Epigenetic Fine Mapping and Functional Dissection of Inherited Non-Coding Variation Impacting the Pharmacogenomics of Acute Lymphoblastic Leukemia Treatment

Acute lymphoblastic leukemia (ALL) is the most prevalent childhood cancer. Despite cure rates exceeding 90%, relapsed ALL is the 5^th most common pediatric cancer. Although chemotherapeutic drug resistance remains the primary cause of treatment failure and relapse, it remains unclear to what extent non-coding inherited DNA sequence variants impact gene regulatory programs that contribute to differences in chemotherapeutic drug resistance and clinical outcome in childhood ALL. To address this knowledge gap we performed epigenetic fine mapping on inherited non-coding DNA sequence variants associated with ex vivo resistance to six antileukemic agents (dexamethasone, prednisolone, l-asparaginase, vincristine, 6-mercaptopurine and 6-thioguanine) in primary ALL cells from pediatric patients and inherited non-coding DNA sequence variants associated with clinical outcome (persistence of minimal residual disease and relapse) in pediatric patient cohorts using chromatin accessibility data from over 130 primary ALL cells, ALL cell lines and ALL xenografts. This analysis fine mapped over 1700 variants (sentinel variants and variants in high linkage disequilibrium; r²>0.8) to cis-regulatory elements in the ALL genome. To comprehensively dissect the functional effects of these fine mapped variants on gene regulatory activity we performed massively parallel reporter assays (MPRAs) in 10 human ALL cell lines of both B- and T-cell lineages. MPRAs utilized 23 DNA barcodes per variant allele and tested variants at bidirectional promoters using both sequence orientations. In total, 98,168 oligonucleotides each containing 175-bp of genomic sequence centered on each variant allele and a unique 10-bp barcode sequence were evaluated for gene regulatory activity. MPRA identified 558 variants with significant and concordant allele-specific activities in three or more ALL cell lines. Expression quantitative trait loci (eQTL) mapping and HiChIP three-dimensional promoter loop profiling identified candidate target genes for a subset of functional variants. We additionally evaluated the functional effects of top ranked variants using luciferase reporter assays and CRISPR/Cas9 genome editing. These variants were associated with the expression of B3GNT5, CACUL1, HERC1, KDM4C and VRK3. Notably, two of these functional variants (rs10411204 and rs1247117) impacted transcription factor (TF) consensus DNA motifs for AP-1 family TFs and PU.1. Collectively, these data suggest that many inherited non-coding variants associated with pharmacogenomic traits in childhood ALL have significant gene regulatory effects. Importantly, the identification of chemotherapeutic drug resistance-associated molecular pathways perturbed by inherited non-coding variants can improve our ability to circumvent drug resistance with novel therapeutic approaches.

Stock:Kite: Honoraria; Servier: Honoraria; Newave Pharmaceuticals: Consultancy; Syndax: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Pluristem: Consultancy, Honoraria; Jazz Pharmaceuticals: Honoraria; Amgen: Honoraria; MorphoSys: Honoraria; Kura Oncology: Honoraria; Agios: Honoraria. Inaba:Servier: Other: Grants and Personal Fees; Amgen: Other: Grants and Personal Fees; Incyte: Other: Grants; JAZZ: Other: Personal Fees; Chugai: Other: Personal Fees. Pui:Novartis: Other: Data monitoring committee; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees. Relling:Servier: Research Funding; BioSkryb: Other: Spouse has equity interest in bioskryb. Evans:Princess Maxima Centre for Childhood Cancer: Membership on an entity's Board of Directors or advisory committees; BioSkryb Genomics Inc: Membership on an entity's Board of Directors or advisory committees. Yang:Takeda Pharmaceutical Company: Research Funding.