Genome-Wide CRISPR/Cas9 Knockout Screen Reveals Novel Gilteritinib Combinations Targeting Oxidative Phosphorylation and De-Novo Purine Biosynthesis Pathways for FLT3-ITD⁺ Acute Myeloid Leukemia

Background: Acute Myeloid Leukemia (AML) is a malignancy of immature leukocytes of the myeloid lineage with a heterogeneous genetic landscape. 30% of AML patients will present with an internal tandem duplication of the fms-like tyrosine kinase 3 (FLT3). Targeted therapies have been developed against the FLT3-ITD; however, a subset of patients are poor responders or will develop refractory disease. FLT3-ITD⁺ patients have shown improved disease status when treated with the FLT3 inhibitor gilteritinib in combination with the BH3-mimetic venetoclax. To explore additional gilteritinib combination strategies, we employed a genome-wide CRISPR/Cas9 Knockout Screen with subsequent genetic and pharmacologic validation.

Methods: Genome-wide CRISPR/Cas9 knockout screening was performed in the FLT3-ITD^+/- MOLM-13 cell line with analysis executed using the MAGeCK pipeline. Differentially abundant sgRNAs between gilteritinib and DMSO group were submitted for Ingenuity Pathway Analysis. Validation of screen hits was accomplished using genetic validation (shRNA knockdown) and pharmacologic inhibition in the context of MTS assays, Annexin V/Propidium Iodide flow cytometry, colony forming assays, and western blot. Transcriptional and metabolic perturbations associated with genetic knockdown and gilteritinib treatment were interrogated utilizing RNA sequencing and liquid chromatography/mass spectrometry analyses respectively. In-vivo survival studies were performed using MOLM-13 Luciferase xenografts into NCG mice with subsequent randomization into vehicle, monotherapy, or gilteritinib combination treatment arms.

Results: The CRISPR/Cas9 screen revealed 1191 genes whose sgRNAs were depleted by at least 1.5-fold between the gilteritinib and DMSO groups. Pathway analysis revealed these genes to be enriched in pathways related to oxidative phosphorylation, mitochondrial dysfunction, and purine biosynthesis. Among the significantly depleted genes from our screen for which pharmacologic inhibitors were available we selected CDK9, DHODH, and PRMT5 for further analysis. Genetic knockdown or pharmacological inhibition of these genes in combination with gilteritinib treatment show synergistic reductions in viability, Ki-67 staining, and colony formation in both cell lines and FLT3-ITD⁺ patient samples. By using RNA-seq and metabolomics, we showed that the knockdown of CDK9, PRMT5, or DHODH plus gilteritinib treatment each had in common the ability to cooperatively shut down oxidative phosphorylation and purine biosynthesis. Supplementation of purine nucleosides rescued the synergistic effect of gilteritinib and CDK9, PRMT5, or DHODH inhibition. Lastly, as a proof of concept, we examined in vivo combinatorial approaches utilizing the 1/2/5/9 CDK inhibitor, dinaciclib, which has been used in conjunction with venetoclax in clinical trials for relapsed/refractory AML, DHODH inhibitor, brequinar, and a novel PRMT5 inhibitor, PRT808. Our human FLT3-ITD AML xenograft model showed a promising survival benefit and tumor burden reduction provided by combination therapies of dinaciclib/gilteritinib, brequinar/gilteritinib, and PRT808/gilteritinib over monotherapies, suggesting that the three combinations may improve the outcome of AML patients with FLT3 mutations.

Conclusion: Our data showcase a novel convergence of oxidative phosphorylation and nucleotide synthesis on the FLT3 axis in FLT3-ITD⁺ AML. We believe that our findings provide the basis for maximizing therapeutic impact of FLT3-ITD inhibitors and a rationale for a clinical trial of these novel combinations.

Scherle:Prelude Therapeutics: Current Employment. Wang:Prelude Therapeutics: Current Employment. Vaddi:Prelude Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Baiocchi:Viracta Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; eLife (Journal): Other: Editorial board; CODIAK Biosciences: Research Funding; Atara Biotherapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sampath:Newwave: Honoraria. Blachly:Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; MingSight Pharmaceuticals: Research Funding; KITE Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees; INNATE Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees.