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

BCL-X_L and BCL-2 (BCL-X_L/2 ) are key antiapoptotic proteins and validated cancer targets. 753B is a novel BCL-X_L/2 proteolysis targeting chimera (PROTAC) that targets both BCL-X_L and BCL-2 to the Von Hippel-Lindau (VHL) E3 ligase, leading to BCL-X_L/2 ubiquitination and degradation (Lv et al. Nat. Comm. 2021). It exhibits an improved potency against BCL-X_L/2 leukemia cells while reduced toxicity to platelets compared to the dual BCL-X_L/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-X_L selective PROTAC) (Khan et.al Nat. Med, 2019) and 753B. 753B induced time and dose-dependent BCL-X_L 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-X_L/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-X_L 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-X_L/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-X_L 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-X_L 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-X_L/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-X_L may eliminate senescent AML cells surviving Ara-C chemotherapy, supporting the utility of future combination studies in leukemia.

Colla:Amgen: Research Funding. Andreeff:Reata: Current holder of stock options in a privately-held company; Brooklyn ITX: Research Funding; Oxford Biomedical UK: Research Funding; Glycomimetics: Consultancy; Aptose: Consultancy, Membership on an entity's Board of Directors or advisory committees; NCI: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Current holder of stock options in a privately-held company; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; Kintor Pharmaceutical: Research Funding; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo Inc.: Consultancy, Research Funding; Chimerix: Current holder of stock options in a privately-held company; Syndax: Consultancy, Research Funding; Pinot Bio: Research Funding; AstraZeneca: Research Funding; German Research Council: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Medicxi: Consultancy; Senti Bio: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding. Daver:Agios, Celgene, SOBI and STAR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kartos and Jazz Pharmaceuticals: Other: Data monitoring committee member; Karyopham Therapeutics and Newave Pharmaceutical: Research Funding; Astellas, AbbVie, Genentech, Daiichi-Sankyo, Novartis, Jazz, Amgen, Servier, Karyopharm, Trovagene, Trillium, Syndax, Gilead, Pfizer, Bristol Myers Squibb, Kite, Actinium, Arog, Immunogen, Arcellx, and Shattuck: Consultancy, Other: Advisory Role; Astellas, AbbVie, Genentech, Daiichi-Sankyo, Gilead, Immunogen, Pfizer, Bristol Myers Squibb, Trovagene, Servier, Novimmune, Incyte, Hanmi, Fate, Amgen, Kite, Novartis, Astex, KAHR, Shattuck, Sobi, Glycomimetics, Trillium: Research Funding. Jain:AbbVie: Consultancy, Honoraria, Other: Travel Support, Research Funding; Mingsight: Research Funding; Janssen Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support; Loxo Oncology: Research Funding; Servier Pharmaceuticals LLC: Research Funding; Newave: Research Funding; Dialectic Therapeutics: Research Funding; Medisix: Research Funding; ADC Therapeutics: Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel Support, Research Funding; Novalgen: Research Funding; TransThera Sciences: Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Other: Travel Support, Research Funding; Cellectis: Honoraria, Research Funding; Beigene: Honoraria; Pharmacyclics, Inc.: Consultancy, Honoraria, Other: Travel Support, Research Funding; Pfizer: Research Funding; CareDx: Honoraria; Genentech, Inc.: Consultancy, Honoraria, Other: Travel Support, Research Funding; Aprea Therapeutics: Research Funding; MEI Pharma: Honoraria; TG Therapeutics: Honoraria; Takeda: Research Funding; BMS: Consultancy, Honoraria, Other: Travel Support, Research Funding; Precision Biosciences: Consultancy, Honoraria, Other: Travel Support, Research Funding; Ipsen: Honoraria; Fate Therapeutics: Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Cellectis: Honoraria, Research Funding; Incyte Corporation: Research Funding. Pemmaraju:stemline: Consultancy; abbvie: Consultancy; immunogen: Consultancy; mustangbio: Research Funding; incyte: Consultancy; novartis: Research Funding; pacylex: Consultancy, Research Funding; samus: Research Funding; daiichi sankyo: Research Funding; cellectis: Research Funding; cellularity: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.