BACKGROUND:Metabolic dysfunction-associated steatotic liver disease (MASLD) progresses to metabolic dysfunction-associated steatohepatitis (MASH) and is a major cause of liver cirrhosis. Although liver inflammation is the hallmark feature of MASH versus MASLD, the involvement of the peripheral immune cell compartments in disease progression is poorly understood, and single-cell profiles of peripheral immune cells in MASLD/MASH are not known. METHODS:Patients with MASLD/MASH and healthy volunteers have been prospectively enrolled in a cross-sectional study. Patients have been histologically stratified and further characterized by liver bulk RNA sequencing (RNA-Seq). Peripheral immune cells from patients and control blood samples have been comprehensively profiled using bulk and single RNA-Seq. RESULTS:Twenty-two patients with fibrosis stage less than F3 have been histologically stratified into patients with low, medium, and high disease activity scores (NAFLD activity score [NAS]). In contrast to fibrosis, the NAS group correlated with noninvasive imaging readouts and blood biomarkers of liver damage and inflammation (ALT, AST). The prevalence of type 2 diabetes and obesity increased with the NAS stage. Bulk RNA-seq profiling of patient liver biopsies revealed gene signatures that were positively and negatively associated with NAS. Known marker genes for liver fibrosis where upregulated on RNA level. Blood bulk RNA-seq showed only moderate differences in patients versus healthy controls. In contrast, single-cell analysis of white blood cells revealed multiple alterations of immune (sub-)populations, including an increased abundance of immature B cells and myeloid suppressor cells in patients with MASLD/MASH as compared to healthy controls. CONCLUSIONS:The study gives new insights into the pathophysiology of MASLD/MASH already manifesting relatively early in peripheral immune cell compartments. This opens new avenues for the development of new biomarker diagnostics and disease therapies.

Chronic liver diseases, such as non-alcoholic steatohepatitis (NASH)-induced cirrhosis, are characterized by an increasing accumulation of stressed, damaged, or dying hepatocytes. Hepatocyte damage triggers the activation of resident immune cells, such as Kupffer cells (KC), as well as the recruitment of immune cells from the circulation toward areas of inflammation. After infiltration, monocytes differentiate into monocyte-derived macrophages (MoMF) which are functionally distinct from resident KC. We herein aim to compare the in vitro signatures of polarized macrophages and activated hepatic stellate cells (HSC) with ex vivo-derived disease signatures from human NASH. Furthermore, to shed more light on HSC activation and liver fibrosis progression, we investigate the effects of the secretome from primary human monocytes, macrophages, and NK cells on HSC activation. Interleukin (IL)-4 and IL-13 treatment induced transforming growth factor beta 1 (TGF-β1) secretion by macrophages. However, the supernatant transfer did not induce HSC activation. Interestingly, PMA-activated macrophages showed strong induction of the fibrosis response genes COL10A1 and CTGF, while the supernatant of IL-4/IL-13-treated monocytes induced the upregulation of COL3A1 in HSC. The supernatant of PMA-activated NK cells had the strongest effect on COL10A1 induction in HSC, while IL-15-stimulated NK cells reduced the expression of COL1A1 and CTGF. These data indicate that other factors, aside from the well-known cytokines and chemokines, might potentially be stronger contributors to the activation of HSCs and induction of a fibrotic response, indicating a more diverse and complex role of monocytes, macrophages, and NK cells in liver fibrosis progression.

BACKGROUND:Acute leukemias represent deadly malignancies that require better treatment. As a challenge, treatment is counteracted by a microenvironment protecting dormant leukemia stem cells.METHODS:To identify responsible surface proteins, we performed deep proteome profiling on minute numbers of dormant patient-derived xenograft (PDX) leukemia stem cells isolated from mice. Candidates were functionally screened by establishing a comprehensive CRISPR‒Cas9 pipeline in PDX models in vivo.RESULTS:A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) was identified as an essential vulnerability required for the survival and growth of different types of acute leukemias in vivo, and reconstitution assays in PDX models confirmed the relevance of its sheddase activity. Of translational importance, molecular or pharmacological targeting of ADAM10 reduced PDX leukemia burden, cell homing to the murine bone marrow and stem cell frequency, and increased leukemia response to conventional chemotherapy in vivo.CONCLUSIONS:These findings identify ADAM10 as an attractive therapeutic target for the future treatment of acute leukemias.

Immune dysregulation and inflammation by hepatic-resident leukocytes is considered a key step in disease progression of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis toward cirrhosis and hepatocellular carcinoma. Here, we provide a protocol for isolation and characterization of liver-resident immune cells from fine-needle biopsies obtained from a rodent model and humans. We describe steps for isolating leukocytes, cell sorting, and RNA extraction and sequencing. We then detail procedures for low-input mRNA sequencing analyses.

Letter to Blood| February 23, 2023 WT1 and DNMT3A play essential roles in the growth of certain patient AML cells in mice Maryam Ghalandary, Maryam Ghalandary ∗ Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany Search for other works by this author on: This Site PubMed Google Scholar Yuqiao Gao, Yuqiao Gao ∗ Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany Search for other works by this author on: This Site PubMed Google Scholar Diana Amend, Diana Amend Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany Search for other works by this author on: This Site PubMed Google Scholar Ginte Kutkaite, Ginte Kutkaite Institute of Computational Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, GermanyDepartment of Biology, Ludwig-Maximilians University Munich, Martinsried, Germany https://orcid.org/0000-0002-2918-294X Search for other works by this author on: This Site PubMed Google Scholar Binje Vick, Binje Vick Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, GermanyGerman Cancer Consortium, Partner Site Munich, Munich, Germany https://orcid.org/0000-0003-1956-2778 Search for other works by this author on: This Site PubMed Google Scholar Karsten Spiekermann, Karsten Spiekermann Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, Ludwig Maximilians University, Munich, Germany https://orcid.org/0000-0002-5139-4957 Search for other works by this author on: This Site PubMed Google Scholar Maja Rothenberg-Thurley, Maja Rothenberg-Thurley Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, Ludwig Maximilians University, Munich, Germany Search for other works by this author on: This Site PubMed Google Scholar Klaus H. Metzeler, Klaus H. Metzeler Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, Ludwig Maximilians University, Munich, GermanyDepartment of Hematology and Cell Therapy, University Hospital Leipzig, Leipzig, Germany https://orcid.org/0000-0003-3920-7490 Search for other works by this author on: This Site PubMed Google Scholar Anetta Marcinek, Anetta Marcinek Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, Ludwig Maximilians University, Munich, Germany Search for other works by this author on: This Site PubMed Google Scholar Marion Subklewe, Marion Subklewe Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, Ludwig Maximilians University, Munich, Germany https://orcid.org/0000-0003-3905-0251 Search for other works by this author on: This Site PubMed Google Scholar Michael P. Menden, Michael P. Menden Institute of Computational Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, GermanyDepartment of Biology, Ludwig-Maximilians University Munich, Martinsried, GermanyGerman Centre for Diabetes Research, Neuherberg, Germany https://orcid.org/0000-0003-0267-5792 Search for other works by this author on: This Site PubMed Google Scholar Vindi Jurinovic, Vindi Jurinovic Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany Search for other works by this author on: This Site PubMed Google Scholar Ehsan Bahrami, Ehsan Bahrami Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany https://orcid.org/0000-0002-1672-5503 Search for other works by this author on: This Site PubMed Google Scholar Irmela Jeremias Irmela Jeremias Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, GermanyGerman Cancer Consortium, Partner Site Munich, Munich, GermanyDepartment of Pediatrics, University Hospital, Ludwig Maximilians University, Munich, Germany https://orcid.org/0000-0003-1773-7677 Search for other works by this author on: This Site PubMed Google Scholar Blood (2023) 141 (8): 955–960. https://doi.org/10.1182/blood.2022016411 Article history Submitted: March 23, 2022 Accepted: September 25, 2022 Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Request Permissions Cite Icon Cite Search Site Citation Maryam Ghalandary, Yuqiao Gao, Diana Amend, Ginte Kutkaite, Binje Vick, Karsten Spiekermann, Maja Rothenberg-Thurley, Klaus H. Metzeler, Anetta Marcinek, Marion Subklewe, Michael P. Menden, Vindi Jurinovic, Ehsan Bahrami, Irmela Jeremias; WT1 and DNMT3A play essential roles in the growth of certain patient AML cells in mice. Blood 2023; 141 (8): 955–960. doi: https://doi.org/10.1182/blood.2022016411 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBlood Search Subjects: Myeloid Neoplasia TO THE EDITOR: Patients with acute myeloid leukemia (AML) experience poor prognosis, and precision oncology represents an attractive therapeutic option, applying targeted therapies against so-called dependencies.1-4 Dependencies are essential components required for cell growth and survival; they represent attractive therapeutic targets as their inhibition reduces tumor burden.1-4 Many genes recurrently mutated in AML contribute to oncogenesis,5,6 which may imply a role as dependency and allow precision therapy, based on genetic profiling. Examples already in routine clinical practice include AML with mutated FMS related receptor tyrosine kinase 3 treated with midostaurin and AML with mutated isocitrate dehydrogenase responding to ivosidenib.2 Herein, we asked whether additional recurrently mutated genes might represent dependencies in established AML. Previous efforts to identify dependencies used established cell lines, including large-scale functional genomic screens; WT1 and DNMT3A were shown to be dispensable... References 1.Kantarjian H, Kadia T, DiNardo C, et al. Acute myeloid leukemia: current progress and future directions. Blood Cancer J. 2021;11(2):41.Google ScholarCrossrefSearch ADS PubMed 2.Short NJ, Konopleva M, Kadia TM, et al. Advances in the treatment of acute myeloid leukemia: new drugs and new challenges. Cancer Discov. 2020;10(4):506-525.Google ScholarCrossrefSearch ADS PubMed 3.Park JJH, Hsu G, Siden EG, Thorlund K, Mills EJ. 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Cancer Discov. 2022;12(2):432-449.Google ScholarCrossrefSearch ADS PubMed 18.Liu WH, Mrozek-Gorska P, Wirth AK, et al. Inducible transgene expression in PDX models in vivo identifies KLF4 as a therapeutic target for B-ALL. Biomark Res. 2020;8:46.Google ScholarCrossrefSearch ADS PubMed 19.Rampal R, Figueroa ME. Wilms tumor 1 mutations in the pathogenesis of acute myeloid leukemia. Haematologica. 2016;101(6):672-679.Google ScholarCrossrefSearch ADS PubMed 20.Pronier E, Bowman RL, Ahn J, et al. Genetic and epigenetic evolution as a contributor to WT1-mutant leukemogenesis. Blood. 2018;132(12):1265-1278.Google ScholarCrossrefSearch ADS PubMed 21.Mayle A, Yang L, Rodriguez B, et al. Dnmt3a loss predisposes murine hematopoietic stem cells to malignant transformation. Blood. 2015;125(4):629-638.Google ScholarCrossrefSearch ADS PubMed 22.Celik H, Mallaney C, Kothari A, et al. Enforced differentiation of Dnmt3a-null bone marrow leads to failure with c-Kit mutations driving leukemic transformation. Blood. 2015;125(4):619-628.Google ScholarCrossrefSearch ADS PubMed 23.Huang YH, Chen CW, Sundaramurthy V, et al. Systematic profiling of DNMT3A variants reveals protein instability mediated by the DCAF8 E3 ubiquitin ligase adaptor. Cancer Discov. 2022;12(1):220-235.Google ScholarCrossrefSearch ADS PubMed 24.Challen GA, Sun D, Jeong M, et al. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet. 2011;44(1):23-31.Google ScholarCrossrefSearch ADS PubMed 25.Bera R, Chiu MC, Huang YJ, Huang G, Lee YS, Shih LY. DNMT3A mutants provide proliferating advantage with augmentation of self-renewal activity in the pathogenesis of AML in KMT2A-PTD-positive leukemic cells. Oncogenesis. 2020;9(2):7.Google ScholarCrossrefSearch ADS PubMed © 2023 by The American Society of Hematology2023 © 2023 by The American Society of Hematology2023 You do not currently have access to this content. Sign in via your Institution

Resistance towards cancer treatment represents a major clinical obstacle, preventing cure of cancer patients. To gain mechanistic insights, we developed a model for acquired resistance to chemotherapy by treating mice carrying patient derived xenografts (PDX) of acute lymphoblastic leukemia with widely-used cytotoxic drugs for 18 consecutive weeks. In two distinct PDX samples, tumors initially responded to treatment, until stable disease and eventually tumor re-growth evolved under therapy, at highly similar kinetics between replicate mice. Notably, replicate tumors developed different mutations in TP53 and individual sets of chromosomal alterations, suggesting independent parallel clonal evolution rather than selection, driven by a combination of stochastic and deterministic processes. Transcriptome and proteome showed shared dysregulations between replicate tumors providing putative targets to overcome resistance. In vivo CRISPR/Cas9 dropout screens in PDX revealed broad dependency on BCL2, BRIP1 and COPS2. Accordingly, venetoclax re-sensitized derivative tumors towards chemotherapy, despite genomic heterogeneity, demonstrating direct translatability of the approach. Hence, despite the presence of multiple resistance-associated genomic alterations, effective rescue treatment for polychemotherapy-resistant tumors can be identified using functional testing in preclinical models.

Background: Acute leukemias require better treatment and targeted therapies represent interesting future therapeutic options. Such therapies precisely inhibit molecules with essential function, also called vulnerabilities or dependencies, so that leukemia cells die once the target is inhibited. Aims: Here, we aimed at identifying therapeutic targets on a patient individual level and in the surrounding of a living organism. Towards this aim, we established CRISPR Cas9 dropout screens in PDX models of acute leukemias in vivo. Methods: Primary patient acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) samples were transplanted into immunocompromised NSG mice and re-passaged to develop serially transplantable PDX models. To overcome limited transduction efficiency inherent to PDX leukemia cells, lentiviruses were used. Results: We first elaborated on the maximum library size to be used in PDX models in vivo. As only a limited number of PDX cells home into mice, library size is limited to ensure the coverage of the library. We used, screening sgRNAs as genetic barcodes in Cas9 negative samples to determine the maximum library size upon next generation sequencing (NGS); a regular distribution of all sgRNAs recovered from mice at the end of the experiments indicated a suitable library size. We found that PDX ALL models tolerated a larger library compared to PDX AML models that might reflect lower intra-sample heterogeneity and higher leukemia stem cell frequency in ALL compared to AML. To perform knockout screens, a split-construct for Cas9 was used to reduce plasmid size and optimize lentiviral transduction efficiency into PDX cells; concomitantly split-GFP was used to enrich cells expressing both Cas9 split plasmids by flow cytometry. A customized library consisting of 146 target genes combined with positive and negative control genes was designed at 5 sgRNAs per gene using the CLUE platform (www.crispr-clue.de) (Becker et al., Nucleic Acids Res. 2020) and cloned into a lentiviral vector backbone. The sgRNA library vectors include either a puromycin-resistance cassette or an H-2Kk surface marker, each combined with a BFP fluorescent marker. This allows to determine transduction efficiency by FACS-based detection of BFP and to enrich the transduced PDX cells either by puromycin selection or by H-2Kk-MACS selection. Input samples were collected after library transduction and enrichment to >90% CRISPR/Cas9 sgRNA library positive cells, while remaining cells were injected into immunocompromised NSG mice, grown in vivo and re-isolated at advanced disease stage. Analysis of input and endpoint samples using NGS enabled to determine the frequency of sgRNAs and bioinformatically compare the abundance of different sgRNAs between the control and the samples of interest using MAGeCK (Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout). A significant sgRNA dropout was characterized by a p-value below 0.05 and a FDR below 0.1. The screening experiments led to the top 10 depleted dropout genes for ALL and AML PDX samples. Besides individual dropouts of the different samples, also shared dropouts were detected. Especially sample overlapping dropouts might represent interesting starting points for new therapeutic options. Summary/Conclusion: In summary, we have established a CRISPR Cas9 screening pipeline, which allows investigating therapeutic targets on a patient-individual level in ALL PDX models and, for the first time, in AML PDX models in vivo.

Interrupting tumor-microenvironment interactions is an attractive therapeutic strategy. We developed a CRISPR-Cas9 screening approach for functional analysis of surface molecules in patient-derived xenograft (PDX) acute leukemia (AL) models in vivo in order to decipher tumor specific vulnerabilities. A customized library was run in 2 AL PDX samples and candidates were confirmed using a competitive in vivo approach. ADAM10 was depleted in both and validated in 6 PDX models. Treating PDX cells with ADAM10 inhibitor reduced the bone marrow (BM) engraftment capacity, while KO of ADAM10 reduced the leukemia stem cell frequency. Both AML and ALL ADAM10 KO PDX samples showed increased sensitivity towards routine chemotherapy in vivo. Reconstitution of ADAM10 KO PDX cells with a WT variant in vivo rescued the phenotype, while an enzymatic domain lacking variant did not, highlighting the importance of ADAM10’s sheddase function. In conclusion, we established CRISPR-Cas9 drop-out screens in PDX models in vivo to explore patient-specific tumor dependencies. Our data revealed ADAM10’s role in maintaining leukemic cells in the BM niche, thus representing an attractive future therapeutic target.

Background: Tumor-microenvironment interactions are critically important determinants contributing to leukemia formation and maintenance. Interrupting the leukemia-bone marrow interaction represents an attractive therapeutic approach in acute leukemia (AL). Functional genomics significantly increases our understanding of the vulnerabilities and gene dependencies of individual tumors. Aims: Here, we developed a CRISPR-Cas9 screening approach for functional analysis of surface molecules in patient-derived xenograft (PDX) AL models in vivo. Methods: Size of CRISPR library was determined by genetic barcoding. Stable expression of fluorescently labelled Cas9 and sgRNA constructs in two PDX samples. Enrichment of double positive cells by MACS and injection into NSG mice. Gene depletion analysis using MAGeCK algorithm to screen and functional competitive in vivo assays to validate the candidates. Characterization of the ADAM10 KO or inhibitor (GI254023X) treated cells for engraftment capacity by homing assay, frequency of leukemic stem cells by competitive limiting dilution transplantation assay (LDTA), sensitivity towards routine chemotherapy by in vivo competitive chemotherapy trials in both lineages. Rescue assay by reconstitution of ADAM10 variants in functional competitive in vivo assays. Results: When running a customized CRISPR-Cas9 screen targeting about 100 cell surface candidates in two AL PDX samples, several sample-specific, but also commonly depleted candidates were identified. CRISPR screen findings were confirmed on the level of single molecules, using a competitive molecular in vivo approach and testing the PDX cells with and without knockout in the same mouse. These experiments validated an essential function for the two well-known depleted candidates CXCR4 and ITGB1 in both PDX models in vivo. Of note, various members of the Solute Carrier Family (SLC) were among the list of drop-out candidates. ADAM10 was identified as a commonly depleted candidate in both PDX models. In vivo competitive experiments confirmed the essential role of ADAM10 in PDX models from 6 additional patients with either acute lymphoblastic leukemia (ALL) or acute myeloblastic leukemia (AML), indicating a broad essential role of ADAM10 in both, ALL and AML, independent from their oncogenic-driver mutations and chromosomal abnormalities. Moreover, treating PDX cells with an ADAM10 chemical inhibitor resulted in significantly reduced engraftment capacity into the bone marrow (BM), indicating a role for ADAM10 in the early engraftment and homing process in the BM microenvironment. Knockout of ADAM10 reduced the frequency of leukemia stem cells, indicating that a relevant fraction of stem cells depends on ADAM10. ADAM10 KO ALL and AML PDX samples showed increased sensitivity towards routine chemotherapy treatments, indicating that inhibition of ADAM10 sensitizes AL towards conventional chemotherapy. When ADAM10 knockout cells were reconstituted with different recombinant ADAM10 variants, PDX in vivo experiments revealed that wildtype ADAM10 rescued the phenotype, while an ADAM10 variant lacking the enzymatic domain did not, highlighting the importance of the sheddase activity for ADAM10 function in leukemia maintenance. Summary/Conclusion: In summary, we established CRISPR-Cas9 drop-out screens in PDX models in vivo as technology to explore patient-specific tumor dependencies. Our data revealed a yet unknown function of ADAM10 to maintain patient leukemic cells in the bone marrow microenvironment niche. ADAM10 thus represents an attractive future therapeutic target for the treatment of acute leukemia.

Acute leukemias require more accurate and effective treatments, especially upon disease relapse. In search for novel therapeutic targets for acute leukemias, we established a pipeline for CRISPR-Cas9 mediated functional genomic screens which harbor the ability to elegantly increase our knowledge about vulnerabilities and gene dependencies. For a highly patient-related setting, we performed CRISPR knockout (KO) dropout screens in patient-derived xenograft (PDX) models in vivo, combining the advantages of studying an individual patient´s tumor cell in the physiologic in vivo bone marrow microenvironment.

Background: Functional genomic screens elegantly increase our understanding of biology of leukemias. So far, CRISPR/Cas9 screens are widely performed in cell lines and in genetically engineered mouse models, in vitro and in vivo; here, we extended their use to patient-derived leukemia cells in vivo. Methods Serially transplantable patient-derived xenograft (PDX) models were generated from children and adults with acute lymphoblastic leukemia (ALL). Cas9 was stably expressed in PDX ALL cells using a split form of Cas9 assembled by inteins, facilitating lentiviral-mediated gene delivery. Customized sgRNA library was generated using golden gate cloning, at 5 sgRNAs per target gene. The sgRNA vector additionally expressed a fluorochrome marker and a tag, for sequential magnetic-activated cell sorting (MACS) and flow cytometry (FACS) enrichment of sgRNA transduced PDX cells. Highly enriched Cas9/sgRNA double transgenic cells were transplanted into NSG mice and animals sacrificed after different periods of time. Cells were re-isolated from bone marrow, purified and subjected to PCR-based amplification of sgRNA library followed by next generation sequencing. Differential sgRNA distributions were analysed using a MAGeCK pipeline. Results We aimed to establish a comprehensive CRISPR screen pipeline allowing functional genomic screens in patients' acute leukemia cells. We investigated surface molecules required for cell homing and growth in mice, using a distinct customized sgRNA library. Quality controls of the sgRNA plasmid pool as well as transgenic PDX input samples verified standard distribution of all sgRNAs. As knockout was required at the time point of transplantation, conditions for prolonged culture of PDX ALL cells in vitro were optimized. Before injection into NSG mice, transduced PDX ALL cells were successfully enriched to above 95% using MACS and FACS. Over time in vivo, deep sequencing of re-isolated PDX cells revealed unchanged distribution of control sgRNAs, but strong loss of sgRNAs targeting CXCR4 and ITGB1, suggesting that CXCR4 and ITGB1 might be required for PDX ALL cell homing and engraftment. To validate the findings of drop-out CRISPR screen, we analyzed single sgRNAs targeting CXCR4 and ITGB1 in PDX cells. Competitive in vivo assays monitored by recombinant fluorochrome markers showed that the cells with CXCR4 or ITGB1 knockout had a significant disadvantage in vivo with respect to homing and growth in mice, compared to the control population. Taken together, we established a comprehensive workflow for CRISPR screen in PDX model of ALL in vivo. Our data identify and validate that CXCR4 and ITGB1 are required for homing and growth of PDX ALL cells in mice. Conclusion We show that CRISPR/Cas9 functional genetic screens are feasible in PDX acute leukemia models in vivo and report the first such screen, as far as to our knowledge. Extending CRISPR/Cas9 screens to patients' cells will greatly facilitate our understanding of individual leukemia biology and therapeutic targets in the future. Becker: AVA Lifescience GmbH: Consultancy.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a critical regulator of cell death and inflammation, but its relevance for human disease pathogenesis remains elusive. Studies of monogenic disorders might provide critical insights into disease mechanisms and therapeutic targeting of RIPK1 for common diseases. Here, we report on eight patients from six unrelated pedigrees with biallelic loss-of-function mutations in RIPK1 presenting with primary immunodeficiency and/or intestinal inflammation. Mutations in RIPK1 were associated with reduced NF-κB activity, defective differentiation of T and B cells, increased inflammasome activity, and impaired response to TNFR1-mediated cell death in intestinal epithelial cells. The characterization of RIPK1-deficient patients highlights the essential role of RIPK1 in controlling human immune and intestinal homeostasis, and might have critical implications for therapies targeting RIPK1.

We identify SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2), also known as BAF60b (BRG1/Brahma-associated factor 60b), as a critical regulator of myeloid differentiation in humans, mice, and zebrafish. Studying patients from three unrelated pedigrees characterized by neutropenia, specific granule deficiency, myelodysplasia with excess of blast cells, and various developmental aberrations, we identified three homozygous loss-of-function mutations in SMARCD2. Using mice and zebrafish as model systems, we showed that SMARCD2 controls early steps in the differentiation of myeloid-erythroid progenitor cells. In vitro, SMARCD2 interacts with the transcription factor CEBPɛ and controls expression of neutrophil proteins stored in specific granules. Defective expression of SMARCD2 leads to transcriptional and chromatin changes in acute myeloid leukemia (AML) human promyelocytic cells. In summary, SMARCD2 is a key factor controlling myelopoiesis and is a potential tumor suppressor in leukemia.

BACKGROUND:Myb-like, SWIRM, and MPN domains 1 (MYSM1) is a transcriptional regulator mediating histone deubiquitination. Its role in human immunity and hematopoiesis is poorly understood.OBJECTIVES:We sought to investigate the clinical, cellular, and molecular features in 2 siblings presenting with progressive bone marrow failure (BMF), immunodeficiency, and developmental aberrations.METHODS:We performed genome-wide homozygosity mapping, whole-exome and Sanger sequencing, immunophenotyping studies, and analysis of genotoxic stress responses. p38 activation, reactive oxygen species levels, rate of apoptosis and clonogenic survival, and growth in immune and nonimmune cells were assessed. The outcome of allogeneic hematopoietic stem cell transplantation (HSCT) was monitored.RESULTS:We report 2 patients with progressive BMF associated with myelodysplastic features, immunodeficiency affecting B cells and neutrophil granulocytes, and complex developmental aberrations, including mild skeletal anomalies, neurocognitive developmental delay, and cataracts. Whole-exome sequencing revealed a homozygous premature stop codon mutation in the gene encoding MYSM1. MYSM1-deficient cells are characterized by increased sensitivity to genotoxic stress associated with sustained induction of phosphorylated p38 protein, increased reactive oxygen species production, and decreased survival following UV light-induced DNA damage. Both patients were successfully treated with allogeneic HSCT with sustained reconstitution of hematopoietic defects.CONCLUSIONS:Here we show that MYSM1 deficiency is associated with developmental aberrations, progressive BMF with myelodysplastic features, and increased susceptibility to genotoxic stress. HSCT represents a curative therapy for patients with MYSM1 deficiency.

Differentiation of hematopoietic stem cells follows a hierarchical program of transcriptional-regulated events. We here identify SMARCD2 (Swi/Snf-related matrix associated actin dependent regulator of chromatin, subfamily D, member 2) as critical regulator of myelopoiesis in humans, mice, and zebrafish. We studied four patients from three unrelated pedigrees presenting with a novel syndromatic phenotype comprising congenital neutropenia, specific granule deficiency, susceptibility to myelodysplasia with excess of blasts, and various skeletal anomalies. All patients had homozygous loss-of-function mutations in SMARCD2. In contrast to wildtype alleles, the variant alleles did not give rise to proteins with capacity to interact with the SWI/SNF subunits BRG1, BAF170, BAF155, and BAF47, as shown by co-immunoprecipitation experiments. In vitro, knockdown of SMARCD2 in promyelocytic NB4 cells, differentiated in the presence of ATRA, led to decreased expression of genes encoding the primary granule proteins cathelicidin (CAMP) and alpha-1-antitrypsin (AAT) as well as specific granule proteins matrix metalloproteinase-8 (MMP8), transcobalamin (TCN1) and lactoferrin (LTF). This phenotype is reminiscent of patients with specific granule deficiency, characterized by mutations in CEBPE, a known transcription factor controlling terminal neutrophil development. We therefore hypothesized that SMARCD2 may act via CEBPe and performed immunoprecipitation studies in transfected cells. Upon pull-down of SMARCD2, CEPBE could be detected, and vice versa, suggesting that both proteins physically interact to control transcriptional networks. To interrogate effects of SMARCD2 deficiency on global chromatin accessibility we made use of ATAC sequencing of undifferentiated and ATRA-differentiated NB4 cells and compared this data with comprehensive RNA-sequencing results. A specific subset of genes was found deregulated in both assays, affecting vesicular trafficking, migration and signalling pathways. To validate a role for SMARCD2 in hematopoiesis in vivo, we generated murine and zebrafish model systems. We generated Smarcd2-/- mice by injection of Smarcd2+/- murine ES cells into blastocysts, transfer into pseudo-pregnant mice and interbreeding of heterozygous Smarcd2+/- offsprings. The mutant allele was inherited in a Mendelian fashion but no viable mice were born. 14.5dpc embryos were characterized by anemia and reduced size compared to their littermates. Analysis of fetal liver hematopoiesis revealed a complete absence of CD11b+Gr1+ and CD11b+Ly6c+ cells, whereas the number of LSK stem cells was not affected. Futhermore, Smarcd2-/- embryos showed aberrations in erythroid cells such as extensive anisocytosis, multinucleated cells, and perturbed mitosis. In cytokine-driven colony forming unit assays, GM-CSF, M-CSF, and G-CSF induced myeloid cell differentiation was decreased. Transcriptional profiling of LSK stem cells revealed a striking dysbalance affecting genes involved in signaling pathways and host defence, including CEBPE-dependent genes. Among a total of 12362 detected genes, we found 4290 to be differentially expressed (DESeq2, FDR<10%). Interestingly, the majority (79%) of the 605 genes with a relatively large difference (fold-change > 1.4, FDR<1%) were up- and not downregulated. Next, we generated three Smarcd2-deficient zebrafish models using a) morpholino-mediated knockdown in Tg(mpx:EGFP)i114 and Tg(lyz:dsRed)nz50 strains of the orthologous gene or b) Crisp/Cas9-mediated genomic engineering of this locus in Tg(mpx:EGFP)i114. In all models, the numbers of neutrophil granulocytes were significantly reduced. We conclude that SMARCD2 is a critical factor orchestrating transcriptional networks controlling hematopoiesis across species, in particular regulation and maintenance of neutrophil differentation and prevention of leukemogenesis.

Inherited bone marrow failure syndromes comprise a heterogeneous group of genetic disorders characterized by dysfunction of hematopoietic stem or progenitor cells. We present a consanguineous pedigree with two siblings with early-onset, progressive bone marrow failure (neutropenia, anemia, thrombocytopenia) associated with trigonocephaply, hyperplastic gingiva, cataract, accessory mammilla, dental changes and mild neurocognitive developmental delay. Pancytopenia at birth as well as non-compaction cardiomyopathy was observed in one of the patients.

Background: In this study, we aim to compare insulin and leptin levels in adolescents with or without overweight and in those with or without abdominal obesity. Materials and Methods: This case-control study was conducted among 486 samples. We randomly selected 243 overweight and an equal number of normal-weight adolescents from among participants of the third survey of a national surveillance program entitled “Childhood and Adolescence Surveillance and PreventIon of Adult Non-communicable diseases study.” Serum insulin and leptin were compared between two groups and their correlation was determined with other variables. Results: The mean age and body mass index (BMI) of participants were 14.10 ± 2.82 years and 22.12 ± 6.49 kg/m2, respectively. Leptin and insulin levels were higher in overweight than in normal-weight adolescents (P < 0.05). Leptin level was higher in children with abdominal obesity than in their other counterparts (P < 0.001). Leptin level was correlated with age, fasting blood glucose, BMI, and insulin level. Conclusion: Insulin and leptin levels were higher among overweight and obese children, which may reflect insulin and leptin-resistance. Given the complications of excess weight from early life, prevention and controlling childhood obesity should be considered as a health priority. Key words: Children, insulin, leptin, obesity, overweight

A growing body of evidence shows that leptin acts as a pro-inflammatory cytokine in autoimmune disorders and is related to multiple sclerosis (MS) pathogenesis. The present study was an analysis of serum leptin levels among healthy volunteers and patients with different subtypes of MS, opticospinal MS (OSMS) and neuromyelitis optica (NMO). Leptin concentrations in the sera of 121 healthy volunteers and 201 patients with different subtypes of MS, as well as in 27 NMO and 27 OSMS, were measured. Significant differences in leptin serum levels were observed between healthy volunteers, and MS, OSMS and NMO patients ( P  < 0.001). Furthermore, leptin serum concentration was in correlation with expanded disability status scale (EDSS) in primary progressive MS and secondary progressive MS groups. Interestingly, while the female-to-male ratio of leptin was approximately 2 in each group, the NMO female patients showed sevenfold higher levels of leptin than males. The present results show that leptin concentration is important in the pathogenesis of different neuroinflammatory diseases of the central nervous system, in particular NMO.