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2662 A Genome-Wide CRISPR-Cas9 Screen Reveals GATOR1 Complex Is a Critical Regulator of Glucocorticoid Sensitivity in B-Cell Precursor Acute Lymphoblastic Leukemia

Program: Oral and Poster Abstracts
Session: 605. Molecular Pharmacology and Drug Resistance: Lymphoid Neoplasms: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Lymphoid Leukemias, ALL, Non-Biological therapies, Chemotherapy, Diseases, Therapies, Lymphoid Malignancies, Biological Processes, molecular biology
Sunday, December 11, 2022, 6:00 PM-8:00 PM

Hiroshi Imanaga1*, Yuichiro Semba, MD1*, Kensuke Sasaki, MD, PhD1*, Kiyoko Miyata, PhD2*, Takuji Yamauchi, MD, PhD1*, Tatsuya Terasaki, MD1*, Fumihiko Nakao, MD1*, Shigeki Hirabayashi, MD, PhD2*, Jumpei Nogami, PhD1*, Koichi Akashi, MD, PhD1 and Takahiro Maeda, MD, PhD2

1Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
2Division of Precision Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan

Glucocorticoids (GCs) are essential components of B-cell precursor acute lymphoblastic leukemia (B-ALL) therapy. Upon GC binding, GC receptor (GR) translocates to the nucleus and activates GR target genes. While GCs reportedly induce cell death via apoptosis and/or cell cycle arrest, their molecular mechanisms remain largely unknown.

To identify molecules/pathways governing GC sensitivity in B-ALL cells, we performed genome-wide CRISPR-Cas9 dropout screens in the presence or absence of dexamethasone (Dex) using two human B-ALL lines (NALM-6 and MUTZ-5). To do so, we employed a baboon envelope pseudotyped lentiviral vector system, which enabled highly efficient transduction of human B cells (Sasaki et al. Blood 2022).

We identified a total of 90 and 46 genes, whose single-guide RNAs (sgRNAs) were significantly enriched upon Dex treatment in NALM-6 and MUTZ-5, respectively. A total of 15 genes were identified in both lines, and among them included those previously implicated in GR resistance, such as NR3C1 and TBL1XR1. Strikingly, sgRNAs targeting DEPDC5, NPRL2 and NPRL3, each encoding components of the GATOR1 complex, a negative regulator of mTORC1, were significantly enriched in the presence of Dex in both lines.

To validate these findings, we depleted each gene via lentivirus-based E2-Crimson-tagged sgRNA and assessed effects on cell growth over 11 days with or without Dex. Cells expressing sgRNA targeting either DEPDC5, NPRL2 or NPRL3, but not those transduced with empty vector, demonstrated a proliferative advantage relative to non-transduced cells in the presence of Dex. Importantly, Dex treatment significantly reduced phospho-P70S6K levels, a readout of the mTOR pathway activation, in GATOR1-intact cells, but not in NPRL2 or DEPDC5 knockout cells, suggesting that Dex suppresses mTORC1-mediated growth signals via the GATOR1 complex.

We next asked whether Dex/GATOR1-mediated mTORC1 suppression is mediated by transcriptional activity of GR. To do so, we established a NALM-6 line deficient in endogenous GR in which a GR mutant lacking DNA binding capacity was exogenously expressed. Dex treatment did not reduce phospho-P70S6K levels in these cells, suggesting that the Dex/GATOR1-mediated mTORC1 suppression seen in GATOR1-intact cells primarily depends on transcriptional capacity of GR.

Given that loss-of-function mutations of DEPDC5 were reported in therapy-refractory B-ALL cases and that GATOR1 complex reportedly functions in amino acid metabolisms, our findings suggest a potential link between GC sensitivity and amino acid metabolism in B-ALL cells.

Disclosures: Hirabayashi: Revorf Co.,ltd.: Current equity holder in private company.

*signifies non-member of ASH