There is growing evidence supporting an inherited basis for susceptibility to acute lymphoblastic leukemia (ALL) in children. In particular, we and others reported recurrent germline ETV6 variants linked to ALL risk, which collectively represent a novel leukemia predisposition syndrome. To understand the influence of ETV6 variation on ALL pathogenesis, we comprehensively characterized a cohort of 32 childhood leukemia cases arising from this rare syndrome. Of 34 nonsynonymous germline ETV6 variants in ALL, we identified 22 variants with impaired transcription repressor activity, loss of DNA binding, and altered nuclear localization. Missense variants retained dimerization with WT ETV6 with potentially dominate negative effects. Whole transcriptome and whole genome sequencing of this cohort of leukemia cases revealed a profound influence of germline ETV6 variants on leukemia transcriptional landscape, with distinct ALL subsets invoking unique patterns of somatic cooperating mutations. 70% of ALL cases with damaging germline ETV6 variants exhibited hyperdiploid karyotype with characteristic recurrent mutations in NRAS, KRAS, and PTPN11. In contrast, the remaining 30% cases had a diploid leukemia genome and an exceedingly high frequency of somatic copy number loss of PAX5 and ETV6, with a gene expression pattern that strikingly mirrored that of ALL with somatic ETV6-RUNX1 fusion. Two ETV6 germline variants gave rise to both AML and ALL, with lineage-specific genetic lesions in the leukemia genomes. ETV6 variants compromise its tumor suppressor activity in vitro with specific molecular targets identified by ATAC-seq profiling. ETV6-mediated ALL predisposition exemplifies the intricate interactions between inherited and acquired genomic variations in leukemia pathogenesis.

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ETS Variant 6 (ETV6) encodes an essential transcriptional repressor abundantly expressed in hematopoietic stem and progenitor cells (HSPCs), where it is required for adult hematopoiesis. Heterozygous pathogenic germline ETV6 variants are associated with Thrombocytopenia 5 (T5), a poorly understood genetic condition predisposing to thrombocytopenia and hematologic malignancies. To elucidate how germline ETV6 variants impact the HSPC compartment and contribute to disease, we generated a knock-in mouse model harboring an Etv6R355X loss-of-function variant, which is equivalent to the T5-associated variant ETV6R359X. Under homeostatic conditions, all HSPC subpopulations are present in the bone marrow (BM) of Etv6R355X/+ mice; however, these animals display subtle shifts in the proportions and/or numbers of specific progenitor subtypes. To examine whether the Etv6R355X/+ mutation impacts HSPC function, we carried out serial competitive transplantation and observed that Etv6R355X/+ lineage-sca1+cKit+ (LSK) cells exhibit significantly impaired reconstitution compared to Etv6+/+ LSK cells with near complete failure to repopulate irradiated-recipients by the tertiary transplant. Mechanistic studies incorporating CUT&RUN, ATAC-Seq and Hi-C identify ETV6 binding at inflammatory gene loci, including those within the TNF signaling pathway, in Etv6+/+ HSPCs, mouse BM-progenitor-derived HPC5 cells, and human CD34+ cells. Further, single-cell RNA-Seq of BM cells isolated post-competitive transplantation reveals upregulation of inflammatory genes in Etv6R355X/+ compared to Etv6+/+ progenitors. Corroborating these findings, Etv6R355X/+ HSPCs produce significantly more TNF than Etv6+/+ cells post-transplantation. From these studies, we conclude that ETV6 is required to repress inflammatory gene expression in HSPCs under conditions of hematopoietic stress and this mechanism may be critical to sustain HSPC function.