Comprehensive CRISPR-enabled functional genomics profiling to identify targetable cancer genes in primary acute myeloid leukemia (AML).
Authors: Corbel Stephane , Hodel Karl , Tran Kathleen , Meyers Maci , Tong Jing , Baltay Michele , Kilfeather Peter , Li Yali , Schmedt Christian , Sampath Srinath , Sampath Srihari 
Published: June 2023

3088 Background: Acute myeloid leukemia (AML) is a life-threatening hematopoietic malignancy affecting both older and younger patient populations. Despite the approval of multiple new therapeutics during the past decade, including new targeted agents, treatment outcomes for AML remain poor, with < 10% 5-year survival in patients over 60 years of age, and < 50% 5-year survival in younger patients. Also, comprehensive genomic profiling (CGP) for common molecular lesions (e.g., FLT3 mutation) incompletely stratifies the patient population, indicating that a more comprehensive approach to personalized medicine could be of significant clinical benefit. To this end we developed Fx Heme, an in vitro functional genomics platform using CRISPR/Cas9 technology in primary cancer patient samples. Previously, we have shown it to stratify clinical response in AML patients treated with a combination of the tyrosine kinase inhibitor sorafenib plus chemotherapy (e.g., GCLAM). Here we profile an expanded cohort to systematically characterize the AML functional genomics landscape and identify potential new targets for AML drug development. Methods: Unsorted primary tumor cells from patient samples were transduced with lentivirus harboring Cas9 enzyme and an sgRNA library which was designed to inhibit expression of genes encoding the targets of FDA-approved targeted oncology drugs, as well as internal negative references. Transduced cells were harvested at multiple timepoints, and sgRNA distribution was assessed by amplicon sequencing of DNA barcodes. Changes in barcode abundance were quantitated and aggregated for each gene to generate gene-level statistics, enabling identification of gene dependencies. Results: Comprehensive CRISPR/Cas9 functional genomics profiling was carried out retrospectively on pre-therapy samples from 59 AML patients. There were no false positives identified among negative controls. Functional dependencies on multiple genes were observed, with approximately half of patients (n=31; 52.5%) showing at least one gene dependency. We observed on average 6.7 dependencies per positive sample, with 5 of them revealing more than 10 dependencies. In addition to the targets of drugs approved to treat AML (FLT3, 10.2%; IDH1/2, 3.4%; SMO, 3.4%), a diversity of targetable genes for new therapeutic approaches to AML were identified across multiple known oncogenic signaling pathways. The prevalence of these recurrent gene dependencies ranged from 3.3% to 23.7% in our AML cohort. Conclusions: In summary, comprehensive functional genomic profiling using Fx Heme provides an effective platform to identify therapeutic opportunities in AML based on dependencies identified for targets of drugs not currently used to treat AML. These insights are now being utilized to develop additional treatment options and more effective personalization of treatment for AML.