Paper Details

ABSTRACT

Background: Genome-wide association studies (GWAS) have identified numerous germline variants associated with pediatric B-cell acute lymphoblastic leukemia (B-ALL), yet most lie in noncoding DNA without a defined mechanism linking sequence to cellular phenotype. Three common B-ALL risk variants (rs75777619, rs28665337, and rs4617118; odds ratios, 1.17 to 1.47) reside within 8q24, a gene-sparse domain approximately 1.4 Mb from MYC. We hypothesized that individual risk nucleotides alter transcription factor binding within endogenous enhancer chromatin and tune MYC expression and leukemic cell behavior.

Significance: Linking a noncoding risk nucleotide to a molecular mechanism, target gene, and cellular consequence within native chromatin remains a central challenge in cancer genetics. Even modest changes in MYC output can reshape lymphoid proliferation, differentiation, and susceptibility to transformation. Defining this sequence-to-phenotype path would show how an inherited allele contributes to B-ALL and provides a framework for dissecting other noncoding leukemia risk loci within distal enhancers.

Results: To nominate a functional variant and its effector, we performed allele-aware position weight matrix (PWM) scanning across the 8q24 risk alleles and integrated motif changes with transcription factor expression across normal B-lineage development and B-ALL cell lines. This analysis prioritized rs75777619 A>G and RUNX1, a master hematopoietic regulator, as the leading variant-factor pair, with RUNX1 predicted to gain binding at the risk (G) allele. Analysis of published B-ALL H3K27ac HiChIP places this element within the MYC regulatory architecture. Across analyzed B-ALL cell lines, contact strength between the rs75777619 loci and the MYC promoter correlated with risk-allele dosage, consistent with modulation of a MYC enhancer-promoter network.

To test this mechanism in native chromatin, we generated isogenic rs75777619 clones in two molecularly distinct B-ALL models (NALM6 and RS4;11). In risk-homozygous NALM6 cells (G/G), we generated G/G, A/G, and A/A clones representing an array of allele dosages. In risk-heterozygous RS4;11 cells (A/G), we generated corrected reference homozygous A/A and risk heterozygous A/G clones. CUT&RUN identified a discrete RUNX1 peak centered on rs75777619 in G/G cells, which was markedly reduced in isogenic corrected A/A cells. Similarly, corrections from G/G to A/A also reduced H3K27ac signal, linking the risk nucleotide to RUNX1 occupancy and local enhancer acetylation.

Given the strong interaction between the rs75777619 locus and the MYC promoter seen in B-ALL H3K27ac HiChIP we then tested MYC output and leukemic cell growth. By RT-qPCR, correction of the G/G risk alleles to A/A reference alleles in NALM6 cells resulted in a 76.3% reduction in MYC expression (P<0.0001, ordinary one-way ANOVA with multiple comparison adjustment), whereas the edited G/G PAM-disrupting control and A/G isogenic clones retained parental-level expression. Similarly, correction of the RS4;11 A/G risk allele cells to A/A reduced MYC expression by 10.9% (P=0.016, unpaired t-test). To assess risk-allele impact on leukemic fitness, we assayed ATP-dependent luminescent signal 72 hours after each clone was plated at equal density as an inference of cell growth. Correction of the NALM-6 G/G risk allele cells to A/A reference alleles resulted in a 31% decrease in growth (P=0.0040, ordinary one-way ANOVA with multiple comparison adjustment), and correction of the RS4;11 A/G risk allele cells to A/A reference alleles similarly reduced growth by 22.9% (P=0.018, unpaired t-test).

Conclusions: Together, these data support rs75777619 as a functional B-ALL susceptibility allele and a sequence-to-phenotype mechanism in which the risk G allele creates a cryptic RUNX1 site within a pre-existing MYC regulatory domain, increases local enhancer acetylation, sustains MYC transcription, and supports leukemic cell growth. More broadly, this work demonstrates how a single inherited nucleotide within a distal enhancer can translate noncoding genetic risk into altered oncogene output and leukemia-relevant cellular behavior, providing a framework for identifying cryptic transcription factor sites introduced by risk alleles in the noncoding leukemia genome.