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. Remarkably, we observed a significant enrichment for hematopoietic SEC target genes upon CTR9 perturbation in HSCs by gene set enrichment analysis (normalized enrichment score = 3.29, p-value < 0.001). In light of these findings suggesting that SEC activity may be increased with partial CTR9 loss-of-function, as occurs in individuals harboring myeloid malignancy variants, we sought to functionally validate these observations. Using the inhibitors of the SEC, including SR-0813 that targets MLLT3 or with an inhibitor of CDK9, we noted rescue of the CTR9-mediated expansion of phenotypic LT- and ST-HSCs without a significant impact on the bulk HSPC population. To further elucidate underlying mechanisms, we performed immunoprecipitation of PAF1 or SEC component MLLT3 in HSPCs with control or CTR9 editing. While we continued to pull down all PAF1 complex components with PAF1, we also noted pulldown of MLLT3, which increased with CTR9 editing. MLLT3 immunoprecipitation revealed selective pulldowns of PAF1 and CDC73, which also increased with CTR9 editing. These findings show how PAF1 complex components PAF1 and CDC73 interact with and stimulate SEC activity. Our findings reveal how CTR9 usually restricts this activity and constrains transcriptional elongation to limit HSC self-renewal. We functionally validated these findings through selective editing of different PAF1 complex components in HSPCs: we observed reduced HSCs upon editing of PAF1 and CDC73, but increases with editing of other PAF1 complex components. Our findings collectively demonstrate a mechanism by which a previously undefined myeloid malignancy predisposition occurs. We demonstrate that CTR9 loss-of-function stimulates SEC activity and thereby results in HSC expansion to confer risk for acquiring MPNs and other myeloid malignancies. Disclosures Armstrong: Neomorph Inc: Consultancy, Current holder of individual stocks in a privately-held company; Imago Biosciences: Consultancy; Vitae/Allergan Pharma: Consultancy; Cyteir Therapeutics: Consultancy; C4 Therapeutics: Consultancy; OxStem Oncology: Consultancy; Accent Therapeutics: Consultancy; Mana Therapeutics: Consultancy; Janssen: Research Funding; Novartis: Research Funding; Syndax: Research Funding; AstraZeneca: Research Funding. Sankaran: Ensoma: Consultancy; Forma: Consultancy; Cellarity: Consultancy; Novartis: Consultancy; Branch Biosciences: Consultancy.

Transcription recycling assays identify PAF1 as a driver for RNA Pol II recycling

RNA Polymerase II (Pol II) transcriptional recycling is a mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of putative RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. Proof-of-principle experiments identified PAF1 complex components among recycling factors and detected defective transcriptional output from Pol II recycling following PAF1 depletion. Dynamic ChIP-seq confirmed PAF1 silencing triggered defective Pol II recycling in human cells. Prostate tumors exhibited enhanced transcriptional recycling, which was attenuated by antibody-based PAF1 depletion. These findings identify Pol II recycling as a potential target in cancer and demonstrate the applicability of in vitro and cellular transcription assays to characterize Pol II recycling in other disease states.

Biochemical insights into Paf1 complex–induced stimulation of Rad6/Bre1-mediated H2B monoubiquitination

The highly conserved multifunctional polymerase-associated factor 1 (Paf1) complex (PAF1C), composed of five core subunits Paf1, Leo1, Ctr9, Cdc73, and Rtf1, participates in all stages of transcription and is required for the Rad6/Bre1-mediated monoubiquitination of histone H2B (H2Bub). However, the molecular mechanisms underlying the contributions of the PAF1C subunits to H2Bub are not fully understood. Here, we report that Ctr9, acting as a hub, interacts with the carboxyl-terminal acidic tail of Rad6, which is required for PAF1C-induced stimulation of H2Bub. Importantly, we found that the Ras-like domain of Cdc73 has the potential to accelerate ubiquitin discharge from Rad6 and thus facilitates H2Bub, a process that might be conserved from yeast to humans. Moreover, we found that Rtf1 HMD stimulates H2Bub, probably through accelerating ubiquitin discharge from Rad6 alone or in cooperation with Cdc73 and Bre1, and that the Paf1/Leo1 heterodimer in PAF1C specifically recognizes the histone H3 tail of nucleosomal substrates, stimulating H2Bub. Collectively, our biochemical results indicate that intact PAF1C is required to efficiently stimulate Rad6/Bre1-mediated H2Bub.

R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

WEE1 inhibitors have now advanced into clinical studies as monotherapy or in combination with chemoradiotherapy in TP53, RAS, BRAF, and SETD2 mutation carriers across several tumour types, yet mechanisms of resistance are still poorly understood. Here, we further elucidate the mechanisms by which AZD1775, the most potent WEE1 inhibitor, kills cells and reveal additional genetic interactions that can result in resistance, but could be used to optimise its clinical utility. We identified RNA Polymerase II-associated factor 1 (PAF1) complex members, CDC73, CTR9, and PAF1 as major determinants of WEE1-inhibitor sensitivity in isogenic SETD2-positive and negative cell lines. PAF1-knockdown cells resist higher doses of the WEE1 inhibitor, which we show is due to reduced DNA damage induction (γH2AX) and delayed G1 checkpoint activation, ultimately protecting cells against replicative catastrophe. Investigations into the molecular mechanisms responsible for PAF1-mediated resistance identify involvement of R-loops and subsequent activation of the cyclin-dependent kinase inhibitor p21Cip1/Waf1, which in addition to causing prolonged G1 arrest in the following cell cycle, also regulates CDK activity, therefore limiting replication. These results provide evidence that the PAF1 complex and p21 are important regulators of proliferation under increased DNA replication stress and their expression levels might be useful biomarkers to predict clinical response to WEE1 inhibitors and other ribonucleotide reductase inhibitors.