Highly immunosuppressive myeloid cells correlate with early relapse after allogeneic stem cell transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for myeloid malignancies such as some acute myeloid leukemias (AML) and high-risk myelodysplastic syndromes (MDS). It aims to eradicate the malignant clone using immunocompetent donor cells (graft-versus-leukemia effect, GVL). Unfortunately, relapse is the primary cause of transplant failure mainly related on HLA loss or downregulation and upregulation of inhibitory ligands on blasts which result in donor immune effector dysfunctions. METHODS: Between 2018 and 2021, we conducted a monocentric prospective study including 61 consecutive patients transplanted for AML or high-risk MDS. We longitudinally investigated immune cells at days + 30, + 90 and + 180 post-transplant from bone marrow and peripheral blood. We assessed the dynamics between myeloid derived suppressor cells (MDSCs) and T-cells. RESULTS: Among the 61 patients, 45 did not relapse over the first 12 months while 16 relapsed during the first year post-transplant. Through months 1 to 6, comparison with healthy donors revealed an heterogenous increase in MDSC frequency. In all recipients, the predominant MDSC subset was granulocytic with no specific phenotypic relapse signature. However, in relapsed patients, in vitro and in vivo functional analyses revealed that MDSCs from peripheral blood were highly immunosuppressive from day + 30 onwards, with an activated NLRP3 inflammasome signature. Only circulating immunosuppressive MDSCs were statistically correlated to circulating double-positive Tim3+LAG3+ exhausted T cells. CONCLUSION: Our simple in vitro functional assay defining MDSC immunosuppressive properties might serve as an early biomarker of relapse and raise the question of new preventive treatments targeting MDSCs in the future. Trial registration NCT03357172.

ZNF683 marks a CD8+ T cell population associated with anti-tumor immunity following anti-PD-1 therapy for Richter syndrome.

Unlike many other hematologic malignancies, Richter syndrome (RS), an aggressive B cell lymphoma originating from indolent chronic lymphocytic leukemia, is responsive to PD-1 blockade. To discover the determinants of response, we analyze single-cell transcriptome data generated from 17 bone marrow samples longitudinally collected from 6 patients with RS. Response is associated with intermediate exhausted CD8 effector/effector memory T cells marked by high expression of the transcription factor ZNF683, determined to be evolving from stem-like memory cells and divergent from terminally exhausted cells. This signature overlaps with that of tumor-infiltrating populations from anti-PD-1 responsive solid tumors. ZNF683 is found to directly target key T cell genes (TCF7, LMO2, CD69) and impact pathways of T cell cytotoxicity and activation. Analysis of pre-treatment peripheral blood from 10 independent patients with RS treated with anti-PD-1, as well as patients with solid tumors treated with anti-PD-1, supports an association of ZNF683high T cells with response.

MicroRNAs miR-16 and miR-519 control meningioma cell proliferation via overlapping transcriptomic programs shared with the RNA-binding protein HuR.

Introduction: Meningiomas are the most common type of primary central nervous system tumors. In about 80% cases, these tumors are benign and grow very slowly, but the remainder 20% can unlock higher proliferation rates and become malignant. In this study we examined two miRs, miR-16 and miR-519, and evaluated their role in tumorigenesis and cell growth in human meningioma. Methods: A cohort of 60 intracranial grade 1 and grade 2 human meningioma plus 20 healthy meningeal tissues was used to quantify miR-16 and miR-519 expressions. Cell growth and dose-response assays were performed in two human meningioma cell lines, Ben-Men-1 (benign) and IOMM-Lee (aggressive). Transcriptomes of IOMM-lee cells were measured after both miR-mimics transfection, followed by integrative bioinformatics to expand on available data. Results: In tumoral tissues, we detected decreased levels of miR-16 and miR-519 when compared with arachnoid cells of healthy patients (miR-16: P=8.7e-04; miR-519: P=3.5e-07). When individually overexpressing these miRs in Ben-Men-1 and IOMM-Lee, we observed that each showed reduced growth (P<0.001). In IOMM-Lee cell transcriptomes, downregulated genes, among which ELAVL1/HuR (miR-16: P=6.1e-06; miR-519:P=9.38e-03), were linked to biological processes such as mitotic cell cycle regulation, pre-replicative complex, and brain development (FDR<1e-05). Additionally, we uncovered a specific transcriptomic signature of miR-16/miR-519-dysregulated genes which was highly enriched in HuR targets (>6-fold; 79.6% of target genes). Discussion: These results were confirmed on several public transcriptomic and microRNA datasets of human meningiomas, hinting that the putative tumor suppressor effect of these miRs is mediated, at least in part, via HuR direct or indirect inhibition.

Leukemia relapse via genetic immune escape after allogeneic hematopoietic cell transplantation.

Graft-versus-leukemia (GvL) reactions are responsible for the effectiveness of allogeneic hematopoietic cell transplantation as a treatment modality for myeloid neoplasia, whereby donor T- effector cells recognize leukemia neoantigens. However, a substantial fraction of patients experiences relapses because of the failure of the immunological responses to control leukemic outgrowth. Here, through a broad immunogenetic study, we demonstrate that germline and somatic reduction of human leucocyte antigen (HLA) heterogeneity enhances the risk of leukemic recurrence. We show that preexistent germline-encoded low evolutionary divergence of class II HLA genotypes constitutes an independent factor associated with disease relapse and that acquisition of clonal somatic defects in HLA alleles may lead to escape from GvL control. Both class I and II HLA genes are targeted by somatic mutations as clonal selection factors potentially impairing cellular immune responses and response to immunomodulatory strategies. These findings define key molecular modes of post-transplant leukemia escape contributing to relapse.

Leukemia relapse via genetic immune escape after allogeneic hematopoietic cell transplantation.

Graft-versus-leukemia (GvL) reactions are responsible for the effectiveness of allogeneic hematopoietic cell transplantation as a treatment modality for myeloid neoplasia, whereby donor T- effector cells recognize leukemia neoantigens. However, a substantial fraction of patients experience relapses because of the failure of the immunological responses to control leukemic outgrowth. Here, through a broad immunogenetic study, we demonstrate that germline and somatic reduction of human leucocyte antigen (HLA) heterogeneity enhances the risk of leukemic recurrence. We show that preexistent germline-encoded low evolutionary divergence of class II HLA genotypes constitutes an independent factor associated with disease relapse and that acquisition of clonal somatic defects in HLA alleles may lead to escape from GvL control. Both class I and II HLA genes are targeted by somatic mutations as clonal selection factors potentially impairing cellular immune reactions and response to immunomodulatory strategies. These findings define key molecular modes of post-transplant leukemia escape contributing to relapse.

Molecular characterization of Richter syndrome identifies de novo diffuse large B-cell lymphomas with poor prognosis.

Richter syndrome (RS) is the transformation of chronic lymphocytic leukemia (CLL) into aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). We characterize 58 primary human RS samples by genome-wide DNA methylation and whole-transcriptome profiling. Our comprehensive approach determines RS DNA methylation profile and unravels a CLL epigenetic imprint, allowing CLL-RS clonal relationship assessment without the need of the initial CLL tumor DNA. DNA methylation- and transcriptomic-based classifiers were developed, and testing on landmark DLBCL datasets identifies a poor-prognosis, activated B-cell-like DLBCL subset in 111/1772 samples. The classification robustly identifies phenotypes very similar to RS with a specific genomic profile, accounting for 4.3-8.3% of de novo DLBCLs. In this work, RS multi-omics characterization determines oncogenic mechanisms, establishes a surrogate marker for CLL-RS clonal relationship, and provides a clinically relevant classifier for a subset of primary "RS-type DLBCL" with unfavorable prognosis.

A High MCM6 Proliferative Index in Atypical Meningioma Is Associated with Shorter Progression Free and Overall Survivals.

The aim of this study was to evaluate the prognostic value of MCM6, in comparison with Ki-67, in two series of grade 1 and 2 meningiomas, and to evaluate its correlation with methylation classes. The first cohort included 100 benign (grade 1, World Health Organization 2021) meningiomas, and the second 69 atypical meningiomas (grade 2). Immunohistochemical Ki-67 and MCM6 labeling indices (LI) were evaluated independently by two observers. Among the atypical meningiomas, 33 cases were also studied by genome-wide DNA methylation. In grade 2 meningiomas, but not grade 1, both Ki-67 and MCM6 LIs were correlated with PFS (p = 0.004 and p = 0.005, respectively; Cox univariate analyses). Additionally, MCM6 was correlated with overall survival only in univariate analysis. In a multivariate model, including mitotic index, Ki-67, MCM6, age, sex, and the quality of surgical resection, only MCM6 was correlated with PFS (p = 0.046). Additionally, we found a significant correlation between PTEN loss and high MCM6 or Ki-67 LIs. Although no correlation was found with the methylation classes and subtypes returned by the meningioma algorithm MNGv2.4., MCM6 LI was significantly correlated with the methylation of 2 MCM6 gene body loci. In conclusion, MCM6 is a relevant prognostic marker in atypical meningiomas. This reproducible and easy-to-use marker allows the identification of a highly aggressive subtype of proliferative meningiomas, characterized notably by frequent PTEN losses, which was previously reported to be sensitive to histone deacetylase inhibitors.

Evolutionary history of transformation from chronic lymphocytic leukemia to Richter syndrome.

Richter syndrome (RS) arising from chronic lymphocytic leukemia (CLL) exemplifies an aggressive malignancy that develops from an indolent neoplasm. To decipher the genetics underlying this transformation, we computationally deconvoluted admixtures of CLL and RS cells from 52 patients with RS, evaluating paired CLL-RS whole-exome sequencing data. We discovered RS-specific somatic driver mutations (including IRF2BP2, SRSF1, B2M, DNMT3A and CCND3), recurrent copy-number alterations beyond del(9p21)(CDKN2A/B), whole-genome duplication and chromothripsis, which were confirmed in 45 independent RS cases and in an external set of RS whole genomes. Through unsupervised clustering, clonally related RS was largely distinct from diffuse large B cell lymphoma. We distinguished pathways that were dysregulated in RS versus CLL, and detected clonal evolution of transformation at single-cell resolution, identifying intermediate cell states. Our study defines distinct molecular subtypes of RS and highlights cell-free DNA analysis as a potential tool for early diagnosis and monitoring.

In Vivo Modeling of CLL Transformation to Richter Syndrome Reveals Convergent Evolutionary Paths and Therapeutic Vulnerabilities.

Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy. SIGNIFICANCE: Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease. This article is highlighted in the In This Issue feature, p. 101.

Molecular landscape of immune pressure and escape in aplastic anemia.

Idiopathic aplastic anemia (IAA) pathophysiology is dominated by autoreactivity of human leukocyte antigen (HLA)-restricted T-cells against antigens presented by hematopoietic stem and progenitor cells (HSPCs). Expansion of PIGA and HLA class I mutant HSPCs have been linked to immune evasion from T-cell mediated pressures. We hypothesized that in analogy with antitumor immunity, the pathophysiological cascade of immune escape in IAA is initiated by immunoediting pressures and culminates with mechanisms of clonal evolution characterized by hits in immune recognition and response genes. To that end, we studied the genetic and transcriptomic make-up of the antigen presentation complexes in a large cohort of patients with IAA and paroxysmal nocturnal hemoglobinuria (PNH) by using single-cell RNA, high throughput DNA sequencing and single nucleotide polymorphism (SNP)-array platforms. At disease onset, HSPCs displayed activation of selected HLA class I and II-restricted mechanisms, without extensive inhibition of immune checkpoint apparatus. Using a newly implemented bioinformatic framework we found that not only class I but also class II genes were often impaired by acquisition of genetic aberrations. We also demonstrated the presence of novel somatic alterations in immune genes possibly contributing to the evasion from the autoimmune T-cells. In contrast, these hits were absent in myeloid neoplasia. These aberrations were not mutually exclusive with PNH and did not correlate with the accumulation of myeloid-driver hits. Our findings shed light on the mechanisms of immune activation and escape in IAA and define alternative modes of clonal hematopoiesis.

Correlation between DNA Methylation and Cell Proliferation Identifies New Candidate Predictive Markers in Meningioma.

Meningiomas are the most common primary tumors of the central nervous system. Based on the 2021 WHO classification, they are classified into three grades reflecting recurrence risk and aggressiveness. However, the WHO's histopathological criteria defining these grades are somewhat subjective. Together with reliable immunohistochemical proliferation indices, other molecular markers such as those studied with genome-wide epigenetics promise to revamp the current prognostic classification. In this study, 48 meningiomas of various grades were randomly included and explored for DNA methylation with the Infinium MethylationEPIC microarray over 850k CpG sites. We conducted differential and correlative analyses on grade and several proliferation indices and markers, such as mitotic index and Ki-67 or MCM6 immunohistochemistry. We also set up Cox proportional hazard models for extensive associations between CpG methylation and survival. We identified loci highly correlated with cell growth and a targeted methylation signature of regulatory regions persistently associated with proliferation, grade, and survival. Candidate genes under the control of these regions include SMC4, ESRRG, PAX6, DOK7, VAV2, OTX1, and PCDHA-PCDHB-PCDHG, i.e., the protocadherin gene clusters. This study highlights the crucial role played by epigenetic mechanisms in shaping dysregulated cellular proliferation and provides potential biomarkers bearing prognostic and therapeutic value for the clinical management of meningioma.

ALDH1L2 Knockout in U251 Glioblastoma Cells Reduces Tumor Sphere Formation by Increasing Oxidative Stress and Suppressing Methionine Dependency.

Previously, the in vitro growth of cancer stem cells in the form of tumor spheres from five different brain cancer cell lines was found to be methionine-dependent. As this earlier work indicated that ALDH1L2, a folate-dependent mitochondria aldehyde dehydrogenase gene, is upregulated in glioblastoma stem cells, we invalidated this gene using CRISPR-cas 9 technique in this present work. We reported here that this invalidation was effective in U251 glioblastoma cells, and no cas9 off target site could be detected by genome sequencing of the two independent knockout targeting either exon I or exon III. The knockout of ALDH1L2 gene in U251 cells rendered the growth of the cancer stem cells of U251 methionine independent. In addition, a much higher ROS (reactive oxygen radicals) level can be detected in the knockout cells compared to the wild type cells. Our evidence here linked the excessive ROS level of the knockout cells to reduced total cellular NADPH. Our evidence suggested also that the cause of the slower growth of the knockout turmor sphere may be related to its partial differentiation.

Epimutations in both the TESK2 and MMACHC promoters in the Epi-cblC inherited disorder of intracellular metabolism of vitamin B12.

BACKGROUND: epi-cblC is a recently discovered inherited disorder of intracellular vitamin B12 metabolism associating hematological, neurological, and cardiometabolic outcomes. It is produced by an epimutation at the promoter common to CCDC163P and MMACHC, which results from an aberrant antisense transcription due to splicing mutations in the antisense PRDX1 gene neighboring MMACHC. We studied whether the aberrant transcription produced a second epimutation by encompassing the CpG island of the TESK2 gene neighboring CCDC163P. METHODS: We unraveled the methylome architecture of the CCDC163P-MMACHC CpG island (CpG:33) and the TESK2 CpG island (CpG:51) of 17 epi-cblC cases. We performed an integrative analysis of the DNA methylome profiling, transcriptome reconstruction of RNA-sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-Seq) of histone H3, and transcription expression of MMACHC and TESK2. RESULTS: The PRDX1 splice mutations and activation of numerous cryptic splice sites produced antisense readthrough transcripts encompassing the bidirectional MMACHC/CCDC163P promoter and the TESK2 promoter, resulting in the silencing of both the MMACHC and TESK2 genes through the deposition of SETD2-dependent H3K36me3 marks and the generation of epimutations in the CpG islands of the two promoters. CONCLUSIONS: The antisense readthrough transcription of the mutated PRDX1 produces an epigenetic silencing of MMACHC and TESK2. We propose using the term 'epi-digenism' to define this epigenetic disorder that affects two genes. Epi-cblC is an entity that differs from cblC. Indeed, the PRDX1 and TESK2 altered expressions are observed in epi-cblC but not in cblC, suggesting further evaluating the potential consequences on cancer risk and spermatogenesis.

Low-frequency Coding Variants Associated With Body Mass Index Affect the Success of Bariatric Surgery.

CONTEXT: A recent study identified 14 low-frequency coding variants associated with body mass index (BMI) in 718 734 individuals predominantly of European ancestry. OBJECTIVE: We investigated the association of 2 genetic scores (GS) with i) the risk of severe/morbid obesity, ii) BMI variation before weight-loss intervention, iii) BMI change in response to an 18-month lifestyle/behavioral intervention program, and iv) BMI change up to 24 months after bariatric surgery. METHODS: The 14 low-frequency coding variants were genotyped or sequenced in 342 French adults with severe/morbid obesity and 574 French adult controls from the general population. We built risk and protective GS based on 6 BMI-increasing and 5 BMI-decreasing low-frequency coding variants that were polymorphic in our study. RESULTS: While the risk GS was not associated with severe/morbid obesity status, BMI-decreasing low-frequency coding variants were significantly less frequent in patients with severe/morbid obesity than in French adults from the general population. Neither the risk nor the protective GS was associated with BMI before intervention in patients with severe/morbid obesity, nor did they affect BMI change in response to a lifestyle/behavioral modification program. The protective GS was associated with a greater BMI decrease following bariatric surgery. The risk and protective GS were associated with a higher and lower risk of BMI regain after bariatric surgery. CONCLUSION: Our data indicate that in populations of European descent, low-frequency coding variants associated with BMI in the general population also affect the outcomes of bariatric surgery in patients with severe/morbid obesity.

Long-Term Overconsumption of Fat and Sugar Causes a Partially Reversible Pre-inflammatory Bowel Disease State.

Nutrition appears to be an important environmental factor involved in the onset of inflammatory bowel diseases (IBD) through yet poorly understood biological mechanisms. Most studies focused on fat content in high caloric diets, while refined sugars represent up to 40% of caloric intake within industrialized countries and contribute to the growing epidemics of inflammatory diseases. Herein we aim to better understand the impact of a high-fat-high-sucrose diet on intestinal homeostasis in healthy conditions and the subsequent colitis risk. We investigated the early events and the potential reversibility of high caloric diet-induced damage in mice before experimental colitis. C57BL/6 mice were fed with a high-fat or high-fat high-sucrose or control diet before experimental colitis. In healthy mice, a high-fat high-sucrose diet induces a pre-IBD state characterized by gut microbiota dysbiosis with a total depletion of bacteria belonging to Barnesiella that is associated with subclinical endoscopic lesions. An overall down-regulation of the colonic transcriptome converged with broadly decreased immune cell populations in the mesenteric lymph nodes leading to the inability to respond to tissue injury. Such in-vivo effects on microbiome and transcriptome were partially restored when returning to normal chow. Long-term consumption of diet enriched in sucrose and fat predisposes mice to colitis. This enhanced risk is preceded by gut microbiota dysbiosis and transcriptional reprogramming of colonic genes related to IBD. Importantly, diet-induced transcriptome and microbiome disturbances are partially reversible after switching back to normal chow with persistent sequelae that may contribute to IBD predisposition in the general population.

Ionizing radiations induce shared epigenomic signatures unraveling adaptive mechanisms of cancerous cell lines with or without methionine dependency.

BACKGROUND: Although radiation therapy represents a core cancer treatment modality, its efficacy is hampered by radioresistance. The effect of ionizing radiations (IRs) is well known regarding their ability to induce genetic alterations; however, their impact on the epigenome landscape in cancer, notably at the CpG dinucleotide resolution, remains to be further deciphered. In addition, no evidence is available regarding the effect of IRs on the DNA methylome profile according to the methionine dependency phenotype, which represents a hallmark of metabolic adaptation in cancer. METHODS: We used a case-control study design with a fractionated irradiation regimen on four cancerous cell lines representative of HCC (HepG2), melanoma (MeWo and MeWo-LC1, which exhibit opposed methionine dependency phenotypes), and glioblastoma (U251). We performed high-resolution genome-wide DNA methylome profiling using the MethylationEPIC BeadChip on baseline conditions, irradiated cell lines (cumulative dose of 10 Gy), and non-irradiated counterparts. We performed epigenome-wide association studies to assess the effect of IRs and methionine-dependency-oriented analysis by carrying out epigenome-wide conditional logistic regression. We looked for epigenome signatures at the locus and single-probe (CpG dinucleotide) levels and through enrichment analyses of gene ontologies (GO). The EpiMet project was registered under the ID#AAP-BMS_003_211. RESULTS: EWASs revealed shared GO annotation pathways associated with increased methylation signatures for several biological processes in response to IRs, including blood circulation, plasma membrane-bounded cell projection organization, cell projection organization, multicellular organismal process, developmental process, and animal organ morphogenesis. Epigenome-wide conditional logistic regression analysis on the methionine dependency phenotype highlighted several epigenome signatures related to cell cycle and division and responses to IR and ultraviolet light. CONCLUSIONS: IRs generated a variation in the methylation level of a high number of CpG probes with shared biological pathways, including those associated with cell cycle and division, responses to IRs, sustained angiogenesis, tissue invasion, and metastasis. These results provide insight on shared adaptive mechanisms of the epigenome in cancerous cell lines in response to IR. Future experiments should focus on the tryptic association between IRs, the initiation of a radioresistance phenotype, and their interaction with methionine dependency as a hallmark of metabolic adaptation in cancer.

Vitamin B12 Deficiency Dysregulates m6A mRNA Methylation of Genes Involved in Neurological Functions.

INTRODUCTION: Vitamin B12 deficiency presents various neurological manifestations, such as cognitive dysfunction, mental retardation, or memory impairment. However, the involved molecular mechanisms remain to date unclear. Vitamin B12 is essential for synthesizing S-adenosyl methionine (SAM), the methyl group donor used for almost all transmethylation reactions. Here, we investigate the m6A methylation of mRNAs and their related gene expression in models of vitamin B12 deficiency. METHODS AND RESULTS: This study observes two cellular models deficient in vitamin B12 and hippocampi of mice knock-out for the CD320 receptor. The decrease in SAM levels resulting from vitamin B12 deficiency is associated with m6 A reduced levels in mRNAs. This is also potentially mediated by the overexpression of the eraser FTO. We further investigate mRNA methylation of some genes involved in neurological functions targeted by the m6A reader YTH proteins. We notably observe a m6A hypermethylation of Prkca mRNA and a consistently increased expression of PKCα, a kinase involved in brain development and neuroplasticity, in the two cellular models. CONCLUSION: Our data show that m6A methylation in mRNA could be one of the contributing mechanisms that underlie the neurological manifestations produced by vitamin B12 deficiency.

Fetal Programming by Methyl Donor Deficiency Produces Pathological Remodeling of the Ascending Aorta.

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