Background: The Bcl-2 inhibitor venetoclax is approved in combination with hypomethylating agents such as azacitidine for the treatment of newly diagnosed elderly patients with AML; however, most patients eventually relapse, especially those with TP53 mutations who have poor prognosis. Wee1 is a crucial cell cycle checkpoint kinase that regulates the G2/M checkpoint in response to DNA damage, and its inhibition can cause mitotic catastrophe and apoptosis. ZN-d5 and ZN-c3 are highly selective and potent inhibitors of Bcl-2 and Wee1, respectively. We show here that the combination is highly active in preclinical models of AML.

Methods: Cell proliferation in tumor cell lines and AML patient’s samples were measured using CellTiter-Glo® (Promega). Relevant markers were measured by Western blotting. Anti-tumor efficacy was determined in cell line derived subcutaneous xenograft models of acute myeloid leukemia (AML) and in an AML patient-derived systemic xenograft (PDX) model. Flow cytometry was used for analysis of the PDX model to measure human AML blast populations.

Results: The in vitro combination of ZN-d5 and ZN-c3 was tested in AML cell lines and showed synergistic or additive anti-proliferative activity which was independent of BCL-2 sensitivity in some cases. In addition, ZN-d5 in combination with ZN-c3 had a synergistic anti-tumor activity in vivo in cell line-derived AML models, such as MV4-11 and HL-60. Also, the triple combination of ZN-d5 + ZN-c3 + azacitidine in the HL-60 in vivo model resulted in synergistic anti-tumor efficacy and significant tumor growth inhibition compared to single- and double-agent treatments. The ZN-d5 + ZN-c3 combination was also tested in vitro in 29 AML patient-derived samples. Response to ZN-c3 alone was observed in 23/29 samples (IC50 < 450 nM), independent of TP53 mutation status. Response to ZN-d5 alone was observed in 17/29 samples (IC50 <200 nM). The combination resulted in synergy being observed in 12/29 models and had greater anti-proliferative activity than either single agent alone in all samples including those insensitive to Bcl-2 inhibition (IC50 > 10 μM). Combination of ZN-d5 and ZN-c3 was also highly active in an AML patient-derived PDX model, resulting in a 99.5% decrease in human CD45+ blast population in the bone marrow. Finally, the combination of ZN-d5 and ZN-c3 was active in 3/3 ex vivo experiments of patients derived AML samples who had progressed on venetoclax.

Conclusions: These results justify testing the ZN-d5 and ZN-c3 combination or possibly the triple combination including azacitidine in AML patients independently of TP53 status. Due to synergism observed with these combination(s) and activity in samples from patients resistant to venetoclax, activity may be seen in patients with low sensitivity to Bcl-2 inhibitors or even in patients who progressed on venetoclax plus azacitidine.

Citation Format: Hooman Izadi, Tiffany Hoang, Noah Ibrahim, Joseph Pinchman, Brant C. Boren, Jianhui Ma, Petrus R. de Jong, Jiali Li, Kevin D. Bunker, Ahmed A. Samatar, Fernando Doñate. Combination of the BCL-2 inhibitor ZN-d5 with the WEE1 inhibitor ZN-c3 shows additive or synergistic anti-tumor activity in acute myeloid leukemia (AML) models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2591.