Dual BCL-XL/2 Protac 753B Not Only Potently Induces Apoptosis in Venetoclax-Resistant Primary AML Cells but Also Effectively Eliminates Chemotherapy-Induced Senescent AML Cells (2022)

BCL-XL and BCL-2 (BCL-XL/2 ) are key antiapoptotic proteins and validated cancer targets. 753B is a novel BCL-XL/2 proteolysis targeting chimera (PROTAC) that targets both BCL-XL and BCL-2 to the Von Hippel-Lindau (VHL) E3 ligase, leading to BCL-XL/2 ubiquitination and degradation (Lv et al. Nat. Comm. 2021). It exhibits an improved potency against BCL-XL/2 leukemia cells while reduced toxicity to platelets compared to the dual BCL-XL/2 inhibitor navitoclax or ABT263 in vitro. Here we examined the potential therapeutic efficacy of 753B against venetoclax-resistant primary AML cells and chemotherapy-induced senescent AML cells in vitro and in vivo. We first evaluated the sensitivity of 24 genetically diverse leukemia cell lines (17 AML, 5 T-ALL, and 2 AML secondary to myeloproliferative neoplasms (MPN-AML) cell lines) to ABT263, DT2216 (a BCL-XL selective PROTAC) (Khan et.al Nat. Med, 2019) and 753B. 753B induced time and dose-dependent BCL-XL degradation in all lines with DC50 (the concentrations with 50% protein degradation) ranging from 0.01 μM to 0.54 μM. BCL-2 was degraded in all except Loucy cells, with DC50 ranging from 0.02 μM to >1 μM. 753B caused dose-dependent reduction of viability in AML cell lines by CellTiter-Glo (CTG) assay at 24hr (IC50 range 0.01μM - 27.35 μM). 753B was more potent than ABT-263 in 12/17 AML and 3/5 T-ALL lines, with the mean IC50 values of 0.10 μM and 0.48 μM, respectively. The degradation of BCL-XL/2 by 753B was accompanied with MCL-1 upregulation in 10/17 cell lines tested, which was reversely correlated with the IC50. Recent findings indicate that chemotherapy (Ara-C)-induced senescence is associated with chemoresistance (Duy et al., Cancer Discovery 2021), and BCL-XL was previously reported to be the primary survival factor of senescent cells (Chang et al.,Nat. Med.2016). As previously reported, Ara-C indeed induced cellular senescence (SnCs) in MOLM-14 and Kasumi-1 AML cells, with increased cell size, higher senescence-associated β-galactosidase activity, upregulation of cell cycle regulator proteins (p16, p21, p53), and expression of senescence-associated secretory phenotype (IL-8 and CCR5). 753B cleared chemotherapy-induced SnCs by induction of apoptosis. To explore the senolytic mechanism of 753B, we FACS-sorted senescence-high or -low populations based on C12-FDG positivity (fluorogenic substrate di-β-D-galactopyranoside). C12-FDG high senescent AML population expressed higher levels of BCL-XL/2, priming the cells to 753B-induced apoptosis. We next isolated blasts from 16 primary AML samples and tested their sensitivity to 753B. 753B potently reduced cells viability with median IC50 value of 0.23 mM (range 0.02 - 2.29 mM). Similar to cell lines, 753B was more potent in degrading BCL-XL and inducing apoptosis than DT2216 in primary AML cells. 753B showed potency comparable to ABT-263 in all tested AML samples, including seven venetoclax-resistant samples (median IC50, 753B 0.22 mM; ABT-263 0.13 mM). 753B degraded both BCL-XL and BCL-2 in primary myeloblasts in association with the induction of apoptosis. BCL-2 degradation was observed in 4 of 6 samples tested. To investigate the antileukemia activity of 753B in vivo, we developed a patient-derived xenograft (PDX) model using NSG mice injected with AML PDX #8550 (harboring FLT3-ITD, DNMT3A, IDH1, KIT, and NPM1 mutations). Mice were randomized into 2 groups to receive either vehicle or 753B (5 mg/kg intraperitoneally every 4 days) for 3 weeks after leukemia engraftment was confirmed (day 33). Mice tolerated 753B therapy well with no significant changes in body weight and no significant platelet toxicity ascertained by blood counts. 753B as a single agent reduced the circulating leukemia cell burden, serially measured by flow cytometry, and extended median survival time from 61 days to 71 days (P = 0.0023)(Fig. 1A). In summary, the novel BCL-XL/2 PROTAC 753B potently reduced cell viability in leukemia cell lines with diverse genetic background, in primary AML cells including venetoclax-resistant AML blasts, and extended survival of mice harboring human AML PDX in vivo without hematologic toxicity. Our data indicate that co-targeting of BCL-2 and BCL-XL may eliminate senescent AML cells surviving Ara-C chemotherapy, supporting the utility of future combination studies in leukemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal