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Summary Background Data to inform surveillance and treatment for leukaemia predisposition syndromes are scarce and recommendations are largely based on expert opinion. This study aimed to investigate the clinical features and outcomes of patients with myelodysplastic syndrome or acute myeloid leukaemia and Shwachman-Diamond syndrome, an inherited bone marrow failure disorder with high risk of developing myeloid malignancies. Methods We did a multicentre, retrospective, cohort study in collaboration with the North American Shwachman-Diamond Syndrome Registry. We reviewed patient medical records from 17 centres in the USA and Canada. Patients with a genetic (biallelic mutations in the SBDS gene) or clinical diagnosis (cytopenias and pancreatic dysfunction) of Shwachman-Diamond syndrome who developed myelodysplastic syndrome or acute myeloid leukaemia were eligible without additional restriction. Medical records were reviewed between March 1, 2001, and Oct 5, 2017. Masked central review of bone marrow pathology was done if available to confirm leukaemia or myelodysplastic syndrome diagnosis. We describe the clinical features and overall survival of these patients. Findings We initially identified 37 patients with Shwachman-Diamond syndrome and myelodysplastic syndrome or acute myeloid leukaemia. 27 patients had samples available for central pathology review and were reclassified accordingly (central diagnosis concurred with local in 15 [56%] cases), 10 had no samples available and were classified based on the local review data, and 1 patient was excluded at this stage as not eligible. 36 patients were included in the analysis, of whom 10 (28%) initially presented with acute myeloid leukaemia and 26 (72%) initially presented with myelodysplastic syndrome. With a median follow-up of 4·9 years (IQR 3·9–8·4), median overall survival for patients with myelodysplastic syndrome was 7·7 years (95% CI 0·8–not reached) and 0·99 years (95% CI 0·2–2·4) for patients with acute myeloid leukaemia. Overall survival at 3 years was 11% (95% CI 1–39) for patients with leukaemia and 51% (29–68) for patients with myelodysplastic syndrome. Management and surveillance were variable. 18 (69%) of 26 patients with myelodysplastic syndrome received upfront therapy (14 haematopoietic stem cell transplantation and 4 chemotherapy), 4 (15%) patients received no treatment, 2 (8%) had unavailable data, and 2 (8%) progressed to acute myeloid leukaemia before receiving treatment. 12 patients received treatment for acute myeloid leukaemia—including the two patients initially diagnosed with myelodysplastic who progressed— two (16%) received HSCT as initial therapy and ten (83%) received chemotherapy with intent to proceed with HSCT. 33 (92%) of 36 patients (eight of ten with leukaemia and 25 of 26 with myelodysplastic syndrome) were known to have Shwachman-Diamond syndrome before development of a myeloid malignancy and could have been monitored with bone marrow surveillance. Bone marrow surveillance before myeloid malignancy diagnosis was done in three (33%) of nine patients with leukaemia for whom surveillance status was confirmed and 11 (46%) of 24 patients with myelodysplastic syndrome. Patients monitored had a 3-year overall survival of 62% (95% CI 32–82; n=14) compared with 28% (95% CI 10–50; n=19; p=0·13) without surveillance. Six (40%) of 15 patients with available longitudinal data developed myelodysplastic syndrome in the setting of stable blood counts. Interpretation Our results suggest that prognosis is poor for patients with Shwachman-Diamond syndrome and myelodysplastic syndrome or acute myeloid leukaemia owing to both therapy-resistant disease and treatment-related toxicities. Improved surveillance algorithms and risk stratification tools, studies of clonal evolution, and prospective trials are needed to inform effective prevention and treatment strategies for leukaemia predisposition in patients with Shwachman-Diamond syndrome. Funding National Institute of Health.

Background Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, short stature, congenital anomalies, and cancer predisposition. Most cases are due to mutations in genes encoding ribosomal proteins (RP) leading to RP haploinsufficiency. Effective treatments for the anemia of DBA include chronic red cell transfusions, long-term corticosteroid therapy, or hematopoietic stem cell transplantation. In a small patient series and in animal models, there have been hematologic responses to L-leucine with amelioration of anemia. The study objectives of this clinical trial were to determine feasibility, safety, and efficacy of L-leucine in transfusion-dependent patients with DBA. Procedure Patients ≥2 years of age received L-leucine 700 mg/m2 orally three times daily for nine months to determine a hematologic response and any improvement in growth (NCT01362595). Results This multicenter, phase I/II study enrolled 55 subjects; 43 were evaluable. There were 21 males; the median age at enrollment was 10.4 years (range, 2.5-46.1 years). No significant adverse events were attributable to L-leucine. Two subjects had a complete erythroid response and five had a partial response. Nine of 25, and 11 of 25, subjects experienced a positive weight and height percentile change, respectively, at the end of therapy. Conclusions L-leucine is safe, resulted in an erythroid response in 16% of subjects with DBA, and led to an increase in weight and linear growth velocity in 36% and 44% of evaluable subjects, respectively. Further studies will be critical to understand the role of L-leucine in the management of patients with DBA.

Humanized mouse model is a powerful tool for the study of human hematopoiesis and hematologic diseases in vivo. However, existing models continue to be limited by absent engraftment of stem cells with inherent growth defects, especially in bone marrow failure (BMF) disorders such as low-grade myelodysplastic syndromes (MDS) and Fanconi Anemia (FA). Previously, we presented a highly efficient MDS/AML PDX model in cytokine-humanized MISTRG mice, that express human M-CSF, IL-3, GM-CSF and THPO in the background of the SIRPα - humanized rag -/- IL2Rg -/- mice. MISTRG mice support efficient, faithful and long-term MDS hematopoietic stem and progenitor cell (HSPC) engraftment with multi-lineage representation. Similar to MDS, FA stem cells are defective and sensitive to toxic stress conferred by inflammation or irradiation. Irradiation, required in conventional mice as pre-conditioning, increases the risk of secondary malignancies, induces activation of inflammatory pathways and may damage the bone marrow microenvironment. In this study, we present MISTRG6kit W41 mice that in addition to the above cytokines express human IL-6, which improves lymphoid development, and that were CRISPR modified to introduce the white spotting 41 mutation in c-Kit (c-kit W41), which results in partial loss of c-kit function in the murine host stem and progenitor cells. To characterize this mouse model, we compare the engraftment capacity between MISTRG6 and MISTRG6kit W41 mice. We find that the HSPCs from both healthy donor and low grade MDS patient can be more efficiently propagated in MISTRG6Kit W41 mice and that they can be serially transplanted. Human cells are detectable at higher levels in PB of MISTRG6Kit W41 mice as early as 8 weeks post transplantation. As previously described, c-kit mutation enhances human erythroid engraftment while multi-lineage engraftment is maintained in engrafted MISTRG6Kit W41 mice. Therefore, MISTRG6Kit W41 mice offer a better host environment that supports human hematopoietic expansion without irradiation. FA is an inherited genetic disease carrying FA gene mutations associated with DNA damage and cancer predisposition and marked by BMF, MDS and AML. FA HSPCs cannot be expanded or grown for sufficient periods in vitro, and existing mouse models have failed to demonstrate engraftment of primary cells from FA patient. We transplanted a MDS sample from a FANCC mutant patient into MISTRG6Kit W41 mice giving rise to a myeloid predominant graft that could be successfully serially transplanted. Moreover, non-transformed FA HSPCs from 2 FANCA and 2 FANCG mutant patients were successfully engrafted in MISTRG6Kit W41 mice. In summary, MISTRG mice with human IL-6 expression and c-Kit W41 mutation provide a more efficient and convenient way for pre-clinical studies of BMFs such as FA and low-grade MDS with maintenance of an intact humanized stem cell niche, and this is the first time we saw primary cells from FA patient propagate in mouse model. We believe that our model would be highly beneficial to the study of additional diseases associated with DNA damage or cancer predisposition, such as dyskeratosis congenita or Schwachman-Diamond syndrome. Disclosures No relevant conflicts of interest to declare.

Fanconi anaemia (FA) is a genetic disorder that is characterized by bone marrow failure (BMF), developmental abnormalities and predisposition to cancer. Together with other proteins involved in DNA repair processes and cell division, the FA proteins maintain genome homeostasis, and germline mutation of any one of the genes that encode FA proteins causes FA. Monoallelic inactivation of some FA genes, such as FA complementation group D1 (FANCD1; also known as the breast and ovarian cancer susceptibility gene BRCA2), leads to adult-onset cancer predisposition but does not cause FA, and somatic mutations in FA genes occur in cancers in the general population. Carcinogenesis resulting from a dysregulated FA pathway is multifaceted, as FA proteins monitor multiple complementary genome-surveillance checkpoints throughout interphase, where monoubiquitylation of the FANCD2-FANCI heterodimer by the FA core complex promotes recruitment of DNA repair effectors to chromatin lesions to resolve DNA damage and mitosis. In this Review, we discuss how the FA pathway safeguards genome integrity throughout the cell cycle and show how studies of FA have revealed opportunities to develop rational therapeutics for this genetic disease and for malignancies that acquire somatic mutations within the FA pathway.

1. Richa Sharma, MD* 2. Grzegorz Nalepa, MD, PhD*,† 1. *Department of Pediatrics, Division of Pediatric Hematology-Oncology, Indiana University School of Medicine, Indianapolis, IN. 2. †Department of Medical and Molecular Genetics and Department of Biochemistry, Indiana University School of Medicine; Bone Marrow Failure Program, Riley Hospital for Children at IU Health, Indianapolis, IN. Diagnostic evaluation and management of children with chronic pancytopenia is highly complex. (1)(2)(3) Clinicians should be aware of the subtle signs and symptoms of acquired and congenital childhood pancytopenias to maintain a high index of suspicion, streamline the referral process, and function within their patients’ multidisciplinary medical homes. After completing this article, readers should be able to: 1. Identify signs and symptoms of pancytopenia. 2. Identify patients who require emergency referral due to a life-threatening illness. 3. Discuss the basics of clinical presentation, diagnostics, and management of childhood severe aplastic anemia. 4. Discuss the basics of clinical presentation, diagnostics, and management of common genetic syndromes associated with pancytopenia. 5. Understand the basic concepts behind laboratory tests and be aware of which tests are appropriate in specific clinical situations. Pancytopenia is characterized by a decreased number of at least two blood cell lines. Pancytopenia may progress acutely, such as with decreased blood cell counts in fulminant sepsis, disseminated intravascular coagulation, or rapid hemolysis. Alternatively, pancytopenia may evolve insidiously over weeks to months. In this review we focus on the latter group of acquired and congenital pancytopenias in children. Chronic pancytopenia is a diagnostic challenge. The range of potential causes is bewildering, signs and symptoms overlap substantially, and many diseases presenting with pancytopenia are life-threatening if not recognized and managed properly. Therefore, clinicians must be familiar with clinical scenarios that should prompt evaluation of blood counts and a pediatric hematology referral. ### Causes Pancytopenia may be …

Correct diagnosis of inherited bone marrow failure syndromes is a challenge because of the significant overlap in clinical presentation of these disorders. Establishing right genetic diagnosis is crucial for patients’ optimal clinical management and family counseling. A nondysmorphic infant reported here developed severe transfusion-dependent anemia and met clinical criteria for diagnosis of Diamond–Blackfan anemia (DBA). However, whole-exome sequencing demonstrated that the child was a compound heterozygote for a paternally inherited pathogenic truncating variant (SPTA1c.4975 C>T) and a novel maternally inherited missense variant of uncertain significance (SPTA1c.5029 G>A) within the spectrin gene, consistent with hereditary hemolytic anemia due to disruption of red blood cell (RBC) cytoskeleton. Ektacytometry demonstrated abnormal membrane flexibility of the child's RBCs. Scanning electron microscopy revealed morphological aberrations of the patient's RBCs. Both parents were found to have mild hereditary elliptocytosis. Importantly, patients with severe RBC membrane defects may be successfully managed with splenectomy to minimize peripheral destruction of misshapen RBCs, whereas patients with DBA require lifelong transfusions, steroid therapy, or hematopoietic stem cell transplantation. As suggested by the WES findings, splenectomy rendered our patient transfusion-independent, improving the family's quality of life and preventing transfusion-related iron overload. This case illustrates the utility of whole-exome sequencing in clinical care of children with genetic disorders of unclear presentation.

We describe a child with dyserythropoietic anemia, thrombocytosis, functional platelet defect, and megakaryocyte dysplasia. We show that (i) this constellation of hematopoietic abnormalities was due to a germline mutation within the 5' untranslated region (5'UTR) of globin transcription factor 1 (GATA1); (ii) the mutation impaired a 5'UTR GATA1 splicing site, with promoted production of the shortened GATA1 isoform lacking the N-terminus; and (iii) expression of the GATA1 N-terminus is restricted to erythroblasts and megakaryocytes in normal marrow, consistent with the patient's abnormal erythropoiesis and megakaryopoiesis. Our findings provide insights into the clinically relevant in vivo function of the N-terminal domain of GATA1 in human hematopoiesis.

The partially understood phosphoinositide signaling cascade regulates multiple aspects of cellular metabolism. Previous studies revealed that INPP5E, the inositol polyphosphate-5-phosphatase that is mutated in the developmental disorders Joubert and MORM syndromes, is essential for the function of the primary cilium and maintenance of phosphoinositide balance in nondividing cells. Here, we report that INPP5E further contributes to cellular homeostasis by regulating cell division. We found that silencing or genetic knockout of

The partially understood phosphoinositide signaling cascade regulates multiple aspects of cellular metabolism. Previous studies revealed that INPP5E, the inositol polyphosphate-5-phosphatase that is mutated in the developmental disorders Joubert and MORM syndromes, is essential for the function of the primary cilium and maintenance of phosphoinositide balance in nondividing cells. Here, we report that INPP5E further contributes to cellular homeostasis by regulating cell division. We found that silencing or genetic knockout of INPP5E in human and murine cells impairs the spindle assembly checkpoint, centrosome and spindle function, and maintenance of chromosomal integrity. Consistent with a cell cycle regulatory role, we found that INPP5E expression is cell cycle dependent, peaking at mitotic entry. INPP5E localizes to centrosomes, chromosomes, and kinetochores in early mitosis and shuttles to the midzone spindle at mitotic exit. Our findings identify the previously unknown, essential role of INPP5E in mitosis and prevention of aneuploidy, providing a new perspective on the function of this phosphoinositide phosphatase in health and development.

Fanconi anemia (FA) is a heterogenous genetic disease with a high risk of cancer. The FA proteins are essential for interphase DNA damage repair; however, it is incompletely understood why FA-deficient cells also develop gross aneuploidy, leading to cancer. Here, we systematically evaluated the role of the FA proteins in chromosome segregation through functional RNAi screens and analysis of primary cells from patients with FA. We found that FA signaling is essential for the spindle assembly checkpoint and is therefore required for high-fidelity chromosome segregation and prevention of aneuploidy. Furthermore, we discovered that FA proteins differentially localize to key structures of the mitotic apparatus in a cell cycle–dependent manner. The essential role of the FA pathway in mitosis offers a mechanistic explanation for the aneuploidy and malignant transformation known to occur after disruption of FA signaling. Collectively, our findings provide insight into the genetically unstable cancers resulting from inactivation of the FA/BRCA pathway.

Fanconi anemia is a complex heterogeneous genetic disorder with a high incidence of bone marrow failure, clonal evolution to acute myeloid leukemia and mesenchymal-derived congenital anomalies. Increasing evidence in Fanconi anemia and other genetic disorders points towards an interdependence of skeletal and hematopoietic development, yet the impact of the marrow microenvironment in the pathogenesis of the bone marrow failure in Fanconi anemia remains unclear. Here we demonstrated that mice with double knockout of both Fancc and Fancg genes had decreased bone formation at least partially due to impaired osteoblast differentiation from mesenchymal stem/progenitor cells. Mesenchymal stem/progenitor cells from the double knockout mice showed impaired hematopoietic supportive activity. Mesenchymal stem/progenitor cells of patients with Fanconi anemia exhibited similar cellular deficits, including increased senescence, reduced proliferation, impaired osteoblast differentiation and defective hematopoietic stem/progenitor cell supportive activity. Collectively, these studies provide unique insights into the physiological significance of mesenchymal stem/progenitor cells in supporting the marrow microenvironment, which is potentially of broad relevance in hematopoietic stem cell transplantation.

Background Atopic dermatitis (AD) is characterized by intense pruritis and is a common childhood inflammatory disease. Many factors are known to affect AD development, including the pleiotropic cytokine IL-4. Yet little is known regarding the direct effects of IL-4 on keratinocyte function. Objective and Methods In this report RNA sequencing and functional assays were used to define the effect of the allergic environment on primary keratinocyte function and wound repair in mice. Results Acute or chronic stimulation by IL-4 modified expression of more than 1000 genes expressed in human keratinocytes that are involved in a broad spectrum of nonoverlapping functions. Among the IL-4–induced changes, repression of fibronectin critically impaired the human keratinocyte wound response. Moreover, in mouse models of spontaneous and induced AD-like lesions, there was delayed re-epithelialization. Importantly, topical treatment with fibronectin restored the epidermal repair response. Conclusion Keratinocyte gene expression is critically shaped by IL-4, altering cell fate decisions, which are likely important for the clinical manifestations and pathology of allergic skin disease.

Fanconi anemia (FA) is an inherited disorder of genomic instability associated with high risk of myelodysplasia and acute myeloid leukemia (AML). Young mice deficient in FA core complex genes do not naturally develop cancer, hampering preclinical studies on malignant hematopoiesis in FA. Here we describe that aging Fancc −/− mice are prone to genomically unstable AML and other hematologic neoplasms. We report that aneuploidy precedes malignant transformation during Fancc −/− hematopoiesis. Our observations reveal that Fancc −/− mice develop hematopoietic chromosomal instability followed by leukemia in an age-dependent manner, recapitulating the clinical phenotype of human FA and providing a proof of concept for future development of preclinical models of FA-associated leukemogenesis.

Neurofibromatosis type 1 (NF1) predisposes individuals to the development of juvenile myelomonocytic leukemia (JMML), a fatal myeloproliferative disease (MPD). In genetically engineered murine models, nullizygosity of Nf1, a tumor suppressor gene that encodes a Ras-GTPase-activating protein, results in hyperactivity of Raf/Mek/Erk in hematopoietic stem and progenitor cells (HSPCs). Activated Erk1/2 phosphorylate kinases and transcription factors with myriad mitogenic roles in diverse cell types. However, genetic studies examining Erk1/2's differential and/or combined control of normal and Nf1-deficient myelopoiesis are lacking. Moreover, prior studies relying on chemical Mek/Erk inhibitors have reached conflicting conclusions in normal and Nf1-deficient mice. Here, we show that while single Erk1 or Erk2 disruption did not grossly compromise myelopoiesis, dual Erk1/2 disruption rapidly ablated granulocyte and monocyte production in vivo, diminished progenitor cell number, and prevented HSPC proliferation in vitro. Genetic disruption of Erk1/2 in the context of Nf1 nullizygosity (Mx1Cre(+)Nf1(flox/flox)Erk1(-/-)Erk2(flox/flox)) fully protects against the development of MPD. Collectively, we identified a fundamental requirement for Erk1/2 signaling in normal and Nf1-deficient hematopoiesis, elucidating a critical hematopoietic function for Erk1/2 while genetically validating highly selective Mek/Erk inhibitors in a leukemia that is otherwise resistant to traditional therapy.

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.

Diamond Blackfan anemia (DBA) is a rare, inherited bone marrow failure syndrome characterized by anemia, congenital anomalies and a predisposition to cancer. The patients usually present during infancy or early childhood, but can also present in adulthood. In the majority of cases DBA is due to a mutation in a small or large ribosomal protein (RP) subunit leading to RP haploinsufficiency. The only treatments for the anemia of DBA are red cell transfusions (accompanied by iron chelation), oral corticosteroid therapy or stem cell transplantation. Pospisilova et al. (Haematologica 2007; 92(5):e66-67) reported one complete and two partial erythroid responses after the use of the branched chain amino acid L-leucine in 6 select patients. In skeletal muscle, leucine supplementation can upregulate components of the protein synthetic machinery, including ribosomal proteins, promoting protein translation. Mouse and fish models of DBA respond with amelioration of anemia to L-leucine. We therefore proposed to study the effect of L-leucine on transfusion dependence and growth in subjects with DBA. Methods: The primary objectives were to determine the feasibility of administering L-leucine in subjects with DBA who are red cell transfusion-dependent and to determine the efficacy of L-leucine to produce a hematologic and growth response. The secondary objective was to determine the safety profile of L-leucine. Twelve study sites were involved in this multi-center, Phase I/II study with an anticipated accrual of 50 subjects. A dose of 700 mg/M2 orally three times per day for 9 months was used. Inclusion criteria included age > 2 years, the diagnosis of DBA and transfusion dependence with adequate kidney and liver function. Response was evaluated at 9 months with Complete Response (CR) defined as no further transfusions required and Hb >9; Partial Response (PR): Hb < 9 gm/dL with an increase in reticulocyte count and transfusion interval; and No Response (NR): no change in transfusion needs, Hb or reticulocyte count . Growth percentiles were evaluated at baseline and at completion of 9 months of treatment and the growth velocity change was calculated. Results: The study opened July 2014 and closed February 2017; 55 subjects were consented; 12 were screen failures; 43 subjects were evaluable. There were 21 males; the median age was 9 years 1 month (2 years 5 months - 46 years 1 month). There were no untoward side effects experienced by any subject that were attributable to the L-leucine. Two subjects had an erythroid CR and 1 subject had a PR. The CRs occurred at 1 month and 3 months after start of L-leucine. The subject with PR had an elevated reticulocyte count but was not able to maintain a Hb >9 gm/dL without a transfusion and thus was not transfusion independent. Of the 30 subjects with growth potential who received L-leucine 10 experienced a positive growth velocity change at 9 months of therapy compared to baseline. At a median age of 7.5 years, the mean pre-leucine height percentile was 27 +/- 17.9 and the post-leucine height percentile was 35 +/- 19.9 (p Conclusions: The administration of L-leucine is safe. L-leucine administration resulted in an erythroid response in 7% of subjects and an increased growth velocity in 33% of growing subjects. Based upon extrapolation from animal models, it is likely that this dose was suboptimal. We hypothesize that higher doses of L-leucine will lead to hematologic responses in more subjects who are transfusion dependent. The potential benefit of added growth in children with DBA may improve final adult height. Disclosures Glader: Agios: Consultancy, Research Funding.

A 23 year old man with short stature, congenital malformation of the left thumb and both ears, and a single kidney presented with back pain, shortness of breath, fatigue, and easy bruising that had developed over several months. Physical examination revealed hepatosplenomegaly. His brother had died in early childhood, more than 20 years ago. The patient’s parents reported that his brother, “had similar but worse congenital malformations, including bad kidney problems.” Full blood count showed pancytopaenia, which prompted further laboratory investigations (table 1). View this table: Table 1 Initial laboratory investigations Radiography of the patient’s left hand (fig 1) revealed a single fused phalanx, anomalous carpal bones, and a hypoplastic radial styloid. Fig 1 (A) Dysmorphic left thumb. …

Fanconi anemia (FA) is an inherited chromosomal instability syndrome characterized by bone marrow failure, myelodysplasia (MDS), and acute myeloid leukemia (AML). Eight FA proteins associate in a nuclear core complex to monoubiquitinate FANCD2/FANCI in response to DNA damage. Additional functions have been described for some of the core complex proteins; however, in vivo genetic proof has been lacking. Here we show that double-mutant Fancc−/−;Fancg−/− mice develop spontaneous hematologic sequelae including bone marrow failure, AML, MDS and complex random chromosomal abnormalities that the single-mutant mice do not. This genetic model provides evidence for unique core complex protein function independent of their ability to monoubiquitinate FANCD2/FANCI. Importantly, this model closely recapitulates the phenotypes found in FA patients and may be useful as a preclinical platform to evaluate the molecular pathogenesis of spontaneous bone marrow failure, MDS and AML in FA.

Healthy cells are kept that way in part by molecular safety mechanisms that prevent genomic instability. When these guards are disrupted, malignant cells are born; however, the force behind these DNA injuries remains mysterious. Do our cells go awry due to assaults from genotoxic environmental

Defects in chromosome–microtubule attachment trigger spindle-checkpoint activation and delay mitotic progression1,2. How microtubule attachment is sensed and integrated into the steps of checkpoint-signal amplification is poorly understood. In a functional genomic screen targeting human kinases and phosphatases, we identified a microtubule affinity-regulating kinase kinase, TAO1 (also known as MARKK) as an important regulator of mitotic progression, required for both chromosome congression and checkpoint-induced anaphase delay. TAO1 interacts with the checkpoint kinase BubR1 and promotes enrichment of the checkpoint protein Mad2 at sites of defective attachment, providing evidence for a regulatory step that precedes the proposed Mad2–Mad1 dependent checkpoint-signal amplification step3. We propose that the dual functions of TAO1 in regulating microtubule dynamics and checkpoint signalling may help to coordinate the establishment and monitoring of correct congression of chromosomes, thereby protecting genomic stability in human cells.

The p53 tumor suppressor, nicknamed “guardian of the genome,” orchestrates the transcriptional response to DNA damage to arrest growth of injured cells. Cells whose genetic damage is irreparable are killed in a p53-dependent manner to prevent genomic instability and malignant transformation. Consequently, inactivation of p53 unleashes an avalanche of accumulating mutations, ultimately resulting in cancer. Cancers may also arise from gain-of-function (GOF) point mutations within p53, although it is not clear how single mutations can turn this mild-mannered tumor suppressor into a cancer-driving oncogene. To solve this puzzle, Zhu et al . audited genome-wide DNA binding locations for wild-type p53 and several GOF p53 mutants. All cancer-associated point mutations redirected p53 from activating the DNA damage response toward promoting expression of histone methyltransferases, master regulators of chromatin structure. Importantly, these p53 mutants stimulated expression of MLL1 (mixed-lineage leukemia), the histone methyltransferase whose dysregulation causes aggressive childhood leukemia. Abnormal MLL1 activity opens chromatin and haphazardly boosts expression of multiple genes, instantly transforming the cellular landscape without any further genetic mutations. Could MLL1 be responsible for the malignant phenotype of cells driven by mutant p53? Indeed, the authors discovered that silencing MLL1 reduced proliferation and tumor formation by cancer cells fueled by GOF p53 mutants but not tumor cells with normal or absent p53. Moreover, drugs attacking the MLL1-activating multiprotein COMPASS complex selectively killed cells harboring GOF p53 mutations, suggesting a possible pharmacological approach to the treatment of cancers bearing these mutations. Analysis of publicly available tumor databases revealed MLL1 activation in tumors with mutant p53, independently validating the functional connection between p53 point mutants and MLL1 activity. Zhu et al. not only discovered how GOF mutations force p53 to activate the MLL1-dependent malignant transcriptional program but also demonstrated how silencing the key regulator, MLL1, shuts down this elaborate gene-activation assault. Their findings strongly suggest that precision medicine therapeutics originally developed to target MLL-driven leukemia may be effective against other cancers driven by the p53 guardian harboring activating mutations. J. Zhu et al ., Gain-of-function p53 mutants co-opt chromatin pathways to drive cancer growth. Nature 525 , 206–211 (2015). [[Abstract]][1] [1]: http://www.nature.com/nature/journal/v525/n7568/abs/nature15251.html

Human cells employ intricate regulatory networks to promptly adapt the activity of genes in response to control signals. Transcriptional coactivators impact gene activity by reorganizing the transcriptional machinery to simultaneously fine-tune multiple genes. Because coactivators coincidentally affect dozens of cancer-related genes, the consequences of coactivator malfunction are dire. Unrestrained oncogene activity allows cells to escape the safety checkpoints and rush through the cell cycle, leading to genomic instability and cancer. The steroid receptor (SRC) coactivators are frequently amplified in human tumors. If tumor survival requires that the SRC switch is stuck in the “on” position, silencing SRC should selectively kill the coactivator-addicted cancers. With this in mind, Wang et al . embarked on a high-throughput chemical screen for modulators of SRC activity. The authors identified several SRC inhibitors in an ocean of more than 300,000 candidates but were intrigued by one molecule (MCB-613) in particular, which acted in the opposite way: MCB-613 dramatically boosted SRC activity in gene reporter assays. At the molecular level, the authors found that the drug bound SRC proteins and augmented SRC interaction with other transcriptional activators such as CBP and CARM1. Most physicians would be reluctant to prescribe an oncogene-activating drug. However, Wang et al . noted that cancer cells died upon in vitro exposure to MCB-613, whereas healthy cells appeared more resistant. MCB-613–exposed cancer cells suffered from reactive oxygen overload and succumbed to paraptosis, a peculiar kind of stress-related death hallmarked by the ballooning of organelles. Thus, MCB-613 amplified oncogenic signals to the level that cancer cells could not tolerate. Further, MCB-613 arrested growth of human breast cancers transplanted into mice without apparent side effects during the 2 months of therapy. More work is needed to confirm that long-term exposure to MCB-613 does not induce cancer. Nevertheless, killing cancer with its own weapon may provide the opportunity to selectively eradicate neoplastic cells with minimal collateral damage. Other oncogene-activating molecules might deserve a similarly detailed evaluation in preclinical studies. L. Wang et al ., Characterization of a steroid receptor coactivator small molecule stimulator that overstimulates cancer cells and leads to cell stress and death. Cancer Cell 28 , 240–252 (2015). [[Abstract]][1] [1]: http://dx.doi.org/10.1016/j.ccell.2015.07.005

We describe a child with dyserythropoietic anemia, thrombocytosis, functional platelet defect, and megakaryocyte dysplasia. We show that (i) this constellation of hematopoietic abnormalities was due to a germline mutation within the 5' untranslated region (5'UTR) of globin transcription factor 1 (GATA1); (ii) the mutation impaired a 5'UTR GATA1 splicing site, with promoted production of the shortened GATA1 isoform lacking the N-terminus; and (iii) expression of the GATA1 N-terminus is restricted to erythroblasts and megakaryocytes in normal marrow, consistent with the patient's abnormal erythropoiesis and megakaryopoiesis. Our findings provide insights into the clinically relevant in vivo function of the N-terminal domain of GATA1 in human hematopoiesis.

Chromosome-capping telomere sequences shorten with each DNA replication cycle. Excessive telomere attrition due to aging or inactivation of telomerase causes genetic damage, myelodysplastic syndrome (MDS), and cancer. Inexplicably, cancers often contain mutations affecting the spliceosome, a molecular machine that regulates splicing of nascent mRNAs. Recently published work revealed a therapeutically relevant signaling network that connects telomere shortening to faulty mRNA maturation, MDS, and cancer. Colla et al . discovered that mice deficient in Tert (the catalytic telomerase subunit) develop MDS and leukemia after a few generations of telomerase-free breeding. Of note, the net timing of symptoms roughly recapitulates the onset of clinical issues in children with inherited telomerase disorders. Surprisingly, telomerase reactivation rescued some of these abnormalities in vivo, indicating that bone marrow with short telomeres can be successfully repaired. How do short telomeres hamper this repair process? According to Colla et al. , telomere erosion impairs splicing of a collection of genes essential for spliceosome activity, DNA repair, and mitotic chromosome segregation. These genes represent potential therapeutic targets in MDS, leukemia, and inherited bone marrow failure syndromes resulting from telomere attrition. This signaling circuit may be exploited therapeutically with more precision if spliceosome mutations impose the malignant behavior on stem cells through selective disruption of downstream pathways. Indeed, this may be the case. Shirai et al. generated MDS-prone transgenic mice that express the disease-associated mutant U2af1 S34F spliceosome subunit. The UAF1S34F mutation impaired the spliceosome’s ability to recognize a defined splicing site, causing erroneous maturation of multiple transcripts associated with MDS and leukemia. Similarly, Kim et al. showed that mice expressing another MDS-linked spliceosome mutation, Srsf2 P95H, recapitulate myelodysplasia and hematopoietic stem cell dysfunction seen in SRSFP95H patients. Comprehensive evaluation of RNA splicing abnormalities in SRSFP95H humans and mice identified a large set of disease-relevant transcripts affected by the SRSFP95H mutation, including another key MDS-associated gene, EZH2 . Together, erosion of telomeres and spliceosome malfunction unleash a self-perpetuating cascade of impaired gene expression, culminating in genome instability, bone marrow failure, and cancer. Future research will determine if this vicious cycle can be broken with a precision therapeutic attack against the signaling pathways identified in this work and follow-up studies. S. Colla et al ., Telomere dysfunction drives aberrant hematopoietic differentiation and myelodysplastic syndrome. Cancer Cell 27 , 644–657 (2015). [[Abstract]][1] C. L. Shirai et al ., Mutant U2AF1 expression alters hematopoiesis and pre-mRNA splicing in vivo. Cancer Cell 27 , 631–643 (2015). [[Abstract]][2] E. Kim et al ., SRSF2 mutations contribute to myelodysplasia by mutant-specific effects on exon recognition. Cancer Cell 27 , 617–630 (2015). [[Abstract]][3] [1]: http://dx.doi.org/10.1016/j.ccell.2015.04.007 [2]: http://dx.doi.org/10.1016/j.ccell.2015.04.008%20%20 [3]: http://dx.doi.org/10.1016/j.ccell.2015.04.006

At least 17 genes—including the famous BRCA tumor-suppressor genes—play a role in Fanconi anemia (FA), an inherited syndrome of bone marrow failure, developmental abnormalities, and predisposition to cancer. As expected with such bewildering genetic complexity, not all FA patients are created equally. Most are diagnosed in childhood due to physical malformations or bone marrow failure. However, intrigued by the peculiar late presentation of a young-adult who was diagnosed with FA at age 33, Xie et al. zeroed in on a new pathway of possible clinical relevance. The patient’s physicians correctly suspected underlying FA because of the patient’s short stature, bone marrow dysfunction, and early-onset breast cancer. Genomic sequencing revealed two mutations in the most common FA-associated gene, FA complementation group A ( FANCA ). The FANCA protein is part of a nuclear core complex essential for function of the FA-BRCA signaling network, which maintains genomic stability by orchestrating DNA-damage repair during interphase, resolving entangled replication forks, and governing mitosis. One of the patient’s mutations, FANCAI939S, affected the protein domain that interacts with FANCA-associated protein 20 (FAAP20), which delivers FANCA to sites of DNA damage. FANCAI939S was unable to recruit FAAP20 and its downstream target, the DNA repair protein REV1. Still, FANCAI939S was able to activate other essential branches of the FA network, including monoubiquitination of the FA group D2 protein (FANCD2), which is essential for the recruitment of a cascade of DNA repair protein. Thus, the FANCAI939S mutation only partially crippled the multifaceted genome protector, perhaps explaining why the patient was not diagnosed with FA until adulthood. In transfection experiments, the mutated FANCAI939S protein was consistently expressed at lower levels relative to wild-type FANCA. Furthermore, loss of FAAP20 binding exposed FANCA to sumoylation on lysine 921, which triggers ring finger protein 4 (RNF4)–dependent polyubiquitination of FANCA—a modification that earmarks FANCA for proteasomal degradation. Thus, the I939S mutation rendered FANCA unstable, likely because FAAP20 binding did not protect FANCA from proteolysis-promoting posttranslational modifications and unrestrained destruction through the RNF4 pathway. Xie et al. propose that this signaling cascade silences the FA network under physiological conditions once DNA damage has been repaired. This work reminds us that mechanistic dissection of patient-derived mutations can be translationally rewarding. Functional dissection of the new FANCAI939S “separation-of-function” mutant unveiled new FA regulatory pathways and provided a plausible explanation for the patient’s clinical course. Together, these advances suggest that the manipulation of proteasomal degradation might prove clinically useful for selected FA patients. J. Xie et al ., RNF4-mediated polyubiquitination regulates the Fanconi anemia/BRCA pathway. J. Clin. Invest. 125 , 1523–1532 (2015). [[Full Text]][1] [1]: http://www.jci.org/articles/view/79325

Dense fibrotic stroma associated with pancreatic ductal adenocarcinoma (PDAC) is a major obstacle for drug delivery to the tumor bed and plays a crucial role in pancreatic cancer progression. Current, anti-stromal therapies have failed to improve tumor response to chemotherapy and patient survival. Furthermore, recent studies show that stroma impedes tumor progression and its complete ablation accelerates PDAC progression. In an effort to understand the molecular mechanisms associated with tumor-stromal interactions, using in vitro and in vivo models and PDAC patient biopsies, we show that the loss of miR-29 is a common phenomenon of activated pancreatic stellate cells (PSCs)/fibroblasts, the major stromal cells responsible for fibrotic stromal reaction. Loss of miR-29 is correlated with a significant increase in extracellular matrix (ECM) deposition, a major component in PDAC stroma. Our in vitro miR-29 gain/loss-of-function studies document the role of miR-29 in PSC-mediated ECM stromal protein accumulation. Overexpression of miR-29 in activated stellate cells reduced stromal deposition, cancer cell viability and cancer growth in co-culture. Furthermore, the loss of miR-29 in TGF-β1 activated PSCs is SMAD3 dependent. These results provide insights into the mechanistic role of miR-29 in PDAC stroma and its potential use as a therapeutic agent to target PDAC.

For 30 years, the mammalian cell nucleus has been hypothesized to contain a filamentous framework, the nuclear matrix or karyoskeleton, which regulates nuclear structure and function. However, such an organized network of filaments has never been observed in living cells. Here we show that human Cdc14B phosphatase in living cells tightly associates with long filaments that begin at the nucleolar periphery and extend to the nuclear envelope, frequently making close connections with nuclear pore complexes. We demonstrate that Cdc14B contains a bipartite signal that directs it to the intranuclear filaments, and we also detect a small amount of Cdc14B on interphase and mitotic centrosomes. Furthermore, we show that Cdc14B is critical for the maintenance of proper nuclear structure together with polo-like kinase Plk1. This work provides the first direct evidence for the existence of an intranuclear filamentous framework in living mammalian cells and implicates Cdc14B in the control of mammalian nuclear architecture. Cell Motil. Cytoskeleton 59:94–108, 2004. © 2004 Wiley-Liss, Inc.

Barth syndrome (BTHS; OMIM# 302060) is an X-linked disorder of neutropenia, cardiomyopathy and muscular weakness due to mitochondrial dysfunction secondary to inherited tafazzin (TAZ) mutations. Neutropenia occurs in the majority of BTHS patients, leading to high risk of recurrent life-threatening bacterial infections. The etiology of the G-CSF-responsive neutropenia in BTHS is incompletely understood and appears to reflect impaired granulopoiesis. Long-term risk of myelodysplasia (MDS) in BTHS individuals requiring chronic G-CSF therapy is unknown due to the rarity of the disorder; further, genotype-phenotype correlations between the severity of neutropenia and TAZ mutations remain to be elucidated. Genetic Taz knockout is embryonic lethal in the mouse. Therefore, a genetic BTHS mouse model is urgently needed to improve our mechanistic understanding of this disease and develop clinical studies based on preclinical evidence. We employed CRISPR/Cas system to edit our BTHS patient's tafazzin point mutation (D75H) into the endogenous murine Taz gene. The resulting mice recapitulated hematopoietic and systemic manifestations of Barth syndrome. TazD75H males are born at less than expected Mendelian frequencies (p Given the role of tafazzin in mitochondrial cardiolipin metabolism, we examined the structure and function of mitochondria in TazD75H mice. Strikingly, transmission electron microscopy revealed accumulation of swollen mitochondria with fractured cristae in TazD75H hematopoietic cells, cardiomyocytes and skeletal muscle, reflecting the mitochondrial phenotype of BTHS patients. Total mitochondrial mass quantified by MitoTracker flow was unchanged in TazD75H hematopoietic cells, but flow cytometry using the JC-1 mitochondrial membrane potential probe revealed disrupted mitochondrial potential during live TazD75H hematopoiesis in multi-potential progenitors (MPPs, p=0.0025), Lin- Sca-1+ c-kit+ cells (p In summary, our work demonstrates that impaired hematopoiesis in Barth syndrome, which is embryonic lethal in the mouse, can be recapitulated in vivo through editing a patient-specific hypomorphic tafazzin mutation into the murine genome. The precision-medicine TazD75H mouse model recapitulates clinical hallmarks of BTHS in hematopoietic and non-hematopoietic tissues, accompanied by structural and functional mitochondrial defects. This work enables in vivo studies to enhance our mechanistic understanding and treatment of hematopoetic and other defects in Barth syndrome. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Diagnosis of bone marrow failure (BMF) disorders is challenging but essential for optimal patient management. Here, we report a young adult from nonconsanguineous parents with progressive pancytopenia since childhood, bone pain, increased bone density, and haphazard ossification replacing hematopoiesis within the bone marrow. Sequencing revealed two novel biallelic variants of unknown significance within the thromboxane A synthase gene, TBXAS1 (c.266T > C; c.989T > C), bioinformatically predicted to disrupt the protein. TBXAS1 mutations result in Ghosal hematodiaphyseal dysplasia (OMIM 231095), the autosomal recessive syndrome associated with abnormal bone structure and BMF. Identification of the genetic defect prompted steroid therapy leading to resolution of symptoms.

Cyclin E-CDK2 is a key regulator in G1/S transition. Previously, we identified a number of CDK2-interacting proteins, including PHF8 (plant homeodomain finger protein 8). In this report, we confirmed that PHF8 is a novel cyclin E-CDK2 substrate. By taking the approach of mass spectrometry, we identified that PHF8 Ser-844 is phosphorylated by cyclin E-CDK2. Immunoblotting analysis indicated that WT PHF8 demethylates histone H3K9me2 more efficiently than the cyclin E-CDK2 phosphorylation-deficient PHF8-S844A mutant. Furthermore, flow cytometry analysis showed that WT PHF8 promotes S phase progression more robustly than PHF8-S844A. Real-time PCR results demonstrated that PHF8 increases transcription of cyclin E, E2F3, and E2F7 to significantly higher levels compared with PHF8-S844A. Further analysis by ChIP assay indicated that PHF8 binds to the cyclin E promoter stronger than PHF8-S844A and reduces the H3K9me2 level at the cyclin E promoter more efficiently than PHF8-S844A. In addition, we found that cyclin E-CDK2-mediated phosphorylation of PHF8 Ser-844 promotes PHF8-dependent rRNA transcription in luciferase reporter assays and real-time PCR. Taken together, these results indicate that cyclin E-CDK2 phosphorylates PHF8 to stimulate its demethylase activity to promote rRNA transcription and cell cycle progression.

Neurofibromatosis type 2 (NF2) is an autosomal dominant genetic disorder resulting from germline mutations in the NF2 gene. Bilateral vestibular schwannomas, tumors on cranial nerve VIII, are pathognomonic for NF2 disease. Furthermore, schwannomas also commonly develop in other cranial nerves, dorsal root ganglia and peripheral nerves. These tumors are a major cause of morbidity and mortality, and medical therapies to treat them are limited. Animal models that accurately recapitulate the full anatomical spectrum of human NF2-related schwannomas, including the characteristic functional deficits in hearing and balance associated with cranial nerve VIII tumors, would allow systematic evaluation of experimental therapeutics prior to clinical use. Here, we present a genetically engineered NF2 mouse model generated through excision of the Nf2 gene driven by Cre expression under control of a tissue-restricted 3.9kbPeriostin promoter element. By 10 months of age, 100% of Postn-Cre; Nf2(flox/flox) mice develop spinal, peripheral and cranial nerve tumors histologically identical to human schwannomas. In addition, the development of cranial nerve VIII tumors correlates with functional impairments in hearing and balance, as measured by auditory brainstem response and vestibular testing. Overall, the Postn-Cre; Nf2(flox/flox) tumor model provides a novel tool for future mechanistic and therapeutic studies of NF2-associated schwannomas.

Fanconi anemia (FA) is a complex heterogenic disorder of genomic instability, bone marrow failure, cancer predisposition, and congenital malformations. The FA signaling network orchestrates the DNA damage recognition and repair in interphase as well as proper execution of mitosis. Loss of FA signaling causes chromosome instability by weakening the spindle assembly checkpoint, disrupting centrosome maintenance, disturbing resolution of ultrafine anaphase bridges, and dysregulating cytokinesis. Thus, the FA genes function as guardians of genome stability throughout the cell cycle. This review discusses recent advances in diagnosis and clinical management of Fanconi anemia and presents the new insights into the origins of genomic instability in FA. These new discoveries may facilitate the development of rational therapeutic strategies for FA and for FA-deficient malignancies in the general population.

Non-dysmorphic 32-year old Caucasian female developed malaise, fluctuating body aches and progressive transfusion-dependent pancytopenia with peripheral blood morphology consistent with myelodysplastic syndrome (MDS). Reportedly, she had an episode of "low blood counts" of unclear etiology 23 years prior. Multiple attempts at bone marrow biopsy were unsuccessful due to technical difficulties with penetrating the bone, necessitating surgical biopsy by an orthopedic surgeon. Bone densitometry revealed increased bone density with Z-score of 4.5, consistent with mild thickening of long bone diaphysis noted via x-ray. Strikingly, the bone marrow biopsy revealed haphazard ossification, immature cartilage formation and thickened trabeculae replacing hematopoiesis within the marrow cavity. Next-generation sequencing revealed two novel biallelic mutations within the thromboxane synthase gene TBXAS1 (c.266T>C; c.989T>C). Both mutations affected conserved amino acid residues and were predicted to be disruptive to the protein function by two independent bioinformatics algorithms (Provean and PolyPhen-2). Thromboxane cascade regulates pathways of inflammation, coagulation and osteoclast activation, suggesting a mechanistic explanation of how thromboxane synthase malfunction causes abnormal bone remodeling and bone marrow failure. TBXAS1 disruption had been described in a rare autosomal recessive syndrome Ghosal hematodiaphyseal dysplasia (GHDD) associated with increased bone density and bone marrow failure. GHDD due to TBXAS1 mutations was genetically confirmed only in four families of Northern African and Middle Eastern descent (Nat Genet 2008; 40: 284-286). To our knowledge, this is the first genetically validated case of TBXAS1-/- Ghosal syndrome in the Caucasian population. Consistent with the well-established role of thromboxane in platelet function, our patient's platelets displayed impaired aggregation in response to arachidonic acid and structural abnormalities by transmission electron microscopy, revealing a functional platelet defect. Steroid therapy rendered the patient transfusion-independent and alleviated chronic pain and inflammation. A long-term follow-up will be needed to determine whether steroid treatment will result in stable improvement in hematopoiesis and decreased bone density, similar to other GHDD patients. This case illustrates how establishing the diagnosis of an underlying rare inherited bone marrow failure syndrome allows personalized, evidence-based treatment with lower risk of morbidity compared to cytotoxic chemotherapy and hematopoietic stem cell transplantation. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Fanconi anemia (FA) is an inherited bone marrow failure syndrome associated with genomic instability, high risk of acute myeloid leukemia (AML) and other malignancies. Somatic mutations within the FA/BRCA signaling network occur in AML in the general population, reflecting the importance of FA genes in tumor suppression. While the role of FA signaling in DNA damage repair and replication is well-established, we and others found that the FA network is essential for error-free chromosome segregation during cell division. Both interphase and mitotic errors contribute to the evolution of genomic instability during FA-/- human and murine hematopoiesis in vivo. However, the molecular mechanisms of FA pathway-dependent genome housekeeping during mitosis are incompletely understood. Through a synthetic lethal kinome-wide shRNA screen in FANCA patient cells, we discovered interphase and mitotic phosphosignaling networks that FANCA-/- cells depend on for survival, including the BUB1-BUBR1 axis of the spindle assembly checkpoint (SAC). BUB1 and BUBR1 are essential SAC kinases that prevent premature anaphase onset and chromosome mis-segregation by inhibiting the APC (anaphase-promoting complex) ubiquitin ligase at the centromeres until all kinetochores achieve correct attachment to the spindle microtubules. Our super-resolution microscopy and biochemistry experiments revealed that FANCA shuttles to kinetochores upon mitotic entry and physically interacts with BUB1 and BUBR1 at the kinetochore-microtubule attachment sites in attachment- and tension-dependent manner. Consistent with impaired SAC, we found that that anaphase onset as well as APC-mediated degradation of cyclin B1, BUBR1 and CDC20 all occur prematurely in FANCA-/- cells. We found that FANCA is essential for BUBR1 lysine 250 (K-250) acetylation at prometaphase kinetochores, and we confirmed that endogenous BUBR1K250 acetylation is disrupted in FANCA-/- primary patient cells using a validated acetyl-specific antibody. BUBR1K250 acetylation event works as a molecular switch in which BUBR1 is converted from a degradation target to a potent inhibitor of the APC ligase. Further, we observed that loss of FANCA disrupts kinetochore recruitment of the BUBR1K250 acetyltransferase PCAF and its upstream regulator, FANCD1/BRCA2. Our findings establish the first mechanistic connection between FANCA, the canonical SAC tumor suppressor cascade and the FA effector FANCD1/BRCA2. These findings further our understanding of the mechanisms of genomic instability and carcinogenesis resulting from loss of FA signaling. Since impaired BUBR1K250 acetylation causes chromosomal instability and cancer in vivo, our results have a direct translational relevance. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Summary Background Plexiform neurofibromas are slow-growing chemoradiotherapy-resistant tumours arising in patients with neurofibromatosis type 1 (NF1). Currently, there are no viable therapeutic options for patients with plexiform neurofibromas that cannot be surgically removed because of their proximity to vital body structures. We undertook an open-label phase 2 trial to test whether treatment with imatinib mesylate can decrease the volume burden of clinically significant plexiform neurofibromas in patients with NF1. Methods Eligible patients had to be aged 3–65 years, and to have NF1 and a clinically significant plexiform neurofibroma. Patients were treated with daily oral imatinib mesylate at 220 mg/m 2 twice a day for children and 400 mg twice a day for adults for 6 months. The primary endpoint was a 20% or more reduction in plexiform size by sequential volumetric MRI imaging. Clinical data were analysed on an intention-to-treat basis; a secondary analysis was also done for those patients able to take imatinib mesylate for 6 months. This trial is registered with ClinicalTrials.gov, number NCT01673009. Findings Six of 36 patients (17%, 95% CI 6–33), enrolled on an intention-to-treat basis, had an objective response to imatinib mesylate, with a 20% or more decrease in tumour volume. Of the 23 patients who received imatinib mesylate for at least 6 months, six (26%, 95% CI 10–48) had a 20% or more decrease in volume of one or more plexiform tumours. The most common adverse events were skin rash (five patients) and oedema with weight gain (six). More serious adverse events included reversible grade 3 neutropenia (two), grade 4 hyperglycaemia (one), and grade 4 increases in aminotransferase concentrations (one). Interpretation Imatinib mesylate could be used to treat plexiform neurofibromas in patients with NF1. A multi-institutional clinical trial is warranted to confirm these results. Funding Novartis Pharmaceuticals, the Indiana University Simon Cancer Centre, and the Indiana University Herman B Wells Center for Pediatric Research.

Germline mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a complex genetic disorder with a high predisposition of numerous skeletal dysplasias including short stature, osteoporosis, kyphoscoliosis, and fracture non-union (pseudoarthrosis). We have developed murine models that phenocopy many of the skeletal dysplasias observed in NF1 patients, including reduced bone mass and fracture non-union. We also show that the development of these skeletal manifestations requires an Nf1 haploinsufficient background in addition to nullizygous loss of Nf1 in mesenchymal stem/progenitor cells (MSCs) and/or their progenies. This is replicated in two animal models of NF1, PeriCre(+);Nf1(flox/-) and Col2.3Cre(+);Nf1(flox/-) mice. Adoptive transfer experiments demonstrate a critical role of the Nf1+/- marrow microenvironment in the impaired fracture healing in both models and adoptive transfer of WT bone marrow cells improves fracture healing in these mice. To our knowledge, this is the first demonstration of a non-cell autonomous mechanism in non-malignant NF1 manifestations. Collectively, these data provide evidence of a combinatory effect between nullizygous loss of Nf1 in osteoblast progenitors and haploinsufficiency in hematopoietic cells in the development of non-malignant NF1 manifestations.

Fanconi anemia (FA) is a genetic disorder characterized by progressive bone marrow failure, congenital abnormalities and predilection towards development of hematopoietic malignancies, including acute myeloid leukemia (AML). Congenital biallelic disruption of the FA/BRCA signaling network causes Fanconi anemia and somatic mutations within the same genes are increasingly identified in a variety of malignancies in non-FA individuals, consistent with the critical role of this signaling pathway in FA and in the general population. The FA/BRCA tumor suppressor network orchestrates interphase DNA-damage repair (DDR) and DNA replication to maintain genomic stability. Additionally, we and others have demonstrated that the genome housekeeping function of FA/BRCA signaling extends beyond interphase: loss of FA/BRCA signaling perturbs execution of mitosis, including the spindle assembly checkpoint (SAC), centrosome maintenance, cytokinesis and resolution of anaphase DNA bridges. Interphase errors exacerbate mitotic abnormalities and mitotic failure promotes interphase mutagenesis. Consequently, we had demonstrated that primary FA patients' cells accumulate genomic abnormalities consistent with a dual mechanism of impaired interphase DDR/replication and defective mitosis. Previous detailed studies had elucidated multiple mechanisms of interphase DDR-dependent assembly and activation of the FA complex at DNA damage sites to arrest the cell cycle and repair DNA lesions. However, the signaling cross-talk nodes between the FA and mitotic checkpoint pathways remain to be discovered. In this study, we identified functionally relevant mitotic signaling defects resulting from FANCA deficiency via a synthetic lethal kinome-wide pooled shRNA screen in primary patient-derived FANCA -deficient cells compared to isogenic FANCA -corrected cell line. Bioinformatics analysis of our screen results followed by secondary validation of selected hits with alternative shRNAs and small-molecule inhibitors revealed conserved mitotic signal transduction pathways regulating the SAC and centrosome maintenance. Our super-resolution structured illumination (SR-SIM) microscopy coupled with deconvolution imaging revealed that a fraction of FANCA co-localizes with key SAC kinases at mitotic centrosomes and kinetochores, consistent with the role of FANCA in centrosome maintenance and the SAC. Co-immunoprecipitation assays identified the biochemical interaction between FANCA and an essential SAC kinase whose loss is synthetic lethal with FANCA deficiency, providing first insights into the interactions between FA signaling and the canonical SAC network. Together, our study has unraveled functional and biochemical connections between FANCA and the centrosome/SAC kinases, consistent with the essential role of FANCA in cell division. Our ongoing work is aimed at mechanistically dissecting molecular links between these two key tumor suppressor signaling pathways in more detail. We hypothesize that impaired FANCA/SAC cross-talk may contribute to genomic instability in FA-deficient cells and provide opportunities to selectively kill FANCA-/- cells. Disclosures No relevant conflicts of interest to declare.

Diamond-Blackfan anemia (DBA) is a rare disorder of impaired erythropoiesis belonging to a broader family of inherited and acquired anemias known as pure red cell aplasias (PRCA). The cardinal feature of DBA is aregenerative anemia accompanied by macrocytosis, reticulocytopenia and decreased or absent erythroid precursors in an otherwise normocellular marrow. Classic DBA is associated with mutations of genes encoding ribosome subunits. Advancements in molecular techniques have prompted discussion regarding the genetic basis of the disease and its prognostic implications as non-ribosomal genes are beginning to be implicated in DBA. Acquired mutations within the X-linked transcription factor GATA1 have been described in Down syndrome-related transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AKML), while germline GATA1 mutations affecting both isoforms are associated with congenital thrombocytopenia. Normal alternative splicing of GATA1 produces a full-length GATA1 isoform (flGATA1) as well as a shortened GATA1 isoform (GATA1s), which lacks the N-terminal domain that activates GATA1-driven erythropoiesis program and recruits flGATA1 to a subset of megakaryocyte and erythroblast genes. Rare germline defects known as "GATA1s mutations" are characterized by an unbalanced production of the GATA1s isoform at the expense of a flGATA1 transcript due to disrupting exon 2 splice sites or the ATG initiation codon. GATA1s mutations had been reported to phenocopy Diamond-Blackfan anemia by causing isolated red blood cell aplasia in some patients, although thrombocytopenia with structural platelet abnormalities and dyserythropoiesis as well as DBA-like picture progressing into MDS had been described in other GATA1s families. Therefore, while GATA1s mutations uniformly disrupt erythropoiesis, the impact of defective flGATA production on megakaryopoiesis in non-trisomy 21 individuals is less clear. Here we describe a child with dyserthropoietic anemia, marked megakaryocyte dysplasia, peripheral thrombocytosis and platelet dysfunction due to a novel disease-causing mutation within the 5' UTR of GATA1. DBA was initially suspected due to progressive macrocytic anemia beginning in infancy. Bone marrow analysis revealed not only a paucity of erythroid precursor cells and dyserythropoiesis, but also prominent megakaryocytosis and megakaryocyte dysplasia, not typically associated with classic DBA. Ribosome mutational analysis was unremarkable, as were cytogenetics and MDS-FISH analysis. Given aregenerative anemia mimicking DBA as well as megakaryocyte dysplasia, the question was raised whether the findings could be associated with a germline mutation in GATA1. Indeed, sequencing revealed a novel, pathogenic mutation in the 5'UTR of GATA1 (c.-21 A>G). We explored the impact of this novel mutation in silico, found it to disrupt a consensus splice site, and further demonstrated that this mutation conferred a striking predominance of the GATA1s isoform at the transcript and protein level. Two-color immunohistochemistry confirmed loss of full-length GATA1 protein in the patient's bone marrow and showed that full-length GATA1 expression is restricted to megakaryocytes and erythroblasts in healthy bone marrow, consistent with dysmegakaryopoiesis and decreased erythropoiesis in our patient. Our findings support a role for the N-terminus of GATA1 in both erythroblast and megakaryocyte maturation and function in vivo. Furthermore, the findings of prominent megakaryocytes in the bone marrow and resultant functional platelet abnormalities may provide subtle clinical clues to differentiate Diamond-Blackfan anemia due to ribosomopathy from dyserythropoiesis secondary to GATA1s mutations. We conclude that GATA1 sequencing, including non-coding GATA1 regions, should be considered in males with congenital multi-lineage dysplasia and/or DBA-like clinical presentation. Disclosures No relevant conflicts of interest to declare.

The Fanconi anemia (FA/BRCA) signaling network prevents bone marrow failure and cancer by protecting genomic integrity. Biallelic germline mutations within this gene network result in Fanconi anemia, an inherited bone marrow failure syndrome characterized by genomic instability and a predisposition to bone marrow failure, myelodysplasia and cancer, particularly acute myelogenous leukemia (AML). Heterozygous inborn mutations in the BRCA branch of FA network increase risk of breast and ovarian cancers as well as other tumors, and somatic mutations of FA/BRCA genes occur in malignancies in non-Fanconi patients. Thus, disruption of FA/BRCA signaling promotes malignancies in the inherited genetic syndromes and in the general population. The FA/BRCA network functions as a genome gatekeeper throughout the cell cycle. In interphase, the FA/BRCA network provides a crucial line of defense against mutagenesis by coordinating DNA damage response to a variety of genotoxic insults, from endogenous aldehydes to replication errors and mutagen exposure. Less is known about the role of the FA/BRCA pathway during mitosis. However, FA signaling has recently been implicated in multiple aspects of cell division, including the spindle assembly checkpoint (SAC) that ensures high-fidelity chromosome segregation at metaphase to anaphase transition; cytokinesis; centrosome maintenance and repair of ultrafine anaphase bridges. Although chromosomal instability due to mitotic errors is a hallmark of cancer, the in vivo contribution of abnormal mitosis to malignant transformation of FA-deficient hematopoietic cells remains unknown. To determine whether error-prone chromosome segregation upon loss of FA signaling contributes to abnormal hematopoiesis and cancer, we generated a novel murine FA model by genetically weakening the SAC in the FA-deficient background. The resulting mice were viable and born at expected Mendelian ratios, but exhibited increased baseline in vivo chromosomal instability evidenced by elevated red blood cell micronucleation, increased frequency of chromosome missegregation and DNA breakage in microscopy-based cytome assays, and augmented bone marrow karyotype instability. Importantly, unlike FA or SAC control animals, the FA-SAC mice were prone to premature death due to the development of myelodysplasia and AML at young age, recapitulating disease manifestations of human Fanconi anemia. This study provides the in vivo evidence supporting the essential role of compromised chromosome segregation in the development of myelodysplasia and acute leukemia due to impaired FA signaling. Our observations provide novel insights into complex mechanisms of genomic instability and carcinogenesis due to FA deficiency. Impaired mitosis is a well-established therapeutic target, and our independent ex vivo experiments using FA patient-derived primary cells show that exposure to antimitotic chemotherapeutics is synthetic lethal with loss of the FA network. Thus, our findings may have implications for future precision strategies against FA-deficient, chromosomally unstable hematopoietic cancers. The FA-SAC mouse model offers a preclinical platform to systematically test this hypothesis in vivo. Disclosures No relevant conflicts of interest to declare.

Dense fibrotic stroma associated with pancreatic ductal adenocarcinoma (PDAC) has been a major obstacle for drug delivery to the tumor bed and may impede attempts to slow down PDAC progression and metastasis. However, current anti-stromal drugs have not improved tumor response to chemotherapy or patient survival. Thus, a better understanding of the molecular mechanisms associated with tumor-stromal interactions is desperately needed to develop novel anti-stromal therapeutic approaches. MicroRNAs (miRNAs) are an abundant class of highly conserved, small non-coding RNAs that function as key regulators of eukaryotic gene expression and cellular homeostasis. miR-29 is known to play a paramount role in the fibrotic process of several organs by providing crucial functions downstream of pro-fibrotic signaling pathways such as TGF-β1 and regulating the expression of extracellular matrix (ECM) proteins, a major component in the PDAC stroma. Upregulation of TGF-β1 is commonly associated with PDAC pathogenesis and is known to activate stromal cells. Furthermore, vascular endothelial growth factor (VEGF) that stimulates tumor angiogenesis is a predicted target of miR-29. We hypothesize that miR-29 may be misregulated in TGF-β1 activated PDAC stromal cells and lead to excessive accumulation of ECM proteins and VEGF. Restored expression of miR-29 could be therapeutically beneficial to modulate tumor-stromal interactions. To understand the role of miR-29 in PDAC stroma, we examined miR-29 expression patterns in TGF-β1 activated stromal cells using qPCR/northern blot analysis and determined ECM and VEGF protein expression. In activated stromal cells, we observed loss of miR-29 in correlation with a significant increase in ECM and VEGF protein expression. In addition, in both murine and human PDAC samples, loss of miR-29 expression is associated with an increase in stromal percentage estimated by Sirius red stain. To evaluate the physiological role of miR-29 in stroma, we performed gain and loss-of-function studies by transfecting stromal cells with synthetic miR-29 mimics or locked nucleic acid, a miR-29 inhibitor. Overexpression of miR-29 in stromal cells suppressed matrix and VEGF protein expression, and conversely, depletion of miR-29 lead to their significant increase. Finally, to evaluate the effect of miR-29 overexpression in stromal cells on cancer colony growth, we directly co-cultured miR-29 transfected stromal cells with pancreatic cancer cells for 10 days, and subsequently, cancer colony number and stromal deposition was determined by crystal violet and Sirius red stains respectively. We observed a significant decrease in the number of cancer colonies and stromal accumulation in co-cultures. In conclusion, our results provide insight into the mechanistic role of miR-29 in PDAC stroma and its potential use as a novel anti-stromal therapeutic agent. Citation Format: Jason J. Kwon, Sarah C. Nabinger, Ravi K. Alluri, Zachary Vega, Smiti S. Sahu, Zahi Abdul Sater, Zhangsheng Yu, A Jesse Gore, Grzegorz Nalepa, Romil Saxena, Murray Korc, Janaiah Kota. Pathophysiological role of microRNA-29 in pancreatic cancer stroma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 172. doi:10.1158/1538-7445.AM2015-172