A Genome-Wide CRISPR Screen Identifies Genes Critical for Resistance to FLT3 Inhibitor AC220

Abstract

Acute myeloid leukemia (AML) is a malignant hematopoietic disease and the most common type of acute leukemia in adults. The mechanisms underlying drug resistance in AML are poorly understood. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are the most common molecular abnormality in AML. Quizartinib (AC220) is a potent and selective second-generation inhibitor of FLT3. It is in clinical trials for the treatment of relapsed or refractory FLT3-ITD-positive and -negative AML patients and as maintenance therapy. To understand the mechanisms of drug resistance to AC220, we undertook an unbiased approach with a novel CRISPR pooled library to screen new genes whose loss of function confers resistance to AC220. We identified SPRY3, an intracellular inhibitor of FGF signaling, and GSK3, a canonical Wnt signaling antagonist, and demonstrated that re-activation of downstream FGF/Ras/ERK and Wnt signaling as major mechanisms of resistance to AC220. We confirmed these findings in primary AML patient samples. Expression of SPRY3 and GSK3A was dramatically reduced in AC220-resistant AML samples, and SPRY3-deleted primary AML cells were resistant to AC220. Intriguingly, expression of SPRY3 was greatly reduced in GSK3 knockout AML cells, which positioned SPRY3 downstream of GSK3 in the resistance pathway. Taken together, our study identified novel genes whose loss of function conferred resistance to a selective FLT3 inhibitor, providing new insight into signaling pathways that contribute to acquired resistance in AML.

<p>Profile of Quizartinib for the Treatment of Adult Patients with Relapsed/Refractory FLT3-ITD-Positive Acute Myeloid Leukemia: Evidence to Date</p>

“…In a separate but similar finding, a genome-wide CRISPR screen identified that loss of SPRY3, an intracellular inhibitor of FGF signaling, and GSK3, a canonical Wnt signaling antagonist, can also induce quizartinib resistance. 63 Deletion of these genes in the FLT3-ITD AML cell line MV4-11, conferred quizartinib resistance as evidenced by increased cell viability and increased downstream MAPK and Wnt signaling. 63 These findings were further confirmed in quizartinib-resistant AML patient samples.…”

“…63 Deletion of these genes in the FLT3-ITD AML cell line MV4-11, conferred quizartinib resistance as evidenced by increased cell viability and increased downstream MAPK and Wnt signaling. 63 These findings were further confirmed in quizartinib-resistant AML patient samples.…”

Phenotypic screening using large‑scale genomic libraries to identify drug targets for the treatment of cancer (Review)

“…Previously, studies using CRISPR-Cas9 libraries were published. Most of these studies used the same type of genome-wide library, GeCKO CRISPR Library version 1 or 2, comprising of >120,000 sgRNAs targeting nearly the entire genome (53)(54)(55)(56). For example, Sustic et al identified the endoplasmic reticulum to nucleus signaling 1 (ERN1)-JNK-JUN pathway as a potential target for improving the anti-cancer effects of MET inhibitors in KRAS-mutated colon cancer (56).…”

CRISPR Screens Identify Essential Cell Growth Mediators in BRAF-inhibitor Resistant Melanoma

“…We next investigated whether CDK6 upregulation might be associated with clinical 291 resistance in some cases. To facilitate this, we generated a 10-gene CDK6 expression 292 "signature" (Table S6) Several genome-wide CRISPR pooled screens have uncovered mediators of drug 325 resistance [53,54]. In this study, we used CRISPR screens to systematically characterize 326 resistance to BRAF inhibitor PLX-4720 in melanoma.…”