Functional characterization of a multi-cancer risk locus on chromosome band 2q33.1 near CASP8

Reviewer #1 (Public review):

Summary:

In this manuscript, Kong et al. conduct a systematic analysis of the multi-cancer risk locus at 2q33. The authors start with a careful analysis of co-localization between the melanoma risk SNPs and several other cancers and conclude that a subset of credible causal SNPs is shared among different cancers, including breast cancer. Next, they define a starting list of 27 SNPs as potential credible causal SNPs and analysis of TADs (topologically associating domains) to zoom in on CASP8 and FLA CC1 as potential target genes. They then systematically rule out coding and splicing variants in the set and focus on a smaller set of three SNPs constituting a melanocyte enhancer element. Using a combination of mass spectrometry, reporter assays, and electrophoretic mobility shift assays, the authors define a role for transcription factors IRF2 and E4F1 in the regulatory network driving risk at the locus.

This work represents a high-quality tour de force, using multiple tools, to zoom in on a gene expression regulatory network associated with risk for multiple cancers. It provides a detailed framework for analyses of other multi-cancer risk loci. Limitations of the work, which is rather a current limitation of the field, is the lack of a model to study how the identified network of regulatory elements, transcription factors, and target genes mechanistically drive risk at the organismal level. Advances such as those described in this manuscript contribute significantly to our knowledge of how common risk variants drive risk.

Reviewer #2 (Public review):

Summary:

Kong et al. investigate a well-validated risk locus at chromosome band 2q33.1 adjacent to CASP8, a ubiquitously expressed and central initiator caspase in the extrinsic apoptotic pathway. Importantly, this region is a multi-cancer risk locus harboring multiple highly correlated risk alleles that are confounded by linkage. In addition to protein coding and splicing variants, further evaluation of eQTL and TWAS results for the locus suggests a cis-regulatory effect is present for CASP8 and nearby FLACC1. The authors prioritize variants using orthogonal statistical fine-mapping approaches and triage top candidates for functional assays. Luciferase reporter assays demonstrated convincing allele-specific regulatory activity of rs3769823 variant as well as suggestive evidence for rs3769821 and rs59308963. These three variants lie in close proximity within a melanocyte regulatory element marked by overlapping promoter and enhancer chromatin state signals. The authors employ a haplotype reporter assay, which shows that the combination of risk alleles in the forward direction has additive effects compared to the protective haplotype. These effects are also cell type specific among melanocytes, melanoma, and breast cancer cell states. Utilizing electron mobility shift assays, the authors convincingly show augmented nuclear protein binding of the rs3769823-A risk allele, and mass spectrometry of allele-specific rs3769823 binding proteins revealed specific activity of E4F1 and IRF2, whose motif score is strengthened by the risk allele. Correlation of these transcription factors' expression with CASP8 expression suggested repressive effects of E4F1 and activating effects of IRF2, which were confirmed in siRNA assays across multiple cell types. These data provide important evidence towards the molecular mechanisms governing disease susceptibility at the 2q33.1 risk locus and nominate s3769823 as a causal variant through cis-regulatory activity by E4F1 and IRF2.

Strengths:

Major strengths of the work include the authors' employment of orthogonal fine-mapping approaches and functional assays in multiple cell types. These help to fortify a novel molecular mechanism of rs3769823 and also work together to propose a complicated multi-variant and cell-type-specific effect at this locus, which is worth future investigation.

Weaknesses:

The rs3769823 variant is a protein-coding variant for CASP8. While the authors conclude that this is likely neutral to CASP8 function, their evidence is suggestive at best and does not close the door on a protein-coding function for this variant.

Similarly, another variant, rs10804111, is associated with alternative splicing of CASP8. The authors do well to include the potent rs10804111 sQTL effect on CASP8 and further confirm it by a minigene assay. However, its exclusion from the fine-mapping results may be due to a potent bias towards active chromatin marks. Therefore, rs10804111 still requires further investigation.
Some attention is given to FLACC1, whose promoter may be in contact with multiple variants. However, little is known about FLACC1 function, and the authors don't provide meaningful supporting data to illustrate whether FLACC1 is relevant in the context of melanocyte, melanoma, or other cancer types that share this risk locus (breast, prostate). Showing the absolute expression levels in the eQTL analysis would be helpful towards this.

Phenotypic assays interrogating the rs3769823-E4F1-IRF2 relevance to melanocyte biology and melanoma pathogenesis are not included.

Finally, the segmented figure organization negatively impacts the readability of the paper.