Abstract 4763: Genome-wide CRISPR screening identifies TSC1 as a regulator of sorafenib resistance in acute myeloid leukemia
Authors: Damnernsawad Alisa , Nechiporuk Tamilla , Kurtz Steve E. , Horton Wesley R. , Nikolova Olga , McWeeney Shannon K. , Tyner Jeffrey W. 
Published: August 2019

Abstract
Hematologic malignancies represent the fifth most common type of cancer and fourth most common cause of cancer-related deaths. These malignancies are characterized by a clonal expansion of hematopoietic progenitor cells that proliferate in the bone marrow, peripheral blood, lymph nodes and other organs. Genetic analyses of these diseases have led to the identification of specific genetic lesions that drive leukemogenesis, which has subsequently led to the development of targeted drugs. 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. AML is known for its molecular and biological heterogeneity, and a variety of genetic lesions have been implicated in the disease. FMS-like tyrosine kinase 3 (FLT3) mutations including FLT3 internal tandem duplication (ITD) or point mutations in the tyrosine kinase domain (TKD) 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 sorafenib to standard treatment prolonged AML patient survival with or without FMS- like tyrosine kinase 3 (FLT3) mutations, although relapse caused by drug-resistance has limited its usefulness. Understanding the mechanism of resistance to targeted drugs, therefore, is necessary to improve treatment regimens for AML patients. We aim to elucidate resistance mechanisms to sorafenib in AML cells using genome-wide CRISPR screening. Two genome-scale human CRISPR knockout (KO) libraries were used to identify genes whose loss-of-function contribute to lower sensitivity to sorafenib in the MOLM13 AML cells. We verified 10 genes from the top hits showing consistency between the two CRISPR libraries, one of which was Tuberous Sclerosis 1 (TSC1). To validate these findings, TSC1 KO cells were generated using lentiCRISPRv2 system and single sgRNA sequence. Drug sensitivity assays confirmed increase in sorafenib resistance of TSC1 KO cells compared to parental cells or cells harboring non-targeting sgRNA. In addition to the sorafenib resistant phenotype, TSC1 KO cells were resistant to a panel of FLT3 inhibitors, quizartinib, crenolanib, gilteritinib, and UNC2025A. Moreover, RNA sequencing results from 271 AML patient peripheral blood or bone marrow samples revealed that lower RNA expression of Tuberous Sclerosis 1 (TSC1) correlated with lower sensitivity to sorafenib. TSC2 was also observed as a hit gene in both CRISPR libraries and lower TSC2 RNA levels also correlated with lower sensitivity to sorafenib, emphasizing TSC1/2 as an important pathway in sorafenib resistance.
Citation Format: Alisa Damnernsawad, Tamilla Nechiporuk, Steve E. Kurtz, Wesley R. Horton, Olga Nikolova, Shannon K. McWeeney, Jeffrey W. Tyner. Genome-wide CRISPR screening identifies TSC1 as a regulator of sorafenib resistance in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4763.