Olverembatinib (HQP1351) in Combination with Lisaftoclax Overcomes Venetoclax Resistance in Preclinical Model of Acute Myeloid Leukemia (AML)
Authors: Xiong Yan , Liang Zhiyan , Min Ping , Wu Bingxing , Yu Huidan , Yang Dajun , Zhai Yifan 
Published: December 2024

Introduction
The combination of BCL-2 inhibitor venetoclax with hypomethylating agents (HMAs) or low-dose cytarabine is widely used in treatment of patients with newly diagnosed AML who are elderly or ineligible for intensive chemotherapy. However, resistance to venetoclax remains a major clinical challenge, and efforts are being undertaken to understand mechanisms of resistance and explore alternative therapeutic strategies to overcome resistance. Olverembatinib is a novel multikinase inhibitor that targets a broad spectrum of kinases, such as FMS-like tyrosine kinase 3 (FLT3), KIT, PDGFR, SRC family kinases, PI3K, and FGFR, which are associated with leukemogenesis and AML development. Previously olverembatinib has been shown to synergize with investigational BCL-2 inhibitor lisaftoclax (APG-275) to potentiate cellular apoptosis in FLT3-ITD mutant AML by downregulating MCL-1. Here, we report that olverembatinib combined with lisaftoclax overcomes venetoclax resistance in various preclinical AML models by regulating signaling pathways involved in cell proliferation, survival, and apoptosis.
Methods
The primary venetoclax-resistant AML cell line OCI-AML-3 was obtained from Cobioer Biosciences Co., Ltd. (Nanjing, China). MOLM13 and MV-4-11 venetoclax-resistant (VEN-RES) FLT3-ITD mutant AML cell lines were developed internally by intermittently exposing the cells with increasing concentrations of venetoclax, and resistance was determined by IC50. Cell-based antiproliferation assays to evaluate the activities of olverembatinib or lisaftoclax alone and in combination were conducted by the CellTiter-Glo® luminescent cell viability assay. Drug-induced apoptosis was measured by flow cytometry. Western blot analyses were used to elucidate potential mechanisms of action.
Results
The synergistic activities of olverembatinib and lisaftoclax (compared to single agents) on antiproliferation were observed in all 3 VEN-RES AML cells in vitro post 72 hours of treatments, including FLT3-ITD mutant MOLM13-VEN-RES, MV-4-11-VEN-RES, and OCI-AML-3 (primary resistant). After a 24-hour treatment, the percentages of apoptotic cells induced by DMSO (vehicle), olverembatinib (10 nM), lisaftoclax (3 μM), and the combination were 5.16%, 34.04%, 5.99%, and 58.40%, respectively, in MOLM-13-VEN-RES cells, and 7.79%, 18.36%, 9.04%, and 68.73%, respectively, in MV-4-11- VEN-RES cells. In OCI-AML-3 cells, percentages of apoptotic cells induced after 24-hour treatment with DMSO, olverembatinib (0.5 μM), lisaftoclax (0.5 μM), and the combination were 3.82%, 8.50%, 8.82%, and 75.13%, respectively. Compared to either single agent, olverembatinib in combination with lisaftoclax significantly increased apoptosis in all 3 VEN-RES AML cells (p &lt; 0.0001).
Mechanistically, western blot results showed that olverembatinib plus lisaftoclax synergistically inhibited expression and phosphorylation of FLT3 and proteins in downstream signaling pathways such as STAT, AKT, and ERK, and their phosphorylation in MOLM13-VEN-RES and MV-4-11-VEN-RES cell lines. The levels of antiapoptotic proteins BCL-2, BCL-xL, and MCL-1 were suppressed by the combination as well. Notably, significant upregulation of phosphorylated FLT-3, AKT and MCL-1 observed in VEN-RES cells (relative to parental cells) may be associated with mechanisms of resistance to BCL-2 inhibition. The combination also significantly increased expression levels of proapoptotic proteins BAX, BAK, BID, PUMA, and Noxa in a dose-dependent manner, resulting in remarkable augmentation of cleaved caspase-3 and poly(ADP-ribose) polymerase 1 (PARP). Similar patterns were observed in the primary VEN-RES OCI-AML3 cell line. Ongoing studies in venetoclax-resistant animal models are further validating the synergistic effects of olverembatinib and lisaftoclax in vivo.
Conclusion
The results demonstrate that olverembatinib in combination with lisaftoclax overcame venetoclax resistance in preclinical AML models. This combination downregulated signaling pathways shown to mediate venetoclax resistance. These promising findings suggest that this strategy may provide a new therapeutic option for patients with venetoclax-resistant AML, an urgent unmet medical need.