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RNA-binding proteins (RBPs) form a large and diverse class of factors many members of which are overexpressed in hematological malignancies. RBPs participate in various processes of mRNA metabolism and prevent harmful DNA:RNA hybrids or R-loops. Here we report that PIWIL4, a germ stem cell-associated RBP belonging to the RNase H-like superfamily, is overexpressed in acute myeloid leukemia patients and is essential for leukemic stem cell function and AML growth, but dispensable for healthy human hematopoietic stem cells. In AML cells, PIWIL4 binds to a small number of known piwi-interacting RNA. It instead largely interacts with mRNA annotated to protein-coding genic regions and enhancers that are enriched for genes associated with cancer and human myeloid progenitor gene signatures. PIWIL4 depletion in AML cells downregulates human myeloid progenitor signature and LSC-associated genes and upregulates DNA damage signalling. We demonstrate that PIWIL4 is an R-loop resolving enzyme that prevents R-loop accumulation on a subset of AML and LSC-associated genes, and maintains their expression. It also prevents DNA damage, replication stress, and activation of the ATR pathway in AML cells. PIWIL4 depletion potentiates sensitivity to pharmacological inhibition of the ATR pathway and creates a pharmacologically actionable dependency in AML cells.

Supplementary Figures & Table

Immune checkpoint inhibitors (ICI), which target immune regulatory pathways to unleash antitumor responses, have revolutionized cancer immunotherapy. Despite the remarkable success of ICI immunotherapy, a significant proportion of patients whose tumors respond to these treatments develop immune-related adverse events (irAE) resembling autoimmune diseases. Although the clinical spectrum of irAEs is well characterized, their successful management remains empiric. This is in part because the pathogenic mechanisms involved in the breakdown of peripheral tolerance and induction of irAEs remain elusive. Herein, we focused on regulatory T cells (Treg) in individuals with irAEs because these cells are vital for maintenance of peripheral tolerance, appear expanded in the peripheral blood of individuals with cancer, and abundantly express checkpoint molecules, hence representing direct targets of ICI immunotherapy. Our data demonstrate an intense transcriptomic reprogramming of CD4+CD25+CD127− Tregs in the blood of individuals with advanced metastatic melanoma who develop irAEs following ICI immunotherapy, with a characteristic inflammatory, apoptotic, and metabolic signature. This inflammatory signature was shared by Tregs from individuals with different types of cancer developing irAEs and individuals with autoimmune diseases. Our findings suggest that inflammatory Treg reprogramming is a feature of immunotherapy-induced irAEs, and this may facilitate translational approaches aiming to induce robust antitumor immunity without disturbing peripheral tolerance.

The BCL2 family plays important roles in acute myeloid leukemia (AML). Venetoclax, a selective BCL2 inhibitor, has received FDA approval for the treatment of AML. However, drug resistance ensues after prolonged treatment, highlighting the need for a greater understanding of the underlying mechanisms. Using a genome-wide CRISPR/Cas9 screen in human AML, we identified genes whose inactivation sensitizes AML blasts to venetoclax. Genes involved in mitochondrial organization and function were significantly depleted throughout our screen, including the mitochondrial chaperonin CLPB. We demonstrated that CLPB is upregulated in human AML, it is further induced upon acquisition of venetoclax resistance, and its ablation sensitizes AML to venetoclax. Mechanistically, CLPB maintains the mitochondrial cristae structure via its interaction with the cristae-shaping protein OPA1, whereas its loss promotes apoptosis by inducing cristae remodeling and mitochondrial stress responses. Overall, our data suggest that targeting mitochondrial architecture may provide a promising approach to circumvent venetoclax resistance.Significance:A genome-wide CRISPR/Cas9 screen reveals genes involved in mitochondrial biological processes participate in the acquisition of venetoclax resistance. Loss of the mitochondrial protein CLPB leads to structural and functional defects of mitochondria, hence sensitizing AML cells to apoptosis. Targeting CLPB synergizes with venetoclax and the venetoclax/azacitidine combination in AML in a p53-independent manner.See related commentary by Savona and Rathmell, p. 831.This article is highlighted in the In This Issue feature, p. 813

sgRNA guide sequences, shRNA sequences and qPCR primers used in this study

Raw counts and gene-based CRISPR score analysis of the genome wide CRIPR/Cas9 loss-of-function screen

Relative quantification of metabolites in the hybrid panel in CLPB knockout MOLM-13

Supplementary Tables and Methods

Supplementary Figures and Figure Legends

CLPB interacting mitochondrial proteins in AML

Although deregulation of transfer RNA (tRNA) biogenesis promotes the translation of pro-tumorigenic mRNAs in cancers

Motivation B-cell epitopes (BCEs) play a pivotal role in the development of peptide vaccines, immuno-diagnostic reagents and antibody production, and thus in infectious disease prevention and diagnostics in general. Experimental methods used to determine BCEs are costly and time-consuming. Therefore, it is essential to develop computational methods for the rapid identification of BCEs. Although several computational methods have been developed for this task, generalizability is still a major concern, where cross-testing of the classifiers trained and tested on different datasets has revealed accuracies of 51–53%. Results We describe a new method called EpitopeVec, which uses a combination of residue properties, modified antigenicity scales, and protein language model-based representations (protein vectors) as features of peptides for linear BCE predictions. Extensive benchmarking of EpitopeVec and other state-of-the-art methods for linear BCE prediction on several large and small datasets, as well as cross-testing, demonstrated an improvement in the performance of EpitopeVec over other methods in terms of accuracy and area under the curve. As the predictive performance depended on the species origin of the respective antigens (viral, bacterial and eukaryotic), we also trained our method on a large viral dataset to create a dedicated linear viral BCE predictor with improved cross-testing performance. Availability and implementation The software is available at https://github.com/hzi-bifo/epitope-prediction. Supplementary information Supplementary data are available at Bioinformatics online.

Purpose: Non-squamous non-small cell lung cancer (nsqNSCLC) patients without detectable actionable alterations in EGFR, ALK, or ROS1, have a diverse genetic background. These patients are eligible to and usually receive untargeted, first line immune checkpoint inhibitors (ICI) and/or chemotherapy with heterogeneous outcomes. To identify potential predictive biomarkers for the stratification of nsqNSCLC patients without actionable genetic alterations, a comprehensive molecular profiling of this subpopulation was performed using the Foundation Medicine genomic database. Methods: Molecular profiles from 53,119 nsqNSCLC patients were analyzed using the FoundationInsightsTM web platform. After the patients with known and likely functional short variants for EGFR and fusions for ALK and ROS1 were excluded, the prevalences for commonly altered genes were determined. Pathway analysis was conducted with Metascape. Statistical significance and enrichment in PD-L1 and tumor mutation burden (TMB) biomarker overlap with altered genes was calculated using a fisher exact test against an alteration negative cohort. Results: nsqNSCLC patients without actionable genetic alterations can be described as a heterogenous population with different genetic alterations. We found a total of 103 genes with an individual, minimal prevalence of >1% to be the major descriptors that collectively were found in most patients of the nsqNSCLC subpopulation. These genes relate to pathways involved in cell cycle, cell proliferation, response to growth factors or chromatin organization. Out of the most frequently altered genes, we identified those that are associated with PD-L1 or TMB high status for which ICI treatment is most effective in nsqNSCLC. Conclusions: The results supported the lack of a general association with high PD-L1 and high TMB calls for biomarker informed treatment options for nsqNSCLC patients. We reported a subpopulation of nsqNSCLC patients that can be described by certain genetic alteration, of which some were associated with ICI-related biomarkers for which patients might benefit from a future precision oncology approach utilizing combined targeted and ICI treatment. Citation Format: Andreas Kloetgen, Danyi Wang, Anna Coenen-Stass, Ioannis Gounaris, Julia F. Hopkins, Giuseppe Locatelli, Juergen Scheuenpflug, Zheng Feng. Comprehensive real-world-data based molecular profiling and mapping of non-squamous NSCLC patients to immune-checkpoint-inhibitor biomarkers. [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 6070.

During the germinal center (GC) reaction, B cells undergo extensive redistribution of cohesin complex and three-dimensional reorganization of their genomes. Yet, the significance of cohesin and architectural programming in the humoral immune response is unknown. Herein we report that homozygous deletion of Smc3, encoding the cohesin ATPase subunit, abrogated GC formation, while, in marked contrast, Smc3 haploinsufficiency resulted in GC hyperplasia, skewing of GC polarity and impaired plasma cell (PC) differentiation. Genome-wide chromosomal conformation and transcriptional profiling revealed defects in GC B cell terminal differentiation programs controlled by the lymphoma epigenetic tumor suppressors Tet2 and Kmt2d and failure of Smc3-haploinsufficient GC B cells to switch from B cell- to PC-defining transcription factors. Smc3 haploinsufficiency preferentially impaired the connectivity of enhancer elements controlling various lymphoma tumor suppressor genes, and, accordingly, Smc3 haploinsufficiency accelerated lymphomagenesis in mice with constitutive Bcl6 expression. Collectively, our data indicate a dose-dependent function for cohesin in humoral immunity to facilitate the B cell to PC phenotypic switch while restricting malignant transformation. Melnick and colleagues show that the cohesin complex exhibits dose-dependent regulation of chromatin remodeling that accompanies the transition of germinal center B cells to plasma cells, which is necessary to prevent lymphomagenesis.

Linker histone H1 proteins bind to nucleosomes and facilitate chromatin compaction1, although their biological functions are poorly understood. Mutations in the genes that encode H1 isoforms B–E (H1B, H1C, H1D and H1E; also known as H1-5, H1-2, H1-3 and H1-4, respectively) are highly recurrent in B cell lymphomas, but the pathogenic relevance of these mutations to cancer and the mechanisms that are involved are unknown. Here we show that lymphoma-associated H1 alleles are genetic driver mutations in lymphomas. Disruption of H1 function results in a profound architectural remodelling of the genome, which is characterized by large-scale yet focal shifts of chromatin from a compacted to a relaxed state. This decompaction drives distinct changes in epigenetic states, primarily owing to a gain of histone H3 dimethylation at lysine 36 (H3K36me2) and/or loss of repressive H3 trimethylation at lysine 27 (H3K27me3). These changes unlock the expression of stem cell genes that are normally silenced during early development. In mice, loss of H1c and H1e (also known as H1f2 and H1f4, respectively) conferred germinal centre B cells with enhanced fitness and self-renewal properties, ultimately leading to aggressive lymphomas with an increased repopulating potential. Collectively, our data indicate that H1 proteins are normally required to sequester early developmental genes into architecturally inaccessible genomic compartments. We also establish H1 as a bona fide tumour suppressor and show that mutations in H1 drive malignant transformation primarily through three-dimensional genome reorganization, which leads to epigenetic reprogramming and derepression of developmentally silenced genes. Mutations in histone H1 induce the remodelling of chromatin architecture to a more relaxed state, which leads to malignant transformation through changes in histone modifications and the expression of stem cell genes.

Summary Loss of imprinting (LOI) results in severe developmental defects, but the mechanisms preventing LOI remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) in pluripotent stem cells. We demonstrate that the IG-DMR consists of two antagonistic elements: a paternally methylated CpG island that prevents recruitment of TET dioxygenases and a maternally unmethylated non-canonical enhancer that ensures expression of the Gtl2 lncRNA by counteracting de novo DNA methyltransferases. Genetic or epigenetic editing of these elements leads to distinct LOI phenotypes with characteristic alternations of allele-specific gene expression, DNA methylation, and 3D chromatin topology. Although repression of the Gtl2 promoter results in dysregulated imprinting, the stability of LOI phenotypes depends on the IG-DMR, suggesting a functional hierarchy. These findings establish the IG-DMR as a bipartite control element that maintains imprinting by allele-specific restriction of the DNA (de)methylation machinery.

Influenza during pregnancy can affect the health of offspring in later life, among which neurocognitive disorders are among the best described. Here, we investigate whether maternal influenza infection has adverse effects on immune responses in offspring. We establish a two-hit mouse model to study the effect of maternal influenza A virus infection (first hit) on vulnerability of offspring to heterologous infections (second hit) in later life. Offspring born to influenza A virus infected mothers are stunted in growth and more vulnerable to heterologous infections (influenza B virus and MRSA) than those born to PBS- or poly(I:C)-treated mothers. Enhanced vulnerability to infection in neonates is associated with reduced haematopoetic development and immune responses. In particular, alveolar macrophages of offspring exposed to maternal influenza have reduced capacity to clear second hit pathogens. This impaired pathogen clearance is partially reversed by adoptive transfer of alveolar macrophages from healthy offspring born to uninfected dams. These findings suggest that maternal influenza infection may impair immune ontogeny and increase susceptibility to early life infections of offspring., Influenza infection during pregnancy can affect health of offspring but it is not clear how this affects immune responses. Here the authors use a mouse model to show that influenza infection during pregnancy can increase susceptibility to secondary infection and alter immune cell function in offspring.

Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors, as is exemplified by reprogramming somatic cells to pluripotent stem cells through the expression of OCT4, KLF4, SOX2 and cMYC. How transcription factors orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a transcription-factor-centric view of chromatin reorganization and its association with three-dimensional enhancer rewiring and transcriptional changes during the reprogramming of mouse embryonic fibroblasts to pluripotent stem cells. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in enhancer connectivity, whereas disruption of individual KLF4 binding sites within pluripotent-stem-cell-specific enhancers was sufficient to impair enhancer-promoter contacts and reduce the expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying transcription factor and offers novel insights into the nature of the molecular events that follow transcription factor binding.

Immune checkpoint inhibitors (ICI), which target immune regulatory pathways to unleash antitumor responses, have revolutionized cancer immunotherapy. Despite the remarkable success of ICI immunotherapy, a significant proportion of patients whose tumors respond to these treatments develop immune-related adverse events (irAE) resembling autoimmune diseases. Although the clinical spectrum of irAEs is well characterized, their successful management remains empiric. This is in part because the pathogenic mechanisms involved in the breakdown of peripheral tolerance and induction of irAEs remain elusive. Herein, we focused on regulatory T cells (Treg) in individuals with irAEs because these cells are vital for maintenance of peripheral tolerance, appear expanded in the peripheral blood of individuals with cancer, and abundantly express checkpoint molecules, hence representing direct targets of ICI immunotherapy. Our data demonstrate an intense transcriptomic reprogramming of CD4+CD25+CD127− Tregs in the blood of individuals with advanced metastatic melanoma who develop irAEs following ICI immunotherapy, with a characteristic inflammatory, apoptotic, and metabolic signature. This inflammatory signature was shared by Tregs from individuals with different types of cancer developing irAEs and individuals with autoimmune diseases. Our findings suggest that inflammatory Treg reprogramming is a feature of immunotherapy-induced irAEs, and this may facilitate translational approaches aiming to induce robust antitumor immunity without disturbing peripheral tolerance.

The identity of dividing cells is challenged during mitosis, as transcription is halted and chromatin architecture drastically altered. How cell type-specific gene expression and genomic organization are faithfully reset upon G1 entry in daughter cells remains elusive. To address this issue, we characterized at a genome-wide scale the dynamic transcriptional and architectural resetting of mouse pluripotent stem cells (PSCs) upon mitotic exit. This revealed distinct patterns of transcriptional reactivation with rapid induction of stem cell genes and their enhancers, a more gradual recovery of metabolic and cell cycle genes, and a weak and transient activation of lineage-specific genes only during G1. Topological reorganization also occurred in an asynchronous manner and associated with the levels and kinetics of transcriptional reactivation. Chromatin interactions around active promoters and enhancers, and particularly super enhancers, reformed at a faster rate than CTCF/Cohesin-bound structural loops. Interestingly, regions with mitotic retention of the active histone mark H3K27ac and/or specific DNA binding factors showed faster transcriptional and architectural resetting, and chemical inhibition of H3K27 acetylation specifically during mitosis abrogated rapid reactivation of H3K27ac-bookmarked genes. Finally, we observed a contact between the promoter of an endoderm master regulator, Gata6, and a novel enhancer which was preestablished in PSCs and preserved during mitosis. Our study provides an integrative map of the topological and transcriptional changes that lead to the resetting of pluripotent stem cell identity during mitotic exit, and reveals distinct patterns and features that balance the dual requirements for self-renewal and differentiation.

Medulloblastomas arise from undifferentiated precursor cells in the cerebellum and account for about 20% of all solid brain tumors during childhood, standard therapies include radiation and chemotherapy, which oftentimes come with severe impairment of the cognitive development of the young patients. Here, we show that the posttranscriptional regulator Y-box binding protein 1 (YBX1), a DNA- and RNA-binding protein, acts as an oncogene in medulloblastomas by regulating cellular survival and apoptosis. We observed different cellular responses upon YBX1 knockdown in several medulloblastoma cell lines, with significantly altered transcription and subsequent apoptosis rates. Mechanistically, PAR-CLIP for YBX1 and integration with RNA-Seq data uncovered direct posttranscriptional control of the heterochromatin-associated gene CBX5, upon YBX1 knockdown and subsequent CBX5 mRNA instability, heterochromatin-regulated genes involved in inflammatory response, apoptosis and death receptor signaling were de-repressed. Thus, YBX1 acts as an oncogene in medulloblastoma through indirect transcriptional regulation of inflammatory genes regulating apoptosis and represents a promising novel therapeutic target in this tumor entity.

Author(s): Nishana, Mayilaadumveettil; Ha, Caryn; Rodriguez-Hernaez, Javier; Ranjbaran, Ali; Chio, Erica; Nora, Elphege P; Badri, Sana B; Kloetgen, Andreas; Bruneau, Benoit G; Tsirigos, Aristotelis; Skok, Jane A | Abstract: An amendment to this paper has been published and can be accessed via the original article.

SUMMARYDysregulation of imprinted gene loci also referred to as loss of imprinting (LOI) can result in severe developmental defects and other diseases, but the molecular mechanisms that ensure imprint stability remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) and the mechanism by which they ensure imprinting maintenance. Using pluripotent stem cells carrying an allele-specific reporter system, we demonstrate that the IG-DMR consists of two antagonistic regulatory elements: a paternally methylated CpG-island that prevents the activity of Tet dioxygenases and a maternally unmethylated regulatory element, which serves as a non-canonical enhancer and maintains expression of the maternal Gtl2 lncRNA by precluding de novo DNA methyltransferase function. Targeted genetic or epigenetic editing of these elements leads to LOI with either bi-paternal or bi-maternal expression patterns and respective allelic changes in DNA methylation and 3D chromatin topology of the entire Dlk1-Dio3 locus. Although the targeted repression of either IG-DMR or Gtl2 promoter is sufficient to cause LOI, the stability of LOI phenotype depends on the IG-DMR status, suggesting a functional hierarchy. These findings establish the IG-DMR as a novel type of bipartite control element and provide mechanistic insights into the control of Dlk1-Dio3 imprinting by allele-specific restriction of the DNA (de)methylation machinery.HIGHLIGHTSThe IG-DMR is a bipartite element with distinct allele-specific functionsA non-canonical enhancer within the IG-DMR prevents DNA methyltransferase activityTargeted epigenome editing allows induction of specific imprinting phenotypesCRISPRi reveals a functional hierarchy between DMRs that dictates imprint stability

Background. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread worldwide and pose a major public health burden. There is increasing evidence that men are more likely to die from SARS-CoV-2 infection than women. However, underlying factors that mediate the observed sex bias in coronavirus disease 2019 (COVID-19) remain unknown. Methods. In this retrospective cohort, we included COVID-19 patients who were admitted to an Intensive Care Unit at the University Hospital Hamburg-Eppendorf, Germany. We obtained demographic data of all patients who were discharged or had died by 29th April 2020. We systematically analyzed sex hormones as well as cytokine and chemokine responses in male and female patients with laboratory-confirmed SARS-CoV-2 infections upon hospital admission. We used uni- and multivariable linear regression methods to identify potential risk factors for disease severity in males and females. Findings. All enrolled patients (n=45; n=35 males and n=10 females) presented comorbidities with hypertension being the most common (45.7% in males; 40% in females), followed by cancer (35% in males; 40% in females), obesity (34.3% in males and 30% in females), type II diabetes (25.7% in males and 20% in females) and chronic heart diseases (8.6% in males and 0% in females). We detected that the vast majority of male COVID-19 patients present low testosterone (68.6%) and low dihydrotestosterone (48.6%) levels. In contrast, most female COVID-19 patients have elevated testosterone levels (60%) without alterations in dihydrotestosterone levels. Both, female and male COVID-19 patients may present elevated estradiol levels (45.7% in males and 40% in females). Disease severity defined by SOFA score correlates with elevated cytokine responses (e.g. IL-6) in males and IL-2 in females. In male COVID-19 patients, testosterone levels negatively correlate with inflammatory IL-2 and IFN-γ, whereas estradiol levels positively correlate with the inflammatory cytokine IL-6. Vice versa, in female COVID-19 patients, testosterone levels positively correlate with inflammatory cytokines (e.g. IL-6). Interpretation. We here show that critically ill male COVID-19 patients suffer from severe testosterone and dihydrotestosterone deficiencies. Both androgens are required to mount antiviral immune responses to combat infection in males.

Timed degradation of the cyclin-dependent kinase inhibitor p27Kip1 by the E3 ubiquitin ligase F-box protein SKP2 is critical for T-cell progression into cell cycle, coordinating proliferation and differentiation processes. SKP2 expression is regulated by mitogenic stimuli and by Notch signaling, a key pathway in T-cell development and in T-cell acute lymphoblastic leukemia (T-ALL); however, it is not known whether SKP2 plays a role in the development of T-ALL. Here, we determined that SKP2 function is relevant for T-ALL leukemogenesis, whereas is dispensable for T-cell development. Targeted inhibition of SKP2 by genetic deletion or pharmacological blockade markedly inhibited proliferation of human T-ALL cells in vitro and antagonized disease in vivo in murine and xenograft leukemia models, with little effect on normal tissues. We also demonstrate a novel feed forward feedback loop by which Notch and IL-7 signaling cooperatively converge on SKP2 induction and cell cycle activation. These studies show that the Notch/SKP2/p27Kip1 pathway plays a unique role in T-ALL development and provide a proof-of-concept for the use of SKP2 as a new therapeutic target in T-cell acute lymphoblastic leukemia (T-ALL).

Lack of cellular differentiation is a hallmark of many human cancers, including acute myeloid leukemia (AML). Strategies to overcome such a differentiation blockade are an approach for treating AML. To identify targets for differentiation-based therapies, we applied an integrated cell surface-based CRISPR platform to assess genes involved in maintaining the undifferentiated state of leukemia cells. Here we identify the RNA-binding protein ZFP36L2 as a critical regulator of AML maintenance and differentiation. Mechanistically, ZFP36L2 interacts with the 3' untranslated region of key myeloid maturation genes, including the ZFP36 paralogs, to promote their mRNA degradation and suppress terminal myeloid cell differentiation. Genetic inhibition of ZFP36L2 restores the mRNA stability of these targeted transcripts and ultimately triggers myeloid differentiation in leukemia cells. Epigenome profiling of several individuals with primary AML revealed enhancer modules near ZFP36L2 that associated with distinct AML cell states, establishing a coordinated epigenetic and post-transcriptional mechanism that shapes leukemic differentiation.

The contribution of microRNA-mediated posttranscriptional regulation on the final proteome in differentiating cells remains elusive. Here, we evaluated the impact of microRNAs (miRNAs) on the proteome of human umbilical cord blood-derived unrestricted somatic stem cells (USSC) during retinoic acid (RA) differentiation by a systemic approach using next generation sequencing analysing mRNA and miRNA expression and quantitative mass spectrometry-based proteome analyses. Interestingly, regulation of mRNAs and their dedicated proteins highly correlated during RA-incubation. Additionally, RA-induced USSC demonstrated a clear separation from native USSC thereby shifting from a proliferating to a metabolic phenotype. Bioinformatic integration of up- and downregulated miRNAs and proteins initially implied a strong impact of the miRNome on the XXL-USSC proteome. However, quantitative proteome analysis of the miRNA contribution on the final proteome after ectopic overexpression of downregulated miR-27a-5p and miR-221-5p or inhibition of upregulated miR-34a-5p, respectively, followed by RA-induction revealed only minor proportions of differentially abundant proteins. In addition, only small overlaps of these regulated proteins with inversely abundant proteins in non-transfected RA-treated USSC were observed. Hence, mRNA transcription rather than miRNA-mediated regulation is the driving force for protein regulation upon RA-incubation, strongly suggesting that miRNAs are fine-tuning regulators rather than active primary switches during RA-induction of USSC.

General control nonderepressible 2 (GCN2) is an environmental sensor controlling transcription and translation in response to nutrient availability. Although GCN2 is a putative therapeutic target for immuno-oncology, its role in shaping the immune response to tumors is poorly understood. Here, we used mass cytometry, transcriptomics, and transcription factor-binding analysis to determine the functional impact of GCN2 on the myeloid phenotype and immune responses in melanoma. We found that myeloid-lineage deletion of GCN2 drives a shift in the phenotype of tumor-associated macrophages and myeloid-derived suppressor cells (MDSCs) that promotes antitumor immunity. Time-of-flight mass cytometry (CyTOF) and single-cell RNA sequencing showed that this was due to changes in the immune microenvironment with increased proinflammatory activation of macrophages and MDSCs and interferon-γ expression in intratumoral CD8+ T cells. Mechanistically, GCN2 altered myeloid function by promoting increased translation of the transcription factor CREB-2/ATF4, which was required for maturation and polarization of macrophages and MDSCs in both mice and humans, whereas targeting Atf4 by small interfering RNA knockdown reduced tumor growth. Last, analysis of patients with cutaneous melanoma showed that GCN2-dependent transcriptional signatures correlated with macrophage polarization, T cell infiltrates, and overall survival. Thus, these data reveal a previously unknown dependence of tumors on myeloid GCN2 signals for protection from immune attack.

The homeobox gene HLXB9 encodes for the transcription factor HB9, which is essential for pancreatic as well as motor neuronal development. Beside its physiological expression pattern, aberrant HB9 expression has been observed in several neoplasias. Especially in infant translocation t(7;12) acute myeloid leukemia, aberrant HB9 expression is the only known molecular hallmark and is assumed to be a key factor in leukemic transformation. However, so far, only poor functional data exist addressing the oncogenic potential of HB9 or its influence on hematopoiesis. We investigated the influence of HB9 on cell proliferation and cell cycle in vitro, as well as on hematopoietic stem cell differentiation in vivo using murine and human model systems. In vitro, HB9 expression led to premature senescence in human HT1080 and murine NIH3T3 cells, providing for the first time evidence for an oncogenic potential of HB9. Onset of senescence was characterized by induction of the p53–p21 tumor suppressor network, resulting in growth arrest, accompanied by morphological transformation and expression of senescence-associated β-galactosidase. In vivo, HB9-transduced primary murine hematopoietic stem and progenitor cells underwent a profound differentiation arrest and accumulated at the megakaryocyte/erythrocyte progenitor stage. In line, gene expression analyses revealed de novo expression of erythropoiesis-related genes in human CD34+hematopoietic stem and progenitor cells upon HB9 expression. In summary, the novel findings of HB9-dependent premature senescence and myeloid-biased perturbed hematopoietic differentiation, for the first time shed light on the oncogenic properties of HB9 in translocation t(7;12) acute myeloid leukemia.

The transcription factor AhR modulates immunity at multiple levels. Here we report that phagocytes exposed to apoptotic cells exhibited rapid activation of AhR, which drove production of the cytokine IL-10. Activation of AhR was dependent on interactions between apoptotic-cell DNA and the pattern-recognition receptor TLR9 that was required for the prevention of immune responses to DNA and histones in vivo. Moreover, disease progression in mouse systemic lupus erythematosus (SLE) correlated with strength of the AhR signal, and the disease course could be altered by modulation of AhR activity. Deletion of AhR in the myeloid lineage caused systemic autoimmunity in mice, and an enhanced AhR transcriptional signature correlated with disease in patients with SLE. Thus, AhR activity induced by apoptotic cell phagocytes maintains peripheral tolerance.

Osteonecrosis (ON) is a debilitating side effect of anti-leukemic treatment. Thus far, the role of leukemic infiltration (LI) of bone is unclear. The first 30 children aged ≥10 years, who were enrolled in the ongoing OPAL trial and had MRI studies at diagnosis and at 6 months, were analyzed. MRI revealed extensive LIs in 24 (80%) patients. The signal abnormalities changed back to a physiological signal in 29 out of 30 children at 6 months. Of the 24 children with LIs at diagnosis, 3 (12.5%) developed ON ≥ II, whereas 4 (66.7%) patients without LIs subsequently developed ON ≥ II (p = .016). No differences between children initially presenting with/without LIs were observed concerning age, pubertal stage, white blood count, immunophenotype, and clinical presentation. Initial radiological LI of bone and, thus, single MRI at diagnosis cannot identify children at high risk of developing radiological ON at 6 months into treatment.

IDH1/2 mutations are early drivers present in diverse human cancer types arising in various tissue sites. IDH1/2 mutation is known to induce a global hypermethylator phenotype. However, the effects on DNA methylation across IDH mutant cancers and functionally different genome regions, remain unknown. We analyzed DNA methylation data from IDH1/2 mutant acute myeloid leukemia, oligodendroglioma, astrocytoma, solid papillary breast carcinoma with reverse polarity, sinonasal undifferentiated carcinoma and cholangiocarcinoma, which clustered by their embryonal origin. Hypermethylated common probes affect predominantly gene bodies while promoters in IDH1/2 mutant cancers remain unmethylated. Enhancers showed global hypermethylation, however commonly hypomethylated enhancers were associated with tissue differentiation and cell fate determination. We demonstrate that some chromosomes, chromosomal arms and chromosomal regions are more affected by IDH1/2 mutations while others remain resistant to IDH1/2 mutation induced methylation changes. Therefore IDH1/2 mutations have different methylation effect on different parts of the genome, which may be regulated by different mechanisms.

CTCF and cohesin play a key role in organizing chromatin into topologically associating domain (TAD) structures. Disruption of a single CTCF binding site is sufficient to change chromosomal interactions leading to alterations in chromatin modifications and gene regulation. However, the extent to which alterations in chromatin modifications can disrupt 3D chromosome organization leading to transcriptional changes is unknown. In multiple myeloma, a 4;14 translocation induces overexpression of the histone methyltransferase, NSD2, resulting in expansion of H3K36me2 and shrinkage of antagonistic H3K27me3 domains. Using isogenic cell lines producing high and low levels of NSD2, here we find oncogene activation is linked to alterations in H3K27ac and CTCF within H3K36me2 enriched chromatin. A logistic regression model reveals that differentially expressed genes are significantly enriched within the same insulated domain as altered H3K27ac and CTCF peaks. These results identify a bidirectional relationship between 2D chromatin and 3D genome organization in gene regulation., In multiple myeloma, a 4;14 translocation induces overexpression of histone methyltransferase NSD2, resulting in expansion of H3K36me2 and shrinkage of H3K27me3 domains. Here the authors find that CTCF, H3K27ac and gene expression changes cluster within a subset of insulated domains implicating 3D chromosome organization as a key factor in the NSD2-mediated phenotype.

Epigenetic dysregulation plays a profound role in the pathogenesis of hematological malignancies, which is often the result of somatic mutations of chromatin regulators. Previously, these mutations were largely considered to alter gene expression in two dimensions, by activating or repressing chromatin states; however, research in the last decade has highlighted the increasing impact of the three-dimensional (3D) organization of the genome in gene regulation and disease pathogenesis. Here, we summarize the current principles of 3D chromatin organization, how the integrity of the 3D genome governs immune cell development and malignant transformation, as well as how underlying (epi-)genetic drivers of 3D chromatin alterations might act as potential novel therapeutic targets for hematological malignancies.

The BCL2 family plays important roles in acute myeloid leukemia (AML). Venetoclax, a selective BCL2 inhibitor, has received FDA approval for the treatment of AML. However, drug resistance ensues after prolonged treatment, highlighting the need for a greater understanding of the underlying mechanisms. Using a genome-wide CRISPR/Cas9 screen in human AML, we identified genes whose inactivation sensitizes AML blasts to venetoclax. Genes involved in mitochondrial organization and function were significantly depleted throughout our screen, including the mitochondrial chaperonin CLPB. We demonstrated that CLPB is upregulated in human AML, it is further induced upon acquisition of venetoclax resistance, and its ablation sensitizes AML to venetoclax. Mechanistically, CLPB maintains the mitochondrial cristae structure via its interaction with the cristae-shaping protein OPA1, whereas its loss promotes apoptosis by inducing cristae remodeling and mitochondrial stress responses. Overall, our data suggest that targeting mitochondrial architecture may provide a promising approach to circumvent venetoclax resistance. Significance: A genome-wide CRISPR/Cas9 screen reveals genes involved in mitochondrial biological processes participate in the acquisition of venetoclax resistance. Loss of the mitochondrial protein CLPB leads to structural and functional defects of mitochondria, hence sensitizing AML cells to apoptosis. Targeting CLPB synergizes with venetoclax and the venetoclax/azacitidine combination in AML in a p53-independent manner. See related commentary by Savona and Rathmell, p. 831. This article is highlighted in the In This Issue feature, p. 813

RNA-binding proteins (RBPs) are essential modulators of transcription and translation frequently dysregulated in cancer. We systematically interrogated RBP dependencies in human cancers using a comprehensive CRISPR/Cas9 domain-focused screen targeting RNA-binding domains of 490 classical RBPs. This uncovered a network of physically interacting RBPs upregulated in acute myeloid leukemia (AML) and crucial for maintaining RNA splicing and AML survival. Genetic or pharmacologic targeting of one key member of this network, RBM39, repressed cassette exon inclusion and promoted intron retention within mRNAs encoding HOXA9 targets as well as in other RBPs preferentially required in AML. The effects of RBM39 loss on splicing further resulted in preferential lethality of spliceosomal mutant AML, providing a strategy for treatment of AML bearing RBP splicing mutations.

Survival of patients with pediatric acute lymphoblastic leukemia (ALL) after allogeneic hematopoietic stem cell transplantation (allo-SCT) is mainly compromised by leukemia relapse, carrying dismal prognosis. As novel individualized therapeutic approaches are urgently needed, we performed whole-exome sequencing of leukemic blasts of 10 children with post-allo-SCT relapses with the aim of thoroughly characterizing the mutational landscape and identifying druggable mutations. We found that post-allo-SCT ALL relapses display highly diverse and mostly patient-individual genetic lesions. Moreover, mutational cluster analysis showed substantial clonal dynamics during leukemia progression from initial diagnosis to relapse after allo-SCT. Only very few alterations stayed constant over time. This dynamic clonality was exemplified by the detection of thiopurine resistance-mediating mutations in the nucleotidase NT5C2 in 3 patients' first relapses, which disappeared in the post-allo-SCT relapses on relief of selective pressure of maintenance chemotherapy. Moreover, we identified TP53 mutations in 4 of 10 patients after allo-SCT, reflecting acquired chemoresistance associated with selective pressure of prior antineoplastic treatment. Finally, in 9 of 10 children's post-allo-SCT relapse, we found alterations in genes for which targeted therapies with novel agents are readily available. We could show efficient targeting of leukemic blasts by APR-246 in 2 patients carrying TP53 mutations. Our findings shed light on the genetic basis of post-allo-SCT relapse and may pave the way for unraveling novel therapeutic strategies in this challenging situation.