Inherited Blood Cancer Predisposition through Altered Transcription Elongation

Despite considerable advances in defining the somatic driver mutations underlying myeloid malignancies, including the myeloproliferative neoplasms (MPNs), a significant heritable component for these diseases remains poorly understood. While common genetic variant association studies have been valuable, they fail to explain the majority of heritable variation. We reasoned that rare variant association studies could provide a valuable complementary approach to identify additional inherited risk factors. We therefore utilized exome sequencing data from 166,953 UK Biobank participants and performed a gene-based burden analysis for germline genetic variants conferring risk for acquiring a myeloid malignancy. CTR9, which encodes a key component of the PAF1 transcription elongation complex, was among the significant genes identified (SKAT-O p-value = 5.47x10 -7). The deleterious variants in CTR9 collectively exhibit a 9.6 (95%CI = 4.86-19.04) increased odds of acquiring a myeloid malignancy and this risk was largely driven by the MPNs. We replicated this association in an independent cohort of 211 MPN patients using external controls. We could show through structural and biochemical analyses that the identified deleterious variants perturbed assembly of the PAF1 complex but did not display dominant negative activity. Given that increased hematopoietic stem cell (HSC) self-renewal has been shown to predispose to the risk of acquiring MPNs, we sought to define whether CTR9 perturbation could alter HSC self-renewal or function. We achieved predominantly heterozygous loss-of-function in human hematopoietic stem and progenitor cells (HSPCs) by titrating Cas9 ribonucleoprotein delivery with several independent guide RNAs. Partial loss of CTR9 in HSPCs resulted in expansion of phenotypic long-term HSCs (LT-HSCs) and more differentiated short-term HSCs (ST-HSCs). We additionally could show through single cell RNA-sequencing (scRNA-seq) that there was an expansion of molecularly defined HSCs upon partial loss of CTR9. The observed increase in HSCs appeared paradoxical, given that the PAF1 complex has been suggested to be crucial for HSC maintenance. To explore how the observed HSC expansion with CTR9 perturbation may arise, as well as given known interactions between the PAF1 complex and the competing transcriptional super elongation complex (SEC), we examined whether SEC target genes in HSCs, such as mid to posterior HOXA genes, may be activated with partial CTR9 loss. R