among specimens from 100 unique ALL patients, we also observed that IBR+VEN demonstrated significantly enhanced efficacy by AUC and IC50 compared to either single agent (p<0.001; median IC50=0.018 µM). In contrast, evaluation of this combination on primary mononuclear cells from two healthy donors showed little to no sensitivity. Breakdown of combination sensitivity (as measured by AUC) by a variety of clinical and genetic features revealed no associations with gender or specimen type. Among continuous variables tested, age was modestly correlated with combination AUC (Spearman r = 0.26) and increased blasts in the bone marrow were associated with increased sensitivity to the combination (Spearman r = -0.41; p = 0.0068). More broadly, specimens from patients with B-cell precursor disease (B-ALL) were more sensitive to IBR+VEN than those with T-cell precursor leukemia (T-ALL) (p = 0.0063). Within the B-ALL patient samples, those harboring the BCR-ABL1 fusion were significantly less sensitive to IBR+VEN than other subtypes of B-ALL (p = 0.0031). Within the T-ALL subset, there was a trend toward reduced sensitivity in patients with evidence of mutations in NOTCH1, though statistical significance was not reached. Evaluation of the combination using an expanded 7x7 concentration matrix in human ALL cell lines revealed varying degrees of sensitivity. For example, IBR+VEN showed enhanced efficacy in RCH-ACV B-ALL cells and showed synergy for the majority of drug-pair concentrations by the highest single agent (HSA) method (ibrutinib, venetoclax, and combination IC50: 0.60, 3.4, and 0.28 uM, respectively).
Conclusion: Our findings suggest that the IBR+VEN combination, currently approved for patients with CLL, also demonstrates impressive efficacy against primary leukemia cells from ALL patients, warranting further investigation as a treatment strategy in the clinic to continue to improve outcomes for patients.

Disclosures
Leonard: Amgen: Research Funding. Druker:Cepheid: Consultancy, Honoraria; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees ; Gilead Sciences: Other: former member of Scientific Advisory Board; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Patents & Royalties, Research Funding; Pfizer: Research Funding; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Patient True Talk: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Beat AML LLC: Other: Service on joint steering committee; CureOne: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Monojul: Other: former consultant; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees. Tyner:Petra: Research Funding; Agios: Research Funding; Array: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Syros: Research Funding; Takeda: Research Funding; Seattle Genetics: Research Funding; AstraZeneca: Research Funding; Seattle Genetics: Research Funding; Array: Research Funding; Aptose: Research Funding; Incyte: Research Funding; Syros: Research Funding; Takeda: Research Funding; Petra: Research Funding; Agios: Research Funding; Constellation: Research Funding; Aptose: Research Funding; Gilead: Research Funding; Incyte: Research Funding; AstraZeneca: Research Funding; Constellation: Research Funding; Janssen: Research Funding; Genentech: Research Funding.