APG-2449, a Novel Focal Adhesion Kinase (FAK) Inhibitor, Exhibits Antileukemic Activity and Enhances Lisaftoclax (APG-2575)-Induced Apoptosis in Acute Myeloid Leukemia (AML)

Introduction

High FAK expression is associated with increased blast proliferation and poor prognosis in AML. Preclinical and clinical studies have demonstrated that selective targeting of BCL-2 has broad antitumor activities against hematologic malignancies, including AML, myelodysplastic syndrome, and chronic lymphocytic leukemia. In AML, resistance to apoptosis induced by BCL-2 inhibition (BCL-2i) is partially mediated by preexisting and therapy-induced upregulation of MCL-1 and BCL-xL. Considering the crosstalk between FAK and MCL-1/BCL-xL pathways, we hypothesized that FAK inhibition could downregulate expression of MCL-1 and BCL-xL, and thus enhance activity of a BCL-2 inhibitor. Lisaftoclax (APG-2575) and APG-2449 are novel, investigational BCL-2 and multikinase (ALK/FAK/ROS1) inhibitors, respectively. Both APG-2449 and lisaftoclax display clinical activity and tolerable safety profiles in patients with hematologic and solid malignancies. Here, we evaluated the effects of combined lisaftoclax and APG-2449 in preclinical models of AML.

Methods

AML cell lines and mouse xenograft models were employed. The AML cells were treated with APG-2449 alone or combined with lisaftoclax, and cell viability was measured by CellTiter-Glo® luminescent cell viability assays. Cellular apoptosis was evaluated by annexin V/propidium iodide staining and flow cytometry. Protein expression of FAK- and apoptosis-related signaling pathways was measured by western blot. The antitumor activity of APG-2449 and lisaftoclax alone or in combination were assessed in the AML cell-derived xenografts (CDX) in mice.

Results

APG-2449 showed similar antiproliferative effects as a single agent in both BCL-2 inhibitor (BCL-2i)-sensitive (MOLM-13, MV4-11, ML-2 and HL-60) and -insensitive AML cell lines (OCI-AML-3). Half-maximal inhibitory concentration (IC₅₀) values ranged from 1 to 6 µM. When combined with lisaftoclax, APG-2449 synergistically reduced cell viability and enhanced apoptosis not only in BCL-2i-sensitive (MV4-11, MOLM-13, and ML-2) but also BCL-2i-insensitive (OCI-AML-3) AML cells in vitro. According to findings from western blots and cell viability assay results, APG-2449 likely antagonized lisaftoclax-induced upregulation of MCL-1 and BCL-xL, and enhanced the antiproliferative activity of lisaftoclax in AML cell lines. This effect was confirmed by silencing FAK with short interfering RNA (siRNA). Silencing FAK with siRNA enhanced the antiproliferative effect of lisaftoclax in the AML cells. APG-2449, when used alone or combined with lisaftoclax, decreased activation of FAK and its downstream effectors, including STAT3/5, MAPK, and AKT, which are known to regulate MCL-1 and BCL-xL expression. In addition, the mice bearing MV4-11 or OCI-AML-3 tumors were treated with APG-2449 alone or combined with lisaftoclax for 3 or 2 weeks, respectively. The results showed that APG-2449 synergistically enhanced the antitumor effect of lisaftoclax in both MV4-11 (BCL-2i sensitive) and OCI-AML-3 (BCL-2i- insensitive) AML CDX xenograft models.

Conclusions

Our data suggest that FAK inhibition by APG-2449 augments the antileukemic activity of lisaftoclax in AML. This synergistic effect can be partially attributed to suppression of MCL-1/BCL-xL by FAK downstream pathways. This novel combination showed antileukemic activity in both BCL-2 inhibitor-sensitive and -insensitive AML cells in vitro and in xenograft models. Overall, these promising results provide a novel approach in the clinical development of lisaftoclax for treatment of patients with AML.

Yu:Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment; Ascentage Pharma Group International: Current holder of stock options in a privately-held company. Liang:Ascentage Pharma Group International: Current holder of stock options in a privately-held company; Ascentage Pharma Group Inc.: Current Employment. Yao:Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment; Ascentage Pharma Group International: Current holder of stock options in a privately-held company. Ji:Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment; Ascentage Pharma Group International: Current holder of stock options in a privately-held company. Yang:Ascentage Pharma Group International: Current holder of stock options in a privately-held company, Other: Leadership and fiduciary officer roles; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Other: Leadership and fiduciary officer roles, Patents & Royalties; Ascentage Pharma Group Inc.: Current Employment, Other: Leadership and fiduciary officer roles, Patents & Royalties. Zhai:Ascentage Pharma Group International: Current holder of stock options in a privately-held company; Guangzhou Healthquest Pharma Co. Ltd.: Current Employment, Other: Leadership role, Patents & Royalties; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Other: Leadership role, Patents & Royalties; Ascentage Pharma Group Inc.: Current Employment, Other: Leadership role, Patents & Royalties.