FLT3-ITD-Specific PROTAC: Enhanced Safety and Increased Synergy with Venetoclax in Acute Myeloid Leukemia

Abstract

Abstract Background Patients with acute myeloid leukemia (AML) carrying Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations often face a poor prognosis, high risk of relapse, and short overall survival. While some FLT3 small molecule inhibitors have been used clinically, challenges such as short efficacy, poor specificity, and resistance persist. Proteolytic targeting chimera (PROTAC), with its lower ligand affinity requirement for target proteins, offers higher targeting capability, and the minimal amount of PROTAC can rapidly degrade proteins. This technology may address issues of poor specificity or resistance seen with small molecule inhibitors. Methods Gilteritinib was used as the ligand for the target protein FLT3-ITD, and different E3 ligase ligands were connected to synthesize several series of PROTAC targeting FLT3-ITD. Results Through in vitro and in vivo screening and structural optimization, PROTAC Z29 linked to VHL E3 ligase ligand, the optimal lead compound, was obtained. Kinase screening showed that the synthesized PROTAC Z29 exhibited better specificity and targeting ability than existing small molecule inhibitors. In vitro, Z29 significantly inhibited the proliferation of FLT3-ITD+ MOLM13 and MV-4-11 cells and induced FLT3 protein degradation through the proteasome pathway at the nanomolar level. In vivo, Z29 significantly inhibited tumor growth rate at a dose of 30 mg/kg in subcutaneous xenograft mice while maintaining the stable body weight of mice. Compared to Gilteritinib, Z29, when used in combination with Venetoclax, demonstrated a higher synergy score in MOLM13 and MV-4-11 cells. We also verified Z29's minimal impact on platelets in a patient-derived xenografts (PDX) model, which may be due to the minimal expression of VHL ligase in platelets. The combination of Z29 and Venetoclax showed better anti-tumor effects and lower platelet toxicity and hepatic toxicity. Conclusion PROTAC technology can enhance drug targeting specificity. The rapid degradation ability and higher targeting specificity of FLT3-ITD PROTAC can mitigate the platelet toxicity of small molecule inhibitors, ensuring safety and efficacy in monotherapy and combination therapy. These findings establish a solid foundation for FLT3-ITD-PROTAC as an effective strategy for the treatment of patients with FLT3-ITD mutation.