Abstract 306: A mechanistic rationale for combining acalabrutinib with CDK9 inhibitor, AZD4573, in ABC-DLBCL
Authors: Boiko Scott , Proia Theresa , Martin Maryann San , Cidado Justin , Shao Wenlin , Drew Lisa 
Published: August 2018

Abstract
Cyclin-dependent kinase 9 (CDK9) regulates transcription elongation through phosphorylation of RNA polymerase II at serine 2 (pSer2-RNAPII), and its short-term inhibition downregulates genes with short-lived transcripts and labile proteins, thus providing a mechanism to inhibit key survival proteins like Mcl1 and Myc. We developed a selective CDK9 inhibitor, AZD4573, that exhibits anticancer activity across a diverse set of hematologic cell lines and is being evaluated in patients with hematologic malignancies. Through efforts to identify combinations with AZD4573, we observed that combined treatment with acalabrutinib, our Bruton tyrosine kinase (BTK) inhibitor, led to more robust and durable responses in preclinical ABC-DLBCL models. We demonstrate BTK inhibition with acalabrutinib upregulates the proapoptotic BH3-only protein Bim in BTKi sensitive ABC-DLBCL cell lines, priming cells for apoptosis induction and further sensitizing them to CDK9 inhibition with AZD4573. Specifically, acalabrutinib upregulated proapoptotic BH3-only proteins Bim and Bmf (&gt;3-fold, 24h) in two BTK-dependent ABC-DLBCL cell lines, OCILy10 and TMD8 but not in a BTK-independent GCB-DLBCL cell line Karpas-422. In each model, treatment with 100nM AZD4573 resulted in rapid induction of cleaved caspase-3 (CC3), and subsequent combination with acalabrutinib accelerated induction of CC3 and increased the magnitude of cell death in the BTK-dependent cell lines. Apoptotic priming by acalabrutinib was validated in vitro in OCILy10 cells by siRNA-mediated knockdown of BH3-only proteins followed by a phenotypic comparison of combination dosing schedules. Both inhibitors dosed concurrently (unprimed) resulted in a minimal increase in the percentage of apoptotic cells compared to treatment with AZD4573 alone (49% of population). In contrast, when dosed sequentially with a pretreatment of acalabrutinib (primed) before the addition of AZD4573, nearly all cells were apoptotic (&gt;90%). Consistent with the in vitro results, the combination of AZD4573 with acalabrutinib resulted in rapid and complete tumor growth inhibition in both BTK-dependent ABC-DLBCL xenograft models. In the OCILy10 model, the combination led to complete tumor regressions and 100% of animals treated in the combination arm (8/8) demonstrated a 60-day delay to regrowth off treatment compared to single-agent AZD4573. Similarly in TMD8, the combination resulted in complete regressions in 100% of treated animals with a highly durable response (17/19 animals tumor-free at 100+ days) whereas the combination yielded no additional benefit in Karpas-422. Acalabrutinib was recently approved for relapsed/refractory mantle cell lymphoma and is being evaluated in patients with additional subtypes of lymphomas. Based on our findings, the combination of acalabrutinib with AZD4573 could be an effective treatment option for patients with ABC-DLBCL.
Citation Format: Scott Boiko, Theresa Proia, Maryann San Martin, Justin Cidado, Wenlin Shao, Lisa Drew. A mechanistic rationale for combining acalabrutinib with CDK9 inhibitor, AZD4573, in ABC-DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 306.