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Despite the inclusion of inherited myeloid malignancies as a separate entity in the World Health Organization Classification, many established predisposing loci continue to lack functional characterization. While germline mutations in the DNA repair factor ERCC excision repair 6 like 2 (ERCC6L2) give rise to bone marrow failure and acute myeloid leukaemia, their consequences on normal haematopoiesis remain unclear. To functionally characterise the dual impact of germline ERCC6L2 loss on human primary haematopoietic stem/progenitor cells (HSPCs) and mesenchymal stromal cells (MSCs), we challenged ERCC6L2-silenced and patient-derived cells ex vivo. Here, we show for the first time that ERCC6L2-deficiency in HSPCs significantly impedes their clonogenic potential and leads to delayed erythroid differentiation. This observation was confirmed by CIBERSORTx RNA-sequencing deconvolution performed on ERCC6L2-silenced erythroid-committed cells, which demonstrated higher proportions of polychromatic erythroblasts and reduced orthochromatic erythroblasts versus controls. In parallel, we demonstrate that the consequences of ERCC6L2-deficiency are not limited to HSPCs, as we observe a striking phenotype in patient-derived and ERCC6L2-silenced MSCs, which exhibit enhanced osteogenesis and suppressed adipogenesis. Altogether, our study introduces a valuable surrogate model to study the impact of inherited myeloid mutations and highlights the importance of accounting for the influence of germline mutations in HSPCs and their microenvironment.

The DExD/H-box RNA helicase DHX34 is a Nonsense-mediated decay (NMD) factor that together with core NMD factors co-regulates NMD targets in nematodes and in vertebrates. Here, we show that DHX34 is also associated with the human spliceosomal catalytic C complex. Mapping of DHX34 endogenous binding sites using Cross-Linking Immunoprecipitation (CLIP) revealed that DHX34 is preferentially associated with pre-mRNAs and locates at exon-intron boundaries. Accordingly, we observed that DHX34 regulates a large number of alternative splicing (AS) events in mammalian cells in culture, establishing a dual role for DHX34 in both NMD and pre-mRNA splicing. We previously showed that germline DHX34 mutations associated to familial Myelodysplasia (MDS)/Acute Myeloid Leukemia (AML) predisposition abrogate its activity in NMD. Interestingly, we observe now that DHX34 regulates the splicing of pre-mRNAs that have been linked to AML/MDS predisposition. This is consistent with silencing experiments in hematopoietic stem/progenitor cells (HSPCs) showing that loss of DHX34 results in differentiation blockade of both erythroid and myeloid lineages, which is a hallmark of AML development. Altogether, these data unveil new cellular functions of DHX34 and suggests that alterations in the levels and/or activity of DHX34 could contribute to human disease.

Modern management of acute myeloid leukaemia (AML) relies on the integration of phenotypic and genetic data to assign classification, establish prognosis, enhance monitoring and guide treatment. The prism through which we can now disperse a patient's leukaemia, interpret and apply our understanding has fundamentally changed since the completion of the first whole-genome sequencing (WGS) of an AML patient in 2008 and where possible, many clinicians would now prefer to delay treatment decisions until the karyotype and genetic status of a new patient is known. The success of global sequencing initiatives such as The Cancer Genome Atlas (TCGA) have brought us significantly closer to cataloguing the full spectrum of coding mutations involved in human malignancy. Indeed, genetic capability has raced ahead of our capacity to apply much of this knowledge into clinical practice and we are in the peculiar position of having routine access to genetic information on an individual patient's leukaemia that cannot be reliably interpreted or utilised. This is a measure of how rapid the progress has been, and this rate of change is likely to continue into the foreseeable future as research intensifies on the non-coding genome and the epigenome, as we scrutinise disease at a single cell level, and as initiatives like Beat AML and the Harmony Alliance progress. In this review, we will examine how interrogation of the coding genome is revolutionising our understanding of AML and improving our ability to underscore differences between paediatric and adult onset, sporadic and inherited forms of disease. We will look at how this knowledge is informing improvements in outcome prediction and the development of novel treatments, bringing us a step closer to personalised therapy for myeloid malignancy.

WFDC2/HE4 encodes a poorly characterised secretory protein that shares structural similarity with multifunctional host defence proteins through possession of two conserved Whey Acidic Protein/four disulphide-core (WFDC) domains. WFDC2 is expressed in multiple epithelia and although its’ function remains unresolved, it is also overexpressed in a number of human cancers and has an established role as a cancer marker. Currently, little is known about the distribution of WFDC2 in the mouse and thus we have systematically analysed the mouse wfdc2 gene, its’ expression and distribution. We have used recombinant WFDC2 for functional studies. Wfdc2 is the most highly expressed family member in the lung and is enriched in the nasopharynx. Wfdc2 is the most highly expressed family member in differentiated epithelial cells isolated from the trachea, nasal passages and middle ear. Wfdc2 consists of 5 exons with exon 3 encoding an unstructured linker region that separates the two WFDC domains. This genomic organisation appears to be restricted to the Muridae and Cricetidae families of rodents. Similar to the situation in man, mouse wfdc2 can be alternatively spliced to yield a number of distinct transcripts that have the potential to generate a repertoire of distinct protein isoforms. We used immunohistochemistry to localise the proteins to tissues of the respiratory tract and head and neck regions. Although the protein was limited to epithelial cells of the respiratory tract and nasal and oral cavities, it was expressed in different cells in different regions suggesting expression is governed by a unique regulatory mechanism. Recombinant WFDC2 did not possess antiproteinase activity against trypsin or elastase and had no clear antimicrobial activity.

Introduction Germinal centre (GC) lymphomas Follicular Lymphoma (FL) and Diffuse Large B Cell Lymphoma (DLBCL) are strikingly dependent on mutations affecting chromatin regulators. The majority of FL tumours harbour multiple mutated chromatin-regulatory genes, including loss-of-function mutations in KMT2D (80%), CREBBP (60%) and gain-of-function mutations in EZH2 (25%), whilst these are also frequently mutated within the GC B-cell (GCB) subtype of DLBCL. The FDA approval of EZH2-inhibitor Tazemetostat for relapsed/refractory FL exemplifies the potential and the need to broaden our search for novel avenues targeting epigenetic reprogramming in GC lymphomas. Hence, in this study, we sought to identify novel epigenetic vulnerabilities and biological interactions in GC B cell lymphomas. Methods and Results: 25 compounds targeting 18 histone binding/modifying enzymes were tested across 12 GCB-DLBCL cell lines harbouring differing degrees of epigenetic dysregulation. The two most potent cytotoxic compounds (GSK-J4 and KDOBA67) both target the H3K27me3 demethylase, KDM6. The KDM6 family consists of KDM6A (UTX) and KDM6B (JMJD3), both of which demethylate H3K27me3/H3K27me2 to lower methylation states, and KDM6C (UTY) which has negligible enzymatic activity. Indeed, KDM6 inhibition (KDM6i) with GSK-J4 increased levels of the inhibitory chromatin mark H3K27me3 and simultaneously reduced H3K27me1/me2, while we observed no global changes in H3K4me3. The induction of apoptosis in the cell lines following either inhibition of the 'eraser' KDM6 (leading to increased H3K27me3) or the opposing 'writer' EZH2 (lowering H3K27me3) appears paradoxical at first glance, given the EZH2 gain-of-function mutations seen in FL/DLBCL and known efficacy of EZH2 inhibitors. However, RNA-sequencing analysis of GSK-J4-treated cells revealed no significant overlap with published EZH2 mutation or EZH2 inhibition signatures from GC lymphoma models, implying that KDM6i-regulated H3K27me3 peaks do not overlay gene promoters targeted by EZH2 in the GC reaction. The most significant gene regulation by KDM6i was a significant up-regulation of 6 members of the metallothionein (MT) family within 4-hours of treatment, which was sustained for 72-hours (MT1E, MT1F, MT1G, MT1H, MT1X and MT2A at 4 hours log2FoldChange 5.1-12.1, average p adj 3.4E-06). These metal-binding proteins cluster on chromosome 16 and their up-regulation correlated with up-regulation of the zinc transporter SLC30A1, suggesting a potential biological relationship between KDM6 enzymes and zinc homeostasis, with both isoforms containing a zinc-binding domain. Targeted validation across 6 DLBCL cell lines also showed MT up-regulation correlated with sensitivity to GSK-J4, whilst siRNA-mediated knockdown of KDM6A or KDM6B individually recapitulated the up-regulation of MT genes, albeit to a lower extent. ChIP-qPCR profiling at the MT loci for H3K27me3 and H3K4me3 revealed no significant changes at 4 or 24 hours; considering that expression of MT genes increase within 60 minutes of drug exposure, suggesting these changes may be induced by an indirect mechanism of KDM6A and/or KDM6B activity. Using ENCODE data of 4 cell lines included in our series (DOHH-2, OCI-LY-1, SU-DHL-6, KARPAS-422), we observe a link between GSK-J4 sensitivity, up-regulation of MT genes and significantly lower basal enrichment for H3K27ac at the MT loci. This distinction may reflect altered 3D chromatin conformation that render only sensitive cell lines permissive to regulation by KDM6i, with H3K27ac having recently been shown to modulate enhancer positioning and interactions in DLBCL cell lines. Conclusion: Screening epigenetic-targeting compounds in GC lymphomas has identified KDM6 as a particularly promising candidate target. Overall, we describe up-regulation of MT genes as one potential mechanism of action, which correlates with response in DLBCL cell lines and suggests that KDM6 members may regulate H3K27me3 in a manner different from EZH2. In our current model we propose, that in GC-lymphomas KDM6A may have tumour suppressive roles whilst KDM6B is required for cell survival. Given KDM6A is frequently lost in cancer and Kabuki syndrome, a congenital developmental disorder caused by germline mutations in KMT2D or KDM6A, our results highlight the importance of establishing what roles the KDM6 family play in lymphoma pathogenesis. Disclosures Fitzgibbon: Epizyme: Research Funding.

Background: The poor-risk cytogenetic subgroup of acute myeloid leukaemia (AML) includes various chromosomal aberrations and represents a heterogeneous population of patients with a dismal 10-year overall survival. While the success of genetic landscaping studies is encouraging, it is debatable whether genomics, or indeed any single-omics platform alone, is sufficient to capture the biology of a disease that continues to evade our existing treatments so effectively. Instead, we need to develop a much better understanding of the complexity of this subgroup of AMLs: the relationship and interdependencies across biochemical pathways, how these may differ between patients and their impact on the leukemia and normal stem cell compartments. To launch this process, we have completed a multi-omics profiling programme to shed new light on the genetic and biochemical features of poor-risk AML (https://poor-risk-aml.bham.ac.uk/). Aims: Application of multi-omics and integrative approaches to decipher the complexities of cytogenetically poor-risk AML Methods: Sample inclusion criteria were based on cytogenetics and availability of sufficient diagnostic bone marrow or peripheral blood material for analysis. The 50 primary AMLs included 17 cases with complex karyotype, 13 -7/del(7), 11 KMT2A rearrangements (with the exception of t(9;11)), 4 t(6;9), 3 -5/de(5), 1 del(17) and 1 inv(3). Profiles consisted of a combination of genomics (whole genome sequencing (WGS, 60X for tumour and 30X for germ-line controls), targeted sequencing of 54 myeloid loci, and total RNA-seq (100 million reads per bulk sample), mass spectrometry proteomics and phosphoproteomics (with >6,000 proteins and > 25,000 phosphorylation sites detected and quantified), mass cytometry (CyTOF, 39 markers), drug screening (ranging from 200-500 approved or investigational compounds) and the selective generation of patient-derived xenograft (PDX) models. Results: Near complete datasets have been compiled on all 50 primary AMLs, with the exception of WGS analysis where profiling was restricted to cases where corresponding germline DNA was available. Integration of WGS and RNA-seq data identified 122 genes having notable allele-specific expression (ASE) in ≥ 5 samples supported by ≥ 3 SNPs and these included the transcription factor GATA2 and the DNA topoisomerase TOP1MT. Use of RNA fusion capture tools resolved novel inter- and intra- chromosomal gene rearrangements that were confirmed by WGS. The four t(6;9)(p23;q34)/DEK-NUP214 cases, with a mean age of diagnosis of 43.5 years and all harboring FLT3-ITD mutations, arose from the most immature hematopoietic compartment (CD34+CD117+ enrichment) and demonstrated a unique transcriptomic signature, which included upregulation of FOXO3 and GRP56. Collectively, t(6;9) primary samples also showed a selective drug sensitivity to XPO1 (selinexor and eltanexor) and JAK inhibitors (ruxolitinib, tofacitinib and momelotinib) compared to other cytogenetic risk groups. On the other hand, a comparison of in vitro drug sensitivity data with genomic data of our entire cohort of patients demonstrated that TP53 wt AMLs (n=37) were more sensitive to all four MDM2 inhibitors (AMG-232, idasanutlin, SAR405838 and NVP-CGM097) compared to TP53 mutated cases (n=13). Comparisons of transcriptomics with the in vitro sensitivity to drugs included in early/late phase AML clinical trials, identified signatures of response associated with MDM2 and Aurora B kinase (AZD1152-HQPA) inhibitors. Phosphoproteomics analysis and machine learning modeling separated KMT2A rearranged leukemias into 2 discrete groups (group A: MLLT4, MLLT10 and TET1; group B with MLLT6, ELL and SEP9 fusion partners). Functionally, group A presented with elevated HOXA10 protein expression and enhanced in vitro response to genotoxic drugs and cell cycle inhibitors when compared to group B leukemia. Conclusions: Our study demonstrates the feasibility of simultaneously generating omics data from several different platforms and highlights that a combination of genetic and proteomic profiles may help to inform the choice of therapies based on the underlying biology of a patient's AML. Disclosures Wennerberg: Novartis: Research Funding; Pfizer: Honoraria. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding; Innovative Mediicines Initiative project Harmony: Research Funding.