Abstract
Cytarabine, daunorubicin, and etoposide (ADE) remain the standard backbone chemotherapy in pediatric AML despite newly approved drugs. However, many pediatric AML develop treatment resistance and relapse, resulting in poor survival. To overcome treatment resistance, it is crucial to understand the molecular mechanisms behind ADE treatment outcomes. In this study, we performed a genome-wide CRISPR/Cas9 synthetic-lethal screening to identify ADE response modulators and integrated CRISPR-screen results with gene expression and clinical outcomes in pediatric AML treated with ADE. K-562 cells were transduced with the Brunello CRISPR/Cas9 library and puromycin selected before the transduced cell population was treated with DMSO, IC30 of cytarabine, daunorubicin, or etoposide (37uM, 0.1uM, and 0.85uM, respectively) for four days at 400x coverage. Genomic DNA was collected for all samples, sgRNAs were PCR amplified, and then sequenced on Illumina Hiseq 4000. The abundance of sgRNA was analyzed using MAGeCK-MLE to obtain normalized beta score per gene to identify treatment-related (treatment minus control beta values [diff-beta]) genes. Genes with diff-beta <2 standard deviations (SD) were associated with drug resistance (negative selection), and genes with diff-beta >2 SD were associated with increased drug sensitivity (positive selection). For translational impacts of CRISPR screen results with outcomes in ADE-treated AML patients, leukemic cell gene expression was evaluated for association with clinical endpoints such as event-free survival (EFS), overall survival (OS), minimal residual disease after induction 1 (MRD1) and relapsed/refractory disease (RR) in pediatric AML from two clinical cohorts, which multisite AML02 trial (n=163) and publicly available TARGET database (only patients treated with the ADE arm of AAML0531, n=213) were included.
At the SD cut-off, 888, 114, and 878 are identified as drug sensitive genes, while 306, 1301, and 412 are resistant genes to cytarabine, daunorubicin, or etoposide, respectively (Figure 1). These significant genes are further tested for associations between expression levels and clinical endpoints in ADE-treated pediatric AML in two independent cohorts. Venn diagram in Figure 2 shows the overlapped genes between CRISPR hits and genes with significant clinical outcome associations in AML02 and TARGET-AAML0531. Of particular interest, five genes are significant CRISPR hits for all three drugs (all are sensitive to cytarabine and etoposide but resistant to daunorubicin), as well as for the clinical outcome associations. Among these, GABARAP, GYPA, and SUCLG2 are associated with better outcomes, while C11orf58 and MATR3 are associated with detrimental outcomes. Furthermore, Figure 2 also shows a heat map that illustrates the association between expression levels and clinical outcomes of these five genes with the available EFS, OS, MRD1, and RR (with and without risk adjustment) in two cohorts. Notably, GYPA (CD235a) is an erythroid cell membrane protein, and high leukemic cell GYPA expression is shown to be associated with favorable EFS and OS in AML02 (EFS HR=0.48, p=0.004 with risk-adjusted HR=0.45 p=0.002; OS HR=0.46, p=0.012 with risk-adjusted HR=0.43, p=0.006) and EFS in TARGET-AAML0531 (HR= 0.67, p=0.0038 with risk-adjusted HR=0.72 p=0.08). Another notable gene with detrimental outcome associations is MATR3, which has been shown to have a deleterious effect on cell fate transitions, chromatin structure, and cell differentiation (PMID: 34716321). Overall, our study coupled the results of a genome-wide CRISPR/Cas9 screen with leukemic cell gene expression and clinical outcomes identified clinically and functionally significant ADE genes. C11orf58, MATR3, GABARAP, GYPA, and SUCLG2 are potential novel biomarkers associated with prognosis in the context of pediatric AML treated with ADE. Our future work is focused on identifying the most optimal combination of induction regimens based on the transcriptomic signature of patients’ leukemic cells.
Acknowledgments: NIH-R01-CA132946 (Lamba and Pounds), NIH-T32-HG008958 (UF PARADIGM Training Program), University of Florida Opportunity Seed Grant (Lamba), American Lebanese Syrian Associated Charities (ALSAC). We thank Dr. Roya Rafiee for CRISPR/Cas9 drug screen contribution; Drs. Campana, Coustan-Smith, and Susana Raimondi for MRD and cytogenetic data.
Disclosures
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
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