Abstract 371: Synthetic lethal and resistance pathways in midostaurin-treated AML
Authors: Brinton Lindsey T. , Williams Katie , Orwick Shelley , Beaver Larry , Canfield Daniel , Cempre Casey , Skinner Jordan , Wasmuth Ronni , Hassan Quais , Byrd John C. , Lapalombella Rosa , Blachly James S. 
Published: August 2019

Abstract
Introduction: Mutations in fms-like tyrosine kinase 3 (FLT3) are common genomic abnormalities in adult acute myeloid leukemia (AML). When FLT3 is present as a dominant internal tandem duplication (ITD) AML clone, it is associated with poor treatment outcome and short overall survival (OS). A phase 3 study with the multi-kinase (including FLT3) inhibitor midostaurin improved OS of FLT3+ AML prompting widespread use, yet little is understood about resistance pathways and synergistic pharmacologic targets. We conducted a genome-wide CRISPR-KO (loss-of-function) screen in a FLT3+ AML cell line treated with midostaurin, validated results in vitro and in vivo, and identified distinct findings relative to limited CRISPR screening previously reported (Cancer Res 77:4402, 2017) with a very selective FLT3-ITD inhibitor (quizartinib).
Methods: CRISPR pooled screens (GeCKOv2 library, &gt;500 cells/sgRNA) of midostaurin versus vehicle were analyzed via MAGeCK. Gene essentiality was derived from guide abundance after 7-day selection of transduced cells compared to plasmid levels. In vitro validation included shRNA knockdown and/or inhibition followed by proliferation and clonogenic assays in cell lines and primary AML blasts. NSG mice (n=7/group) engrafted with MOLM-13-luciferase cells were treated with vehicle, single agents, or combination and evaluated for disease burden (IVIS) and survival (Kaplan-Meier).
Results: We first confirmed our screening approach correlated well with previously reported essential genes (Cell Rep 17:1193, 2016; Cancer Cell 33:386, 2017;Genome Biol 16:281, 2015). We then identified &gt;500 genes (FDR&lt;2%) with significant depletion (synergistic) or enrichment (resistance) after midostaurin treatment. Top synergistic genes included BCL2, MEF2D, CCNG1, and RPS14, with no overlap to reported quizartinib results. We selected BCL2 for immediate validation based on demonstrated efficacy targeting this gene in AML. Indeed, shRNA knockdown of BCL2 sensitized FLT3+ AML cell lines to midostaurin. Similarly, combination treatment of FLT3+ cell lines and AML patient tumor samples with midostaurin and venetoclax displayed synergy at clinically relevant doses. In a xenograft model, combination therapy decreased disease burden and improved survival over single agents supporting synergy of these agents in vivo.
Conclusion: FLT3 inhibitors represent the first targeted therapeutic to impact AML survival and herein we use genome-wide CRIPSR screening to demonstrate that resistance mechanisms of broad-spectrum kinase inhibitors are distinct from those previously reported with specific inhibitors (quizartinib). Furthermore, we validate the select target BCL2 genetically and with the therapeutic venetoclax demonstrating in vitro and in vivo synergy, thereby justifying pursuit of this combination in future clinical trials. Ongoing efforts include validation of other targets that will be presented.
Citation Format: Lindsey T. Brinton, Katie Williams, Shelley Orwick, Larry Beaver, Daniel Canfield, Casey Cempre, Jordan Skinner, Ronni Wasmuth, Quais Hassan, John C. Byrd, Rosa Lapalombella, James S. Blachly. Synthetic lethal and resistance pathways in midostaurin-treated AML [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 371.