Inhibition of Unc-51-like Kinase 1 (ULK1) with Novel Small Molecular Inhibitor MRT68921 Preferentially Induces Apoptosis and Autophagy in FLT3-ITD- Mutated Acute Myeloid Leukemia

Abstract

In normal karyotype acute myeloid leukemia (AML), FLT3-ITD mutation is associated with dismal prognosis with early relapse even after allogeneic stem cell transplantation. Unfortunately, to date small-molecule inhibitors of FLT3 have resulted in only partial and transient clinical responses with residual leukemic blasts acquiring resistance to FLT3 inhibitors. Therefore, elucidation of novel molecular targets should be necessary for effective eradication of FLT3-ITD AML cells. Evidences are accumulating on the functional roles of autophagy in the initiation and maintenance of AML as well as the development of drug resistance. Unc-51-like kinase 1 (ULK1) is a conserved serine-threonine kinase that plays a central role in the initiation of autophagy. Our group demonstrated that ULK1 is potentially involved in the development of resistance of AML leukemia stem cells to BET inhibitor, JQ1. The fact that ULK1 is the only conserved serine/threonine kinase in the autophagy cascade makes it a very attractive target for therapeutic development. However, the role of Ulk1 in FLT3-ITD AML remains unclear. In this study, we observed that MRT68921, a potent inhibitor of both ULK1 and ULK2, induced apoptotic cell death in FLT3-ITD-mutated AML cell lines (MV4-11, Molm13, U937/FLT3-ITD-muated) in a dose-dependent manner. However, apoptosis-inducing effect of MRT68921 was significantly lower in FLT3-WT AML (HL-60, U937). Cell death was accompanied with cleavage of caspases and PARP, which were partially blocked with caspase inhibitor z-VAD-fmk, indicating the caspase-dependent mechanism exists. MRT68921 treatment led to a notable decrease in the levels of phosphorylated (p) ATG13 (Ser 318) as well as total ULK1 and p-ULK1 (Ser 555). Interestingly, MRT68921 induced LC3-II lipidation, autophagosome, and GFP/LC3 punta formation, indicating autophagy was paradoxically activated in FLT3-ITD-mutated AML cells. AMPKa phosphorylation (T712) was increased in MTR68921-responsive cells. In contrast, autophagy induction was negligible to modest in FLT3-WT AML cells. Treatment of FLT3-ITD cells with autophagy inhibitors, 3-MA, bafilomycin A1, and hydroxychloroquine, markedly enhanced the MRT68921-induced apoptosis, strongly suggesting that prosurvival autophagy activation occurred with MRT68921 in FLT3-ITD cells. Reduction in the levels of total FLT3 and p-FLT3 protein were observed concurrently with downregulation of p-STAT5 in FLT3-ITD cells. Endoplasmic reticulum stress-associated proteins, p-PERK and p-eIF2a were also downregulated with MRT68921 in FLT3-ITD cells. Taken together, targeting the ULK1 pathway could be an effective therapeutic strategy for combating FLT3-ITD AML. Inhibition of prosurvival autophagy pathway could enhance the anti-leukemia effects of MRT68921. DisclosuresNo relevant conflicts of interest to declare.