Abstract CC09-01: Targeting BCL-XL/BCL-2 by PROTAC 753b effectively eliminates AML cells and enhances efficacy of chemotherapy by targeting senescent cells

BCL-XL and BCL-2 are key anti-apoptotic proteins facilitating cancer survival, senescence and chemoresistance. 753b is a novel BCL-XL/BCL-2 proteolysis targeting chimera (PROTAC) that targets both BCL-XL and BCL-2 to the Von Hippel-Lindau (VHL) E3 ligase, sparing platelets that lack VHL expression. Here we studied the efficacy of 753b and its senolytic activity in leukemia. We first evaluated the sensitivity of genetically diverse 17 leukemia cell lines to BCL-XL/2 dual inhibitor ABT-263, BCL-XL PROTAC DT2216 and BCL-XL/2 PROTAC 753b. 753b induced time-dependent BCL-XL degradation within 6 hours, at concentrations lower than that of DT2216. BCL-XL was degraded by 753b in all 17 cell lines tested; BCL-2 was partially degraded in 12/17 cell lines. 753b treatment (24hrs) caused a dose-dependent reduction of viability in AML cell lines by CellTiter-Glo (CTG) assay, with IC₅₀ ranging from 0.06-27umol/L. Six AML (KG-1, Kasumi-1, OCI-AML3, U937, TF-1; MPN-AML HEL-92) and three T-ALL (Jurkat, PF832, CCRF-CEM) were sensitive to 753b with average IC₅₀ 812nM and to ABT263 (average IC₅₀ 2,232 nM). Recent findings indicate that chemoresistance in AML is associated with chemotherapy (Ara-C)-induced senescence (Duy et al., Cancer Discovery 2021). In vitro, Ara-C induced cellular senescence (SnCs) in AML cell lines Molm14 and Kasumi-1, as manifested by increased cell size, induction of senescence-associated β-galactosidase activity, upregulation of cycle regulator proteins (p16, p21, p53) and mRNA expression of senescence-associated secretory phenotype (IL-6, IL-8, IL-1β).Conversely, 753b combined with Ara-C largely reversed chemotherapy-induced SnCs phenotype and facilitated induction of cell death in senescent AML cells. To explore the senolytic mechanism of 753b, we FACS-sorted viable leukemia cells into senescence-high or -low populations based on C¹²-FDG (fluorogenic substrate di-β-D-galactopyranoside). Consistent with prior data in normal HSC (Chang et al., Nat. Med. 2016), C¹²-FDG high population expressed higher levels of BCL-XL/2, representing therapeutic target for 753b. To study pre-clinical activity of 753b in primary AML, we exposed freshly isolated blasts from 16 AML patients to concentration range of 753b. 753b potently reduced viability of primary AML with median IC₅₀ values of 228 nmol/L, ranging between 18 - 2,291 nmol/L (13/16 with IC₅₀ below 450 nmol/L). Consistent with cell line data, 753b was more potent degrader of BCL-XL and apoptosis inducer compared with DT2216. BCL-2 degradation was seen in 3 out of 5 samples tested. 753b at 1 umol/L induced apoptotic cell death in both, bulk AML blasts and CD34+ stem/progenitor cells (mean specific apoptosis: 63.9% vs 52.4%, n=7). In summary, BCL-XL/2 PROTAC 753b reduced viability by inducing apoptosis in AML cell lines and in the majority of primary AML samples. Targeting BCL-XL effectively eliminated chemotherapy-induced senolytic cells. In vivo experiments in the cell line- and patient-derived xenografts of 753b combined with chemotherapy are ongoing and will be presented. Citation Format: Yannan Jia, Qi Zhang, Weiguo Zhang, Michael Andreeff, Nitin Jain, Helen Ma, Peiyi Zhang, Guangrong Zheng, Daohong Zhou, Marina Konopleva. Targeting BCL-XL/BCL-2 by PROTAC 753b effectively eliminates AML cells and enhances efficacy of chemotherapy by targeting senescent cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr CC09-01.