Abstract 1097: Mechanisms of primary and acquired resistance to venetoclax in chronic lymphocytic leukemia (CLL)
Authors: Murali Ishwarya , Cha Justin , Leshchiner Ignaty , Kuang Yanan , Vasquez Kevin , Khalsa Jasneet , Fernandes Stacey M. , Utro Filippo , Rhrissorrakrai Kahn , Levovitz Chaya , Danysh Brian P. , Slowik Kara , Jacobs Raquel A. , Paweletz Cloud P. , Parida Laxmi , Getz Gad , Brown Jennifer R. 
Published: July 2021

Abstract
Mechanisms of resistance to venetoclax, a highly selective oral BCL2 inhibitor approved for therapy of CLL, remain poorly understood and we sought to characterize the clonal evolution of resistance in patients developing progressive disease on venetoclax. We performed whole-exome sequencing (WES) on 7 relapsed/refractory CLL patients with disease progressing on venetoclax. We observed del17p in 43% (3/7), TP53 mutation in 86% (6/7) and unmutated IGHV in 71% (5/7) of patients. A median of four longitudinal tumor samples were sequenced per patient (total Nsamples= 25). We performed digital droplet PCR (ddPCR) analysis to look at the emergence of BCL2 G101V mutations that have been previously linked with venetoclax resistance after long times on therapy. G101V mutation was detected by ddPCR in two patients at low variant allelic fractions (VAF) of 0.03% and 4.68% respectively, and not detected by WES. We further detected one droplet positive for G101V mutation in two other patients in venetoclax progression bone marrow samples. Due to the very low VAF of the BCL2 G101V mutations, we suspected that other mechanisms of acquired resistance were more significant in this cohort of relatively early relapses. Analysis of WES data showed no somatic single nucleotide variants (sSNVs) selected in more than one patient with resistance. However, copy number analysis revealed acquired del8p in 3 patients, resulting in large subclonal expansions. Furthermore, del 8p co-occurred with amp 1q21.2-21.3 affecting the MCL1 gene in 2 patients, and with amp 8q, del 17p, del 18q22.1-23 and del 9p23-21.2 (containing the MYC, TP53, BCL2 and CDKN2A/B genes respectively) in a third. Two other patients showed expansion of clones harboring del 10q23.31-24.1, that includes PTEN, while a third contains an expanding clone with two IRF8 mutations (S283C and SFF416fs). Resistance in the final patient is likely associated with marked expansion of clones with TP53 and SF3B1 mutations. RNA-seq analysis comparing pre-venetoclax to post-venetoclax resistant samples from 8 CLL patients showed downregulation of the BCR, FCGR and MAPK signaling pathways, with upregulation of mitochondrial translation, oxidative phosphorylation and the TCA/citric acid cycle at the time of progression. RNA-seq analysis focusing on the patients with del 8p shows significant downregulation of the TNFRSF10A/10B genes (TRAIL-Rs), with gene set enrichment analysis (GSEA) showing positive enrichment for WNT5A/FZD4 signaling and CREB signaling via the PKC and MAPK pathways, concomitant with downregulation of the BCR and FCGR pathways at progression. Our data suggest several mechanisms of venetoclax resistance in CLL, including loss of TNFRSF10A/B, sometimes with MCL1 upregulation, as well as WNT pathway upregulation and BCR pathway downregulation, which we are now validating in primary patient PBMCs and relevant cell line models.
Citation Format: Ishwarya Murali, Justin Cha, Ignaty Leshchiner, Yanan Kuang, Kevin Vasquez, Jasneet Khalsa, Stacey M. Fernandes, Filippo Utro, Kahn Rhrissorrakrai, Chaya Levovitz, Brian P. Danysh, Kara Slowik, Raquel A. Jacobs, Cloud P. Paweletz, Laxmi Parida, Gad Getz, Jennifer R. Brown. Mechanisms of primary and acquired resistance to venetoclax in chronic lymphocytic leukemia (CLL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1097.