A patient-derived CRISPR platform reveals selective dependencies in Acute Myeloid Leukemia

Abstract Introduction: CRISPR/Cas9 screening is a powerful tool used to delineate genes critical for AML progression and drug resistance. However, most of the studies have been performed in cell lines and PDX models. While cell lines fail to represent the genetic, epigenetic, and phenotypic heterogeneity of primary leukemic cells, PDX models are limited by variable engraftment efficiency, time-consuming processes, and high costs. To overcome these challenges, we pioneered an integrated approach combining bioinformatics and CRISPR/Cas9 screening to uncover genetic drivers and vulnerabilities in AML using patient-derived primary cells in culture. Insights from our study could inform the development of targeted therapies and improve patient outcomes. Methods Custom CRISPR/Cas9 screen targeting 2,440 genes associated with AML, leukemic stemness, and the druggable genome was performed using mononuclear cells (MNCs) isolated from peripheral blood or bone marrow from 9 AML patients. Briefly, the cells were transduced with custom library and cultured for 24-30 days. After an initial few days in suspension, a subset of MNCs from 5 of 9 patients transitioned to an adherent state 5-7 days post-transduction. Both adherent and suspension cells were collected at different time points, sgRNA was quantified and sequenced using the Illumina NovaSeq X Plus. sgRNA abundance was analyzed using the RRA algorithm of the MAGeCK computational tool and genes with significant negative or positive enrichment in each patient was identified by comparing early versus late time points and adherent versus suspension cell fractions. Results In a subset of patient samples, AML cells transitioned from suspension to an adherent phenotype during long-term culture, a state previously associated with increased chemotherapy resistance due to interactions with endothelial niches. Our CRISPR/Cas9 screen revealed that this adherent cell population harbored distinct genetic dependencies, including consistent negative enrichment of EDNRA and SOD1.These genes were selectively essential in the adherent fraction but not in suspension cells, suggesting context-specific vulnerabilities. The identification of these targets offers new opportunities to therapeutically disrupt chemo resistant AML niches, potentially enhancing the efficacy of existing treatments and reducing relapse risk. EDNRA has been implicated in many cancers and in context of AML, it is a downstream target of HOXA9 and MEIS1. Our results indicate that EDNRA knockout inhibits AML cell proliferation and growth, highlighting its potential as a therapeutic target. Furthermore, EDNRA is overexpressed in AML compared to normal cells, and since FDA approved EDNRA inhibitors such as Macitentan, Bosentan, Ambrisentan, and Clazosentan are already being used to treat pulmonary arterial hypertension, this presents a promising opportunity to evaluate these inhibitors as potential chemotherapy-sensitizing agents for high-risk AML.SOD1 plays a critical role in eliminating toxic radicals generated within biological systems and has been associated with poor outcomes in AML. SOD1 has been identified as a synthetic lethal target in PPM1D-mutant leukemia cells, corroborating our findings. TRAPPC9, SUZ12, and ERG were significant genes with negative enrichment when comparing adherent and suspension fractions across 5 patient samples. These genes have a higher expression in primary AML cells compared to normal controls, implying potential roles in disease progression. SDHA was identified as a significant negatively enriched gene in the suspension cell fraction in 4 of 9 patients. It exhibited elevated expression in AML cells compared to controls, and its deficiency has been linked to the development of drug resistance in AML, emphasizing its role in both drug resistance and disease progression. Conclusion This study demonstrates the establishment of a scalable platform for CRISPR screening in primary AML cells for identification of more conserved vulnerabilities that may be exploited therapeutically, with EDNRA as a potential target. Ongoing work includes validating key targets with the goal of advancing therapeutic strategies. Future studies will expand CRISPR screening to a broader set of patient samples with diverse cytogenetic profiles and incorporate CROP-seq technology to perform single-cell CRISPR screens, providing deeper insights into the molecular mechanisms underlying AML progression and relapse.