Translatome Proteomics Identifies Autophagy As a Resistance Mechanism to on-Target FLT3 Inhibitors in Acute Myeloid Leukemia

Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 are among the most frequent mutational targets in acute myeloid leukemia (AML), drive leukemia progression and confer a poor prognosis. Primary resistance to FLT3 kinase inhibitors (FLT3i) such as quizartinib, crenolanib and gilteritinib is a frequent clinical challenge and often occurs in the absence of identifiable genetic causes, suggesting that adaptive cellular mechanisms mediate primary resistance to on-target FLT3i therapy.

We therefore systematically investigated acute cellular responses to on-target therapy with multiple FLT3i in FLT3-ITD+ AML using recently developed translatome proteomics to measure changes in the nascent proteome in combination with kinase signaling mapping by phosphoproteomics to identify therapy resistance mechanisms to FLT3 inhibitors in vitro. This systems-biology approach showed that the autophagy network escapes global translation repression caused by FLT3i and pinpointed autophagy induction mediated by the AKT-mTORC1-ULK1 axis as the most dominant stress response mechanism to on-target FLT3i therapy. FLT3i induced autophagy in a concentration- and time-dependent manner specifically in FLT3-ITD+ cells in vitro and in primary human AML cells ex vivo. Inhibition of FLT3-ITD signaling led to decreased phosphorylation at AKT S473, decreased phosphorylation at the AKT substrate mTOR S2448, and decreased phosphorylation at the mTORC1 substrate ULK1 S757. CRISPR/Cas9-targeting of TSC2, a negative mTORC1 regulator, ULK1 or ATG3, a core autophagy protein required for autophagosome formation, significantly attenuated or largely abolished autophagy induction by FLT3i. Pharmacological autophagy inhibition (ROC-325, Lys05) or genetic ablation of autophagy (shRNA-mediated knockdown of ATG3, ULK1 or its target Beclin1) increased the sensitivity to FLT3-targeted therapy in cell lines, patient-derived xenografts and primary AML cells ex vivo. In immunodeficient mice xenografted with human FLT3-ITD+ AML cells, oral co-treatment with gilteritinib and autophagy inhibitor ROC-325 synergistically impaired leukemia progression and extended overall survival.

Our findings show the utility of functional translatome proteomics to systematically investigate cell-autonomous therapy resistance mechanisms, identify a molecular mechanism responsible for primary on-target FLT3i treatment resistance and demonstrate the pre-clinical efficacy of a rational combination treatment strategy targeting both FLT3 and autophagy induction.

Meyer:Parthenon Therapeutics: Current equity holder in private company. Ullrich:Phialogics: Honoraria; BMS: Honoraria.