George S. Vassiliou

The N7-methylguanosine (m ⁷ G) modification is one of the most prevalent tRNA modifications, mainly catalysed by the METTL1–WDR4 protein complex ^1–4 . The m ⁷ G methyltransferase METTL1 has been linked to oncogenic transformation and the maintenance of several aggressive malignancies; however, the clinical implications of therapeutically targeting a tRNA-modifying enzyme have not been established. Here we present the development and characterization of STM9005, a highly selective and potent first-in-class tRNA methyltransferase inhibitor of METTL1. Treatment of numerous cancer cell models (including leukaemia, melanoma, pancreatic carcinoma, oesophageal cancer and small cell lung carcinoma) with STM9005 blocks cell growth, causes differentiation changes and dysregulates cell cycle progression. These cellular effects were accompanied by a selective decrease in m ⁷ G on a subset of tRNAs and a reduction in their expression levels, which impacted the translation of oncogenic mRNAs. We show that pharmacological inhibition of METTL1 in vivo impairs tumour growth and significantly prolongs survival in multiple mouse cancer models of different tissue origin. Collectively, our results provide a strong proof of concept that targeting of tRNA methylation is a promising avenue of anti-cancer therapy and illustrate that catalytic inhibition of METTL1 represents a therapeutic strategy against a number of aggressive malignancies.

Summary: Haematological malignancies are rare in primary care and are hard to predict. Existing prediction models often require a specific working diagnosis and are based on extensive clinical information, access to which may be limited at initial work‐up in primary care. We aimed to develop a tool for estimating overall risk of haematological malignancy in primary care patients using simple data inputs. Including all full blood cell counts (FBCs) measured from 2000 to 2016 in 856 403 adult primary care patients in Eastern Denmark, we used machine learning to develop CBC‐HEMA, a risk assessment tool using FBCs, age, sex and C‐reactive protein measures to stratify ‘any haematological malignancy’ risk within 6 months, 1 year and 3 years with area under the curve (AUC) of 0.81, 0.78 and 0.71 respectively. Predictions of chronic lymphoid leukaemia and myeloproliferative disease were excellent, while CBC‐HEMA was unable to accurately predict non‐Hodgkin lymphoma and plasma cell dyscrasia. External validation was conducted in 123 015 primary care patients from Western Denmark with similar results and in 418 410 individuals from the background population cohort UK Biobank with inferior performance showing unfitness in screening healthy populations. The CBC‐HEMA https://shiny.sund.ku.dk/CopLab/blood‐cancer‐risk‐prediction/ may support risk assessment in primary care but cannot replace a clinical evaluation.

Background: Clonal hematopoiesis (CH) is associated with increased risks of diverse cardiovascular diseases, hematologic malignancies and mortality, yet no preventive therapies are approved. As emerging data implicate lipid pathways in CH pathogenesis, we investigated the association of statin use and genetically proxied inhibition of HMG-CoA reductase (HMGCR) with CH risk, and validated findings using primary peripheral blood mononuclear cells (PBMCs). Methods: We performed an observational analysis of 416,118 UK Biobank participants of European ancestry using multivariable logistic regression to compare CH prevalence among statin users and nonusers. Mendelian randomization (MR) analyses evaluated the causal association of genetically proxied lowering of low-density lipoprotein cholesterol (LDL-C) with risk of CH using two instruments; (i) the lead HMGCR variant (rs12916) which proxied LDL-C lowering by statins, and, (ii) 303 genome-wide LDL-C-lowering variants representing polygenic mechanisms. Summary statistics were obtained from the Global Lipid Genetics Consortium genome-wide association study (N = 842,634). Experimentally, primary PBMCs from a DNMT3A ^R882 hotspot mutation carrier were cultured in methylcellulose with pravastatin or vehicle control to evaluate colony-forming dynamics. Results: Among 416,118 individuals, 20,488 had CH, including 11,550 with single DNMT3A -mutant and 4,375 with single TET2 -mutant CH. Pre-recruitment statin users had reduced odds of DNMT3A -mutant CH (OR=0.93; 95% CI:0.88-0.98; P =0.009), driven primarily by associations with DNMT3A ^R882 –mutant (OR=0.78; 95% CI:0.66-0.92; P =0.003), but not TET2 -mutant CH (OR=1.05; 95% CI:0.97-1.14; P =0.20). Similarly, genetically predicted HMG-CoA-reductase inhibition equivalent to a 1 SD reduction in circulating LDL-C levels was associated with lower odds of DNMT3A -mutant CH (OR=0.66; 95% CI:0.45-0.95; P =0.03) but not TET2 -mutant CH (OR=1.34; 95% CI:0.76-2.36; P = 0.31). By contrast, polygenic estimation of LDL-C lowering was not associated with DNMT3A -mutant CH (OR=1.05; 95% CI:0.97-1.14; P =0.20), suggesting protective effects were independent of LDL-C lowering per se. Genetically predicted HMG-CoA reductase inhibition had wide effects on blood cell counts and indices, suggesting effects on bone marrow cell dynamics. In vitro , pravastatin selectively suppressed colony formation of primary human DNMT3A ^R882 –mutant relative to wild-type cells (P=0.031). Conclusions: Statin therapy and genetically predicted lifelong inhibition of HMG-CoA reductase were significantly associated with reduced risk of DNMT3A -mutant CH, likely via LDL-C-independent mechanisms, which may be specific to DNMT3A -mutant CH. This provides a strong rationale for prospective trials evaluating the effect of statins on risk of developing DNMT3A -mutant CH, subsequent clonal expansion, and associated clinical sequelae. Clinical Perspectives: What is new?: This study provides the first evidence that statins may protect against DNMT3A -mutant clonal hematopoiesis (CH), with convergent findings of observational analysis of statin users and genetic epidemiology utilizing Mendelian randomization to proxy lifelong HMG-CoA reductase inhibition. Observational analyses suggest the protective effect may be strongest against DNMT3A ^R882 –mutant CH. We provide evidence that pravastatin selectively suppresses colony formation of primary human DNMT3A ^R882 –mutant primary peripheral blood mononuclear cells in vitro , suggesting a possible effect on clonal expansion. What are the clinical implications?: Although DNMT3A -mutant CH is associated with diverse cardiovascular diseases, hematological malignancies and mortality, no preventive treatments are currently established; this study provides a viable avenue for future interventional studies. These data establish a strong rationale for prospective trials evaluating whether statins prevent the development or expansion of DNMT3A -mutant CH and its cardiovascular consequences. Given that statins are widely used with established safety profiles, affordable and have proven cardiovascular benefit, such trials are readily implementable.

Acute myeloid leukemia (AML) is an aggressive leukemia with high rates of chemoresistance and relapse. Patients with AML undergoing induction chemotherapy often have delayed erythropoietic recovery and febrile neutropenia. Infection is a leading cause of mortality in this population. There is an unmet need to improve disease-specific outcomes in patients with AML undergoing cytotoxic chemotherapy. AML, at diagnosis, is characterized by increased levels of circulating iron due to erythroid block and cell death, which is further aggravated upon intensive chemotherapy. We hypothesized that iron, particularly toxic non–transferrin-bound iron (NTBI), can be redistributed away from AML cells and bacteria into nonmalignant transferrin receptor (CD71)–expressing cells by administering exogenous iron-free apotransferrin (apoTF). Using mouse models of AML, we show that mice treated with human apoTF had decreased NTBI and increased bone marrow erythropoiesis and B cell responses. ApoTF treatment resulted in normalization of bone marrow blood vessels and reduction of lipid peroxidation in endothelial cells. Crucially, apoTF combined with chemotherapy resulted in a reduction of AML cells and in improved survival, which was dependent on adaptive immunity. We established a murine model of Escherichia coli sepsis in leukemic mice receiving chemotherapy. We show that apoTF administration increased the survival of E. coli –infected mice. Mechanistically, apoTF treatment decreased the levels of circulating C-C motif chemokine ligand 2 (CCL2) and interleukin-6 through reduced expression of CCL2 in lipopolysaccharide-polarized macrophages. Our results demonstrate an overall benefit of iron redistribution induced by transferrin in combination with cytotoxic chemotherapy in AML.

The terms clonal monocytosis of undetermined significance (CMUS) and clonal cytopenia and monocytosis of undetermined significance (CCMUS) were introduced by the International Consensus Classification of Myeloid Neoplasms to describe cases of clonal hematopoiesis (CH) and concurrent monocytosis that did not meet the diagnostic criteria of chronic myelomonocytic leukemia. To date, their practical relevance as clinicopathological entities at a population level has not been assessed. Here, we assess the prevalence, significance, and natural history of CMUS and CCMUS among 431 531 UK Biobank participants through analysis of clinical, genomic, and health outcome data. We find that CMUS with an absolute monocytosis and CCMUS are high-risk entities strongly associated with incident myeloid neoplasia (MN), cardiovascular disease, and renal disease. Noting the overall higher monocyte counts in men and the low rate of progression of DNMT3A-CMUS, we reveal that amending the definition of CMUS/CCMUS to incorporate sex-specific monocyte thresholds and the exclusion of isolated DNMT3A mutations from the definition significantly strengthens the association with incident MN. Finally, given their association with poor outcomes, we develop MoSAIC, a machine-learning classifier, to infer the presence of SRSF2 mutations (associated with high MN risk) among individuals with monocytosis, based on complete blood count indices alone. We corroborate our findings in an independent cohort of 625 328 Danish primary care patients. Our findings underscore the clinical relevance of CMUS and CCMUS as distinct high-risk states within the spectrum of CH and establish an evidence base to refine their diagnostic definition.

Clonal hematopoiesis (CH), the clonal expansion of a hematopoietic stem cell and its progeny driven by somatic mutations, has been associated with inferior survival outcomes among recipients of autologous stem cell transplants (ASCT). Leukocyte telomere length (LTL) has a complex but well-documented interaction with CH, but the impact of this interaction on stem cell transplantation has not been adequately examined. We measured LTL in graft cell DNA from 452 patients undergoing ASCT for myeloma, for whom targeted DNA sequencing for CH driver gene mutations was available. We interrogated clinical and longitudinal large-scale laboratory data for these patients to understand the impact of graft LTL on progression-free survival (PFS) and overall survival after transplantation, as well as blood count indices and their trajectories. In multivariate analyses, longer LTL was associated with increased PFS among patients without CH. However, this protective association was not seen in patients with CH. We also report that among patients with CH, longer LTL was associated with an increased red cell distribution width before myeloablative chemotherapy and after ASCT. Collectively, these data reveal hitherto undescribed interactions between LTL, CH, and ASCT outcomes.

Clonal haematopoiesis (CH) becomes ubiquitous as humans age. The role of somatic driver mutations in its development has been studied widely, but little is known about CH without identified genetic drivers, also known as “CH with unknown drivers” (CH-UD). A fundamental unresolved question is whether CH-UD is driven by undiscovered somatic genetic drivers or by other cell-heritable traits. Here, to investigate this, we develop a new machine learning classifier to improve CH-UD detection from whole-genome sequencing data. After excluding 77,885 individuals with previously documented driver CH or mosaic chromosomal alterations (mCA), we applied our classifier to 407,512 UK Biobank participants and identified 26,963 (6.6%) with CH-UD. A genome-wide association study (GWAS) of common germline variants identified 31 polymorphic loci associated with predisposition to CH-UD. Of these, 25 were associated with other forms of CH at genome-wide significance. Linkage Disequilibrium Score Regression analyses revealed an unexpectedly high genetic correlation (r _g =0.794) between CH-UD and non- DNMT3A driver CH, indicative of a remarkable overlap between the genetic aetiologies of the two phenomena. Analysis of 2,941 plasma protein measurements in 47,757 individuals revealed that TCL1A was the most significantly elevated plasma protein in CH-UD, mirroring the finding that the TCL1A locus was in the top two most significant associations of CH-UD GWAS and TET2 -CH and ASXL1 -CH GWAS, the two most common forms of non-DNMT3A-CH. Furthermore, TCL1A plasma levels rose steadily with age even in those without detectable CH, particularly among carriers of the common TCL1A risk variant (rs2887399-G), potentially via stochastic promoter demethylation as described in TET2 -CH and ASXL1 -CH. Phenome-wide association analysis of 13,225 binary and 1,682 quantitative traits revealed that, similarly to non- DNMT3A -CH, CH-UD was significantly associated with several malignant (haematological and solid organ) and non-malignant (including cardiovascular and renal) diseases. Our findings reveal striking genetic and phenotypic similarities between CH-UD and non- DNMT3A driver CH, including a strong dependence on TCL1A, a protein recently found to inhibit DNA methylation. Collectively, these observations propose that CH-UD develops through selection acting on ageing-associated epigenetic changes that mirror those of non- DNMT3A -CH, but without the need for somatic genetic drivers.