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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.

Acute myeloid leukemia (AML) is an aggressive hematological disorder comprising a hierarchy of quiescent leukemic stem cells (LSCs) and proliferating blasts with limited self-renewal ability. AML has a dismal prognosis, with extremely low 2-year survival rates in the poorest cytogenetic risk patients, primarily due to the failure of intensive chemotherapy protocols to deplete LSCs and toxicity of therapy toward healthy hematopoietic cells. We studied the role of cyclin-dependent kinase regulatory subunit 1 (CKS1)–dependent protein degradation in primary human AML and healthy hematopoiesis xenograft models in vivo. Using a small-molecule inhibitor (CKS1i), we demonstrate a dual role for CKS1-dependent protein degradation in reducing patient-derived AML blasts in vivo and, importantly, depleting LSCs, whereas inhibition of CKS1 has the opposite effect on normal hematopoiesis, protecting normal hematopoietic stem cells from chemotherapeutic toxicity. Proteomic analysis of responses to CKS1i in our patient-derived xenograft mouse model demonstrate that inhibition of CKS1 in AML leads to hyperactivation of RAC1 and accumulation of lethal reactive oxygen species, whereas healthy hematopoietic cells enter quiescence in response to CKS1i, protecting hematopoietic stem cells. Together, these findings demonstrate that CKS1-dependent proteostasis is a key vulnerability in malignant stem cell biology.

Significance Bone marrow failure (BMF) syndromes are inherited life-threatening conditions characterized by low blood cell production and predisposition to cancer. In this study we report a germ line frameshift variant in the Sp1 transcription factor in a family of patients with BMF and acute leukemia. Sp1 is ubiquitously expressed in human tissues and regulates transcription for blood cell lineage specification. Dissecting the molecular function of this SP1 variant revealed a hypermorphic effect, triggering superactivation of Sp1-mediated transcription in the patients’ blood. To our knowledge, this is the first report of a naturally occurring germ line variant in SP1 that alters transcriptional networks and disrupts hematopoiesis in humans., Inherited bone marrow failure (BMF) syndromes are a heterogeneous group of diseases characterized by defective hematopoiesis and often predisposing to myelodysplastic syndrome (MDS) and acute myelogenous leukemia. We have studied a large family consisting of several affected individuals with hematologic abnormalities, including one family member who died of acute leukemia. By whole-exome sequencing, we identified a novel frameshift variant in the ubiquitously expressed transcription factor specificity protein 1 (SP1). This heterozygous variant (c.1995delA) truncates the canonical Sp1 molecule in the highly conserved C-terminal DNA-binding zinc finger domains. Transcriptomic analysis and gene promoter characterization in patients’ blood revealed a hypermorphic effect of this Sp1 variant, triggering superactivation of Sp1-mediated transcription and driving significant up-regulation of Sp1 target genes. This familial genetic study indicates a central role for Sp1 in causing autosomal dominant transmission of BMF, thereby confirming its critical role in hematopoiesis in humans.

Germline variants in RUNX1, GATA2 and DDX41 may confer a predisposition to hereditary haematopoietic malignancies (HHMs) such as MDS and AML yet have distinct age ranges of malignancy diagnosis and a highly variable overall risk for leukemogenesis. The increased awareness and identification of carriers of these germline variants, particularly before development of malignancy, has changed the way in which individuals and families need to be managed in the clinic. Individuals need lifelong monitoring and may also need modification to treatments when malignancy does develop, compared to sporadic counterparts. Gaps in understanding pre-malignant states in HHM syndromes have hampered efforts to design effective clinical surveillance regimes, provide personalized pre-emptive treatments, and appropriate counselling to patients.

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.

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis with increased incidence in elderly individuals. Genetic alterations do not fully explain the molecular pathogenesis of the disease, indicating that other types of lesions may play a role in its development. In this work, we analyzed the transcriptional lesions of human HSCs, demonstrating how aging and MDS are characterized by a complex transcriptional rewiring that manifests as diverse linear and non-linear transcriptional dynamisms. While aging-associated lesions seemed to predispose elderly HSCs to myeloid transformation, disease-specific alterations may be involved in triggering MDS development. Among MDS-specific lesions, we detected the overexpression of the transcription factor DDIT3. Exogenous upregulation of DDIT3 in human healthy HSCs induced an MDS-like transcriptional state, and a delay in erythropoiesis, with an accumulation of cells in early stages of erythroid differentiation, as determined by single-cell RNA-sequencing. Increased DDIT3 expression was associated with downregulation of transcription factors required for normal erythropoiesis, such as KLF1, TAL1 or SOX6, and with a failure in the activation of their erythroid transcriptional programs. Finally, DDIT3 knockdown in CD34+ cells from MDS patients was able to restore erythropoiesis, as demonstrated by immunophenotypic and transcriptional profiling. These results demonstrate that DDIT3 may be a driver of MDS transformation, and a potential therapeutic target to restore the inefficient erythropoiesis characterizing these patients. KEY POINTSO_LIHuman HSCs undergo a complex transcriptional rewiring in aging and MDS that may contribute to myeloid transformation. C_LIO_LIDDIT3 overexpression induces a failure in the activation of erythroid transcriptional programs, leading to inefficient erythropoiesis. C_LI

Acute myeloid leukemia (AML) is an aggressive hematological disorder comprising a hierarchy of quiescent leukemic stem cells (LSCs) and proliferating blasts with limited self-renewal ability. AML has a dismal prognosis, with extremely low two-year survival rates in the poorest cytogenetic risk patients, primarily due to the failure of intensive chemotherapy protocols unable to deplete LSCs, which reconstitute the diseasein vivo, and the significant toxicity towards healthy hematopoietic cells. Whilst much work has been done to identify genetic and epigenetic vulnerabilities in AML LSCs, little is known about protein dynamics and the role of protein degradation in drug resistance and relapse. Here, using a highly specific inhibitor of the SCFSKP2-CKS1complex, we report a dual role for CKS1-dependent protein degradation in reducing AML blastsin vivo, and importantly depleting LSCs. Whilst many AML LSC targeted therapies show significant toxicity to healthy hematopoiesis, inhibition of CKS1-dependent protein degradation has the opposite effect, protecting normal hematopoietic cells from chemotherapeutic toxicity. Together these findings demonstrate CKS1-dependent proteostasis is key for normal and malignant hematopoiesis.SignificanceCKS1-dependent protein degradation is a specific vulnerability in AML LSCs. Specific inhibition of SCFSKP2-CKS1is lethal toCKS1BhighAML blasts and all AML LSCs. Normal hematopoiesis is protected from chemotherapeutic toxicity by inhibition of CKS1-dependent protein degradation, substantiating a dual role for CKS1-dependent protein degradation in clinical treatment of AML.

Biallelic variants in the ERCC excision repair 6 like 2 gene (ERCC6L2) are known to cause bone marrow failure (BMF) due to defects in DNA repair and mitochondrial function. Here, we report on eight cases of BMF from five families harboring biallelic variants in ERCC6L2, two of whom present with myelodysplasia. We confirm that ERCC6L2 patients' lymphoblastoid cell lines (LCLs) are hypersensitive to DNA-damaging agents that specifically activate the transcription coupled nucleotide excision repair (TCNER) pathway. Interestingly, patients' LCLs are also hypersensitive to transcription inhibitors that interfere with RNA polymerase II (RNA Pol II) and display an abnormal delay in transcription recovery. Using affinity-based mass spectrometry we found that ERCC6L2 interacts with DNA-dependent protein kinase (DNA-PK), a regulatory component of the RNA Pol II transcription complex. Chromatin immunoprecipitation PCR studies revealed ERCC6L2 occupancy on gene bodies along with RNA Pol II and DNA-PK. Patients' LCLs fail to terminate transcript elongation accurately upon DNA damage and display a significant increase in nuclear DNA-RNA hybrids (R loops). Collectively, we conclude that ERCC6L2 is involved in regulating RNA Pol II-mediated transcription via its interaction with DNA-PK to resolve R loops and minimize transcription-associated genome instability. The inherited BMF syndrome caused by biallelic variants in ERCC6L2 can be considered as a primary transcription deficiency rather than a DNA repair defect.

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 wild-type ETV6 with potentially dominant-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 acute myeloid leukemia 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 assay for transposase-accessible chromatin sequencing profiling. ETV6-mediated ALL predisposition exemplifies the intricate interactions between inherited and acquired genomic variations in leukemia pathogenesis.

Familial CEBPA- mutated acute myeloid leukemia (AML) represents a recognized leukemia predisposition syndrome, with several families described in the literature since the initial report in 2004. The pathological features and long-term survival of individuals with familial CEBPA -mutated AML are reminiscent of sporadic CEBPA dm AML. Germline mutations predominantly localize to the N-terminal and are associated with near complete penetrance, with age of AML onset from 2–50 years, frequently accompanied by the acquisition of a second CEBPA mutation in C-terminal domain. Patients appear to have a significant risk of late AML recurrence and these typically represent independent leukemic episodes, characterized by a unique molecular profile that is distinct from that of the preceding tumor. While these patients respond well to salvage therapies, allogeneic hematopoietic stem cell transplantation (HSCT) should be considered for patients with high-risk features at presentation or recurrent disease, with the aim of eradicating the germline mutation and improving long-term survival. In contrast, inherited C-terminal CEBPA mutations occur less frequently and appear to demonstrate reduced penetrance, impeding clinical detection and surveillance.

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.

Acute myeloid leukemia (AML) is characterised by a series of genetic and epigenetic alterations that result in deregulation of transcriptional networks. One understudied source of transcriptional regulators are transposable elements (TEs), whose aberrant usage could contribute to oncogenic transcriptional circuits. However, the regulatory influence of TEs and their links to AML pathogenesis remain unexplored. Here we identify six endogenous retrovirus (ERV) families with AML-associated enhancer chromatin signatures that are enriched in binding of key regulators of hematopoiesis and AML pathogenesis. Using both locus-specific genetic editing and simultaneous epigenetic silencing of multiple ERVs, we demonstrate that ERV deregulation directly alters the expression of adjacent genes in AML. Strikingly, deletion or epigenetic silencing of an ERV-derived enhancer suppresses cell growth by inducing apoptosis in leukemia cell lines. This work reveals that ERVs are a previously unappreciated source of AML enhancers that may be exploited by cancer cells to help drive tumour heterogeneity and evolution., Transposable elements are a potential source of transcriptional regulators, but how these sequences contribute to oncogenesis remains poorly understood. Here, the authors identify endogenous retroviruses (ERVs) with acute myeloid leukemia (AML)-associated enhancer chromatin signatures, and provide evidence that ERV activation provides an additional layer of gene regulation in AML.

Cancers can be best described as genetic diseases, where mutations typically accumulate over a protracted period of time, leading to a cellular shift from normalcy to malignancy and an ever-evolving tumour and its microenvironment. The tools at our disposal to characterise the genetic landscape(s) of these tumours and our appreciation of their complexity have fundamentally changed over the last 10 years, following the first whole-genome sequencing (WGS) of a case of acute myeloid leukaemia (AML) in 2008 and the introduction of global initiatives (e.g. The Cancer Genome Atlas (TCGA)), both with an overarching goal of improving diagnosis, treatment and cancer prevention by setting out to systematically explore the entire spectrum of genomic changes involved in human disease. While this journey is far from complete, modern diagnosis of cancers now relies on the integration of morphological and molecular information that, together, offer the potential to refine classification, establish prognosis and determine the most appropriate treatment for groups of patients. In this chapter, we examine how genomics has revolutionised our understanding of the diagnosis of blood cancers, using the exemplar of AML, and how this new knowledge is set to inform and direct treatment in the near future.

Significance Bone marrow failure (BMF) is an inherited life-threatening condition characterized by defective hematopoiesis, developmental abnormalities, and predisposition to cancer. BMF caused by ERCC6L2 mutations is considered to be a genome instability syndrome, because DNA repair is compromised in patient cells. In this study, we report BMF cases with biallelic disease-causing variants and provide evidence from patients’ cells that transcription deficiency can explain the genome instability. Specifically, we demonstrate that ERCC6L2 participates in RNA polymerase II-mediated transcription via interaction with DNA-dependent protein kinase (DNA-PK) and resolves DNA–RNA hybrids (R loops). Collectively, our data point to a causal mechanism in BMF in which patients with ERCC6L2 mutations are defective in the repair of transcription-associated DNA damage., Biallelic variants in the ERCC excision repair 6 like 2 gene (ERCC6L2) are known to cause bone marrow failure (BMF) due to defects in DNA repair and mitochondrial function. Here, we report on eight cases of BMF from five families harboring biallelic variants in ERCC6L2, two of whom present with myelodysplasia. We confirm that ERCC6L2 patients’ lymphoblastoid cell lines (LCLs) are hypersensitive to DNA-damaging agents that specifically activate the transcription coupled nucleotide excision repair (TCNER) pathway. Interestingly, patients’ LCLs are also hypersensitive to transcription inhibitors that interfere with RNA polymerase II (RNA Pol II) and display an abnormal delay in transcription recovery. Using affinity-based mass spectrometry we found that ERCC6L2 interacts with DNA-dependent protein kinase (DNA-PK), a regulatory component of the RNA Pol II transcription complex. Chromatin immunoprecipitation PCR studies revealed ERCC6L2 occupancy on gene bodies along with RNA Pol II and DNA-PK. Patients’ LCLs fail to terminate transcript elongation accurately upon DNA damage and display a significant increase in nuclear DNA–RNA hybrids (R loops). Collectively, we conclude that ERCC6L2 is involved in regulating RNA Pol II-mediated transcription via its interaction with DNA-PK to resolve R loops and minimize transcription-associated genome instability. The inherited BMF syndrome caused by biallelic variants in ERCC6L2 can be considered as a primary transcription deficiency rather than a DNA repair defect.

c-MYC oncogene is deregulated in most human tumours. Histone marks associated with transcriptionally active genes define high-affinity c-MYC targets. The mechanisms involved in their recognition by c-MYC are unknown. Here we report that c-MYC interacts with BPTF, a core subunit of the NURF chromatin-remodelling complex. BPTF is required for the activation of the full c-MYC transcriptional programme in fibroblasts. BPTF knockdown leads to decreased c-MYC recruitment to DNA and changes in chromatin accessibility. In Bptf-null MEFs, BPTF is necessary for c-MYC-driven proliferation, G1–S progression and replication stress, but not for c-MYC-driven apoptosis. Bioinformatics analyses unveil that BPTF levels correlate positively with c-MYC-driven transcriptional signatures. In vivo, Bptf inactivation in pre-neoplastic pancreatic acinar cells significantly delays tumour development and extends survival. Our findings uncover BPTF as a crucial c-MYC co-factor required for its biological activity and suggest that the BPTF-c-MYC axis is a potential therapeutic target in cancer., c-MYC genomic distribution is dictated by the epigenetic context but the mechanisms are unknown. Here, the authors show that c-MYC requires the chromatin reader BPTF to activate its transcriptional program and promote tumour development in vivo, suggesting that BPTF is a potential target for cancer therapy.

Proteomic and genomic integration identifies kinase and differentiation determinants of kinase inhibitor sensitivity in leukemia cells

Regulator of telomere elongation helicase 1 (RTEL1) is a DNA helicase involved in telomere maintenance.1,2 Germline biallelic RTEL1 variants have been previously reported in a subset of patients with dyskeratosis congenita (DC) and its severe variant Hoyeraal-Hreidarsson syndrome (HH).3–6 Furthermore, germline heterozygous RTEL1 variants have been linked to a subset of patients with pulmonary fibrosis.2,7,8 We have undertaken sequencing analysis (whole exome and targeted9) of RTEL1, using genomic DNA extracted from peripheral blood of 429 patients from our international bone marrow failure registry which includes DC, HH, aplastic anemia (AA), and familial myelodysplasia/leukemia (MDS/AML). This has revealed that 35 out of the 429 patients have RTEL1 variants (Table 1). Based on the minor allele frequency in the population reported on the Exome Aggregation Consortium database (ExAC – http://exac.broadinstitute.org/), the type of variant (missense, nonsense and indels), telomere length, the Combined Annotation Depletion (CADD) score,10 and segregation as well as information found in literature, we classified these variants into four different groups: (1) biallelic variants, (2) heterozygous loss of function (LOF) variants, (3) heterozygous missense variants of unknown significance (VUS) and (4) heterozygous missense bystander variants.

In the original version of this article the authors noted an omission in the author affiliations where the university details: Queen Mary University of London was not included in the original affiliation for the majority of the authors. The correct affiliations are as follows1. Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK3. Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK6. Evolution and Cancer Laboratory, Barts Cancer Institute, Queen Mary University of London, London, UK.

Germline variants within the transcription factor RUNX1 are associated with familial platelet disorder and acute leukemia in over 40% of carriers. At present, the somatic events triggering leukemic transformation appear heterogeneous and profiles of leukemia initiation across family members are poorly defined. We report a new RUNX1 family where three sisters harboring a germline nonsense RUNX1 variant, c.601C>T (p.(Arg201*)), developed acute myelomonocytic leukemia (AML) at 5 years of age. Whole-exome sequencing of tumor samples revealed all three siblings independently acquired variants within the JAK-STAT pathway, specifically targeting JAK2 and SH2B3 (a negative regulator of JAK2), while also sharing the 46/1 haplotype linked with sporadic JAK2-positive myeloproliferative neoplasms. In-depth chromosomal characterization of tumors revealed acquired copy number gains and uniparental disomy amplifying RUNX1, JAK2 and SH2B3 variants, highlighting the significance of co-operation between these disrupted pathways. One sibling, presenting with myelodysplasia at 14 years, had no evidence of clonal or subclonal JAK2 or SH2B3 variants, suggesting the latter were specifically associated with leukemic transformation in her sisters. Collectively, the clinical and molecular homogeneity across these three young siblings provides the first notable example of convergent AML evolution in a RUNX1 pedigree, with the recurrent acquisition of JAK-STAT pathway variants giving rise to high-risk AML, characterized by chemotherapy resistance and relapse.

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16 p.-7 fig.-4 tab. Cabrera, Javier et al., Root-knot nematodes (RKNs) induce inside the vascular cylinder the giant cells (GCs) embedded in the galls. The distinctive gene repression in early-developing GCs could be facilitated by small RNAs (sRNA) such as miRNAs, and/or epigenetic mechanisms mediated by 24nt-sRNAs, rasiRNAs and 21-22nt-sRNAs. Therefore, the sRNA-population together with the role of the miR390/TAS3/ARFs module were studied during early gall/GC formation., Three sRNA libraries from 3-d-post-inoculation (dpi) galls induced by Meloidogyne javanica in Arabidopsis and three from uninfected root segments were sequenced following Illumina-Solexa technology. pMIR390a::GUS and pTAS3::GUS lines were assayed for nematode-dependent promoter activation. A sensor line indicative of TAS3-derived tasiRNAs binding to the ARF3 sequence (pARF3:ARF3-GUS) together with a tasiRNA-resistant ARF3 line (pARF3:ARF3m-GUS) were used for functional analysis. The sRNA population showed significant differences between galls and controls, with high validation rate and correspondence with their target expression: 21-nt sRNAs corresponding mainly to miRNAs were downregulated, whilst 24-nt-sRNAs from the rasiRNA family were mostly upregulated in galls. The promoters of MIR390a and TAS3, active in galls, and the pARF3:ARF3-GUS line, indicated a role of TAS3-derived-tasiRNAs in galls. The regulatory module miR390/TAS3 is necessary for proper gall formation possibly through auxin-responsive factors, and the abundance of 24-nt sRNAs (mostly rasiRNAs) constitutes a gall hallmark., This work was supported by the Spanish Government (AGL2013-48787 to C.E. and PCIN-2013-053 to C.F.) and by the Castilla-la Mancha Government (PEII-2014-020-P to C.F.). J.C. was supported by a fellowship from the Ministry of Education, Spain. C.M. was supported by a PhD grant from INRASPE Dpt and the Conseil R egional Provence-Alpes-C^ote d’Azur(PACA). B.F. was supported by the French Government (National Research Agency, ANR) through the ‘Investments for the Future’ LabEx SIGNALIFE (ANR-11-LABX-0028-01).

Disruption of the Krebs cycle is a hallmark of cancer. IDH1 and IDH2 mutations are found in many neoplasms, and germline alterations in SDH genes and FH predispose to pheochromocytoma/paraganglioma and other cancers. We describe a paraganglioma family carrying a germline mutation in MDH2, which encodes a Krebs cycle enzyme. Whole-exome sequencing was applied to tumor DNA obtained from a man age 55 years diagnosed with multiple malignant paragangliomas. Data were analyzed with the two-sided Student's t and Mann-Whitney U tests with Bonferroni correction for multiple comparisons. Between six- and 14-fold lower levels of MDH2 expression were observed in MDH2-mutated tumors compared with control patients. Knockdown (KD) of MDH2 in HeLa cells by shRNA triggered the accumulation of both malate (mean ± SD: wild-type [WT] = 1±0.18; KD = 2.24±0.17, P = .043) and fumarate (WT = 1±0.06; KD = 2.6±0.25, P = .033), which was reversed by transient introduction of WT MDH2 cDNA. Segregation of the mutation with disease and absence of MDH2 in mutated tumors revealed MDH2 as a novel pheochromocytoma/paraganglioma susceptibility gene.

Introduction Protein kinases play a key role in how cells respond and adapt to intra and extracellular stimuli. By the addition of phosphate groups to serine, threonine or tyrosine residues, these enzymes modify the activity and properties of the targeted proteins which in turn modulate biological processes like proliferation, differentiation and cell death. Kinase signalling pathways are deregulated in most cancer types including haematological malignancies. Indeed, the kinases FLt-3, c-Kit and JAK2 as well as the up-stream kinase signalling regulators KRAS and NRAS are among the most frequently mutated genes in acute myeloid leukaemia (AML). Consequently, protein kinases have attracted the attention of the pharmaceutical and biotechnology companies and inhibitors have been found for one fifth of human kinases. In the case of AML, midostaurin, a multi-kinase inhibitor that targets, among others, the tyrosine kinase Flt-3, has granted a breakthrough therapy designation by the FDA and several other kinase inhibitors are in clinical trials or under preclinical investigation. Molecular profiling of patient samples will play a pivotal role for the development and implementation of personalized therapies including those based on kinase inhibitors. We used a molecular profile generated by a phosphoproteomics approach to rationalize why some primary AML cells respond to treatment with different kinase inhibitors while others are resistant to the same treatments. Methods Label free phosphoproteomics based on trypsin digestion and TiO2 phosphoenrichment was used to quantify > 5,000 phosphorylation sites in mononuclear cells extracted from the peripheral blood of 36 AML patients. KSEA technology was applied to infer kinase activity from the phosphoproteomics data and DAVID software was used to determine gene ontology enrichments based on the genes that code for the proteins where the phosphorylation sites were detected. Guava EasyCyte Flow Cytometry was used to determine cell viability after the treatment of the same patient samples with different kinase inhibitors. Mass cytometry was used to measure the expression at the plasma membrane of 17 surface markers in 30 of the previously analysed AML primary samples. Results The FAB classification subdivide AML cases depending on cytomorphological features. We compared the phosphoproteomes of M1 and M4 classes that are associated with early and late states of differentiation. Based on the 150 phosphopeptides more significantly regulated between FAB-M1 and FAB-M4 groups, hierarchical clustering analysis was used to stratify AML patient samples into two subsets named M1-Like and M4-Like. Phosphoproteome reanalysis showed that the M4-Like set upregulated 1255 phosphopeptides and downregulated 446 when compared with the M1-Like set. The upregulated group comprised regulatory phosphorylation sites in several kinases including PAK1 and PCK delta. Kinase activity analysis using KSEA (Kinase Substrate Enrichment Analysis) also showed an increased activity of PAK, PKCδ and other kinases like P38 alpha in the M4-Like group. Interestingly, the PAK inhibitor PF03758309 reduced more efficiently the viability in M4-Like group than in the M1-Like group (average reduction after a 72h treatment with 1µM of 55.2% for M4-Like compared to 33.8% for M1-Like, p-value = 0.0078). This difference was not observed for other inhibitors such as those targeting CK2 or p38. CyTOF analysis showed that the M4-Like group upregulated the surface expression of several differentiation markers. Discussion Predicting the effectiveness of a drug for a particular patient is a major goal of personalized medicine. In the case of kinase inhibitors, responses may be influenced by several factors including the activity of the targeted kinase as well as the activity of other kinases that act in parallel pro-survival pathways. In this work, we have found that differentiation leads to a particular activation pattern of the signalling networks, a phenomenon that determines the response to signalling inhibitors. Conclusion We found phosphoproteomics signatures in primary AML that are associated with distinct haematopoietic differentiation stages. These signatures are in turn associated with how AML cells respond to kinase inhibitors. Disclosures Fitzgibbon: Epizyme: Research Funding; Gilead: Honoraria; Janssen: Honoraria; Celgene: Honoraria.

Background: The chromosomal translocation t(7;11)(p15,p15) encodes the oncogenic transcription factor NUP98-HOXA9 which results in a fusion of the nucleoporin 98kDa (NUP98) and homeobox A9 (HOXA9) genes. The oncogenic mechanisms underlying this translocation remain poorly understood and patients are currently inadequately served by traditional cytotoxic chemotherapy regimens. Aims:To decipher the underlying biology of the NUP98-HOXA9 fusion protein and develop rational therapeutic strategies targeting its oncogenic mechanism. Methods: Human cellular models expressing NUP98-HOXA9, HOXA9 wt or NUP98 wt were established by retroviral transduction of HEK293FT human cell line and human hematopoietic progenitors (CD34+, hHP) isolated from donor cord blood. Chromatin immunoprecepitation experiments followed by sequencing (ChIP-seq) and quantitative ChIP (qChIP) were used to define fusion specific binding locations. Cloning regulatory regions of selected target genes in a luciferase vectorconfirmed the direct involvement of NUP98-HOXA9 in their regulation. RTQ-PCR and gene expression microarrays were used to evaluate expression levels. Co-Immunoprecipitation experiments validated protein-protein interactions and drug treatments were performed at IC50. Cell viability was analysed by apoptosis, proliferation and Colony Forming Unit assays. Results:Comparison of ChIP-seq data from HEK293FTmodels of NUP98-HOXA9, HOXA9 wt or NUP98 wt respectively, identified 4,471 target genomic regions of the fusion protein (FDR < 0.05), located within +4/-4 kb from the annotated Transcription Start Site (TSS) of 1,363 genes, with 399 genes common to HOXA9 wt and 5 to NUP98 wt. The NUP98-HOXA9 binding sites included enhancers of MEIS1, HOXA9 and PBX3 (PBX3 and HOXA9 were common to NUP98 wt and MEIS1 to HOXA9 wt). Together these transcription factors form a key activator complex that regulates the expression of genes involved in leukemogenesis and its overexpression is significant related to adverse prognosis in AML. Luciferase assays showed that the upregulation of this leukemic axis was directly induced by the interaction of NUP98-HOXA9 with the corresponding enhancer regions of MEIS1, HOXA9 and PBX3. Treatment of cells with HXR9, a specific peptide inhibitor of HOXA9 and PBX3 interaction, led to a selective decrease in the proliferation of hHP expressing NUP98-HOXA9, confirming the relevance of these target genes to its oncogenic mechanism. Combining ChIP-seq and gene expression data of three independent clones of hHP expressing NUP98-HOXA9 and patient samples (n = 5) harbouring t(7;11)(p15,p15) revealed a dual regulatory role of the fusion protein, in both repressing and activating target gene transcription where, for example, MEIS1, HOXA9, PBX3 and AFF3 were found overexpressed and BIRC3, SMAD1, FILIP1L and PTEN downregulated. Interactions of NUP98-HOXA9 with p300 and HDAC1 were shown to drive this transcriptional activation and repression, respectively. We found using qChIP experiments that p300 bound to the regulatory regions of the overexpressed genes only when NUP98-HOXA9 was present, whereas we observed significant enrichment of HDAC1 binding to the promoter regions of the downregulated genes when the fusion protein was expressed. Taking advantage of this latter observation, we demonstrated a dramatic inhibitory effect on the viability of hHP expressing NUP98-HOXA9after the treatment with subtherapeutic doses (IC50 = 4nM) of the HDAC inhibitor LBH-589 (Panobinostat) with no effect in control hHP transduced with an empty vector. Conclusion: An improved understanding of the pathobiology underlying recurrent translocation events in AML is a critical first step for the development of rational, targeted therapies. Here, we identify upregulation of the targetable MEIS1-HOXA9-PBX3 complex underpinning the leukemogenic activity of NUP98-HOXA9. Its activity in repressing transcription mediated through interaction with HDAC1, has been shown to be also a key pathogenic mechanism that can be exploited through use of HDAC inhibitors and potentially lead to a promising new therapy for this high-risk group of patients. Disclosures No relevant conflicts of interest to declare.

Background : While myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are considered sporadic hematopoietic stem cell clonal disorders, there are rare occurrences of familial cases ( Aims:To investigate the molecular mechanisms underlying the variable penetrance and clinical heterogeneity observed in a GATA2-mutated family. Results:Targeted deep-sequencing of 33 genes frequently mutated in MDS/AML revealed a low overall burden of acquired mutations in the symptomatic carriers with no mutations detected in asymptomatic family members. It was noteworthy that an acquired ASXL1 mutation (p.Gly646TrpfsTer12) was identical in all affected individuals (III.1, III.3 and III.7) (Figure 1) although the variant allele frequency was lower (12%) in III.7 and remained stable (range 12-6%) over a 4 year monitoring period. GATA2 expression was lower in III.7 as assessed by quantitative RT-PCR and strikingly this was associated with monoallelic expression of the mutated GATA2 allele with complete loss of the wild-type (WT) allele expression. Temporal analysis of III.7 at yearly intervals demonstrated reactivation of the WT allele 2 years later, coinciding with a marked improvement in hematological parameters (normal monocyte count, neutrophils >1x109/L). These changes in GATA2 expression were not linked to gross changes in methylation, as assessed by methylation specific PCR and bisulphite sequencing, nor acquisition of additional mutations in the WT promoter. Instead, we believe that allele-specific fluctuations in expression are accompanied by changes in chromatin structure at the promoter. Using a SNP (rs1806462 [C/A]) located in the 5'UTR of GATA2, we assessed allele-specific enrichment of H3K4me3 and H3K27me3 chromatin marks by chromatin immunoprecipitation. Sanger sequencing revealed a significant enhancement in the deposition of H3K4me3 activating chromatin mark on the mutated allele compared to the WT allele at diagnosis and this was reversed at later follow-up, correlating with reactivation of the WT allele expression. There were no discernible allele-specific differences in the H3K27me3 mark across the phenotypes at different time-points. Conclusion: Variable penetrance amongst germline mutation carriers is a feature of many families with inherited forms of MDS/AML and this may be related to the nature of secondary genetic events acquired in at-risk individuals. In this study, however, we show that changes in the WT:mutant allele expression ratio as a result of local and allele-specific changes in chromatin deposition may also influence the penetrance of the inherited mutation. Figure 1 Figure 1. Disclosures Cavenagh: Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

Currently, multiple myeloma (MM) patients are broadly grouped into a non-hyperdiploid (nh-MM) group, highly enriched for IgH translocations, or into a hyperdiploid (h-MM) group, which is typically characterized by trisomies of some odd-numbered chromosomes. We compared the micro RNA (miRNA) expression profiles of these two groups and we identified 16 miRNAs that were downregulated in the h-MM group, relative to the nh-MM group. We found that target genes of the most differentially expressed miRNAs are directly involved in the pathogenesis of MM; specifically, the inhibition of hsa-miR-425, hsa-miR-152 and hsa-miR-24, which are all downregulated in h-MM, leads to the overexpression of CCND1, TACC3, MAFB, FGFR3 and MYC, which are the also the oncogenes upregulated by the most frequent IgH chromosomal translocations occurring in nh-MM. Importantly, we showed that the downregulation of these specific miRNAs and the upregulation of their targets also occur simultaneously in primary cases of h-MM. These data provide further evidence on the unifying role of cyclin D pathways deregulation as the key mechanism involved in the development of both groups of MM. Finally, they establish the importance of miRNA deregulation in the context of MM, thereby opening up the potential for future therapeutic approaches based on this molecular mechanism.

The chromosomal translocation t(7;11)(p15,p15), that results in the oncogenic fusion protein Nup98-Hoxa9 (NH), appears in 1% of patients with AML and is associated with very poor prognosis and short overall survival. Despite the large severity of the leukemia induced by this fusion protein, the oncogenic events triggered by NH are poorly understood, although a potential role as an aberrant transcription factor has been proposed. We have generated a human Hematopoietic Progenitors (hHP) cellular model expressing NH constitutively to identify the molecular mechanisms supporting the malignancy of this fusion protein, facilitating the search for therapeutic targets. We identified the DNA binding sites of NH by performing ChIP-seq experiments, which were validated by qRT-PCR analysis on ChIP selected DNA and Luciferase assays. Expression profiling was performed in hHP-NH and co-Immunoprecipitations (Co-IPs) were done to demonstrate the interaction of NH with different transcriptional regulators. Specific drug sensitivity of the hHP-NH model was assessed in cell proliferation assays. Our work provides the first description of the DNA binding sites of NH, most of which are regulatory regions of genes involved in the development of AML. In particular, we demonstrate that NH induces the overexpression of MEIS1, HOXA9 and PBX3, transcription factors forming an activator complex that is a key element in the leukemic onset driven by other chromosome rearrangements. Interestingly, we show that NH directly interacts with this complex through Pbx3. To evaluate the biological relevance of the interaction of the MEIS1-HOXA9-PBX3 complex with NH, we have analyzed the sensitivity of hHP-NH to the HXR9 peptide (an inhibitor of the HOXA9-PBX3 interaction). Supporting our hypothesis, we observed an inhibitory effect on hHP-NH viability after HXR9 treatment. Finally, by combining the expression profile data from hHP-NH and the ChIP-seq results using GSEA analysis, we show that NH is able to induce both overexpression and down-regulation of its target genes. To provide evidences of the activator-repressor role of NH, we performed different Co-IPs that demonstrated its direct interaction with both p300 (transcriptional activator) and HDAC1 (transcriptional inhibitor). Taken together, we show that the direct overexpression of the complex MEIS1-HOXA9-PBX3 is one of the pathogenic mechanisms induced by NH. As expected, the disruption of this complex with the HXR9 peptide in the hHP-NH model has a direct effect on cell viability. Furthermore, we show that NH interacts with this complex via PBX3 and also with p300 and HDAC1. The features and architecture of these interactions need to be further explored, but these findings allow us to consider the use of the HXR9 peptide or some HDAC inhibitors as possible treatments for these patients. Citation Format: Ana Rio-Machin, Alba Maiques-Diaz, Sandra Rodriguez-Perales, Sara Alvarez, Rocio N. Salgado, Álvaro Eguileor, Raul Torres, Juan C. Ramirez, Juan C. Cigudosa. Interactions of the fusion protein Nup98-Hoxa9 with Pbx3, p300 and HDAC1: widening the targeted therapy window in acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 472. doi:10.1158/1538-7445.AM2014-472