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Purpose:Biomarkers of response and resistance to FLT3 tyrosine kinase inhibitors (TKI) are still emerging, and optimal clinical combinations remain unclear. The purpose of this study is to identify co-occurring mutations that influence clinical response to the novel FLT3 inhibitor pexidartinib (PLX3397).Experimental Design:We performed targeted sequencing of pretreatment blasts from 29 patients with FLT3 internal tandem duplication (ITD) mutations treated on the phase I/II trial of pexidartinib in relapsed/refractory FLT3-ITD+ acute myeloid leukemia (AML). We sequenced 37 samples from 29 patients with available material, including 8 responders and 21 non-responders treated at or above the recommended phase II dose of 3,000 mg.Results:Consistent with other studies, we identified mutations in NRAS, TP53, IDH2, and a variety of epigenetic and transcriptional regulators only in non-responders. Among the most frequently mutated genes in non-responders was Cyclin D3 (CCND3). A total of 3 individual mutations in CCND3 (Q276*, S264R, and T283A) were identified in 2 of 21 non-responders (one patient had both Q276* and S264R). No CCND3 mutations were found in pexidartinib responders. Expression of the Q276* and T283A mutations in FLT3-ITD MV4;11 cells conferred resistance to apoptosis, decreased cell-cycle arrest, and increased proliferation in the presence of pexidartinib and other FLT3 inhibitors. Inhibition of CDK4/6 activity in CCND3 mutant MV4;11 cells restored pexidartinib-induced cell-cycle arrest but not apoptosis.Conclusions:Mutations in CCND3, a gene not commonly mutated in AML, are a novel cause of clinical primary resistance to FLT3 inhibitors in AML and may have sensitivity to CDK4/6 inhibition.

Supplemental Figures

Inborn errors of human IFN-γ-dependent macrophagic immunity underlie mycobacterial diseases, whereas inborn errors of IFN-α/β-dependent intrinsic immunity underlie viral diseases. Both types of IFNs induce the transcription factor IRF1. We describe unrelated children with inherited complete IRF1 deficiency and early-onset, multiple, life-threatening diseases caused by weakly virulent mycobacteria and related intramacrophagic pathogens. These children have no history of severe viral disease, despite exposure to many viruses, including SARS-CoV-2, which is life-threatening in individuals with impaired IFN-α/β immunity. In leukocytes or fibroblasts stimulated in vitro, IRF1-dependent responses to IFN-γ are, both quantitatively and qualitatively, much stronger than those to IFN-α/β. Moreover, IRF1-deficient mononuclear phagocytes do not control mycobacteria and related pathogens normally when stimulated with IFN-γ. By contrast, IFN-α/β-dependent intrinsic immunity to nine viruses, including SARS-CoV-2, is almost normal in IRF1-deficient fibroblasts. Human IRF1 is essential for IFN-γ-dependent macrophagic immunity to mycobacteria, but largely redundant for IFN-α/β-dependent antiviral immunity.

Most cell–cell interactions and crosstalks are mediated by ligand–receptor interactions. The advent of single-cell RNA-sequencing (scRNA-seq) techniques has enabled characterizing tissue heterogeneity at single-cell level. In the past few years, several methods have been developed to study ligand–receptor interactions at cell type level using scRNA-seq data. However, there is still no easy way to query the activity of a specific user-defined signaling pathway in a targeted way or to map the interactions of the same subunit with different ligands as part of different receptor complexes. Here, we present DiSiR, a fast and easy-to-use permutation-based software framework to investigate how individual cells are interacting with each other by analyzing signaling pathways of multi-subunit ligand-activated receptors from scRNA-seq data, not only for available curated databases of ligand–receptor interactions, but also for interactions that are not listed in these databases. We show that, when utilized to infer ligand–receptor interactions from both simulated and real datasets, DiSiR outperforms other well-known permutation-based methods, e.g. CellPhoneDB and ICELLNET. Finally, to demonstrate DiSiR’s utility in exploring data and generating biologically relevant hypotheses, we apply it to COVID lung and rheumatoid arthritis (RA) synovium scRNA-seq datasets and highlight potential differences between inflammatory pathways at cell type level for control versus disease samples.

Purpose: Biomarkers of response and resistance to FLT3 tyrosine kinase inhibitors (TKI) are still emerging, and optimal clinical combinations remain unclear. The purpose of this study is to identify co-occurring mutations that influence clinical response to the novel FLT3 inhibitor pexidartinib (PLX3397). Experimental Design: We performed targeted sequencing of pretreatment blasts from 29 patients with FLT3 internal tandem duplication (ITD) mutations treated on the phase I/II trial of pexidartinib in relapsed/refractory FLT3-ITD+ acute myeloid leukemia (AML). We sequenced 37 samples from 29 patients with available material, including 8 responders and 21 non-responders treated at or above the recommended phase II dose of 3,000 mg. Results: Consistent with other studies, we identified mutations in NRAS, TP53, IDH2, and a variety of epigenetic and transcriptional regulators only in non-responders. Among the most frequently mutated genes in non-responders was Cyclin D3 (CCND3). A total of 3 individual mutations in CCND3 (Q276*, S264R, and T283A) were identified in 2 of 21 non-responders (one patient had both Q276* and S264R). No CCND3 mutations were found in pexidartinib responders. Expression of the Q276* and T283A mutations in FLT3-ITD MV4;11 cells conferred resistance to apoptosis, decreased cell-cycle arrest, and increased proliferation in the presence of pexidartinib and other FLT3 inhibitors. Inhibition of CDK4/6 activity in CCND3 mutant MV4;11 cells restored pexidartinib-induced cell-cycle arrest but not apoptosis. Conclusions: Mutations in CCND3, a gene not commonly mutated in AML, are a novel cause of clinical primary resistance to FLT3 inhibitors in AML and may have sensitivity to CDK4/6 inhibition.

Refereed/Peer-reviewed

Mendelian susceptibility to mycobacterial disease (MSMD) is characterized by a selective predisposition to clinical disease caused by the Bacille Calmette-Guérin (BCG) vaccine and environmental mycobacteria. The known genetic etiologies of MSMD are inborn errors of IFN-γ immunity due to mutations of 15 genes controlling the production of or response to IFN-γ. Since the first MSMD-causing mutations were reported in 1996, biallelic mutations in the genes encoding IFN-γ receptor 1 (IFN-γR1) and IFN-γR2 have been reported in many patients of diverse ancestries. Surprisingly, mutations of the gene encoding the IFN-γ cytokine itself have not been reported, raising the remote possibility that there might be other agonists of the IFN-γ receptor. We describe 2 Lebanese cousins with MSMD, living in Kuwait, who are both homozygous for a small deletion within the IFNG gene (c.354_357del), causing a frameshift that generates a premature stop codon (p.T119Ifs4*). The mutant allele is loss of expression and loss of function. We also show that the patients’ herpesvirus Saimiri–immortalized T lymphocytes did not produce IFN-γ, a phenotype that can be rescued by retrotransduction with WT IFNG cDNA. The blood T and NK lymphocytes from these patients also failed to produce and secrete detectable amounts of IFN-γ. Finally, we show that human IFNG has evolved under stronger negative selection than IFNGR1 or IFNGR2, suggesting that it is less tolerant to heterozygous deleterious mutations than IFNGR1 or IFNGR2. This may account for the rarity of patients with autosomal-recessive, complete IFN-γ deficiency relative to patients with complete IFN-γR1 and IFN-γR2 deficiencies.

Most of cell-cell interactions and crosstalks are mediated by ligand-receptor interactions. The advent of single-cell RNA-sequencing (scRNA-seq) techniques has enabled characterizing tissue heterogeneity at single-cell level. Over the past recent years, several methods have been developed to study ligand-receptor interactions at cell type level using scRNA-seq data. However, there is still no easy way to query the activity of a specific user-defined signaling pathway in a targeted way or map the interactions of the same subunit with different ligands as part of different receptor complexes. Here, we present DiSiR, a fast and easy-to-use permutation-based software framework to investigate how individual cells are interacting with each other by analyzing signaling pathways of multi-subunit ligand-activated receptors from scRNA-seq data, not only for available curated databases of ligand-receptor interactions, but also for interactions that are not listed in these databases. We show that, when utilized to infer melanoma disease map on a gold-standard dataset, DiSiR outperforms other well-known permutation-based methods, e.g., CellPhoneDB and ICELLNET. To demonstrate DiSiR’s utility in exploring data and generating biologically relevant hypotheses, we apply it to COVID lung and rheumatoid arthritis (RA) synovium scRNA-seq data and highlight potential differences between inflammatory pathways at cell type level for control vs. disease samples.

The purpose of this study is to identify key regulatory pathways that potentially drive abnormal gene expression program in relapsed Acute Myeloid Leukemia (AML) patients, by integrative computational analyses on multi-omics molecular profiles. Relapsed AML remains a clinical challenge. Epigenetic heterogeneity may contribute to transcriptional dysregulation and disease progression in AML. However, what specific transcriptional programs and potential regulatory mechanisms contribute to disease relapse are not yet well understood. To characterize the global transcriptional landscapes in relapsed AML, we integrated genomics data from two cohorts of matched diagnosis and relapse patient specimens. We identified 5,416 differentially expressed genes (DEGs) between diagnosis and relapse in Cohort I. Unsupervised clustering yielded three distinct DEG groups: group A, B and C genes that were predominantly (88%) down-regulated, divergently regulated, or predominantly (65%) up-regulated, respectively, upon relapse. The expression pattern of all DEGs separated the patients into two clusters, most robustly by Group B genes. Interestingly, the majority of DEGs did not associate with changes in gene promoter methylation. Similar patterns were observed in Cohort II. We used Binding Analysis for Regulation of Transcription (BART) to identify transcriptional regulators (TRs) that potentially regulated the DEGs not associated with DNA methylation changes, and assessed the differential expression of identified TRs during disease progression. PU.1 was identified as a potential TR for Group A genes and was down-regulated upon relapse. GATA1 and TAL1 were identified as regulating Group B genes and were up-regulated in patient cluster1 and down-regulated in cluster2, consistent with the expression pattern of Group B genes. RBBP5 was a top predicted TR for Group C genes and was up-regulated upon relapse. We next validated the potential functionality of those predicted factors. In NSG mice transplanted with a human AML specimen, TAL1 and GATA1 were downregulated in AML cells collected four weeks after chemotherapy treatment, and were inferred as TRs for the down-regulated genes, similar to the patient data. PU.1 was inferred as regulating the up-regulated genes. Furthermore, we found that the level of differential expression of TAL1, GATA1, and PU.1 in each patient specimen associated with the correlation of DEG profiles between the patient specimen and TR perturbation in human-derived hematopoietic cell lines. Our results support the possibility that in some AML patients, TRs with roles in hematopoiesis and leukemia might contribute to disease relapse. Further mechanistic studies deciphering the molecular and phenotypic events facilitated by these TRs will yield significant insight into disease biology and possible therapeutic targeting approaches in relapsed AML. Citation Format: Zhenjia Wang, Yaseswini Neelamraju, Cem Meydan, Nicholas Dunham, Jorge Gandara, Tak Lee, Subhash Prajapati, Franck Rapaport, Caroline Sheridan, Paul Zumbo, Michael Becker, Lars Bullinger, Martin Carroll, Richard D’Andrea, Richard Dillon, Ross Levine, Christopher E. Mason, Ari Melnick, Donna Neuberg, Stefan Bekiranov, Chongzhi Zang, Francine E. Garrett-Bakelman. Gene expression profiles reveal distinct regulatory activities of transcription factors GATA1 and TAL1 upon AML relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3155.

Autosomal dominant (AD) NFKB1 deficiency is thought to be the most common genetic etiology of common variable immunodeficiency (CVID). However, the causal link between NFKB1 variants and CVID has not been demonstrated experimentally and genetically, as there has been insufficient biochemical characterization and enrichment analysis. We show that the cotransfection of NFKB1-deficient HEK293T cells (lacking both p105 and its cleaved form p50) with a κB reporter, NFKB1/p105, and a homodimerization-defective RELA/p65 mutant results in p50:p65 heterodimer–dependent and p65:p65 homodimer–independent transcriptional activation. We found that 59 of the 90 variants in patients with CVID or related conditions were loss of function or hypomorphic. By contrast, 258 of 260 variants in the general population or patients with unrelated conditions were neutral. None of the deleterious variants displayed negative dominance. The enrichment in deleterious NFKB1 variants of patients with CVID was selective and highly significant (P = 2.78 × 10−15). NFKB1 variants disrupting NFKB1/p50 transcriptional activity thus underlie AD CVID by haploinsufficiency, whereas neutral variants in this assay should not be considered causal.

We studied a child with severe viral, bacterial, fungal, and parasitic diseases, who was homozygous for a loss-of-function mutation of REL, encoding c-Rel, which is selectively expressed in lymphoid and myeloid cells. The patient had low frequencies of NK, effector memory cells reexpressing CD45RA (Temra) CD8(+) T cells, memory CD4(+) T cells, including Th1 and Th1*, Tregs, and memory B cells, whereas the counts and proportions of other leukocyte subsets were normal. Functional deficits of myeloid cells included the abolition of IL-12 and IL-23 production by conventional DC1s (cDC1s) and monocytes, but not cDC2s. c-Rel was also required for induction of CD86 expression on, and thus antigen-presenting cell function of, cDCs. Functional deficits of lymphoid cells included reduced IL-2 production by naive T cells, correlating with low proliferation and survival rates and poor production of Th1, Th2, and Th17 cytokines by memory CD4(+) T cells. In naive CD4(+) T cells, c-Rel is dispensable for early IL2 induction but contributes to later phases of IL2 expression. The patient’s naive B cells displayed impaired MYC and BCL2L1 induction, compromising B cell survival and proliferation and preventing their differentiation into Ig-secreting plasmablasts. Inherited c-Rel deficiency disrupts the development and function of multiple myeloid and lymphoid cells, compromising innate and adaptive immunity to multiple infectious agents.

BACKGROUND: Cytomegalovirus (CMV) can cause severe disease in children and adults with a variety of inherited or acquired T-cell immunodeficiencies, who are prone to multiple infections. It can also rarely cause disease in otherwise healthy persons. The pathogenesis of idiopathic CMV disease is unknown. Inbred mice that lack the gene encoding nitric oxide synthase 2 (Nos2) are susceptible to the related murine CMV infection. METHODS: We studied a previously healthy 51-year-old man from Iran who after acute CMV infection had an onset of progressive CMV disease that led to his death 29 months later. We hypothesized that the patient may have had a novel type of inborn error of immunity. Thus, we performed whole-exome sequencing and tested candidate mutant alleles experimentally. RESULTS: We found a homozygous frameshift mutation in NOS2 encoding a truncated NOS2 protein that did not produce nitric oxide, which determined that the patient had autosomal recessive NOS2 deficiency. Moreover, all NOS2 variants that we found in homozygosity in public databases encoded functional proteins, as did all other variants with an allele frequency greater than 0.001. CONCLUSIONS: These findings suggest that inherited NOS2 deficiency was clinically silent in this patient until lethal infection with CMV. Moreover, NOS2 appeared to be redundant for control of other pathogens in this patient. (Funded by the National Center for Advancing Translational Sciences and others.)

Most patients with autosomal dominant hyper-IgE syndrome (AD-HIES) are heterozygous for rare STAT3 variants. The mechanism of dominance was recently questioned. The authors show that both in-frame and out-of-frame STAT3 variants underlie AD-HIES by negative dominance and not haploinsufficiency., Most patients with autosomal dominant hyper-IgE syndrome (AD-HIES) carry rare heterozygous STAT3 variants. Only six of the 135 in-frame variants reported have been experimentally shown to be dominant negative (DN), and it has been recently suggested that eight out-of-frame variants operate by haploinsufficiency. We experimentally tested these 143 variants, 7 novel out-of-frame variants found in HIES patients, and other STAT3 variants from the general population. Strikingly, all 15 out-of-frame variants were DN via their encoded (1) truncated proteins, (2) neoproteins generated from a translation reinitiation codon, and (3) isoforms from alternative transcripts or a combination thereof. Moreover, 128 of the 135 in-frame variants (95%) were also DN. The patients carrying the seven non-DN STAT3 in-frame variants have not been studied for other genetic etiologies. Finally, none of the variants from the general population tested, including an out-of-frame variant, were DN. Overall, our findings show that heterozygous STAT3 variants, whether in or out of frame, underlie AD-HIES through negative dominance rather than haploinsufficiency., Graphical Abstract

International audience; Genetic variants underlying life-threatening diseases, being unlikely to be transmitted to the next generation, are gradually and selectively eliminated from the population through negative selection. We study the determinants of this evolutionary process in human genes underlying monogenic diseases by comparing various negative selection scores and an integrative approach, CoNeS, at 366 loci underlying inborn errors of immunity (IEI). We find that genes underlying autosomal dominant (AD) or X-linked IEI have stronger negative selection scores than those underlying autosomal recessive (AR) IEI, whose scores are not different from those of genes not known to be disease causing. Nevertheless, genes underlying AR IEI that are lethal before reproductive maturity with complete penetrance have stronger negative selection scores than other genes underlying AR IEI. We also show that genes underlying AD IEI by loss of function have stronger negative selection scores than genes underlying AD IEI by gain of function, while genes underlying AD IEI by haploinsufficiency are under stronger negative selection than other genes underlying AD IEI. These results are replicated in 1,140 genes underlying inborn errors of neurodevelopment. Finally, we propose a supervised classifier, SCoNeS, which predicts better than state-of-the-art approaches whether a gene is more likely to underlie an AD or AR disease. The clinical outcomes of monogenic inborn errors, together with their mode and mechanisms of inheritance, determine the levels of negative selection at their corresponding loci. Integrating scores of negative selection may facilitate the prioritization of candidate genes and variants in patients suspected to carry an inborn error.

Purpose Germline heterozygous mutations of GATA2 underlie a variety of hematological and clinical phenotypes. The genetic, immunological, and clinical features of GATA2-deficient patients with mycobacterial diseases in the familial context remain largely unknown. Methods We enrolled 15 GATA2 index cases referred for mycobacterial disease. We describe their genetic and clinical features including their relatives. Results We identified 12 heterozygous GATA2 mutations, two of which had not been reported. Eight of these mutations were loss-of-function, and four were hypomorphic. None was dominant-negative in vitro, and the GATA2 locus was found to be subject to purifying selection, strongly suggesting a mechanism of haploinsufficiency. Three relatives of index cases had mycobacterial disease and were also heterozygous, resulting in 18 patients in total. Mycobacterial infection was the first clinical manifestation in 11 patients, at a mean age of 22.5 years (range: 12 to 42 years). Most patients also suffered from other infections, monocytopenia, or myelodysplasia. Strikingly, the clinical penetrance was incomplete (32.9% by age 40 years), as 16 heterozygous relatives aged between 6 and 78 years, including 4 older than 60 years, were completely asymptomatic. Conclusion Clinical penetrance for mycobacterial disease was found to be similar to other GATA2 deficiency-related manifestations. These observations suggest that other mechanisms contribute to the phenotypic expression of GATA2 deficiency. A diagnosis of autosomal dominant GATA2 deficiency should be considered in patients with mycobacterial infections and/or other GATA2 deficiency-related phenotypes at any age in life. Moreover, all direct relatives should be genotyped at the GATA2 locus., INSERM, University of Paris; Rockefeller University; St. Giles Foundation; National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH)United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Allergy & Infectious Diseases (NIAID) [R37AI095983]; French National Research Agency (ANR) under the "Investments for the future" programFrench National Research Agency (ANR) [ANR-10-IAHU-01, ANR13-ISV3-0001-01, ANR-16-CE17-0005-01]; ECOS-NORD [C19S01-63407, SRC2017]; ANRHGDIFDFrench National Research Agency (ANR) [ANR-14-CE15-006-01]; ANR-IFNGPHOX [ANR-13-ISV3-0001-01]; GENMSMD [ANR-16-CE17-0005-01]; Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2012/11757-2, 2010/51814-0, 2012/51094-2]; Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPQ)National Council for Scientific and Technological Development (CNPq) [303809/2010-8]; Instituto de Salud Carlos IIIInstituto de Salud Carlos IIIEuropean Commission [PI11/01086, PI14/00405]; European Regional Development Fund (ERDF)European Commission; Colombia-France (ECOS-NORD/COLCIENCIAS/MEN/ICETEX) [619-2013]; Diana Garcia de Olarte foundation PID; ColcienciasDepartamento Administrativo de Ciencia, Tecnologia e Innovacion Colciencias [713-2016, 111574455633]; "Poste d'accueil" INSERMInstitut National de la Sante et de la Recherche Medicale (Inserm); Imagine Institute, The Laboratory of Human Genetics of Infectious Diseases is supported in part by institutional grants from INSERM, University of Paris, The Rockefeller University and the St. Giles Foundation, the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) (R37AI095983), and grants from the French National Research Agency (ANR) under the "Investments for the future" program (ANR-10-IAHU-01) and IFNGPHOX (ANR13-ISV3-0001-01 for JB and ACN), GENMSMD (ANR-16-CE17-0005-01 for JB) grants, ECOS-NORD (C19S01-63407 for JB and JFR), and SRC2017 (for JB). CO-Q is supported by ANRHGDIFD (ANR-14-CE15-006-01). AG was supported by the ANR-IFNGPHOX (ANR-13-ISV3-0001-01), GENMSMD (ANR-16-CE17-0005-01), and the Imagine Institute. AC-N and EBO-J are supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP grants 2012/11757-2, 2010/51814-0, and 2012/51094-2) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPQ grant 303809/2010-8). MMG and RC are supported by Instituto de Salud Carlos III, grants PI11/01086 and PI14/00405, co-financed by the European Regional Development Fund (ERDF). JFR and AAA are supported by Colombia-France (ECOS-NORD/COLCIENCIAS/MEN/ICETEX; 619-2013, Diana Garcia de Olarte foundation PID and Colciencias grant 713-2016 #111574455633). JR was supported by "Poste d'accueil" INSERM and Imagine Institute.

The pathophysiology of adverse events following programmed cell death protein 1 (PD-1) blockade, including tuberculosis (TB) and autoimmunity, remains poorly characterized. We studied a patient with inherited PD-1 deficiency and TB who died of pulmonary autoimmunity. The patient’s leukocytes did not express PD-1 or respond to PD-1-mediated suppression. The patient’s lymphocytes produced only small amounts of interferon (IFN)-γ upon mycobacterial stimuli, similarly to patients with inborn errors of IFN-γ production who are vulnerable to TB. This phenotype resulted from a combined depletion of Vδ2+ γδ T, mucosal-associated invariant T and CD56bright natural killer lymphocytes and dysfunction of other T lymphocyte subsets. Moreover, the patient displayed hepatosplenomegaly and an expansion of total, activated and RORγT+ CD4−CD8− double-negative αβ T cells, similar to patients with STAT3 gain-of-function mutations who display lymphoproliferative autoimmunity. This phenotype resulted from excessive amounts of STAT3-activating cytokines interleukin (IL)-6 and IL-23 produced by activated T lymphocytes and monocytes, and the STAT3-dependent expression of RORγT by activated T lymphocytes. Our work highlights the indispensable role of human PD-1 in governing both antimycobacterial immunity and self-tolerance, while identifying potentially actionable molecular targets for the diagnostic and therapeutic management of TB and autoimmunity in patients on PD-1 blockade. Dysregulated immune features in a patient with a homozygous loss-of-function mutation in PDCD1 suggest that IL-6, IL-23, STAT3 and RORγT might be potential targets for treatment of PD-1 blockade-induced autoimmunity.

Humans homozygous or hemizygous for variants predicted to cause a loss of function of the corresponding protein do not necessarily present with overt clinical phenotypes. However, the set of effectively dispensable genes in the human genome has not yet been fully characterized. We report here 190 autosomal genes with 207 predicted loss-of-function variants, for which the frequency of homozygous individuals exceeds 1% in at least one human population from five major ancestry groups. No such genes were identified on the X and Y chromosomes. Manual curation revealed that 28 variants (15%) had been misannotated as loss-of-function, mainly due to linkage disequilibrium with different compensatory variants. Of the 179 remaining variants in 166 genes (0.82% of 20,232 genes), only 11 alleles in 11 genes had previously been confirmed experimentally to be loss-of-function. The set of 166 dispensable genes was enriched in olfactory receptor genes (41 genes), but depleted of genes expressed in a wide range of organs and in leukocytes. The 125 dispensable non-olfactory receptor genes displayed a relaxation of selective constraints both between species and within humans, consistent with greater redundancy. In total, 62 of these 125 genes were found to be dispensable in at least three human populations, suggesting possible evolution toward pseudogenes. Out of the 179 common loss-of-function variants, 72 could be tested for two neutrality selection statistics, and eight displayed robust signals of positive selection. These variants included the knownFUT2mutant allele conferring resistance to intestinal viruses and anAPOL3variant involved in resistance to parasitic infections. Finally, the 41 dispensable olfactory receptor genes also displayed a strong relaxation of selective constraints similar to that observed for the 341 non-dispensable olfactory receptor genes. Overall, the identification of 166 genes for which a sizeable proportion of humans are homozygous for predicted loss-of-function alleles reveals both redundancies and advantages of such deficiencies for human survival.Significance statementHuman genes homozygous for seemingly loss of function (LoF) variants are increasingly reported in a sizeable proportion of individuals without overt clinical phenotypes. Here, we found 166 genes with 179 predicted LoF variants for which the frequency of homozygous individuals exceeds 1% in at least one of the populations present in databases ExAC and gnomAD. This set of putatively dispensable genes showed relaxation of selective constraints suggesting that a large number of these genes are undergoing pseudogenization. Eight of the common LoF variants displayed robust signals of positive selection including two variants located in genes involved in resistance to infectious diseases. The identification of dispensable genes will allow identifying functions that are, at least nowadays, redundant, or possibly advantageous, for human survival.

BackgroundGenetic variants underlying severe diseases are less likely to be transmitted to the next generation, and are thus gradually and selectively eliminated from the population through negative selection. Here, we study the determinants of this evolutionary process in genes underlying severe diseases in humans.ResultsWe propose a novel approach, CoNeS, integrating known negative selection scores through principal component projection. We compare evidence for negative selection at 319 genes underlying inborn errors of immunity (IEI), which are life-threatening monogenic disorders. We find that genes underlying autosomal dominant (AD) or X-linked IEI are under stronger negative selection than those underlying autosomal recessive (AR) IEI, which are under no stronger selection than genes not known to be disease-causing. However, we find that genes with mutations causing AR IEI that are lethal before reproductive maturity and that display complete penetrance are under stronger negative selection than other genes underlying AR IEI. We also find that genes underlying AD IEI by haploinsufficiency are under stronger negative selection than other genes underlying AD IEI. Finally, we replicate these results in a study of 1,140 genes causing inborn errors of neurodevelopment.ConclusionsThese findings collectively show that the clinical outcomes of inborn errors, together with the mode and mechanism of inheritance of these errors, determine the strength of negative selection acting on severe disease-causing genes. These findings suggest that estimating the intensity of negative selection with CoNeS may facilitate the selection of candidate genes in patients suspected to carry an inborn error.

Patients with myeloproliferative neoplasms (MPNs) frequently progress to bone marrow failure or acute myeloid leukemia (AML), and mutations in epigenetic regulators such as the metabolic enzyme isocitrate dehydrogenase (IDH) are associated with poor outcomes. Here, we showed that combined expression of Jak2V617Fand mutant IDH1R132Hor Idh2R140Q induces MPN progression, alters stem/progenitor cell function, and impairs differentiation in mice. Jak2V617FIdh2R140Q–mutant MPNs were sensitive to small-molecule inhibition of IDH. Combined inhibition of JAK2 and IDH2 normalized the stem and progenitor cell compartments in the murine model and reduced disease burden to a greater extent than was seen with JAK inhibition alone. In addition, combined JAK2 and IDH2 inhibitor treatment also reversed aberrant gene expression in MPN stem cells and reversed the metabolite perturbations induced by concurrent JAK2 and IDH2 mutations. Combined JAK2 and IDH2 inhibitor therapy also showed cooperative efficacy in cells from MPN patients with both JAK2mutand IDH2mutmutations. Taken together, these data suggest that combined JAK and IDH inhibition May offer a therapeutic advantage in this high-risk MPN subtype., Damon Runyon Cancer Research Foundation (DRG-2241-15), Howard Hughes Medical Institute (Faculty Scholars Award), Stand Up To Cancer, National Cancer Institute (U.S.) (P50CA165962), National Cancer Institute (U.S.) (P30CA14051), Koch Institute for Integrative Cancer Research ( Dana-Farber Harvard Cancer Center Bridge Project), Leukemia & Lymphoma Society of America. Specialized Center of Research (SCOR) Program, National Institutes of Health (U.S.) (grant U54OD020355-01), National Institutes of Health (U.S.) (grant NCI R01CA172636), National Institutes of Health (U.S.) (grant R35CA197594), National Cancer Institute (U.S.) (Cancer Center Support Grant (P30 CA008747).

Summary Genetic and functional studies underscore the central role of JAK/STAT signaling in myeloproliferative neoplasms (MPNs). However, the mechanisms that mediate transformation in MPNs are not fully delineated, and clinically utilized JAK inhibitors have limited ability to reduce disease burden or reverse myelofibrosis. Here we show that MPN progenitor cells are characterized by marked alterations in gene regulation through differential enhancer utilization, and identify nuclear factor κB (NF-κB) signaling as a key pathway activated in malignant and non-malignant cells in MPN. Inhibition of BET bromodomain proteins attenuated NF-κB signaling and reduced cytokine production in vivo . Most importantly, combined JAK/BET inhibition resulted in a marked reduction in the serum levels of inflammatory cytokines, reduced disease burden, and reversed bone marrow fibrosis in vivo .

International audience; Herpes simplex virus-1 (HSV-1) encephalitis (HSE) is typically sporadic. Inborn errors of TLR3- and DBR1-mediated central nervous system cell-intrinsic immunity can account for forebrain and brainstem HSE, respectively. We report five unrelated patients with forebrain HSE, each heterozygous for one of four rare variants of SNORA31, encoding a small nucleolar RNA of the H/ACA class that are predicted to direct the isomerization of uridine residues to pseudouridine in small nuclear RNA and ribosomal RNA. We show that CRISPR/Cas9-introduced bi- and monoallelic SNORA31 deletions render human pluripotent stem cell (hPSC)-derived cortical neurons susceptible to HSV-1. Accordingly, SNORA31-mutated patient hPSC-derived cortical neurons are susceptible to HSV-1, like those from TLR3- or STAT1-deficient patients. Exogenous interferon (IFN)-β renders SNORA31- and TLR3- but not STAT1-mutated neurons resistant to HSV-1. Finally, transcriptome analysis of SNORA31-mutated neurons revealed normal responses to TLR3 and IFN-α/β stimulation but abnormal responses to HSV-1. Human SNORA31 thus controls central nervous system neuron-intrinsic immunity to HSV-1 by a distinctive mechanism.

Genetic etiologies of chronic mucocutaneous candidiasis (CMC) disrupt human IL-17A/F-dependent immunity at mucosal surfaces, whereas those of connective tissue disorders (CTDs) often impair the TGF-β-dependent homeostasis of connective tissues. The signaling pathways involved are incompletely understood. We report a three-generation family with an autosomal dominant (AD) combination of CMC and a previously undescribed form of CTD that clinically overlaps with Ehlers-Danlos syndrome (EDS). The patients are heterozygous for a private splice-site variant of MAPK8, the gene encoding c-Jun N-terminal kinase 1 (JNK1), a component of the MAPK signaling pathway. This variant is loss-of-expression and loss-of-function in the patients' fibroblasts, which display AD JNK1 deficiency by haploinsufficiency. These cells have impaired, but not abolished, responses to IL-17A and IL-17F. Moreover, the development of the patients' TH17 cells was impaired ex vivo and in vitro, probably due to the involvement of JNK1 in the TGF-β-responsive pathway and further accounting for the patients' CMC. Consistently, the patients' fibroblasts displayed impaired JNK1- and c-Jun/ATF-2-dependent induction of key extracellular matrix (ECM) components and regulators, but not of EDS-causing gene products, in response to TGF-β. Furthermore, they displayed a transcriptional pattern in response to TGF-β different from that of fibroblasts from patients with Loeys-Dietz syndrome caused by mutations of TGFBR2 or SMAD3, further accounting for the patients' complex and unusual CTD phenotype. This experiment of nature indicates that the integrity of the human JNK1-dependent MAPK signaling pathway is essential for IL-17A- and IL-17F-dependent mucocutaneous immunity to Candida and for the TGF-β-dependent homeostasis of connective tissues.

Juvenile-onset recurrent respiratory papillomatosis (JRRP) is a rare and debilitating childhood disease that presents with recurrent growth of papillomas in the upper airway. Two common human papillomaviruses (HPVs), HPV-6 and -11, are implicated in most cases, but it is still not understood why only a small proportion of children develop JRRP following exposure to these common viruses. We report 2 siblings with a syndromic form of JRRP associated with mild dermatologic abnormalities. Whole-exome sequencing of the patients revealed a private homozygous mutation in NLRP1, encoding Nucleotide-Binding Domain Leucine-Rich Repeat Family Pyrin Domain-Containing 1. We find the NLRP1 mutant allele to be gain of function (GOF) for inflammasome activation, as demonstrated by the induction of inflammasome complex oligomerization and IL-1β secretion in an overexpression system. Moreover, patient-derived keratinocytes secrete elevated levels of IL-1β at baseline. Finally, both patients displayed elevated levels of inflammasome-induced cytokines in the serum. Six NLRP1 GOF mutations have previously been described to underlie 3 allelic Mendelian diseases with differing phenotypes and modes of inheritance. Our results demonstrate that an autosomal recessive, syndromic form of JRRP can be associated with an NLRP1 GOF mutation.

Juvenile-onset recurrent respiratory papillomatosis (JRRP) is a rare and debilitating childhood disease that presents with recurrent growth of papillomas in the upper airway. Two common human papillomaviruses (HPVs), HPV-6 and -11, are implicated in most cases, but it is still not understood why only a small proportion of children develop JRRP following exposure to these common viruses. We report 2 siblings with a syndromic form of JRRP associated with mild dermatologic abnormalities. Whole-exome sequencing of the patients revealed a private homozygous mutation in NLRP1, encoding Nucleotide-Binding Domain Leucine-Rich Repeat Family Pyrin Domain-Containing 1. We find the NLRP1 mutant allele to be gain of function (GOF) for inflammasome activation, as demonstrated by the induction of inflammasome complex oligomerization and IL-1β secretion in an overexpression system. Moreover, patient-derived keratinocytes secrete elevated levels of IL-1β at baseline. Finally, both patients displayed elevated levels of inflammasome-induced cytokines in the serum. Six NLRP1 GOF mutations have previously been described to underlie 3 allelic Mendelian diseases with differing phenotypes and modes of inheritance. Our results demonstrate that an autosomal recessive, syndromic form of JRRP can be associated with an NLRP1 GOF mutation., United States Department of Health and Human Services; National Institutes of Health (NIH); NIH National Center for Advancing Translational Sciences (NCATS); French National Research Agency (ANR); Integrative Biology of Emerging Infectious Diseases Laboratoire d'Excellence; French Cancer Institute; Strategic Positioning Fund on Genetic Orphan Diseases from A* STAR, Singapore; Jeffrey Modell Foundation; Bijzonder Onderzoeksfonds-Tenure Grant; Cure-AID; European Union ERA-Net for Research Programmes on Rare Diseases; H2020; European Union (European Union); National Research Foundation; St. Giles Foundation; Rockefeller University; Institut National de la Sante et de la Recherche Medicale (Inserm); Paris Descartes University; Shapiro-Silverberg Fund for the Advancement of Translational Research; American Philosophical Society Daland Fellowship in Clinical Investigation; NIH National Center for Research Resources (NCRR)

International audience; Hundreds of patients with autosomal recessive, complete IL-12p40 or IL-12Rβ1 deficiency have been diagnosed over the last 20 years. They typically suffer from invasive mycobacteriosis and, occasionally, from mucocutaneous candidiasis. Susceptibility to these infections is thought to be due to impairments of IL-12-dependent IFN-γ immunity and IL-23-dependent IL-17A/IL-17F immunity, respectively. We report here patients with autosomal recessive, complete IL-12Rβ2 or IL-23R deficiency, lacking responses to IL-12 or IL-23 only, all of whom, unexpectedly, display mycobacteriosis without candidiasis. We show that αβ T, γδ T, B, NK, ILC1, and ILC2 cells from healthy donors preferentially produce IFN-γ in response to IL-12, whereas NKT cells and MAIT cells preferentially produce IFN-γ in response to IL-23. We also show that the development of IFN-γ-producing CD4 + T cells, including, in particular , mycobacterium-specific TH1* cells (CD45RA− CCR6+), is dependent on both IL-12 and IL-23. Last, we show that IL12RB1, IL12RB2, and IL23R have similar frequencies of deleterious variants in the general population. The comparative rarity of symptomatic patients with IL-12Rβ2 or IL-23R deficiency, relative to IL-12Rβ1 deficiency , is, therefore, due to lower clinical penetrance. There are fewer symptomatic IL-23R-and IL-12Rβ2-deficient than IL-12Rβ1-deficient patients, not because these genetic disorders are rarer, but because the isolated absence of IL-12 or IL-23 is, in part, compensated by the other cytokine for the production of IFN-γ, thereby providing some protection against mycobacteria. These experiments of nature show that human IL-12 and IL-23 are both required for optimal IFN-γ dependent immunity to mycobacteria, both individually and much more so cooperatively.

Mutational profiling has demonstrated utility in predicting the likelihood of disease progression in patients with myelofibrosis (MF). However, there is limited data regarding the prognostic utility of genetic profiling in MF patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT). We performed high-throughput sequencing of 585 genes on pre-transplant samples from 101 patients with MF who underwent allo-HCT and evaluated the association of mutations and clinical variables with transplantation outcomes. Overall survival (OS) at 5 years post-transplantation was 52%, and relapse-free survival (RFS) was 51.1 % for this cohort. Nonrelapse mortality (NRM) accounted for most deaths. Patient's age, donor's age, donor type, and Dynamic International Prognostic Scoring System score at diagnosis did not predict for outcomes. Mutations known to be associated with increased risk of disease progression, such as ASXL1, SRSF2, IDH1/2, EZH2, and TP53, did not impact OS or RFS. The presence of U2AF1 (P = .007) or DNMT3A (P = .034) mutations was associated with worse OS. A Mutation-Enhanced International Prognostic Scoring System 70 score was available for 80 patients (79%), and there were no differences in outcomes between patients with high risk scores and those with intermediate and low risk scores. Collectively, these data identify mutational predictors of outcome in MF patients undergoing allo-HCT. These genetic biomarkers in conjunction with clinical variables may have important utility in guiding transplantation decision making.

SummaryAlthough clinically tested JAK inhibitors reduce splenomegaly and systemic symptoms, molecular responses are not observed in most myeloproliferative neoplasm (MPN) patients. We previously demonstrated that MPN cells become persistent to type I JAK inhibitors that bind the active conformation of JAK2. We investigated whether CHZ868, a type II JAK inhibitor, would demonstrate activity in JAK inhibitor persistent cells, murine MPN models, and MPN patient samples. JAK2 and MPL mutant cell lines were sensitive to CHZ868, including type I JAK inhibitor persistent cells. CHZ868 showed significant activity in murine MPN models and induced reductions in mutant allele burden not observed with type I JAK inhibitors. These data demonstrate that type II JAK inhibition is a viable therapeutic approach for MPN patients.

Background: In the last few decades, theoretical models of cancer growth and progression have long focused on the aberrations of cancer cells alone, such as the abnormal mitotic and invasive characteristics of cancer cells. More recent research across multiple solid tumors suggests a critical interplay between solid tumors and immune regulating cells. Mounting evidence suggests that the immune system can tip the scales of cancer progression, eliciting either an anti-tumor or pro-tumor immune response depending upon varying stimulating and inhibitory factors. Here, we are the first to demonstrate novel mutations including leukemogenic mutations among tumor infiltrating lymphocytes in breast cancer patients. Methods: We obtained 17 primary breast cancer samples from patients who presented for either a lumpectomy or mastectomy as part of an IRB approved biospecimen protocol. Of the 17 patient samples, 13 had triple negative breast cancer, 2 had ER+, HER2+ disease, and 2 had ER+, HER2- disease. In the 17 samples, we used fluorescent activated cell sorting to separate CD45-positive hematopoietic cells from CD45-negative epithelial cells. We then performed exome sequencing of tumor-infiltrating hematopoietic cells to investigate for the presence of pathogenic mutations in tumor-associated leukocytes. In this first step, we identified candidate mutations in known cancer genes, including BCOR, NOTCH2, TET2, NF1, EZH2, and JAK1. As a validation step, we then performed capture-based sequencing of tumor-infiltrating leukocytes in 20 breast cancer samples matched to each patient’s germline DNA sample (buccal swab). In 10 of the 20 patients, we identified and validated somatic mutations. Of note, 6 of these patients harbored mutations known to be associated with leukemia, including DNTM3A, TET2, and BCOR. Most of these mutations were present in at least 5-20% of reads. This suggests that these mutations were present in enriched subclones and were not rare alleles occurring in a minority of hematopoietic stem cells. Lastly, we performed 454 deep sequencing analysis of microdissected tumor DNA samples and confirmed the absence of these mutations in breast cancer cells. Conclusion: Our data demonstrate somatic mutations in tumor infiltrating leukocytes in breast tumors which were not identified in matched germline or tumor DNA samples. Notably, some of these mutations have been implicated in the pathogenesis of lymphoid and myeloid malignancies. This observation suggests a unique relationship between cancer cells and mutant infiltrating leukocytes. We are now investigating the functional interaction between cancer cells and hematopoietic cells. Our findings reframe our understanding of carcinogenesis and offer novel opportunities for cancer detection and treatment. Citation Format: Elizabeth A Comen, Maria Kleppe, Hannah Wen, Britta Weigelt, Lennart Bastian, Brian Blum, Franck T Rapaport, Matt Keller, Nicolas Socci, Agnes Viale, Daoqi You, Robert Benezra, Edi Brogi, Jorge Reis-Filho, Michael Berger, Ross Levine, Larry Norton. Somatic leukemogenic mutations associated with infiltrating white blood cells in breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD1-4.

Specific combinations of acute myeloid leukemia (AML) disease alleles, including FLT3 and TET2 mutations, confer distinct biologic features and adverse outcome. We generated mice with mutations in Tet2 and Flt3, which resulted in fully penetrant, lethal AML. Multipotent Tet2(-/-);Flt3(ITD) progenitors (LSK CD48(+)CD150(-)) propagate disease in secondary recipients and were refractory to standard AML chemotherapy and FLT3-targeted therapy. Flt3(ITD) mutations and Tet2 loss cooperatively remodeled DNA methylation and gene expression to an extent not seen with either mutant allele alone, including at the Gata2 locus. Re-expression of Gata2 induced differentiation in AML stem cells and attenuated leukemogenesis. TET2 and FLT3 mutations cooperatively induce AML, with a defined leukemia stem cell population characterized by site-specific changes in DNA methylation and gene expression.

Deficiency in the cytosolic DNA sensor RNA Polymerase III was recently described in children with severe varicella zoster infection in the CNS or lungs. Here we describe a pair of monozygotic female twins, who both experienced severe recurrent CNS vasculitis caused by VZV reactivation. The clinical presentation and findings included recurrent episodes of headache, dizziness, and neurological deficits, cerebrospinal fluid with pleocytosis and intrathecal VZV antibody production, and magnetic resonance scan of the brain showing ischaemic lesions. We performed whole exome sequencing and identified a rare mutation in the Pol III subunit POLR3F. The identified R50W POLR3F mutation is predicted to be damaging by bioinformatics and when tested in functional assays, patient PBMCs exhibited impaired antiviral and inflammatory responses to the PoL III agonist Poly(dA:dT) as well as increased viral replication in patient cells compared to controls. Altogether, these cases add genetic and immunological evidence to the novel association between defects in sensing of AT-rich DNA present in the VZV genome and increased susceptibility to severe manifestations of VZV infection in the CNS in humans.AbbreviationsCADDcombined annotation dependent depletionCSFcerebrospinal fluidDOCKdedicator of cytokinesisIFNGRinterferon gamma receptorMSCmutation significance cut-offNKnatural killerPOLIIIRNA polymerase IIISCIDsevere combined immunodeficiencyTYKtyrosine kinaseVZVvaricella zoster virusWESwhole exome sequencing

Summary Mutations in epigenetic modifiers and signaling factors often co-occur in myeloid malignancies, including TET2 and NRAS mutations. Concurrent Tet2 loss and Nras G12D expression in hematopoietic cells induced myeloid transformation, with a fully penetrant, lethal chronic myelomonocytic leukemia (CMML), which was serially transplantable. Tet2 loss and Nras mutation cooperatively led to decrease in negative regulators of mitogen-activated protein kinase (MAPK) activation, including Spry2, thereby causing synergistic activation of MAPK signaling by epigenetic silencing. Tet2/Nras double-mutant leukemia showed preferential sensitivity to MAPK kinase (MEK) inhibition in both mouse model and patient samples. These data provide insights into how epigenetic and signaling mutations cooperate in myeloid transformation and provide a rationale for mechanism-based therapy in CMML patients with these high-risk genetic lesions.

Myelofibrosis (MF) is a bone marrow disorder characterized by clonal myeloproliferation, aberrant cytokine production, extramedullary hematopoiesis, and bone marrow fibrosis. Although somatic mutations in JAK2, MPL, and CALR have been identified in the pathogenesis of these diseases, inhibitors of the Jak2 pathway have not demonstrated efficacy in ameliorating MF in patients. TGF-β family members are profibrotic cytokines and we observed significant TGF-β1 isoform overexpression in a large cohort of primary MF patient samples. Significant overexpression of TGF-β1 was also observed in murine clonal MPLW515L megakaryocytic cells. TGF-β1 stimulated the deposition of excessive collagen by mesenchymal stromal cells (MSCs) by activating the TGF-β receptor I kinase (ALK5)/Smad3 pathway. MSCs derived from MPLW515L mice demonstrated sustained overproduction of both collagen I and collagen III, effects that were abrogated by ALK5 inhibition in vitro and in vivo. Importantly, use of galunisertib, a clinically active ALK5 inhibitor, significantly improved MF in both MPLW515L and JAK2V617F mouse models. These data demonstrate the role of malignant hematopoietic stem cell (HSC)/TGF-β/MSC axis in the pathogenesis of MF, and provide a preclinical rationale for ALK5 blockade as a therapeutic strategy in MF.

Recent studies have reported that activation-induced cytidine deaminase (AID) and ten-eleven-translocation (TET) family members regulate active DNA demethylation. Genetic alterations of TET2 occur in myeloid malignancies, and hematopoietic-specific loss of Tet2 induces aberrant hematopoietic stem cell (HSC) self-renewal/differentiation, implicating TET2 as a master regulator of normal and malignant hematopoiesis. Despite the functional link between AID and TET in epigenetic gene regulation, the role of AID loss in hematopoiesis and myeloid transformation remains to be investigated. Here, we show that Aid loss in mice leads to expansion of myeloid cells and reduced erythroid progenitors resulting in anemia, with dysregulated expression of Cebpa and Gata1, myeloid/erythroid lineage-specific transcription factors. Consistent with data in the murine context, silencing of AID in human bone marrow cells skews differentiation toward myelomonocytic lineage. However, in contrast to Tet2 loss, Aid loss does not contribute to enhanced HSC self-renewal or cooperate with Flt3-ITD to induce myeloid transformation. Genome-wide transcription and differential methylation analysis uncover the critical role of Aid as a key epigenetic regulator. These results indicate that AID and TET2 share common effects on myeloid and erythroid lineage differentiation, however, their role is nonredundant in regulating HSC self-renewal and in myeloid transformation.

Central diabetes insipidus (DI) is a rare finding in patients with acute myeloid leukemia (AML), usually occurring in patients with chromosome 3 or 7 abnormalities. We describe four patients with AML and concurrent DI and a fifth patient with AML and panhypopituitarism. Four of five patients had monosomy 7. Three patients had chromosome 3q21q26/EVI-1 gene rearrangements. The molecular genotype of patients with AML and DI is not known. Therefore, we performed gene sequencing of 30 genes commonly mutated in AML in three patients with available leukemia cell DNA. One patient had no identifiable mutations, and two had RUNX1 F158S mutations.

Genome profiling represents a critical pillar for clinical, translational, and basic research studies. Hospitals, core facilities, and research enterprises invest significant resources to generate genomic data sets. Yet, data management and analysis is frequently manual, which demands significant operator time and often results in siloed resources rendering them as single-use assets. Centralization of the genomic capital in a framework that enables automated processing, metadata integration and continuous interrogation maximizes return for investment and serves as the critical catalyst for research innovation, clinical translation and reproducible research. We developed Isabl, a plug-and-play infrastructure for scalable bioinformatics operations. Isabl provides solutions for databasing, assets management, tracking, automated and reproducible data processing. Dynamic reporting and meta-analysis across data assets is enabled. Isabl is built on four main components. First, an individual-centric and extensible relational database with tracking support for samples (temporal, spatial, aliquot), experimental data (assays, platforms, sequencing runs), cohorts (clinical trials, research projects) and versioned bioinformatics applications (assembly aware, tools, results). Second, the database is exposed through a fully featured RESTful API that enables horizontal integration with information systems such as sequencing cores LIMS, variant visualization platforms like cBioPortal, and where applicable, clinical and biospecimen institutional databases. Third, a Software Development Kit (SDK) built for Next Generation Sequencing assets management. The SDK enables automated execution of data import and language-agnostic bioinformatics applications (alignment, variant calling, post-processing) with support for cohort and individual level reporting features. Furthermore, the SDK facilitates dynamic retrieval of results using vertical and horizontal queries (individual and cohort level, respectively). Lastly, Isabl comes with a Single Page Web Application that fosters user interaction with multidisciplinary teams (i.e. researchers, project coordinators, engineers, clinicians) facilitating tracking of analyses, results visualization, and dynamic query processing. Isabl is currently supporting the Memorial Sloan Kettering Genome Pediatrics Precision Medicine Initiative, a prototype platform that delivers integrated, real-time automated reporting of clinical targeted gene re-sequencing, research whole genome and transcriptome profiling data; as well as linked data from pre-clinical models (i.e. PDX) and single cells studies. As an open-source tool, Isabl democratizes access to a purpose built, automated, scalable and fully integrable bioinformatics architecture. Isabl will be available at https://github.com/isabl-io. Citation Format: Juan S. Medina-Martínez, Juan E. Arango-Ossa, Gunes Gundem, Max F. Levine, Minal Patel, Noushin R. Farnoud, Venkata D. Yellapantula, Gao Teng, Joseph G. Mccarter, Elsa Bernard, Franck Rapaport, Dominik Glodzik, Ross L. Levine, Andrew Kung, Elli Papaemmanuil. A plug-and-play infrastructure for scalable bioinformatics operations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5105.

JAK1 is a critical effector of pro-inflammatory cytokine signaling and plays important roles in immune function, while abnormal JAK1 activity has been linked to immunological and neoplastic diseases. Specific functions of JAK1 in the context of hematopoiesis, and specifically within hematopoietic stem cells (HSCs), have not clearly been delineated. Here, we show that conditional Jak1 loss in HSCs reduces their self-renewal and markedly alters lymphoid/myeloid differentiation invivo. Jak1-deficient HSCs exhibit decreased competitiveness invivo and are unable to rescue hematopoiesis in the setting of myelosuppression. They exhibit increased quiescence, an inability to enter the cell cycle in response to hematopoietic stress, and a marked reduction in cytokine sensing, including in response to type I interferons and IL-3. Moreover, Jak1 loss is not fully rescued byexpression of a constitutively active Jak2 allele. Together, these data highlight an essential role for Jak1 in HSC homeostasis and stress responses.

After allogeneic hematopoietic stem cell transplantation (allo-HSCT), intestinal bacteria modulate risks of infection and graft-versus-host disease (GVHD). Neutropenic fever is common and treated with a choice of clinically equivalent antibiotics that target obligately anaerobic bacteria (anaerobes) to varying degrees. We retrospectively examined 857 allo-HSCT recipients and found that treatment of neutropenic fever with imipenem-cilastatin and piperacillin-tazobactam was associated with increased GVHD-related mortality at 5 years (21.5% in imipenem-cilastatin-treated patients vs. 13.1% in untreated patients, p=0.025, and 19.8% in piperacillin-tazobactam-treated patients vs. 11.9% in untreated patients, p=0.007). However, two other antibiotics also used to treat neutropenic fever, aztreonam and cefepime, were not associated with GVHD-related mortality (p=0.78 and p=0.98, respectively). Analysis of stool microbiota composition showed that piperacillin-tazobactam administration was associated with increased compositional perturbation. Studies in mouse models demonstrated similar effects of these antibiotics, as well as aggravated GVHD mortality with imipenem-cilastatin or piperacillin-tazobactm compared to aztreonam (p

Genetic heterogeneity contributes to clinical outcome and progression of most tumors, but little is known about allelic diversity for epigenetic compartments, and almost no data exist for acute myeloid leukemia (AML). We examined epigenetic heterogeneity as assessed by cytosine methylation within defined genomic loci with four CpGs (epialleles), somatic mutations, and transcriptomes of AML patient samples at serial time points. We observed that epigenetic allele burden is linked to inferior outcome and varies considerably during disease progression. Epigenetic and genetic allelic burden and patterning followed different patterns and kinetics during disease progression. We observed a subset of AMLs with high epiallele and low somatic mutation burden at diagnosis, a subset with high somatic mutation and lower epiallele burdens at diagnosis, and a subset with a mixed profile, suggesting distinct modes of tumor heterogeneity. Genes linked to promoter-associated epiallele shifts during tumor progression showed increased single-cell transcriptional variance and differential expression, suggesting functional impact on gene regulation. Thus, genetic and epigenetic heterogeneity can occur with distinct kinetics likely to affect the biological and clinical features of tumors. Refereed/Peer-reviewed