Genome-Wide CRISPR Screening Identifies MAPK and Mtorc Pathways As Regulators of Sorafenib Resistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a fast progressing blood malignancy with impaired differentiation and proliferation of myeloid precursors. It is one of the most common leukemias in adults and is known for its molecular and biological heterogeneity, with a variety of genetic lesions implicated in the disease. Among these variants, internal tandem duplication (ITD) or point mutations in the tyrosine kinase domain (TKD) of FLT3 tyrosine kinase are found in around 30% of AML patients.

Sorafenib, a multi-kinase inhibitor that targets FLT3, RAF, VEGFR, FGFR, KIT and RET, is approved for use in hepatocarcinoma, renal cell carcinoma, and thyroid carcinoma treatments. Addition of different FLT3 inhibitors such as sorafenib to standard-of-care chemotherapy treatment prolongs AML patient survival with or without FLT3 mutations, although relapse caused by drug resistance remains a clinical challenge. Understanding the mechanisms of resistance to FLT3-targeted drugs, therefore, is necessary to improve treatment options and patient outcomes in AML. We aimed to elucidate resistance mechanisms to sorafenib by subjecting MOLM13 AML cells to genome-wide CRISPR screening to identify genes whose loss-of-function contributes to reduced drug sensitivity.

Using Mageck along with an internally developed tiering system for screen hit prioritization, we identified negative regulators of MAPK as well as mTOR pathways as main players in sorafenib resistance. We validated prioritized hit genes using individual sgRNAs to generate single gene deficient cell models for LZTR1, NF1, TSC1 or TSC2. Drug sensitivity assays confirmed an increase in sorafenib resistance in these knockout cells. LZTR1-, TSC1- or TSC2-deficient cells also exhibited reduced sensitivity to a panel of additional FLT3 inhibitors. RNA sequencing results from 271 AML patient peripheral blood or bone marrow samples revealed a correlation between sorafenib sensitivity and lower expression of LZTR1, NF1, TSC1, and TSC2. MOLM13 cell lines resistant to crenolanib, quizartinib, and sorafenib were independently generated by incremental increase in concentration of each drug in cell culture media. Similarly, western blot analysis demonstrated up-regulation of MAPK and/or mTORC1 activity in these resistant cell lines. In addition, these cells were sensitive to MEK inhibitors, and the combination of FLT3 + MEK inhibitors showed synergistic efficacy over single agents in both resistant and parental cells. Taken together, our work identifies the contribution of the MAPK and PI3K/mTOR pathways to FLT3 inhibitor resistance in AML and suggests the combination of FLT3 + MEK inhibitors may be effective for AML patients with FLT3 mutations and those with resistance to FLT3 inhibitors.