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Myeloid neoplasms and acute leukemias derive from the clonal expansion of hematopoietic cells driven by somatic gene mutations. Although assessment of morphology plays a crucial role in the diagnostic evaluation of patients with these malignancies, genomic characterization has become increasingly important for accurate diagnosis, risk assessment, and therapeutic decision making. Conventional cytogenetics, a comprehensive and unbiased method for assessing chromosomal abnormalities, has been the mainstay of genomic testing over the past several decades and remains relevant today. However, more recent advances in sequencing technology have increased our ability to detect somatic mutations through the use of targeted gene panels, whole-exome sequencing, whole-genome sequencing, and whole-transcriptome sequencing or RNA sequencing. In patients with myeloid neoplasms, whole-genome sequencing represents a potential replacement for both conventional cytogenetic and sequencing approaches, providing rapid and accurate comprehensive genomic profiling. DNA sequencing methods are used not only for detecting somatically acquired gene mutations but also for identifying germline gene mutations associated with inherited predisposition to hematologic neoplasms. The 2022 International Consensus Classification of myeloid neoplasms and acute leukemias makes extensive use of genomic data. The aim of this report is to help physicians and laboratorians implement genomic testing for diagnosis, risk stratification, and clinical decision making and illustrates the potential of genomic profiling for enabling personalized medicine in patients with hematologic neoplasms.

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.

Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R–mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.

As germline variants can influence cancer patient treatment decisions, outcomes and counselling, and the level of genetic predisposition for sporadic childhood acute myeloid leukemia (AML) is not clearly established, we undertook a comprehensive analysis of rare germline variants in childhood AML. As childhood AML is rare,1 to date pan-cancer childhood cohorts have included few AML cases and often the germline panels used have not included key genes relevant to myeloid malignancy. We therefore combined data from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) program together with an Australian childhood AML cohort (Supplemental Tables 1 and 2) to identify the rare germline variants in a large panel of cancer predisposition genes (n=216) compiled from literature review, and including genes involved in familial hematological malignancies (HM) and bone marrow failure (BMF) syndromes (Supplemental Table 3). We analyzed whole genome sequencing (WGS) and whole exome sequence (WES) data available through the TARGET program (n=48) (phs000218.v22.p8.c1) and WES data for the Australian cohort (n=24). Given that damaging and disease-causing variants are predicted to have a low population prevalence we identified extremely rare, potentially deleterious germline variants [VAF >30%; MAF (gnomAD) 10] and classified these as shown in Figure 1. All variants passing initial filtering are listed in Supplemental Table 4. The distribution of germline and somatic variants is shown in Supplemental Table 5, Supplemental Figure 1. Given the small cohort size pairwise comparisons of germline variants and clinico-pathological characteristics did not reveal significant associations after applying multiple correction (Supplemental Table 6).

Genomic events associated with poor outcome in chronic myeloid leukemia (CML) are poorly understood. We performed whole-exome sequencing, copy-number variation, and/or RNA sequencing for 65 patients to discover mutations at diagnosis and blast crisis (BC). Forty-six patients with chronic-phase disease with the extremes of outcome were studied at diagnosis. Cancer gene variants were detected in 15 (56%) of 27 patients with subsequent BC or poor outcome and in 3 (16%) of 19 optimal responders (P 5 .007). Frequently mutated genes at diagnosis were ASXL1, IKZF1, and RUNX1. The methyltransferase SETD1B was a novel recurrently mutated gene. A novel class of variant associated with the Philadelphia (Ph) translocation was detected at diagnosis in 11 (24%) of 46 patients comprising fusions and/or rearrangement of genes on the translocated chromosomes, with evidence of fragmentation, inversion, and imperfect sequence reassembly. These were more frequent at diagnosis in patients with poor outcome: 9 (33%) of 27 vs 2 (11%) of 19 optimal responders (P 5 .07). Thirty-nine patients were tested at BC, and all had cancer gene variants, including ABL1 kinase domain mutations in 58%. However, ABL1 mutations cooccurred with other mutated cancer genes in 89% of cases, and these predated ABL1 mutations in 62% of evaluable patients. Gene fusions not associated with the Ph translocation occurred in 42% of patients at BC and commonly involved fusion partners that were known cancer genes (78%). Genomic analysis revealed numerous relevant variants at diagnosis in patients with poor outcome and all patients at BC. Future refined biomarker testing of specific variants will likely provide prognostic information to facilitate a risk-adapted therapeutic approach. Refereed/Peer-reviewed

DEAD-Box helicase 41 (DDX41) is one of the most commonly reported familial hematological malignancy (HM) genes, first reported in 2015. Mutated, it predisposes to both MDS/AML and lymphoma with an age of diagnosis similar to that of sporadic cases. Consequently, unrecognized DDX41 mutated cases are present in 'sporadic' cohorts with families often identified this way. Individuals with DDX41 mutation or deficiency have generally poor outcome with no effective targeted therapies available. The biological mechanism by which mutant DDX41 predisposes to HM is poorly understood making risk assessment for asymptomatic carriers difficult. In over 50% of HM affected individuals with germline DDX41 mutations, an acquired somatic mutation is identified on the other DDX41 allele with R525H being most common. Analysis of other somatic mutations in germline DDX41 mutation carriers reveals a minimal fingerprint of acquired somatic alterations in known myeloid malignancy driver genes with an absence of NPM1 and FLT3 mutations and an under-representation of mutations in DNMT3A, TET2, IDH1/2 and RUNX1. This may indicate a mechanism of leukemia development unique to germline DDX41 cases. Through an integrative combinatorial genomics approach of whole exome sequencing, leukemic panel sequencing and RNA-sequencing, we are in the process of comprehensively assessing six independent DDX41 germline mutated families. We and others have identified that DDX41 mutations are recurrently associated with MDS with erythroid dysplasia and erythroid subtype leukemia consistent with functional in vitro experiments showing that DDX41 mutations including loss-of-function can impact erythroid differentiation. Our preliminary RNA-Seq analysis on peripheral blood mononuclear cells of DDX41 carriers with HM reveals altered expression of genes involved in hemoglobin complex and innate immunity. DDX41 is reported to bind RNA splicing components such as SF3B1 and PRPF8 which are recurrently mutated in MDS/AML and intriguingly, the R525H mutation disrupts this interaction. Further analysis of our RNA-Seq data interrogating alternative splicing, gene expression analysis and biological pathways to uncover mechanisms for mutant DDX41 oncogenicity is currently ongoing. We have used CRISPR-Cas9 technology to generate Ddx41M1I mice as a model for one of the most common germline mutations in familial HM to investigate the direct effect of DDX41 mutation on hematopoiesis. Preliminary results indicate a sub-clinical reduction in hemoglobin levels in mice carrying the M1I mutation in heterozygosity when compared to their wildtype littermates. We are further investigating these mice with particular emphasis on characterizing hematopoiesis longitudinally to better understand mechanisms of disease onset and progression. This integrative genomic and functional approach to evaluate both mutations and biological pathways affected in DDX41 mutated malignancies will provide biological insight facilitating advancement in diagnosis, risk assessment, monitoring and personalized treatment. Disclosures Scott: Celgene: Honoraria.

Introduction. The Australian Familial Haematological Cancer Study (AFHCS) was founded in 2004 when only one familial haematological malignancy (FHM) gene was known, RUNX1 causing thrombocytopenia and predominantly an MDS/AML HM phenotype. Today, there are over a dozen known FHM genes, mostly characterized as predisposing to myeloid malignancies. Within our AFHCS cohort of over 200 families, this is also true with a significant proportion of families with predisposition to myeloid malignancies solved with our identification of germline mutations in genes such as GATA2, RUNX1,CEBPA and DDX41. In contrast the majority of unsolved families have a predominance of lymphoid malignancies, most commonly non-Hodgkin lymphoma and chronic lymphocytic leukaemia. In several of these families we have found known pathogenic mutations in genes involved in regulation of the homologous recombination (HR) DNA damage repair (DDR) pathway (e.g. BRCA2 Y1710fs* and PALB2 K142*) that are previously characterised with regard to their known solid tumour predisposition. In the AFHCS families with these mutations we observe a mixture of hematological tumors of mostly lymphoid origin as well as non-hematological tumours. This led us to hypothesise that there is a subset of families for which the predisposition to malignancy includes a spectrum of different malignancies, i.e. pan-cancer predisposition. Further, we hypothesise that these are associated with mutations in genes with roles in HR DNA repair. In light of this we undertook a review of the phenotypic and genetic characteristics of families in our AFHCS cohort. Methods. Malignancy phenotype data, with regard to the different tumor types observed in each AFHCS family was annotated and aggregated. Covariance matrix analysis was then used to compare the number of times different tumor types were observed occurring together in a family pedigree. Genetic data: we curated whole exome sequence data for damaging mutations in genes with a known role in HR DNA damage repair. Results. A phenotype review of our cohort found 52% of assessable FHM families also had multiple types of solid tumour and/or individuals with diagnosis of multiple malignancies, indicative of a 'pan-cancer' phenotype. Covariance analysis showed that Hodgkin and non-Hodgkin lymphoma most often occurred together and also co-occurred with non-HM tumors breast, melanoma, colorectal, prostate and ovarian subtypes, implying a possible shared genetic etiology (Figure 1A). Assessment of AFHCS families for mutations in DDR genes including DNA repair (e.g. FANC/BRCA pathway genes) and chromosome cohesion (RAD21 and related) identified 18 variants segregating with malignancy including 5 frameshift or stopgain mutations. These high impact mutations included genes previously characterised in the context of autosomal recessive predisposition to hematological malignancy such as PALB2/BRCA2 family (Fanconi Anemia, see Figure 1B), NBN1 (Nijmegen Breakage Syndrome), DDX11 (Warsaw Syndrome) and others. A further 18 variants segregated in families where pan-cancer status is still under investigation. Conclusions. Phenotypic and genetic analysis of unsolved HM predisposed families have identified that familial clustering of lymphoma in actuality identifies families with pan-cancer germline genetic susceptibility, characterised by mutations in DNA damage response genes. Collectively these mutations may have a significant impact on our understanding of germline cancer predisposition, a finding supported by recent genetic population studies on sporadic cohorts, including pediatric cancer cohorts, opening the door for the application of rational therapies as evidenced by the BRCA/PARP inhibitor paradigm. However, as each individual gene mutation is rare, international collaborative studies will be essential for accurate measurements of risk and prognostication to assist individuals in the clinic. Disclosures Branford: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Qiagen: Consultancy, Honoraria; Cepheid: Consultancy, Honoraria. Scott:Celgene: Honoraria.

Approximately 8% of un-selected pediatric (Zhang et al, NEJM 2015) and adult (Huang et al, Cell 2018) cancer patients have a deleterious germline (GL) variant. However, the frequency in patients with >2 non-related cancers, a combination of different hematological malignancies (HM) or a HM and a non-hematological malignancy, is not known. We hypothesize that genetic predisposition is higher in patients with multiple cancers, compared to historical literature for patients with a single cancer. Method: Clinical and laboratory information on 213 cancer patients, enrolled in the South Australian MDS (SA-MDS) registry (n=90) and University of Chicago clinic (n=123) were analyzed. Germline variants were identified by sequencing paired bone marrow and germline samples for 217-300 cancer related genes and annotated using American College of Medical Genetics (ACMG) 2015 guidelines as pathogenic or likely pathogenic (i.e. deleterious). Additionally, somatic mutations in 240 genes were identified in the SA-MDS cohort (n=90). Results: The median age at diagnosis was 64 years (interquartile range, 61-76 years). One-hundred and five patients had therapy-related myeloid neoplasm (T-MN), following treatment with chemo- and/or radiotherapy for an unrelated prior malignancy. While 100 patients had non-related multiple cancers (MC) one of them being HM without prior exposure to chemotherapy or radiotherapy, and, 8 patients had only myelodysplastic syndrome or acute myeloid leukemia. Overall, 20.5% (42/205) patients harbored 46 deleterious GL variants, annotated according to ACMG guidelines (Fig 1A). Most GL variants were in DNA repair pathway (26/46, 57%) followed by TP53, telomere maintenance and drug transport pathways (4/46, 9% each; Fig 1B). Three DDX41 variants were also seen. The frequency of patients with deleterious GL variants was similar in T-MN (21/105; 20%) and MC (21/100, 21%; Fig 1C). Similarly, the frequency of patients with deleterious GL variants in DNA repair pathways was also similar in T-MN (13/105, 12%) and MC (11/100, 11%). Of the patients with available cytogenetic data, complex cytogenetics were more frequent in patients with a deleterious GL variant compared to those without (11/28, 39% vs 28/125, 22%; p=0.06). At the time of analysis, somatic and germline mutation data on 240 genes was available in the SA-MDS cohort only (n=90). Somatic TP53 mutations were frequent in patients with deleterious GL variant (8/15, 53%) compared to patients without deleterious GL variant (11/75, 15%; p=0.003) (Fig 1D). Interestingly, 80% (4/5) patients with deleterious GL variant in a DNA repair pathway had a somatic TP53 mutation compared to 40% (4/10) with deleterious GL variant in non-DNA repair pathways. Conclusions: Our results from a large cohort show that patients with >2 non-related cancers have a higher frequency of deleterious germline variants compared to previous reports from patients with single cancer (20.5% vs 8%). Variants in the DNA repair pathway are the most common. The presence of deleterious germline variants is associated with genetic instability as evidenced by high frequency of complex karyotypes and somatic TP53 mutations. Disclosures Scott: Celgene: Honoraria. Godley:UpToDate, Inc.: Patents & Royalties: receives royalties from a coauthored article on inherited hematopoietic malignancies ; Invitae, Inc.: Membership on an entity's Board of Directors or advisory committees.

The Australian Familial Haematological Cancer Study (AFHCS) was initiated in 2004 with the aim to define genes predisposing to hematological malignancy (HM) to offer better options for clinical decision making and genetic counselling, and to identify therapeutic targets. The study is a referral centre for Australia and New Zealand, and currently has 230 families with multiple cases of myeloid and/or lymphoid malignancies or early onset cases (Figure 1), and is growing as clinical awareness of a germline genetic basis for blood cancers increases. To date, we have identified families with causal germline variants in several predisposition genes (five GATA2, ten RUNX1, one CEBPA, ten DDX41, one SAMD9L) including novel single nucleotide variants, deletions and insertions in coding and intronic sequences using traditional Sanger sequencing and now genomic and transcriptomic technologies. Of these, one GATA2 and four DDX41 germline mutations were identified during the screening of "sporadic" MDS samples. All four DDX41 mutant samples also harbored a somatic DDX41 (R525H) variant on the other allele at a low variant allele frequency. A comprehensive clinical analysis of the RUNX1 families has uncovered segregating phenotypes, in addition to thrombocytopenia and myeloid and lymphoid malignancies, including skin disorders such as psoriasis. In an increasing number of individuals in these families, important clinical decisions have been made dependent on mutation carrier status. Recently, we have identified and characterized a unique myeloproliferative neoplasm (MPN)/acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) family with a germline Chr14q duplication that overlaps with duplications in two other reported MPN/AML families. This appears to be a unique genotypic/phenotypic entity when compared to other myeloid predisposition genes and their associated phenotypes. Interestingly, we have identified several families carrying heterozygous pathogenic/likely pathogenic variants in genes representing autosomal recessive genomic instability syndromes segregating with HM. Here mutations in the genes NBN, RECQL4, DDX11 and RAD21 appear to act in an autosomal dominant manner. Further, we have found DNA damage repair gene predicted pathogenic variants in PALB2 and BARD1 in families with both solid cancers and HM, predominantly lymphomas, implicating an expansion of the major predisposition phenotype of these gene perturbations. Familial cases of chronic lymphocytic leukemia (CLL) have been well recognized, but it has been particularly difficult to identify predisposing variants. We have identified a number of strong candidate genes/variants in CLL families including PRPF8 (Y208C and N400S) and SAMHD1 (R371H) although more families are required to confirm these. An integral part of the AFHCS is the continued generation of cell and animal models to help define mechanisms of action of predicted or known pathogenic variants, and functional model systems for testing of variants of unknown significance. To facilitate the collection of patient samples, we have adopted the use of hair bulbs as the main germline sample as they are easy to collect, can be easily sent long distance by mail at room temperature, require no culture, are quickly and cheaply processed and provide good quality DNA using automated procedures. Overall, collaborative efforts within Australia and New Zealand and internationally have been highly fruitful in solving familial cases of hematopoietic malignancies over the last 15 years, and even more concerted international efforts will be required in the future to uncover the familial basis of unsolved cases, particularly in the lymphoid lineage, and to clarify best approaches for clinical decision making and treatment options. Figure 1. Summary of AFHCS families with associated hematological malignancies. Figure Disclosures Scott: Celgene: Honoraria.

2-Hydroxy-propyl-β-cyclodextrin (HPβCD), a cholesterol scavenger, is currently undergoing Phase 2b/3 clinical trial for treatment of Niemann Pick Type C-1 (NPC1), a fatal neurodegenerative disorder that stems from abnormal cholesterol accumulation in the endo/lysosomes. Unfortunately, the extremely high doses of HPβCD required to prevent progressive neurodegeneration exacerbates ototoxicity, pulmonary toxicity and autophagy-based cellular defects. We present unexpected evidence that a poly (ethylene glycol) (PEG)-lipid conjugate enables cholesterol clearance from endo/lysosomes of Npc1 mutant (Npc1−/−) cells. Herein, we show that distearyl-phosphatidylethanolamine-PEG (DSPE-PEG), which forms 12-nm micelles above the critical micelle concentration, accumulates heavily inside cholesterol-rich late endosomes in Npc1−/− cells. This potentially results in cholesterol solubilization and leakage from lysosomes. High-throughput screening revealed that DSPE-PEG, in combination with HPβCD, acts synergistically to efflux cholesterol without significantly aggravating autophagy defects. These well-known excipients can be used as admixtures to treat NPC1 disorder. Increasing PEG chain lengths from 350 Da-30 kDa in DSPE-PEG micelles, or increasing DSPE-PEG content in an array of liposomes packaged with HPβCD, improved cholesterol egress, while Pluronic block copolymers capable of micelle formation showed slight effects at high concentrations. We postulate that PEG-lipid based nanocarriers can serve as bioactive drug delivery systems for effective treatment of lysosomal storage disorders.

Therapy-related myeloid neoplasm (t-MN) is considered to be a direct stochastic complication of chemotherapy and/or radiotherapy for primary cancer or autoimmune diseases. However, genetic predisposition is reported in 8-12% of sporadic adult cancer patients [Lu et al Nature Communication 2015 and Huang et al Cell 2018]. Similarly, genetic predispositions to t-MN have also been reported in limited single institute studies of small numbers of patients [Churpek et al Cancer 2016]. In this study, we performed comprehensive germline and somatic mutation profiling in t-MN using next generation sequencing. Matched germline material was available for 62/194 (32%) patients. Mutation profiling was correlated with clinical features including family history in 194 patients enrolled in the South Australian MDS (SA-MDS) registry and Cleveland Clinic (CC). An in-house well established filtering pipeline was used for identification of somatic mutations. Only variants with Genome Aggregation Database (gnomAD) minor allele frequency (MAF) of ≤0.01% and variant allele frequency (VAF) of ≥35% were selected for further analysis of germline variants. Variants reported in in the Catalogue of Somatic Mutations in Cancer database and MDS/AML were excluded from further analysis. Variants reported pathogenic in Breast Cancer Information Core (BIC) database and Leiden Open Variation Database (LOVD) were retained. Other variants were included if truncating (nonsense, indels, splice alterations), CADD>20, or predicted deleterious by >4/6 scoring algorithms (GERP>4, PhyloP>2, SIFT, PolyPhen2, MutationTaster and FATHMM). Forty-one (21%) t-MN patients harbored 45 rare (MAF The frequency of these germline mutations in our t-MN cohort is higher than in the general population (gnomAD; Table 1) and in patients with primary malignancies such as breast cancer and lymphoma [Lu et al Nature Communication 2015 and Churpek et al Cancer 2016]. Intriguingly, the frequency of germline FA gene mutations (FAMT) in our therapy-related myelodysplastic syndrome (t-MDS) patients is also higher than those reported in primary MDS patients (18% vs 9%, p=0.02) [Przychodzen et al 2018]. Additionally, of those with available family history, 62% of t-MN patients have first and/or second degree relatives with non-skin cancers. Significantly more patients with FA mutation (FAMT) had first and second degree relatives with cancers compared to patients without FA (FAWT) mutations (82% vs 58%; p=0.03). Additionally, chromosomes 3 and 7 abnormalities, as well as monosomal karyotype, were more frequent in FAMT cases compared to FAWT. Similarly, somatic mutations in GATA2 (10% vs 2%; p=0.02), BCOR (13% vs 4%; p=0.03) and IDH2 (10% vs 2%; p=0.02) were more frequent in FAMT compared to FAWT cases (Figure 1C). In summary, we show that at least one in five t-MN patients harbor deleterious germline mutations, and 82% of FAMT patients have a first or second degree relative with cancers. These findings have implication in management of not only t-MN patients but genetic testing for their family members. Disclosures Branford: Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Cepheid: Honoraria. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy. Hiwase:Celgene: Research Funding; Novartis: Research Funding.

Clinical Genome Resource (ClinGen) is an NIH/NHGRI-funded effort dedicated to building an authoritative central resource that defines the clinical relevance of genes and variants for use in precision medicine and research. ClinGen has developed both gene and variant expert panels to adapt the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for consistent and accurate variant classification of specific genes and diseases. Here, we describe a new effort initiated in 2018 and supported by the American Society of Hematology (ASH) in collaboration with ClinGen to develop expert panels. This effort was motivated by the increasing use of genomics in clinical hematology and the lack of resources containing expert interpretation of germline variation. This panel, named the ClinGen Myeloid Malignancy Variant Curation Expert Panel is focused on the curation and annotation of variants in genes associated with familial/inherited risk for myeloid malignancies. Our team consists of expert clinicians, clinical laboratory diagnosticians, and researchers interested in developing and implementing standardized protocols for sequence variant specific annotations of genes in inherited myeloid malignancies. The optimization of the ACMG/AMP guidelines encompasses disease-/gene-informed specifications or strength adjustments of existing rules, including defining gene-specific population frequency cutoffs, and specifying recommendations for the use of computational/predictive data, as supported by published functional and clinical data in addition to guidance on ACMG/AMP variant interpretation provided by the ClinGen effort. Our initial focus has been to organize sub-groups of teams to develop approaches for evaluating ACMG/AMP codes to interpret germline variants of the RUNX1 gene. Once the curation of RUNX1 variants is underway, we will extend our focus to include CEBPA, DDX41, ETV6, and GATA2. These efforts will be bolstered by encouraging submission of existing variant interpretations to ClinVar or other public variant databases by the Hematology community. In summary, the ClinGen Myeloid Malignancy Variant Curation Expert Panel aims to develop recommendations to optimize ACMG/AMP criteria for standardization of variant interpretation in myeloid leukemia genes and make expert-reviewed and interpreted variants available to the hematology community through ClinVar and the ClinGen website (www.clinicalgenome.org) to support patient care and research. Disclosures DiNardo: Karyopharm: Honoraria; Agios: Consultancy; Medimmune: Honoraria; Celgene: Honoraria; Bayer: Honoraria; Abbvie: Honoraria. Nichols:Incyte: Research Funding; Alpine Immune Sciences: Research Funding. Plon:Baylor Genetics: Membership on an entity's Board of Directors or advisory committees.

Background: It has been known for approximately 19 years that germline mutations in RUNX1, lead to familial platelet disorder with predisposition to myeloid malignancy (FPD-MM, OMIM 601399). Since that time researchers have identified a broad range of different RUNX1 mutations, in over 100 families. In large families, the diagnosis of malignancy shows variable penetrance among family members with the same mutation; some carriers of RUNX1 mutations do not develop malignancy. The causes of this heterogeneity are currently not known, complicating counselling and risk analysis for individual carriers. Recent advances in genetic sequencing technology applied by many FPD-MM research groups around the world have highlighted their value in understanding the somatic genetic changes that are associated with development of malignancies in germline RUNX1 mutation carriers. Collectively this information could lead to powerful insights essential for more precise risk assessment, monitoring, and therapeutic intervention. Specifically, a growing catalogue of somatic mutations associated with germline RUNX1 malignancy offers the opportunity for informed monitoring of asymptomatic RUNX1 carriers for additional high-risk somatic mutations, in turn providing the possibility for early therapeutic intervention to arrest the leukemic process. The challenge in advancing these goals for FPD-MM is the relative rarity of the disorder in individual populations. Global data sharing in a highly interactive FPD-MM research community offers a solution to this problem that benefits all patients world-wide. Aims: To create a global RUNX1 network through identifying and contacting researchers and clinicians with known and novel germline RUNX1 families, seeking their collaboration to share genomics data. To create a RUNX1.db portal to collectively house and analyse genomics data from germline RUNX1 carriers, with associated phenotype and clinical information, such that researchers have an ongoing means by which to combine their data with those generated by other groups around the world. Methods: RUNX1 network : Through existing collaborative networks, a systematic review of the literature, and referrals from initial contacts, we have identified a global network of researchers managing FPD-MM cases and families. RUNX1.db: We have adapted a custom-built variant analysis platform, VariantGrid, that is a visual web application and database designed to help scientists manage and analyse DNA variants that can be used to aggregate and analyse multiple datasets. Results: Preliminary analysis of aggregated data from the literature suggests there are features of germline RUNX1 syndrome that can be ascertained. These include frequent somatic mutation of RUNX1 in malignancy development, as well as mutations in genes associated with clonal hematopoiesis that may precede development of overt leukemia. This analysis also suggests that different types of germline RUNX1 mutations may be associated with different combinations of somatic mutations in the tumour. To further this analysis, we have identified over 70 groups internationally that either manage RUNX1 families or have identified potential germline carriers through genomics initiatives. This represents a large and growing resource for both scientific studies and clinical programs to benefit individuals with FPD-MM. Many of these groups have generated NGS data sets from patient samples which will be available for analysis through this portal. Further details and activities of the network, and results from the genomics aggregation database will be presented. Conclusion: We have created a global RUNX1 network, aggregated their data, and generated a RUNX1.db genomics portal for the continuous curation of genomics data from germline RUNX1 carriers. A preliminary analysis has already identified specific features of germline RUNX1 mutated malignancies that have clinical importance. Ongoing scientific and clinical studies through the RUNX1 network will enhance the power of aggregated data analysis, with RUNX1.db providing a central link, driving new insights that benefit patients. Disclosures Natsoulis: Imago BioSciences, Inc.: Consultancy, Equity Ownership. Guzman:Cellectis: Research Funding. DiNardo:Bayer: Honoraria; Karyopharm: Honoraria; Celgene: Honoraria; Agios: Consultancy; Medimmune: Honoraria; Abbvie: Honoraria. Borate:Novartis: Consultancy; Agios: Consultancy. Wei:Amgen: Honoraria, Other: Advisory committee, Research Funding; Pfizer: Honoraria, Other: Advisory committee; Celgene: Honoraria, Other: Advisory committee, Research Funding; Abbvie: Honoraria, Other: Advisory board, Research Funding, Speakers Bureau; Servier: Consultancy, Honoraria, Other: Advisory committee, Research Funding; Novartis: Honoraria, Other: Advisory committee, Research Funding, Speakers Bureau. Dokal:MRC, Bloodwise, Telomerase Activator Sciences: Research Funding; The Gary Woodward Dyskeratosis Congenita Trust: Membership on an entity's Board of Directors or advisory committees; Action Medical Research, European School of Haematology: Membership on an entity's Board of Directors or advisory committees; Barts and The London School of Medicine and Dentistry, Queen Mary University of London,: Employment, Research Funding; Telomerase Activator Sciences: Research Funding; Barts and The London, Queen Mary University of London: Employment; Gary Woodward Dyskeratois Congenita Trust: Membership on an entity's Board of Directors or advisory committees. Hiwase:Novartis: Research Funding; Celgene: Research Funding. Branford:Cepheid: Honoraria; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Kramer:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Daiichi Sankyo: Consultancy. Owen:Merck: Honoraria; Teva: Honoraria; AbbVie: Research Funding; Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Celgene: Research Funding; AstraZeneca: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Honoraria, Research Funding. Fitzgibbon:Epizyme: Consultancy, Research Funding; Gilead: Consultancy. Rienhoff:Imago BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Kurokawa:Astellas Pharma: Research Funding; Sumitomo Dainippon Pharma: Research Funding; Nippon Sinyaku: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Eizai: Research Funding; MSD: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Pfizer: Research Funding; Takeda Pharmaceutical: Research Funding; Otsuka Pharmaceutical: Research Funding; Teijin Pharma: Research Funding; Chugai Pharmaceutical: Research Funding.

Introduction: Myeloid neoplasms occurring after exposure to chemo- and/or radiotherapy, termed Therapy-related myeloid neoplasms (T-MN), are considered poor prognosis. This perception creates an inherent bias in treatment decision, resulting in either over- (allo-HSCT) or under-treatment (palliation) and exclusion from most clinical trials. The optimal T-MN treatment paradigm is unknown, partly because its mutational architecture is not well defined. Few small studies show mutations in only 50-60% (Ok, Leuk Res 2015; Lindsley, Blood 2015) of T-MN (compared to 80-90% in PMDS). This study compares the mutational architecture of T-MN and PMDS from the South Australian Myelodysplastic Syndrome registry. Methods: Demographic, clinical and laboratory data including cytogenetic profiles of 129 T-MN (95 T-MDS; 34 T-AML [≥20% blasts]) and 108 PMDS patients were analysed. Targeted Massively Parallel Sequencing of a custom panel of 43 myeloid neoplasms associated genes (all coding regions) was performed on diagnosis bone marrow samples. Mutations with VAF ≥3% were selected for further assessment. Overall survival (OS) was calculated from date of diagnosis to date of last follow-up or death and adjusted using time varying covariate to account for disease modifying therapy (DMT) exposure. Results: Compared to PMDS, T-MN patients were younger at diagnosis (71.1 vs 75.3 years, p Although the OS was poorer for T-MN than PMDS (10.6 vs 31.4 mo, p As expected, VL or Low risk T-MN patients were different from T-MN with intermediate, High or VH IPSS-R score (n=74) who showed higher frequency of high-risk karyotypes and poorer OS. Additionally, the mutation profile was also different between the two groups; TP53 mutations were less common while TET2, SF3B1, IDH2 mutations were significantly more frequent in low-risk T-MN. Conclusions: In our cohort, somatic mutations were seen in 93% of T-MN which is higher than published literature. TP53 and spliceosomal mutations were commonest in T-MN and PMDS, respectively. Although, the T-MN survival is poorer than PMDS, the subgroup with IPSS-R Very Low or Low risk mirrors clinical and genetic characteristics of PMDS and has similar outcome. Such patients should be managed at par with PMDS counterparts and, importantly, should not be excluded from appropriate clinical trials based on pre-MDS chemotherapy or radiotherapy exposure. Disclosures Hiwase: Novartis: Research Funding; Celgene: Research Funding.

Krüppel-like factor 5 (Klf5) encodes a zinc-finger transcription factor and has been reported to be a direct target of C/EBPα, a master transcription factor critical for formation of granulocyte-macrophage progenitors (GMP) and leukemic GMP (L-GMP). Using an in vivo hematopoietic-specific gene-ablation model we demonstrate that loss of Klf5 function leads to a progressive increase in peripheral white blood cells, associated with increasing splenomegaly. Long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC) and multipotent progenitors (MPP) were all significantly reduced in Klf5Δ/Δ mice, and knockdown of KLF5 in human CD34+ cells suppressed colony forming potential. ST-HSC, MPP and total numbers of committed progenitors were increased in the spleen of Klf5Δ/Δ mice, and reduced β1- and β2-integrin expression on hematopoietic progenitors suggests that increased splenic hematopoiesis result from increased stem and progenitor mobilization. Klf5Δ/Δ mice show a significant reduction in the fraction of Gr1+Mac1+ cells (neutrophils) in peripheral blood and bone marrow, and increased frequency of eosinophils in the peripheral blood, bone marrow and lung. Thus, these studies demonstrate dual functions of Klf5 in regulating hematopoietic stem and progenitor proliferation and localization in the bone marrow, as well as lineage choice post-GMP, promoting increased neutrophil output at the expense of eosinophil production. Refereed/Peer-reviewed

Recently our group and others have identified DDX41 mutations both as germline and acquired somatic mutations in families with multiple cases of late onset myelodysplastic syndrome and/or acute myeloid leukemia (MDS/AML), suggesting that DDX41 acts as a tumor suppressor. To determine if novel DDX41 mutations could be identified in families with additional types of hematologic malignancies, our groups screened two cohorts of families with a diverse range of hematologic malignancy subtypes. Among 289 families, we identified nine with DDX41 mutations (3%). As previously observed, MDS/AML were the most common malignancies, often of the erythroblastic subtype, and one family displayed early onset follicular lymphoma. Five novel mutations were identified, including missense mutations within important functional domains, and start-loss and splicing mutations predicted to result in truncated proteins. We also show that most asymptomatic mutation carriers have normal blood counts until malignancy develops. This study expands both the mutation and phenotypic spectra observed in families with germline DDX41 mutations. With an increasing number of both inherited and acquired mutations in this gene being identified, further study of how DDX41 disruption leads to hematologic malignancies is critical. Refereed/Peer-reviewed

Activation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) family of receptors promotes the survival, proliferation, and differentiation of cells of the myeloid compartment. Several signaling pathways are activated downstream of the receptor, however it is not clear how these induce specific biologic outcomes. We have previously identified 2 classes of constitutively active mutants of the shared signaling subunit, human (h) βc, of the human GM-CSF/interleukin-3 (IL-3)/IL-5 receptors that exhibit different modes of signaling. In a factor-dependent bipotential myeloid cell line, FDB1, an activated mutant containing a substitution in the transmembrane domain (V449E) induces factor-independent proliferation and survival, while mutants in the extracellular domain induce factor-independent granulocyte-macrophage differentiation. Here we have used further mutational analysis to demonstrate that there are nonredundant functions for several regions of the cytoplasmic domain with regard to mediating proliferation, viability, and differentiation, which have not been revealed by previous studies with the wild-type GM-CSF receptor. This unique lack of redundancy has revealed an association of a conserved membrane-proximal region with viability signaling and a critical but distinct role for tyrosine 577 in the activities of each class of mutant.

Germline predisposition to haematological malignancy is an entity with an increasing awareness amongst hematologists in both clinical and research domains, with new predisposition genes being identified at a rapid rate. Recent advances in sequencing technologies, allowing the molecular profiling of hematological malignancies, have revealed clonal evolution of tumours in germline mutation carriers. RUNX1, as the first autosomal dominant hematological malignancy predisposition gene, is the best characterised in this regard. A consensus of findings from several groups indicates that in tumours of germline RUNX1 mutation carriers, somatic alteration of the second RUNX1 allele is the most frequent event observed. This is similarly true for DDX41 germline mutated tumours, where up to 50% of tumours have mutation of the second DDX41 allele, predominantly p.R525H. Studies on germline CEBPA mutated tumours also indicate a high selection for somatic mutation of the second allele, with temporally distinct leukemias arising in patients each carrying different somatic CEBPA mutations. These findings are in contrast to germline GATA2 mutated malignancy where biallelic mutations are rare and instead monosomy 7 and somatic mutation of ASXL1 are most commonly seen. How acquired mutations and epigenetic changes, as well as additional germline modifiers, can affect malignancy phenotype, age of onset and disease penetrance within families is an emerging theme that will also be discussed. Germline predisposition syndromes also offer a unique opportunity to identify acquired changes that occur prior to overt malignancy development in carriers. Although not yet well understood, this is an area of great interest. Recent studies in germline RUNX1 mutation carries without malignancy, has identified acquired mutations in genes associated with clonal hematopoiesis (e.g. DNMT3A). These mutations occur at an earlier age than that observed in the general population for clonal hematopoiesis, raising the possibility that they may be triggers for early malignancy onset. In contrast to clonal evolution that promotes tumour development; somatic alterations may also act to rescue consequences of germline mutations through somatic revertant mosaicism. This is well established in bone marrow failure syndromes such as Fanconi Anemia and Diamond Blackfan Anemia. More recently, SAMD9 and SAMD9L germline mutated syndromes (MIRAGE syndrome and Ataxia Pancytopenia syndrome, respectively) have allowed the examination of both phenomena operating contemporaneously in families. Selective pressure to remove or correct germline gain of function mutations, that cause growth deficiencies in hematopoietic cells, leads to acquisition of mutations and chromosomal abnormalities that act to rescue cytopenias, but can also predispose to subsequent development of myelodysplastic syndrome (e.g. monosomy 7). Finally, treatment related manipulation of the hematopoietic system offers its own risks for clonal evolution leading to tumour development where germline predisposition is concerned. In severe congenital neutropenia, treatment with G-CSF may lead to an increased risk of MDS and AML, associated with somatic CSF3R and RUNX1 mutations. Additionally, where asymptomatic germline carriers are inadvertently used as stem cell donors in genetic predisposition syndromes, stem cell stress experienced from reconstituting the hematopoietic system of the host may trigger development of donor cell leukemia. A better understanding of the contexts in which clonal haematopoiesis in germline predisposition syndromes occur, offers the hope of more sensitive molecular monitoring techniques and development of improved and earlier treatment interventions. Disclosures No relevant conflicts of interest to declare.

While there have been extensive studies to define the roles of recurrent somatic mutations in AML, the contribution of germline variants to AML initiation and progression is less well established. DNA repair disorders often predispose patients to developing myeloid malignancies. In particular, biallelic mutations affecting FANC genes cause the recessive heritable bone marrow failure syndrome Fanconi Anemia (FA), which is associated with >800-fold increased risk of progression to AML. A recent explosion of cancer predisposition studies has also revealed the importance of germlineFANC variants in elevated cancer risk (Cancer Treat Rev 2012; 38:89). To investigate the role of FANC gene variants in AML we have performed a case-control study, analyzing rare, deleterious somatic and germline variants for the 19 FANC genes in adult AML and healthy controls cohorts. Whole exome sequencing was performed on diagnosis samples from 131 adult Caucasian AML patients from two major Australian centers, and a cohort of 329 healthy females. We identified rare Tier 1 variants using a minor allele frequency (MAF) < 0.001, as reported in common dbSNP137, 1000 Genome and NHLBI-ESP project databases. Combined Annotation Dependent Depletion algorithm (CADD, Nat Genet 2014; 46: 310) >10 was used to filter for FANC gene variants with high probability of pathogenicity. Sanger sequencing of matched tumour/non-tumour DNA showed the large majority of variants tested to be germline (90%), consistent with previous studies reporting that somatic FANC genes variants are extremely rare in AML (< 1%). Overall, we identified 52 FANC gene variants in 44 cases with 34% of AML cases carrying one or more variant. For independent validation we determined the presence of somatic and germline FANC variants in the TCGA AML cohort using an identical pipeline and filtering analysis. In line with our results, we found that 36% of TCGA AML patients carry at least one germline FANC variant. We investigated known disease-causing (D-C) variants in these two AML cohorts using the FA (FAMutdb) and breast cancer (kConFab and BIC) mutation databases. We found 8 D-C FANC variants in the Australian AML cohort and 5 in the TCGA cohort, with 1 variant present in both cohorts. Moreover, the frequency of D-C variants in our cohort of females with AML (n=51) is 13.7%, while the frequency in the healthy female cohort is 4.5%, comparable to that reported in the ESP database for female European-Americans (2.1%, Hum Mol Genet 2014; 23: 6815). Accordingly, we determined that deleterious FANC germline variants confer a significant increased risk of AML (P=0.018, OR=3.3 for the Australian AML cohort). Finally, we performed mutational burden analysis to investigate enrichment of variants associated with particular FANC genes across the AML cohort. This revealed a significant enrichment of FANCL variants in AML vs healthy controls (P=0.008, Figure 1). FANCL is the enzymatic component of the FA core complex that monoubiquitinates the FANCD2/I heterodimer initiating DNA repair, and its down-regulation has been linked to AML (Oncogene 2016; doi:10.1038). Several FANCL variants, found in our AML cohort, affect the catalytic RING domain and are of particular interest. These include a D-C null variant present in 2 patients, a frame shift variant in 2 patients who presented with AML at a very early age (27 and 46 years old), and a variant affecting a critical conserved residue required for monoubiquitination of FANCD2/I. In conclusion, we show enrichment of rare potentially deleterious FANC gene mutations in AML, associated with a 3-fold increased risk of developing the disease. We hypothesise that, in hematopoietic stem/progenitor cells, these variants confer a subtle defect in interstrand cross-link repair leading to an increased accumulation of mutations and subsequent development of AML. Consistent with this there have been several reports of defective DNA damage repair and increased sensitivity to DNA damaging agents in cells from FANC carriers compared to normal controls (Nat Commun 2014; 5:5496; Mutagenesis 2009; 24:67). Importantly, it is possible to target defects in several DNA repair pathways, and our finding identifies a group of AML patients who may benefit from approaches that target defective FA and homologous recombination pathways. Figure 1. A significant increase mutational burden of FANCL was observed in our AML cohort (line represents P=0.05). Figure 1. A significant increase mutational burden of FANCL was observed in our AML cohort (line represents P=0.05). Disclosures Gill: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Background: This year, germline predisposition to haematological malignancy (HM) debuts in the World Health Organization classification of myeloid neoplasms and acute leukemia (Blood, 2016;127:2391). It has been 17 years since germline mutations in RUNX1 were found to lead to familial platelet disorder (FPD) with predisposition to myelodysplastic syndrome and acute myeloid leukaemia (MDS/AML) (Nat Genet. 1999;23:166). Now, nearly 80 families have been reported with damaging germline mutations or deletions affecting RUNX1 function, associated with FPD, making it an increasingly significant clinical presence. Although thrombocytopenia and platelet dysfunction are present in almost all RUNX1 mutant carriers, we and others have observed that the predisposition to HM varies between family members, with respect to age at diagnosis and the type of malignancy, and in some cases RUNX1 mutation carriers have no apparent HM development over their lifespan. The reasons for this heterogeneity are currently unknown. Aims: We are conducting an international collaborative study examining RUNX1 mutated families. The aim of the research project is to classify the range of phenotypes correlated with RUNX1 mutations comprehensively (including non-malignant phenotypes such as skin disorders) and to determine if the type of RUNX1 mutation and the presence of other germline and acquired mutations in relevant HM genes correlate with the likelihood of HM development, or the type of HM that develops. Across all of our data we aim to analyse clinically relevant information that will be used to inform prognosis and clinical management in germline RUNX1 mutation carriers. Results:From a review of the literature for previously characterised RUNX1 mutant families most mutations are predicted to be loss-of-function, with the combination of frameshift, stopgain, splicing and deletion accounting for the majority of alterations (57, 70%) compared to missense mutations (22, Figure 1). The most common sites of mutation are R201 and R204, affected by both missense and stopgain (10 total), which lie within the nuclear localisation signal at the end of the RUNT domain (Figure 1). We also surveyed in detail 12 RUNX1 pedigrees with both novel and previously described missense, frameshift, stopgain and deletion mutations and found that, while all families developed myeloid malignancies, 6 families also had individuals who developed lymphoid malignancy (most often Acute lymphoblastic leukemia (ALL)) which was heritable in sub-families, and subject to anticipation (e.g see IV-5 and V-5 in Figure 2). Consistent with population genome wide association studies identifying RUNX1 as a susceptibility locus for psoriasis (J Autoimmun. 2015;64:66), we find that skin conditions (psoriasis, eczema) are common, and present in germline RUNX1 carriers in 50% of our families; most commonly observed in families with stopgain and frameshift mutations. Genomic analysis of selected samples confirms that mutation of the other RUNX1 allele is the most commonly acquired mutation in germline RUNX1 mutation carriers developing HM. Alterations of chromosomes 21 and 7 are also common. DNMT3A and PHF6 acquired mutations were the next most frequently observed in tumors and mutations in U2AF1 and ASXL1 in the blood of RUNX1 carriers without HM were observed, suggestive of pre-HM clonal expansion. Finally, in a family with a novel R169I RUNX1 mutation, a rare germline ASXL1 variant (E1102D, 1.0% in ExAC) was found in two RUNX1 carriers who developed early onset AML. This variant is also significantly enriched in an MDS cohort unselected for family history compared to the general population (HR 1.3, p=0.02), as well as ASXL1 N986S (0.1% in ExAC, HR 3.3, p=0.0002) suggesting they operate as germline HM risk modifiers. Interestingly RUNX1 and ASXL1 acquired mutations often co-occur in sporadic MDS/AML and our data suggests this collaboration may also occur at the germline level. Conclusions:Annotation of skin phenotypes co-existent with a family history of haematological malignancy may assist in identifying RUNX1 mutant families. Both acquired and germline mutations in known HM genes may modify germline RUNX1 driven HM penetrance and phenotype. Our data suggest that screening of RUNX1 germline mutation carriers for germline and acquired variants in other HM genes could provide an important tool for defining risk and requires further investigation. Disclosures Owen: Pharmacyclics: Research Funding; Janssen: Honoraria; Roche: Honoraria, Research Funding; Novartis: Honoraria; Gilead: Honoraria, Research Funding; Lundbeck: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Abbvie: Honoraria. Godley:UpToDate: Honoraria; Onconova, Inc.: Research Funding.

Granulocyte/macrophage colony-stimulating factor promotes growth, survival, differentiation, and activation of normal myeloid cells and plays an important role in myeloid leukemias. The GM-CSF receptor (GMR) shares a signaling subunit, βc, with interleukin-3 and interleukin-5 receptors and has recently been shown to induce activation of Janus kinase 2 (JAK2) and downstream signaling via formation of a unique dodecameric receptor complex. In this study we use 2 activated βc mutants that display distinct signaling capacity and have differential requirements for the GMR α-subunit (GMR-α) to dissect the signaling pathways associated with the GM-CSF response. The V449E transmembrane mutant selectively activates JAK2/signal transducer and activator of transcription 5 and extracellular signal-regulated kinase (ERK) pathways, resulting in a high level of sensitivity to JAK and ERK inhibitors, whereas the extracellular mutant (FIΔ) selectively activates the phosphoinositide 3-kinase/Akt and IκKβ/nuclear factorκB pathways. We also demonstrate a novel and direct interaction between the SH3 domains of Lyn and Src with a conserved proline-rich motif in GMR-α and show a selective requirement for Src family kinases by the FIΔ mutant. We relate the nonoverlapping nature of signaling by the activated mutants to the structure of the unique GMR complex and propose alternative modes of receptor activation acting synergistically in the mature liganded receptor complex.

Deregulated cell survival programs are a classic hallmark of cancer. We have previously identified a serine residue (Ser585) in the βc subunit of the granulocyte-macrophage colony-stimulating factor receptor that selectively and independently promotes cell survival. We now show that Ser585 phosphorylation is constitutive in 20 (87%) of 23 acute myeloid leukemia (AML) patient samples, indicating that this survival-only pathway is frequently deregulated in leukemia. We performed a global expression screen to identify gene targets of this survival pathway and report a 138-gene βc Ser585-regulated transcriptome. Pathway analysis defines a gene network enriched for PI3-kinase target genes and a cluster of genes involved in cancer and cell survival. We show that one such gene, osteopontin (OPN), is a functionally relevant target of the Ser585-survival pathway as shown by siRNA-mediated knockdown of OPN expression that induces cell death in both AML blasts and CD34+CD38−CD123+ leukemic progenitors. Increased expression of OPN at diagnosis is associated with poor prognosis with multivariate analysis indicating that it is an independent predictor of overall patient survival in normal karyotype AML (n = 60; HR = 2.2; P = .01). These results delineate a novel cytokine-regulated Ser585/PI3-kinase signaling network that is deregulated in AML and identify OPN as a potential prognostic and therapeutic target.

Background: Majority of MDS cases appear to be sporadic in nature, but 10-15% have clear familial basis due to predisposing mutations in genes such as RUNX1, GATA2, CEBPA and DDX41. Contribution of germline variants in sporadic MDS is not studied. This study attempts to address the contribution of germline variants in MDS pathogenesis. Methods: We performed amplicon-based massively parallel sequencing (AmpliSeq custom panel adapted for Illumina HiSeq2500 sequencing) on all coding regions of 29 myeloid genes for 144 MDS samples. After identifying the variants in five genes (TET2, MET, GATA2, ASXL1, NOTCH1), we tested an additional 96 MDS samples including therapy-related myeloid neoplasm (T-MN) using a Sequenom assay. We also analyzed WES data for these variants in 178 AML samples and 758 normal controls and AmpliSeq data for ASXL1 and TET2 variants in 655 CML samples. Results: Collation of all coding variants in the 29 myeloid genes sequenced identified germline variants occurring in primary MDS at frequencies significantly higher than expected when compared to the normal population (ExAC and matched cohort were similar) (Table 1). These variants occurred in 5 genes (TET2, MET, GATA2, ASXL1 and NOTCH1) at increased frequencies of 1.5-16.6 fold. Numerous MDS samples had multiple variants (4 with 4 variants, 4 with 3 variants, 18 with 2 variants) while 70 had 1 variant. The 3 germline MET variants have been previously investigated in solid tumorigenesis and likely generate MET variant proteins that contribute to numerous cancer types including MDS. Interestingly, 7/17 (41%) MDS cases with germline MET variants also had other cancers including pancreatic, gastric and laryngeal cancers. Of the TET2 variants, Y867H and P1723S were concurrent in 5 MDS, 5 AML and 6 CML samples indicative of them being on the same allele (i.e. a haplotype). They were seen at higher than normal frequency in MDS and AML, but were not significantly enriched in CML. We are currently confirming their coexistence on the same allele and assaying for decreased TET2 activity to determine whether one or both variants contribute to the phenotype. Other variants identified in MDS include the rare GATA2 (P161A) variant which is present in 1% of the population and the nearby common GATA2 (A164T) allele (~20%). These were mutually exclusive in our cohort and were seen at 3.9 and 1.5-fold, respectively, above the expected population frequency. We generated the P161A variant using site-directed mutagenesis and assayed for GATA2 transactivation activity in HEK293 cells with a GATA2-responsive LYL1 promoter-Luciferase construct (Figure 1). We also included empty vector (EV), wildtype (WT) GATA2 and T354M which is the most common highly penetrant autosomal dominant mutation leading to familial MDS/AML. As expected, T354M displayed a marked decrease in transactivation ability when compared to WT. The P161A variant similarly displayed loss-of-function in this assay, but not to the same magnitude as T354M. This is consistent with the hypothesis that reduced GATA2 function predisposes to myeloid malignancy where decreasing GATA2 activity correlates with increasing risk of developing malignancy. In our study 10/36 (28%) cases harboring these variants were T-MN cases. Apart from MET (E168D) (11.4-fold), the 2 rare variants with highest frequency in MDS versus controls were ASXL1 (N986S) (16.6-fold) and NOTCH1 (R912W) (6.5-fold). ASXL1 is an epigenetic regulator often mutated in hematopoietic malignancy and aberrant NOTCH1 function has been associated with myeloid and lymphoid malignancies. Conclusions: We have identified common and rare germline variants in genes involved in myeloid malignancy that may contribute to MDS pathogenesis. It remains to be seen whether they contribute to initiation, maintenance and/or progression of MDS and other hematopoietic malignancies. This is the first study reporting higher frequency of germline variants in sporadic MDS cases. Table 1. Frequency of germline variants in MDS, AML and CML in comparison to ExAC. Table 1. Frequency of germline variants in MDS, AML and CML in comparison to ExAC. Disclosures Hiwase: Celgene Corporation: Research Funding.

Acute Myeloid Leukemia (AML) remains a clinical challenge, with most patients dying of relapsed disease. The complete biological basis of relapse remains unclear. Genetic lesions and heterogeneity have been proposed as key drivers of clinical outcome, yet do not fully explain leukemia relapse. Epigenomic dysregulation is a hallmark of newly diagnosed AML. Plasticity is a core property of the epigenome, enabling cells to adapt to stressful conditions, independent of genetic alterations. Hence we asked whether epigenomic plasticity might contribute to AML progression, have functional consequences and be independent of genetic influences in AML (a question that has not been addressed for any tumor type). Methods. We formed an international consortium to collect and profile paired diagnosis and relapse AML specimens. We extracted DNA and RNA from 138 clinically annotated AML patient samples. We obtained matched germline DNA as genetic controls, and fourteen normal CD34+ specimens as DNA methylation and transcriptome controls. We performed methylome sequencing (ERRBS), genomic sequencing (exomes and targeted resequencing) and transcriptomic (RNA-seq) profiling. For a single patient, more intensive multi-layer profiling (whole genome sequencing, ERRBS, RNA-seq and single cell RNA-seq) was performed at five serial time points. We quantified epigenetic allelic heterogeneity (epialleles) using a novel approach that employs entropy equations (MethClone), and validated epiallele composition using orthogonal methods. Some of the major conclusions are: 1) Epigenetic allelic diversity is an independent variable linked to clinical outcome. Statistically significant epiallele shift (ΔS 2) Promoter epialleles are linked to hypervariable transcriptional regulation. We observed substantial change in epiallele burden at relapse versus diagnosis. A subset of the eloci localized to gene promoters. High promoter epiallele variance was significantly associated with high transcriptional variance (p 3) AML patients can be classified according to epigenetic allele progression at relapse. K-means clustering based on epiallele shift at diagnosis versus relapse distributed patients into three classes: those with reduced, increasing or stable epiallele burden. Strikingly, there was no correlation between epiallele changes and the patterns of genomic evolution. Furthermore, there was no correlation between epiallele patterns acquired with mutations in epigenetic modifiers or other recurrently mutated genes in AML. 4) Epigenetic heterogeneity upon disease relapse is divergent from the genetic landscape. Integrating whole genome sequencing and methylome analysis we observed that a) significant increases in epigenetic heterogeneity precede significant changes in the abundance of somatic mutations; b) whereas a high number of somatic mutations were shared across all time points, epialleles exhibited dominance of distinct and unique eloci at each time point; and c) the variant epiallele frequency decreased earlier in progression than somatic mutation variant allele frequency, suggesting that epigenetic clonal diversification can precede genetic clonal evolution. Summary. Based on our results we propose that epigenetic allele diversity allows populations of leukemia cells to sample transcriptional states more freely thus creating the potential for greater evolutionary fitness. This provides an additional independent mechanism of plasticity that can explain the resilient nature of AML to adapt and survive exposure to chemotherapy drugs, independent of genetic heterogeneity. Disclosures Perl: Actinium Pharmaceuticals: Consultancy; Asana Biosciences: Consultancy; Arog Pharmaceuticals: Consultancy; Ambit/Daichi Sankyo: Consultancy; Astellas US Pharma Inc.: Consultancy. Becker:Millenium: Research Funding. Lewis:Roche: Honoraria, Other: Travel; Amgen: Other: Travel. Levine:Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.

BACKGROUND: We have previously shown that one target of hyper-methylation in AML is the promoter of the tumour suppressor and stress-response mediator Growth Arrest and DNA Damage inducible 45A (GADD45A) (GADD45AmeHI; 42% of AML). In mice Gadd45a has recently been shown to play a critical role in HSC stress responses. Gadd45a deficiency leads to enhanced HSC self-renewal, DNA damage accumulation in HSC, increased susceptibility to leukemogenesis, and impairment in HSC apoptosis after genotoxic exposure (Chen et al, Blood 2014). These findings suggest that hypermethylation of the GADD45A gene may play an important role in the altered properties of HSC, leukaemic initiation and progression. Promoter hypermethylation of this gene defines a patient group with poor survival on standard therapy (Perugini et al, Leukaemia 2012). To explore further the molecular basis of the GADD45AmeHI patient group weperformed genetic profiling of diagnosis samples using a Sequenom multiplex mutation panel, or using whole exome sequencing for broader coverage (n=95 patients).Sequenom MassARRAY was used for quantitative detection of GADD45A promoter methylation in patient samples. For a cohort of matched diagnosis and relapse samples we used CpG methylation data for GADD45A determined by ERRBS (Akalin et al, PLoSGenetics 2012). Response to cytotoxic drugs and assessment of drug combinations with 5-Aza-deoxycytidine (decitabine, DAC) and anthracycline (Daunorubicin, DNR) was performed in AML cell lines, and with primary leukemic cell populations. RESULTS: The association of the GADD45AmeHI patient group with poor outcome was validated in an independent AML patient cohort of 48 patients from the Alfred Hospital, Melbourne, Australia (p=0.003; HR3.35). Whole exome sequencing and Sequenom multiplex analysis of 95 AML patients revealed a striking co-occurrence of the GADD45AmeHI phenotype with mutations in IDH1, IDH2, and TET2 (p We next tested whether reactivation of GADD45A expression in GADD45AmeHI patient samples could be achieved through the use of hypo-methylation agents, and whether this is beneficial for response to chemotherapy. DAC treatment has been reported to induce DNA demethylation and GADD45A reactivation in primary AML samples (Klco et al, Blood 2013), and we observe reduced GADD45A promoter methylation and increased expression following DAC treatment of the GADD45AmeHI AML cell line (Mv4;11), consistent with DNA methylation-induced gene silencing of GADD45A. DAC pre-treatment of the GADD45AmeHI AML cell lines MOLM13 and Mv4;11, and three primary AML samples (GADD45AmeHI), resulted in increased GADD45A expression and increased DNR sensitivity. CONCLUSIONS: DNA methylation of the GADD45A proximal promoter marks a large percentage of AML patients at diagnosis including the majority of those with IDH1/2 and TET2 mutations (collectively these occur in 28% of AML (Network CGAR, N Engl J Med, 2013)), and is an independent predictor of poor outcome in two independent patient cohorts. Our data shows that silencing of GADD45A through increased promoter CpG methylation maybe an important early event in leukaemogenesis associated with impaired TET2 activity. Based on recent studies describing the properties of Gadd45a-deficient murine HSC we suggest reduced GADD45A activity in this subset of patients may contribute to the properties of pre-leukaemic HSC that have been associated with IDH1/2 mutation and reported to display clonal expansion, resistance to chemotherapy, and ultimately a high risk of relapse. In vitro drug experiments suggest that a priming schedule of DAC followed by DNR may provide a successful tailored treatment strategy for GADD45AmeHI patients, in combination with GADD45A expression as a biomarker predicting increased DNR sensitivity. Fig 1: Co-association of GADD45AmeHI with IDH1/2 and TET2 mutations in 95 AML patients Fig 1:. Co-association of GADD45AmeHI with IDH1/2 and TET2 mutations in 95 AML patients Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Activating mutations of the FLT3 gene are common in acute myeloid leukemia (AML), with approximately 25-30% of cases containing an internal tandem duplication (ITD) in the juxtamembrane domain. The presence of a FLT3-ITD mutation is associated with a poor prognosis, the severity of which, can be modulated by the combination of co-occuring mutations. In animal models, expression of Flt3-ITD by transgenesis, bone marrow transplantation or gene knock-in does not lead to an acute leukemia but a myeloproliferative disease, resembling CMML, suggesting a requirement for additional co-operating mutations (Lee et al, Cancer Cell. 2007, 12: 367). This is supported by in vivo models which demonstrate that the combination of the Flt3-ITD mutation with other genetic lesions leads to the development of an acute leukemia in mice (Chen et al, Genes Dev. 2013, 27: 1974). AIMS: To identify and characterise novel genes that alter Flt3-ITD induced MPN by using an N-ethyl-N-Nitroso-urea (ENU) mutagenesis strategy in mice with an Flt3-ITD homozygous knock-in background. METHODS: An autosomal dominant screen for Flt3-ENU co-operating mutations was carried out at the Australian Phenomics Facility by mating ENU-mutagenised male mice homozygous for the Flt3-ITD knock-in allele to homozygous Flt3-ITD females. G1 mice were screened for changes in blood cell parameters indicative of an altered disease state (compared to that induced by Flt3-ITD alone). Mice with blood cell parameters outside two standard deviations of the relevant G1 mean were identified as potential mutation carriers and bred to Flt3-ITD homozygous knock-in mice to test for heritability of the phenotype. Where pedigrees were generated demonstrating heritable phenotypes, multiple affected and unaffected littermates were subject to exome sequencing and analysis to identify a list of candidate gene mutations segregating with the disease phenotype. RESULTS: 150 G1 mice were screened, leading to the identification of four pedigrees with heritable phenotypes marked by an exacerbated MPN. Exome sequencing has identified a short list of 3 genes for one pedigree (pedigree 37) that includes a mutation in Neurofibromatosis 1 (Nf1), a gene known to be involved in the induction of juvenile myelomonocytic leukemia and frequently lost in AML (Parkin et al,Clin Cancer Res 2010, 16:4135). In another pedigree (pedigree 24) we identified a mutation in Ndufa10 as the single candidate segregating with the phenotype (Figure 1A-B). Ndufa10 encodes a subunit of the mitochondrial respiratory complex I, which is the first and largest complex in the mitochondrial electron transport chain. Importantly, germline mutations in this gene lead to a complex I deficiency syndrome in humans, indicating that it is a critical subunit of this complex. We hypothesise that mutation of Ndufa10 leads to altered cellular metabolism in hematopoietic stem and progenitor cells which contributes to exacerbation of the MPN, possibly through an alteration in production of reactive oxygen species and a shift in the balance between glycolysis and oxidative phosphorylation. Breeding of the Ndufa10 mutation onto a non Flt3-ITD background shows that action of the mutation is not dependent on the presence of Flt3-ITD, as these mice also have altered blood counts, including increased WBC (Figure 1C). CONCLUSIONS: It is possible to identify mutations that exacerbate Flt3-ITD induced MPN through mutagenesis and an efficient blood screening strategy. In addition, using this strategy, we have identified novel mutations that act independently of Flt3-ITD to induce changes in the haematological compartment. Translation of these findings to human AML may indicate pathways that will be targets for new and complementary treatments in AML. Figure 1. A. Flt3-ITD Pedigree 24 indicating affected mice and genotyping for the Ndufa10 mutation. +/+=Ndufa10 wt, m/+=Ndufa10 heterozygous mutant. B. WBC counts for male mice from Pedigree 24 at 15-17 weeks (+/+, n=3; m/+, n=8). WBC from ENU G1 mice are shown as a comparison (G1, n=75). C. WBC counts for male mice on a wildtype C57 background at 16-18 weeks. +/+=Ndufa10 wt (n=7), m/+=Ndufa10 heterozygous mutant (n=8), m/m=Ndufa10 homozygous mutant (7). Figure 1. A. Flt3-ITD Pedigree 24 indicating affected mice and genotyping for the Ndufa10 mutation. +/+=Ndufa10 wt, m/+=Ndufa10 heterozygous mutant. B. WBC counts for male mice from Pedigree 24 at 15-17 weeks (+/+, n=3; m/+, n=8). WBC from ENU G1 mice are shown as a comparison (G1, n=75). C. WBC counts for male mice on a wildtype C57 background at 16-18 weeks. +/+=Ndufa10 wt (n=7), m/+=Ndufa10 heterozygous mutant (n=8), m/m=Ndufa10 homozygous mutant (7). Disclosures No relevant conflicts of interest to declare.

Introduction - AML is a complex group of malignancies, with heterogeneity in morphology, cytogenetics, molecular characteristics, aggressiveness and importantly, in its response to treatment and survival outcomes. Next generation sequencing by the Cancer Genome Atlas Research Network analysed 200 primary AML cases and identified 23 genes that display recurrent somatic mutations at varying frequency in AML (NEJM 368(22):2059-2074). Defects in DNA repair are frequently identified in treatment-related AML and inherited mutations in genes of DNA repair pathways predispose patients to myeloid malignancies. For example, biallelic mutations in FANC genes, which cause the recessive heritable bone marrow failure syndrome Fanconi Anaemia (FA) are associated with high risk of progression to AML and other cancers (Kutler et al.Blood, 101:1249-1256), suggesting a potential involvement of FANC gene mutations in AML pathogenesis. Methods - In this study we present a two-stage approach to gene discovery in AML: initial unbiased whole genome sequence (WGS) and whole exome sequence (WES) analysis of tumour DNA from a cytogenetically normal AML case at diagnosis and relapse, and corresponding germ-line DNA (prepared from mesenchymal stromal cells). Potential oncogenic mutations and changes associated with disease progression were identified. WES of a further 96 diagnostic AML samples further defined recurrent mutations and allowed identification of affected functional groups and networks in AML. Results – WGS and WES were performed on diagnosis, non-haematopoietic and relapse samples from an index AML patient. Somatic SNVs and indels unique to the tumour samples include a number of variants in genes previously reported as recurrently somatically mutated in AML including FLT3, WT1 and IDH2. Somatic mutations in genes not previously associated with AML were also identified including a mutation in FANCD2 (p.S1412N) present in the index AML tumour DNA at diagnosis and at relapse. Variants in genes recurrently mutated at low frequency in AML can also be disease drivers, however separating such genes from the background level of mutation in AML requires analysis across multiple samples, and sequencing studies to determine recurrence and/or mutations in proteins involved in the same functional pathway or complex. STRING-db v9.05 (Franceschini et al. NAR, 2013(41), Database issue) was used to identify a larger network of proteins, including and associated with the FANC genes, involved in homologous recombination-mediated DNA repair. Known somatic mutations from other AML studies were mapped onto this network; as shown in Figure 1 multiple genes in this extended network are affected by somatic mutation in AML suggesting a potential role in pathogenesis. Analysis of our WES data from diagnosis samples from a further 96 Australian AML cases identified an additional two somatic mutations in genes from the extended STRING-db v9.05 FANC network. In total we identified 18 mutations in the 16 classified FANC genes and 8 variants in the BLM complex as shown in Figure 2. Two of the germline FANC gene mutations, FANCM-Q13333fs and FANCD2-R926X, are known pathogenic mutations in FA. Patients with mutations in the 8 FANC genes of the core complex form a distinct subset from those with mutations in the other 8 FANC genes. 5 of the 8 patients with mutations in the BLM complex also form a separate group while BLM complex mutations are present in 2 patients that also have FANC mutations. For the two patients with acquired changes the allele frequency for these FANC mutations is greater than 25% suggesting an early origin in disease. Discussion. Our findings suggest that germline and somatic mutations affecting function of the FANC DNA repair pathway may be a recurrent abnormality in AML, potentially contributing to leukaemogenesis. FANC/BLM gene mutations frequently co-exist with mutations in DNMT3A and DNMT1; 46% of the patients with DNMT3A/DNMT1 mutations are also mutant for FANC or BLM complex genes representing significant over-representation (p = 0.021). Within the group of FANC and BLM patients there is also significant under-representation of FLT3-ITD mutations and mutations in N-RAS and K-RAS (p = 0.051), raising the possibility that defects in homologous DNA repair may favour cooperation with alternative signalling pathways. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

During recent years, several significant discoveries have been made concerning the function of ETS-domain transcription factors. This family of transcription factors was originally defined on the basis of the conserved primary sequence of their DNA-binding domains. The ETS DNA-binding domain is also conserved at the structural level and is a divergent member of the winged helix-turn-helix superfamily of DNA binding proteins. This sequence conservation is reflected by their overlapping DNA-binding specificities based on the central GGAA/T motif. In addition to DNA-protein interactions, protein-protein interactions with partner proteins often play major roles in targeting ETS-domain proteins to specific promoters. Several such partner proteins have been identified. ETS-domain proteins function as either transcriptional activators or repressors and their activities are often regulated by signal transduction pathways, including the MAP kinase pathways. Specific links between such pathways and ETS-domain proteins have been established in several different experimental systems. ETS-domain transcription factors regulate a diverse array of biological functions including mammalian haematopoiesis and Drosophila eye development. In vertebrates, many ETS-domain proteins regulate embryonic and adult haematopoiesis. Deregulation of ETS-domain protein activity often leads to tumorigenesis. Future work will uncover further details of how these transcription factors work at the molecular level to regulate specific biological processes.

Treatment failure in Acute Myeloid Leukemia (AML) is attributed in many cases to relapsed disease. Relapsed AML is a fundamental clinical challenge since most patients have poor clinical outcomes. The exact biological basis of AML relapse remains unclear. Genetic clonal evolution is widely believed to underlie the emergence of chemotherapy resistant clones. However, only limited, predominantly non-overlapping, somatic mutations and copy number aberrations were found to occur upon AML relapse. Furthermore, in a subset of cases, no relapse specific somatic mutations or copy number aberrations were identified. This suggests a role for other mechanisms in relapsed AML. We hypothesize that epigenetic plasticity and deregulation contributes to the pathogenesis of relapse in AML. To explore this notion, we performed a genome scale epigenetic and genetic analysis of thirty-nine paired diagnosis and relapsed AML human patient samples using exome capture, RNA-seq and ERRBS for DNA methylation sequencing. Exome capture was performed on each patient’s germline DNA as well. Exome capture revealed only a limited number of known recurrent somatic mutations acquired upon disease relapse, in agreement with previous reports. In contrast, upon disease relapse we identified thousands of statistically significant changes in cytosine methylation patterns. Globally, the majority of patients (85%) displayed striking predominance of DNA hypermethylation (p= 1.00433e-05, binomial test for equality of proportions) upon disease relapse. Notably a smaller set of patients displayed the opposite epigenetic phenotype with prominent loss of cytosine methylation. While differential methylation in the hypermethylated group of patients localized predominantly to CpG islands, the majority of differential methylation in the hypomethylated group localized to regions lacking both CpG islands and shores. In spite of these two distinct overall cytosine methylation patterns, the majority of differentially methylated cytosines are located in intergenic regions in all cases, and a subset of promoters were hypermethylated in almost all patients at relapse. A pathway analysis indicated that the commonly hypermethylated gene promoters at relapse are involved in the Hedghog, Wnt and calcium signaling pathways (p In order to determine whether epigenetic events linked to AML relapse could be modeled experimentally we performed a pilot study of a human AML xenograft in immunocompromised mice. Engrafted mice were treated with Ara-C at a clinically relevant dose (60mg/Kg; n=2) or vehicle alone (n=3) for five consecutive days. Human AML cells were collected at various timepoints including 28 days after Ara-C treatment where the AML had frankly relapsed in mice. Cytosine methylation profiles obtained through ERRBS revealed predominantly hypermethylated cytosines when compared to the xenotransplanted diagnostic sample (72% hypermethylated versus 28% hypomethylated). Remarkably, there was a strong overlap with gene promoters that are also aberrantly methylated in relapsed AML patients (p We conclude that there are epigenetically distinct forms of relapsed AML. Nonetheless, there is convergent epigenetic regulation of specific gene pathways that may contribute to relapsed AML pathogenesis and xenotransplanted AML mice can serve as experimental models for further study. Finally, the genomic distribution of reprogrammed methylation suggests a role for epigenetic plasticity at distal regulatory elements. Whereas it remains unclear whether these changes represent clonal selection, their extensive and dynamic range suggest that exposure to chemotherapy may alter the fidelity of mechanisms that control cytosine methylation distribution thus permitting widespread and distant epigenetic reprogramming and contributing to disease relapse. Disclosures: No relevant conflicts of interest to declare.

Abstract 2396 Background: Despite recent advances in understanding the key molecular mechanisms of leukemogenesis, the outcome for patients with Acute Myeloid Leukemia, particularly with a normal karyotype, remains poor. For this large group of patients, genetic alterations in genes such as FLT3, NPM1, CEBPA, IDH1/2, and DNMT3A provide useful prognostic information. However, risk stratification of this group remains only partially resolved and markers of response that can be therapeutically targeted would likely improve outcome for these patients. GADD45A is a tumor suppressor gene that plays cell-type dependent roles in cellular stress coordinating DNA repair and de-methylation, cell cycle arrest, and pro-apoptotic or pro-survival responses (Cancer Ther. 2009;7:268). Methylation of four discrete CpG residues in the proximal promoter of GADD45A is a hallmark of many solid tumours and has been associated with impaired cell stress signalling and reduced drug response (Cancer Res. 2009;69:1527; Oncogene. 2005;24:2705). In AML, GADD45A expression is broadly down-regulated both in normal karyotype and other cytogenetic classes. Down-regulation of GADD45A in AML has been associated with FLT3-ITD (Leukemia. 2009;23:729) and RUNX1 mutations (Satoh et al, Leukemia. 2011;Epub). For those patients without these mutations, the mechanism of GADD45A down-regulation and its prognostic significance remains unknown. We hypothesised that the promoter of GADD45A is methylated in AML and that this methylation is functionally important in patient response. Methods: Using the Sequenom MassARRAY methodology we screened for methylation of four GADD45A promoter CpG dinucleotides (CpG1–4) previously shown to be associated with silencing of GADD45A in breast and prostate cancer, in a retrospective cohort of 222 AML patients collected at diagnosis from the Royal Adelaide Hospital. We then determined association of CpG methylation with outcome and mutation status in our treated patient cohort of 167 patients. In AML cell lines and in primary patient samples we also determined the response to cytotoxic agents in vitro in the presence and absence of demethylating agents. Results: We observed hypermethylation of the CpG1–4 in the proximal promoter of GADD45A in 93 of 222 (42%) of AML patients and in 6 AML cell lines. In the 167 patients treated with standard induction chemotherapy regimes, 61 patients showed methylation of the GADD45A proximal promoter. Of the four CpG residues, methylation of CpG1 was associated with poor overall and event-free survival, in AML overall (Figure 1A) and in normal karyotype AML (Figure 1B). GADD45A CpG1 methylation was significantly associated with IDH1 and IDH2 mutations (p Conclusions: DNA methylation of the GADD45A proximal promoter marks a large percentage AML patients, including those with with IDH1/2 mutations, and is an independent predictor of poor outcome, particularly in AML patients with normal karyotype. Our biological data shows that induction of GADD45A mRNA expression with decitabine in methylated samples is associated with increased response to cytotoxic agents. Thus GADD45A proximal promoter methylation represents a new biomarker that may provide prognostic information in the heterogeneous normal karyotype AML group. Given that recent clinical trials are combining Azacitidine with chemotherapy and other agents for induction therapy in AML (Blood;118:1472), this may represent a marker to help define patients that will benefit from this approach. Disclosures: Wei: Celgene: Research Funding.

Abstract 3508 Background: Krüppel-like factor 5 (KLF5) is a transcription factor with regulatory roles in cell growth, survival, and differentiation. Our previous studies have indicated that KLF5 is required for myeloid blood lineage differentiation and is a target of genomic methylation in Acute Myeloid Leukaemia (AML) which is associated with reduced expression. Further, we have shown that treatment of leukaemia cell lines, displaying KLF5 hypermethylation, with demethylating agents is associated with induction of KLF5 expression, supporting an inhibitory role of methylation on expression (Diakiw et al, Leuk Res 36:110). Other recent studies have shown that KLF5 expression is reduced by the presence of a heterozygous genotype at a promoter SNP (rs3812852) and plays a role in pathogenic processes in heart and brain. We investigated both methylation and SNP genotype as mechanisms of KLF5 gene regulation in AML and their relationship to patient outcome. Methods: We analysed 232 diagnostic bone marrow mononuclear cell samples from a retrospective cohort of patients with AML. The genotype of the rs3812852 SNP was determined by Sequenom MassARRAY genotyping. DNA methylation of the KLF5 gene locus (intron 1) was assessed using Sequenom MassARRAY epityper. KLF5 gene expression was determined for 85 of these samples using quantitative real-time PCR. Of the 232 patients, 161 received treatment with induction chemotherapy and were included in survival analysis. Results: We found that 28 (12%) patients were heterozygous (G/A) for the SNP (rs3812852) in the KLF5 promoter, with one patient having the rare homozygous genotype (G/G) and 204 (88%) patients the common homozygous genotype (A/A). KLF5 gene expression correlated with the SNP genotype, with the heterozygous patients displaying significantly reduced KLF5 expression compared to normal controls (CD34 cells from normal donors, p=0.044). Consistent with our previous study on a smaller test cohort we observed that hypermethylation of this region of KLF5 is common and is present in 50% of samples in our larger cohort. AML patients with high methylation of KLF5 had significantly reduced KLF5 expression compared to normal controls and AML patients with low KLF5 methylation (p=0.032 and p=0.048 respectively). The correlation of gene expression with the SNP genotype and KLF5 methylation groups was also true when considered together, as patients with the heterozygous genotype (A/G) and high methylation had lower KLF5 expression than patients with the common homozygous genotype (A/A) and low KLF5 methylation (Figure 1A). Cox multi-variate analysis (variables of age, WCC and mutational status of FLT3-ITD, IDH1/2, NPM1) of survival of patients treated with induction chemotherapy showed that the presence of a G/A genotype at the KLF5 promoter SNP was not an independent predictor of overall survival (p= 0.502). In contrast hypermethylation of KLF5 intron 1 was a highly significant independent predictor of poorer overall survival (median survival 10.4 months versus 20.7 months, HR 1.5, p=0.001, Figure 1B). Importantly, when the heterozygous SNP genotype (A/G) and high methylation status were combined, this group of patients had even poorer OS (median survival 5.2 months, HR 3.6, Figure 1C) and this was significant compared to the group with homozygous SNP genotype (A/A) and low methylation (p=0.002). Conclusions: We have shown that both methylation of KLF5 intron 1 and a heterozygous genotype at a promoter polymorphism are associated with reduced KLF5 expression in AML. KLF5 hypermethylation is an independent predictor of poor prognosis and combined with the heterozygous SNP genotype defines a group with particularly poor survival indicating a connection with KLF5 gene expression and response to treatment. Importantly, as the majority of the effect is associated with hypermethylation of KLF5, these patients (50%) may benefit from treatment with demethylating agents. Disclosures: Wei: Celgene: Research Funding.

Abstract 2313 Krüppel-like factor 5 (KLF5) is a member of a highly conserved group of transcription factors (the KLF family) that have regulatory roles in development and in the growth and differentiation of many adult tissues. In humans, KLF5 is located at 13q21–22, which is frequently lost in multiple tumour types, including tumours of the breast, cervix, endometrium, ovary and prostate where it is associated with loss of KLF5 expression, however little is known about the potential role of Klf5 in the hematopoietic system. We initially identified that Klf5 mRNA is up-regulated during myeloid differentiation of murine myeloid cell lines (J Leukoc Biol, 80: 433), consistent with published microarray data in human and mouse primary haemopoietic system. Functionally we have shown that knockdown of Klf5 gene expression reduces granulocyte differentiation in response to cytokine simulation (Leuk Res, 2012. 36: 110). Here we show that retroviral over-expression of Klf5 in the murine myeloid FDB1 cell line induces growth arrest associated with differentiation and increased apoptosis. Consistent with this, enforced expression of Klf5 in primary mouse bone marrow progenitors inhibited cell growth in liquid culture, with a reduction in c-Kit+ cells co-incident with an increase in differentiated myeloid cells at day 3 post-transduction. In colony forming assays, Klf5-expressing cells had significantly reduced myeloid colony forming ability in response to a combination of IL-3, IL-6 and SCF as well as M-CSF and G-CSF alone. To extend this functional analysis of Klf5 we have generated an in vivo gene-ablation model. As non-conditional Klf5 knockout (KO) die at embryonic day 8.5 we have generated pan-hematopoietic Klf5 conditional gene KO mice by using the Cre/LoxP recombination system with vav-cre transgenic mice. For this we first generated Klf5 floxed mice (Klf5fl/fl) allowing specific deletion of exon 2, which is the largest exon of Klf5 and encodes the majority of the protein. Klf5 fl/fl , Vav-cre+/− mice were born at the expected Mendelian ratios and showed no difference in overall weight or growth compared to heterozygote Klf5 KO or floxed littermates. We analysed the Klf5fl/fl, Vav-cre+/− mice (n=10) and Klf5fl/fl mice (n=9) at 12 weeks of age for defects in steady state hematopoiesis. Interestingly, our preliminary data shows that the spleen weight of Klf5 KO animals (Klf5fl/fl, Vav-cre+/−) is significantly higher compared to Klf5fl/fl mice (mean 0.097 vs 0.070 gram respectively, p Overall our data in the Klf5 conditional KO model are consistent with Klf5 playing a role in growth and differentiation in the myeloid lineage, as suggested by our in vitro data, and further suggests that Klf5 may have roles in multiple haematopoietic lineages. We will present further data from this model including flow cytometric analysis assessing the stem and progenitor cell compartments from bone marrow, as well as clonal assays to enumerate different haemopoietic progenitors and to assess changes in self renewal capacity of cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 1690 Background – Acute Myeloid Leukaemia (AML) has a worldwide incidence of approximately 3.5 per 100,000 population per year with most cases occurring in adults. Survival at 12 months is less than 30% and at 5 years is less than 10% (NCI, 2010). AML is a complex disease that demonstrates marked heterogeneity morphologically, cytogenetically and molecularly. The largest cytogenetic subgroup of AML has normal cytogenetics and accounts for approximately 45% of de novo AML cases. Furthermore, the response to treatment and survival outcomes of cytogenetically normal AML (cn-AML) is remarkably heterogeneous. Aims – – To undertake whole-genome and exome sequence analysis of a cn-AML case at diagnosis and at relapse to identify subtle, potentially oncogenic mutations at the molecular level that may initiate AML and those that may be responsible for drug resistance or relapse. Specific Aims: i. –Comparison of the AML genomes at diagnosis, remission and relapse to identify genomic regions with changes in copy number and loss of heterozygosity, using single nucleotide polymorphism (SNP) microarray analysis. ii. Identify chromosomal rearrangements such as small insertions, duplications, inversions and deletions, (“indels”) and translocations that may contribute to AML initiation or relapse, using massively parallel, ultra-high throughput (“next generation”), paired-end sequencing of total genomic DNA at low depth. iii. Identify DNA point mutations potentially affecting protein function that may contribute to AML initiation or relapse by using next generation sequencing at high depth of the captured exomes (putative coding regions) of the AML and control genomes. Methods – The PwC (Price-Waterhouse-Coopers) Leukaemia and Lymphoma Tissue Bank, a joint initiative of the Australasian Leukaemia and Lymphoma Group and the Leukaemia Foundation, provided samples from an AML patient with normal cytogenetics and a blast count at diagnosis of 70% and at relapse of 85%. Genomic DNA extracted from autologous mesenchymal stem/stromal cells (MSCs) was used to represent non-leukaemic, germline, control DNA. Primary cell culture of MSCs from cryopreserved bone marrow aspirate cells (50 × 106) of our test patient at remission was achieved with standard tissue culture methods. High molecular weight DNA was extracted from the patient's MSCs and marrow cells at diagnosis, remission and relapse samples. Sonication (Covaris) and libraries appropriate for paired-end high-throughput sequencing (Illumina Genome Analyzer II instrument) were prepared from gel purified DNA fragments approximately 200 bp in size. Results and Discussion – Primary cell culture of MSCs from bone marrow aspirates proved to be a robust source of germline genomic DNA with several advantages over skin. Firstly, tissue banked samples, will not include biopsies of normal skin. Furthermore, skin can be contaminated by circulating leukaemic cells, which is problematic with low-depth genomic sequencing. The homogenous immunophenotype of the cultured MSCs indicate their purity. Preliminary SNP microarray analysis identified a large region of uniparental disomy (copy number neutral loss of heterozygosity) involving most of chromosome 13q, which was not identified by standard cytogenetic analysis. Low-pass or shallow paired-end genomic DNA sequencing has generated the following outputs. MSC genome: 10.7 Gb (3.6X haploid genome coverage) resulting from 152 × 106 paired sequence reads, AML_diagnosis genome: 21.6 Gb (7.2X) resulting from 142 × 106 paired sequence reads, AML_relapse genome: 26.8 Gb (8.9X) resulting from 177 × 106 paired sequence reads. The capture baits representing the human exome span 26,225,870 bp (approximately 0.9% of haploid human genome). Exome capture libraries were prepared from the three sources of DNA (MSC, AML_diagnosis and AML_relapse) and sequenced using a single lane for each library. The outputs for all three were very similar, approximately 23 × 106 paired sequence reads, representing 1.3 Gb (49X haploid exome coverage). The reads for whole genome or exome capture were of high quality and more than 98% could be unambiguously aligned to the human reference genome in the correct orientation and interval distance. Here we will present MSC enrichment results and sequencing output results, quality control and preliminary analysis of genomic alterations and exonic mutations. Disclosures: No relevant conflicts of interest to declare.

Abstract LBA-3 Mutations in the transcription factor genes, RUNX1 and CEBPA, can lead to an autosomal dominant familial predisposition to MDS/AML. Using a candidate gene approach, we have detected domain specific heterozygous mutations in the GATA2 gene in 4 MDS/AML families which predispose to MDS/AML. The same novel heterozygous T354M missense mutation was observed in 3 families and a 355delT mutation in 1 family, all with multigenerational transmission of MDS and/or MDS/AML. Importantly, these genetic variants segregate with all affected members in each of the families. The 2 mutated threonine residues are in 5 consecutive highly conserved threonine residues at the DNA-binding, protein-protein interacting second zinc finger (ZF2) of GATA2. Neither these mutations, nor any other variants in the GATA2 coding sequence, were seen in a population screen of 695 normal individuals. Haplotype analysis suggests that the T354M mutation has multiple ancestral origins. While mutations in RUNX1 and CEBPA, can also lead to familial predisposition to MDS/AML, these patients with GATA2 mutations are unique in that there is no obvious pre-MDS or pre-leukaemic phenotype such as thrombocytopenia (RUNX1) and eosinophilia (CEBPA) in predisposed carriers. Most patients in these families have had a rapid disease course “appearing out of the blue” leading to death, with a variety of ages of onset from teenagers to early 40s. Yet remarkably, there are still asymptomatic carriers in their 60s. One of these carriers, and his 2 children, has had bone marrow prophylactically stored over 15 years ago in case of disease onset. No pathogenic GATA2 coding sequence changes were found in 268 sporadic MDS/AML patient samples. Additionally, GATA2 mutations were not found in germline samples from 35 other families predisposed to AML and various other hematological malignancies. Both the T354M and 355delT mutants appear to localize appropriately to the nucleus and maintain at least some DNA binding in electrophoretic mobility shift assays. We used the known murine Gata3 ZF2 structure bound to DNA to model the effects of the observed mutations and demonstrated that the T354 residue does not contact DNA but makes polar contact with the adjacent threonines, and via its amino group, with C349 which coordinates the zinc atom. Replacement of the T354 side-chain with the bulky methionine moiety may affect the zinc contacts and is predicted to alter the overall structure of this ZF2. In contrast, 355delT will shorten the conserved threonine string which is predicted to impact on the orientation and position of L359 which directly contacts DNA. Thus, 355delT is likely to have an effect on DNA binding. Luciferase reporter assays indicate that T354M and 355delT greatly reduce the transactivation ability of GATA2 on multiple response elements, impacting on downstream target genes such as RUNX1 and CD34. Of note, T354M shows a markedly lesser synergistic effect than wildtype (WT) GATA2 with PU.1 on the CSF1R promoter. Competition assays show that these mutations may be acting in a dominant negative fashion in some biological contexts. In stable promyelocytic HL-60 cell lines expressing regulatable GATA2 (WT or T354M), T354M allows proliferation to proceed even under stimulus to differentiate with all-trans retinoic acid. Microarray studies indicated that the down regulation of proapoptotic BCL-xS by T354M, but not WT, may be responsible for this phenotype. GATA2 is considered to be a hematopoietic “stemness” gene, highly expressed in haematopoietic stem cells and is required for megakaryocyte and mast cell production. GATA2 is down regulated during myeloid differentiation and forced overexpression prevents such differentiation. Discovery of GATA2 mutants in MDS/AML predisposed families provides new tools for probing the mechanism of GATA2 induced leukemogenesis, and possibly also for clarifying its role in maintenance of stemness. Our findings highlight the power of investigating familial predispositions to cancer identifying specific mutations with unique biological effects. They have immediate implications for diagnostic genetic testing, and longer term therapeutic implications through identification of drugable biological pathways such as apoptosis. The poor outcome associated with these mutations may suggest that an aggressive strategy is appropriate in the treatment of affected individuals in families found to be carrying GATA2 mutations. Disclosures: No relevant conflicts of interest to declare.

As a strategy to identify key targets in myeloid leukemia we examined directly the overlap in signaling and downstream events induced by several activated receptor mutants associated with AML. We compared the events induced by a leukemic mutant (V449E) of the GM-CSF receptor with events associated with the activating FLT3 internal tandem duplication mutant (FLT3-ITD) and the D835Y kinase domain mutant (FLT3-TKD). Receptor mutants were introduced by retroviral transduction into the FDB-1 bi-potential myeloid cell line model (McCormack MP et al., Blood.95:120–127, 2000). We used biochemical analysis and pathway inhibitors to demonstrate that the GMR V449E mutant selectively activates the JAK2-STAT5 and p44/42 MAPK pathways which are central to the ability of this mutant to confer continued growth and arrested differentiation in the absence of growth factor. Like the GMR V449E mutant, the FLT3-ITD and -TKD mutants arrest granulocyte-macrophage differentiation and support continued growth in the absence of added growth factor. For both of these mutants we also observed constitutive JAK2 and STAT5 phosphorylation and sensitivity to a selective JAK2 inhibitor (JAK2 Inhibitor II, Merck) suggesting a potential key role for JAK2 in signalling from FLT3 activating mutants. Both FLT3 mutants activated the p44/42 MAPK pathway, resulted in Akt phosphorylation and were sensitive to PI3-kinase and MEK inhibitors. As activation of the p44/42 MAPK pathway is common to all mutants, and has recently been associated with suppressed differentiation in AML (Radomska HS et al, J Exp Med. 203:371–381, 2006), we used the selective MEK inhibitor, U0126 (Merck), to investigate the contribution of this pathway to maintenance of the differentiation block. Treatment of factor-independent FDB-1 cells expressing the GMR V449E mutant, or either FLT3 mutant, with U0126 for 48 hours induced morphological differentiation suggesting that the sustained activation of p44/42 MAPK contributes to maintaining the arrest in myeloid differentiation. To explore further the overlapping nature of signalling by the GMRV449E and FLT3-ITD activated mutants we utilised the Wilcoxan rank sum test to perform gene-set enrichment analysis. Analysis of 119 FLT3-ITD associated genes (126 probes) (Mizuki M et al, Blood101: 3164–3173, 2003) revealed that this gene-set shows evidence of differential expression in response to GMR V449E signaling (p=2.8x10−7). In particular, we identify the tumor suppressor gene, Gadd45α, as a common gene, down-regulated in response to signalling via the FLT3-ITD, FLT3-TKD and GMR V449E activated receptors. Analysis of AML gene expression data (Valk PJ et al, New Engl. J. Med: 350:1617–1628, 2004) indicates that activation of Gadd45α maybe suppressed by multiple pathways in AML blasts.

trk A tyrosine kinase (the high affinity receptor for nerve growth factor) binds to the p53 tumour suppressor protein in vitro and in vivo. Our aim was to determine which regions of p53 are involved in trk A association. In vitro binding experiments using baculovirus expressed trk A and in vitro transcribed and translated C-terminus p53 deletion mutants show amino acids 327–338 critical for association. Also, analysis with mutants at the N-terminus, conserved regions II, III, IV and V or amino acid positions 173, 175, 181, 248 and 249 (which are amino acids frequently mutated in a variety of neoplasms and transformed cell lines), show that these sites are not involved in trk A binding. Importantly, similar results are obtained after immunoprecipitation of lysates from p53 negative fibroblasts expressing trk A and the above p53 mutant proteins. These data suggest that the amino-terminus of the oligomerisation domain of p53 is involved in p53/trk A association.

Background DNA methylation profiling reveals important differentially methylated regions (DMRs) of the genome that are altered during development or that are perturbed by disease. To date, few programs exist for regional analysis of enriched or whole-genome bisulfate conversion sequencing data, even though such data are increasingly common. Here, we describe an open-source, optimized method for determining empirically based DMRs (eDMR) from high-throughput sequence data that is applicable to enriched whole-genome methylation profiling datasets, as well as other globally enriched epigenetic modification data. Results Here we show that our bimodal distribution model and weighted cost function for optimized regional methylation analysis provides accurate boundaries of regions harboring significant epigenetic modifications. Our algorithm takes the spatial distribution of CpGs into account for the enrichment assay, allowing for optimization of the definition of empirical regions for differential methylation. Combined with the dependent adjustment for regional p-value combination and DMR annotation, we provide a method that may be applied to a variety of datasets for rapid DMR analysis. Our method classifies both the directionality of DMRs and their genome-wide distribution, and we have observed that shows clinical relevance through correct stratification of two Acute Myeloid Leukemia (AML) tumor sub-types. Conclusions Our weighted optimization algorithm eDMR for calling DMRs extends an established DMR R pipeline (methylKit) and provides a needed resource in epigenomics. Our method enables an accurate and scalable way of finding DMRs in high-throughput methylation sequencing experiments. eDMR is available for download at http://code.google.com/p/edmr/.