Abstract 4057: Inhibition of polyploidy and senescence induces intrinsic apoptosis in double hit or double expresser DLBCL
Authors: Islam Shariful , Qi Wenqing , Morales Carla , Cooke Laurence , Spier Catherine M. , Mahadevan Daruka 
Published: July 2017

Abstract
Introduction: Double Hit (DH) or Double Expresser (DE) DLBCL are aggressive Non-Hodgkin’s Lymphomas (NHLs) with translocation or over-expression of MYC and BCL-2 which are incurable with current therapies. MYC is a critical driver of oncogenic phenotypes and regulates expression of numerous genes including aurora kinases (AKs), which are validated drug targets. AKs are over-expressed in DLBCL and AK inhibition in two Phase-II trials of relapsed and refractory B/T-NHL resulted in 20%-30% overall response rate. Bruton’s tyrosine kinase (BTK) plays a central role in B-cell receptor (BCR) signaling and inhibitors of BTK have shown efficacy in B-NHL clinical trials. Targeting CD20 (rituximab) in DLBCL inhibits BCL-2 and induces apoptosis via caspase-3 activation. We hypothesized that DH/DE-DLBCL are amenable to a novel therapeutic strategy of AK, BTK and CD20 inhibition.
Methods: Four DH/DE DLBCL cell lines were evaluated for viability by MTS; polyploidy, senescence and apoptosis by flow cytometry; DNA damage by TUNEL assay. Phospho-BCL-2, -AK and -BTK analysis by western. Safety and efficacy of drug combinations in a mouse xenograft model. Analysis of patient samples and mouse tumors by IHC.
Results: DH/DE-DLBCL cell lines and patient samples displayed over-expression of MYC, BCL-2, BTK and AK-A proteins. Cell viability assays with alisertib alone showed ~30% cell death, while majority of remaining ~70% cells were polyploid and senescent at day-4 (a mechanism of treatment failure). These polyploid and senescent cells (PASC) showed increased levels of phospho-BTK indicating increased BCR signaling (a rationale for combination therapy). We show that alisertib + ibrutinib + rituximab significantly reduced PASCs leading to intrinsic cell death compared to single/double combinations. Dual inhibition of AK + BTK reduced phospho-ERK-1/2, -Bcl-6 and -MYC with increased DNA breaks indicated by up regulation of phospho-H2A-X, -Chk-2, and pro-apoptotic Noxa and Puma proteins. Triple therapy reduced Bcl-2, Bcl-xL and increased Noxa and Puma compromising the mitochondrial outer membrane potential, release of Cyt-C, activation of caspase-3 and induction apoptosis by PARP cleavage. A DLBCL SCID mouse xenograft model showed ibrutinib alone is inactive while alisertib + ibrutinib was additive with a tumor growth inhibition (TGI) rate of ~25%. TGI for ibrutinib + rituximab was ~50-60%. However, triple therapy showed a TGI of &gt;90% and was well tolerated. Survival analysis: 67% of mice alive at day-89 with triple therapy versus 20% with ibrutinib + rituximab. Other treatments showed no survival past day-52.
Conclusions: A novel triple therapy consisting of alisertib + ibrutinib + rituximab inhibits PASCs induced by AK inhibition in DH/DE-DLBCL leading to a significant anti-proliferative signal and intrinsic apoptosis. Our work provides a rationale for exploring these combinations in the clinic.
Citation Format: Shariful Islam, Wenqing Qi, Carla Morales, Laurence Cooke, Catherine M. Spier, Daruka Mahadevan. Inhibition of polyploidy and senescence induces intrinsic apoptosis in double hit or double expresser DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4057. doi:10.1158/1538-7445.AM2017-4057