Dual Inhibition of Bruton's Tyrosine Kinase and BCL2: A Promising Therapeutic Strategy for Myeloid and Lymphoid Leukemias

Background:The identification of effective therapies based on targeted interventions for human cancers faces the challenges of genetic and epigenetic heterogeneity underlying the disease. Large-scale sequencing efforts have uncovered a spectrum of mutations in many hematologic malignancies, suggesting that combinations of agents will be required to treat these diseases effectively. Combinatorial approaches will also be critical for combating the emergence of genetically heterogeneous subclones, rescue signals in the microenvironment, and tumor-intrinsic feedback pathways that all contribute to disease relapse.

Methods:We used a functional ex vivo screening assay to identify small-molecule targeted inhibitors and inhibitor combinations demonstrating selective efficacy across broad categories of leukemia. Primary mononuclear cells isolated from leukemia patients (n=588) were plated in the presence of a panel of graded concentrations of over 120 single-agent inhibitors or combinations spanning multiple drug classes. Leukemia specimens from 5 diagnosis subgroups included acute myeloid leukemia (AML; n=293), acute lymphoblastic leukemia (ALL; n=83), chronic lymphocytic leukemia (CLL; n=140), chronic myeloid leukemia (CML; n=26) and myeloproliferative neoplasms/myelodysplastic syndrome (MPN or MDS/MPN; n=46) patients. The combinations were designed as drug pairs that target non-overlapping biological pathways and comprise drugs from different classes, preferably with Food and Drug Administration approval. IC₅₀ and AUC values were derived from probit-based regression for each response curve. Mutational status for FLT3-ITD and NPM1 were compiled from clinical labs or by capillary electrophoresis using a QiaXcel instrument. Disease status was obtained from clinical chart review. Single and combination drug treatment IC₅₀ and AUC values were compared within groups by Friedman test, across groups by Kruskal-Wallis test, and with continuous variables by Spearman rank correlation.

Results:Among 588 unique leukemia patients evaluated, we observed that the combination of venetoclax (a BCL2 inhibitor) with ibrutinib(a BTK inhibitor) showed enhanced benefit above that for either single agent in both myeloid and lymphoid malignancies, including AML, ALL, and CLL. Expanded analyses of combination sensitivity in AML patients showed no significant associations with age, gender, or ELN prognostic risk. Comparison of AML samples by disease treatment status indicated a slightly greater sensitivity in relapsed/refractory (n=42) versus newly diagnosed (n=115) AML samples by both AUC and IC₅₀ (p=0.028 and 0.026, respectively). Among AML samples, FLT3-ITD and NPM1 mutation status was available for 204 and 188 patients, respectively. Venetoclax + ibrutinib was significantly more effective in patient samples harboring FLT3-ITD or NPM1 mutations by both AUC (p=0.0002 and p=0.0032, respectively) and IC₅₀ (p=0.0002 and p=0.021, respectively). Furthermore, among 112 AML samples with RNAseq data available, sensitivity to the combination (AUC) was significantly correlated with gene expression for BCL2 and BTK (Spearman r: -0.48 and -0.21, respectively). Evaluation of the combination using an expanded 7x7 concentration matrix on AML cell lines revealed synergy for the majority of drug-pair concentrations by the Bliss independence model. Analysis to align additional clinical and genetic features for leukemia patient samples with drug sensitivities is in progress and results with be presented at the meeting.

Conclusion:Both myeloid and lymphoid-derived primary leukemia cells show sensitivity to combined inhibition of BCL2 and BTK with the combination of venetoclax and ibrutinib, suggesting this drug pair may have broad therapeutic indications.

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