Human erythroleukemia genetics and transcriptomes identify master transcription factors as functional disease drivers.

Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.

Heterogeneity in the Prevalence of High-Risk Human Papillomavirus Infection in Human Immunodeficiency Virus-Negative and Human Immunodeficiency Virus-Positive Men Who Have Sex With Men in West Africa.

BACKGROUND: Men who have sex with men (MSM) are at high risk of human papillomavirus (HPV) infection. We assessed (i) the prevalence of high-risk HPV (HR-HPV) infection and associated factors, and (ii) the prevalence of vaccine-preventable HPV infections in MSM in Burkina Faso, Côte d'Ivoire, Mali, and Togo. METHODS: A cross-sectional study was conducted in 2017-2018 among MSM ≥18 years old followed in community-based clinics. HPV infection was investigated in oral and anal samples using the e-BRID system. Factors associated with HR-HPV infection were identified using multivariate logistic regressions. RESULTS: Among 631 participants, 425 were HIV-negative and 206 HIV-positive. HR-HPV prevalence ranged from 9.2% to 34.8% in the former, and 33.3% to 71.0% in the latter, according to the study country. In multivariate analysis, HIV infection (adjusted odds ratio (aOR) 3.61, 95% confidence interval (CI) 2.48-5.27) and study country (4.73, 2.66-8.43 for Mali; 3.12, 1.68-5.80 for Burkina Faso; 3.51, 1.92-6.42 for Togo) were associated with HR-HPV infection. Other associated factors were low educational level, self-defined homosexual identity, and condomless anal sex. The prevalence of infections which can be prevented with bivalent, quadrivalent, and nonavalent vaccines was 5.9, 27.1, and 34.6% in HIV-negative participants, and 18.9, 43.7, and 54.9% in HIV-positive participants, respectively. CONCLUSIONS: HR-HPV prevalence was very heterogeneous between the study countries in both HIV-negative and HIV-positive MSM. Vaccine-preventable HPV infections predominated. Vaccination should be proposed to young MSM to reduce the burden of HPV infection in this vulnerable population and their female partners in West Africa.

BCOR and BCORL1 mutations in myelodysplastic syndromes and related disorders.

Patients with low-risk myelodysplastic syndromes (MDS) that rapidly progress to acute myeloid leukemia (AML) remain a challenge in disease management. Using whole-exome sequencing of an MDS patient, we identified a somatic mutation in the BCOR gene also mutated in AML. Sequencing of BCOR and related BCORL1 genes in a cohort of 354 MDS patients identified 4.2% and 0.8% of mutations respectively. BCOR mutations were associated with RUNX1 (P = .002) and DNMT3A mutations (P = .015). BCOR is also mutated in chronic myelomonocytic leukemia patients (7.4%) and BCORL1 in AML patients with myelodysplasia-related changes (9.1%). Using deep sequencing, we show that BCOR mutations arise after mutations affecting genes involved in splicing machinery or epigenetic regulation. In univariate analysis, BCOR mutations were associated with poor prognosis in MDS (overall survival [OS]: P = .013; cumulative incidence of AML transformation: P = .005). Multivariate analysis including age, International Prognostic Scoring System, transfusion dependency, and mutational status confirmed a significant inferior OS to patients with a BCOR mutation (hazard ratio, 3.3; 95% confidence interval, 1.4-8.1; P = .008). These data suggest that BCOR mutations define the clinical course rather than disease initiation. Despite infrequent mutations, BCOR analyses should be considered in risk stratification.

Mutations affecting mRNA splicing define distinct clinical phenotypes and correlate with patient outcome in myelodysplastic syndromes.

A cohort of MDS patients was examined for mutations affecting 4 splice genes (SF3B1, SRSF2, ZRSR2, and U2AF35) and evaluated in the context of clinical and molecular markers. Splice gene mutations were detected in 95 of 221 patients. These mutations were mutually exclusive and less likely to occur in patients with complex cytogenetics or TP53 mutations. SF3B1(mut) patients presented with lower hemoglobin levels, increased WBC and platelet counts, and were more likely to have DNMT3A mutations. SRSF2(mut) patients clustered in RAEB-1 and RAEB-2 subtypes and exhibited pronounced thrombocytopenias. ZRSR2(mut) patients clustered in International Prognostic Scoring System intermediate-1 and intermediate-2 risk groups, had higher percentages of bone marrow blasts, and more often displayed isolated neutropenias. SRSF2 and ZRSR2 mutations were more common in TET2(mut) patients. U2AF35(mut) patients had an increased prevalence of chromosome 20 deletions and ASXL1 mutations. Multivariate analysis revealed an inferior overall survival and a higher AML transformation rate for the genotype ZRSR2(mut)/TET2(wt) (overall survival: hazard ratio = 3.3; 95% CI, 1.4-7.7; P = .006; AML transformation: hazard ratio = 3.6; 95% CI, 2-4.2; P = .026). Our results demonstrate that splice gene mutations are among the most frequent molecular aberrations in myelodysplastic syndrome, define distinct clinical phenotypes, and show preferential associations with mutations targeting transcriptional regulation.

Array comparative genomic hybridization and sequencing of 23 genes in 80 patients with myelofibrosis at chronic or acute phase.

Myelofibrosis is a myeloproliferative neoplasm that occurs de novo (primary myelofibrosis) or results from the progression of polycythemia vera or essential thrombocytemia (hereafter designated as secondary myelofibrosis or post-polycythemia vera/ essential thrombocythemia myelofibrosis). To progress in the understanding of myelofibrosis and to find molecular prognostic markers we studied 104 samples of primary and secondary myelofibrosis at chronic (n=68) and acute phases (n=12) from 80 patients, by using array-comparative genomic hybridization and sequencing of 23 genes (ASXL1, BMI1, CBL, DNMT3A, EZH2, IDH1/2, JAK2, K/NRAS, LNK, MPL, NF1, PPP1R16B, PTPN11, RCOR1, SF3B1, SOCS2, SRSF2, SUZ12, TET2, TP53, TRPS1). We found copy number aberrations in 54% of samples, often involving genes with a known or potential role in leukemogenesis. We show that cases carrying a del(20q), del(17) or del(12p) evolve in acute myeloid leukemia (P=0.03). We found that 88% of the cases were mutated, mainly in signaling pathway (JAK2 69%, NF1 6%) and epigenetic genes (ASXL1 26%, TET2 14%, EZH2 8%). Overall survival was poor in patients with more than one mutation (P=0.001) and in patients with JAK2/ASXL1 mutations (P=0.02). Our study highlights the heterogeneity of myelofibrosis, and points to several interesting copy number aberrations and genes with diagnostic and prognostic impact.

Difference in cytokine production and cell cycle progression induced by Epstein-Barr virus Lmp1 deletion variants in Kmh2, a Hodgkin lymphoma cell line.

BACKGROUND: Epstein-Barr virus (EBV) is associated with 20-40% of Hodgkin's Lymphoma (HL) cases. EBV-encoded latent membrane protein 1 (LMP1) is a well-known oncogenic protein and two C-terminal deletion variants, del30-LMP1 and del69-LMP1, have been described in animal models to be more tumorigenic than the wild-type form. This work aims to detail the implication of LMP1 in the development of HL and to characterize the particular effects of these variants. METHODS: We established HL-derived cell lines stably transfected with the pRT-LMP1 vector coding for the EBNA1 gene and allowing expression of the different LMP1 variants under the control of a doxycyclin-inducible promoter. Communication between cells was assessed by measuring the expression of various pro-inflammatory cytokines by flow cytometry after intracellular LMP1 and cytokine double staining. Proliferative properties of LMP1 variants were also compared by studying the repartition of cells in the different phases of the cell cycle after EdU incorporation combined to LMP1 and DAPI staining. RESULTS: All LMP1 proteins induced the expression of several pro-inflammatory cytokines such as TNF-α, TNF-ß, IL-6, RANTES/CCL5 and IFN-γ. However, the del30-LMP1 variant induced cytokine expression at a lower level than the other variants, especially IFN-γ, while the del69-LMP1 variant stimulated greater cytokine expression. In addition, we measured that all LMP1 proteins greatly impacted the cell cycle progression, triggering a reduction in the number of cells in S-phase and an accumulation of cells in the G2/M phase compared to the HL-non induced cells. Interestingly, the del30-LMP1 variant reduced the number of cells in S-phase in a significantly greater manner and also increased the number of cells in the G0/G1 phase of the cell cycle. CONCLUSION: Weak IFN-γ expression and specific alteration of the cell cycle might be a way for del30-LMP1 infected cells to escape the immune anti-viral response and to promote the development of cancer. The differences observed between the LMP1 variants reflect their own oncogenic properties and eventually impact the development of HL.

Gene mutations differently impact the prognosis of the myelodysplastic and myeloproliferative classes of chronic myelomonocytic leukemia.

Initially classified in the myelodysplastic syndromes (MDSs), chronic myelomonocytic leukemia (CMML) is currently considered as a MDS/myeloproliferative neoplasm. Two classes-myelodysplastic and myeloproliferative-have been distinguished upon the level of the white blood cell count (threshold 13 G/L). We analyzed mutations in 19 genes reported in CMML to determine if and how these mutations impacted the respective prognosis of the two classes. We defined four major mutated pathways (DNA methylation, ASXL1, splicing, and signaling) and determined their prognostic impact. The number of mutated pathways impacted overall survival in the myelodysplastic class but not in the myeloproliferative class. The myeloproliferative class had a worse prognosis than the myelodysplastic class and was impacted by RUNX1 mutations only. Our results argue for a reclassification of CMML based on the myelodysplastic/myeloproliferative status.

Genetic deletion of JAM-C in pre-leukemic cells rewires leukemic stem cell gene expression program in AML.

The leukemic stem cell (LSC) score LSC-17 based on a stemness-related gene expression signature is an indicator of poor disease outcome in acute myeloid leukemia (AML). However, our understanding of the relationships between LSC and pre-leukemic cells is still incomplete. In particular, it is not known whether "niche-anchoring" of pre-leukemic cell affects disease evolution. To address this issue, we conditionally inactivated the adhesion molecule JAM-C expressed by haematopoietic stem cells (HSC) and LSC in an inducible iMLL-AF9-driven AML mouse model. Deletion of Jam3 (encoding JAM-C) before induction of the leukemia-initiating iMLL-AF9 fusion resulted in a shift from long term to short term-HSC expansion, without affecting disease initiation and progression. In vitro experiments showed that JAM-C controlled leukemic cell nesting irrespective of the bone marrow stromal cells used. RNA sequencing performed on leukemic HSC isolated from diseased mice revealed that genes upregulated in Jam3-deficient animals belonged to Activation Protein-1 (AP-1) and TNF-/NFB pathways. Human orthologs of dysregulated genes allowed to identify a score based on AP-1/TNF-a gene expression that was distinct and complementary from LSC-17 score. Sub-stratification of AML patients with LSC-17 and AP-1/TNF-genes signature defined four groups with median survival ranging from below one year to a median not reached after 8 years. Finally, coculture experiments showed that AP-1 activation in leukemic cells was dependent on the nature of stromal cells. Altogether, our results identify the AP-1/TNF- gene signature as a proxy of LSC anchoring in specific bone marrow niches which improves the prognosis value of the LSC-17 score. NCT02320656.

Molecular similarity between myelodysplastic form of chronic myelomonocytic leukemia and refractory anemia with ring sideroblasts.

Chronic myelomonocytic leukemia is similar to but a separate entity from both myeloproliferative neoplasms and myelodysplastic syndromes, and shows either myeloproliferative or myelodysplastic features. We ask whether this distinction may have a molecular basis. We established the gene expression profiles of 39 samples of chronic myelomonocytic leukemia (including 12 CD34-positive) and 32 CD34-positive samples of myelodysplastic syndromes by using Affymetrix microarrays, and studied the status of 18 genes by Sanger sequencing and array-comparative genomic hybridization in 53 samples. Analysis of 12 mRNAS from chronic myelomonocytic leukemia established a gene expression signature of 122 probe sets differentially expressed between proliferative and dysplastic cases of chronic myelomonocytic leukemia. As compared to proliferative cases, dysplastic cases over-expressed genes involved in red blood cell biology. When applied to 32 myelodysplastic syndromes, this gene expression signature was able to discriminate refractory anemias with ring sideroblasts from refractory anemias with excess of blasts. By comparing mRNAS from these two forms of myelodysplastic syndromes we derived a second gene expression signature. This signature separated the myelodysplastic and myeloproliferative forms of chronic myelomonocytic leukemias. These results were validated using two independent gene expression data sets. We found that myelodysplastic chronic myelomonocytic leukemias are characterized by mutations in transcription/epigenetic regulators (ASXL1, RUNX1, TET2) and splicing genes (SRSF2) and the absence of mutations in signaling genes. Myelodysplastic chronic myelomonocytic leukemias and refractory anemias with ring sideroblasts share a common expression program suggesting they are part of a continuum, which is not totally explained by their similar but not, however, identical mutation spectrum.

Identification of new interacting partners of the shuttling protein ubinuclein (Ubn-1).

We have previously characterized ubinuclein (Ubn-1) as a NACos (Nuclear and Adherent junction Complex components) protein which interacts with viral or cellular transcription factors and the tight junction (TJ) protein ZO-1. The purpose of the present study was to get more insights on the binding partners of Ubn-1, notably those present in the epithelial junctions. Using an in vivo assay of fluorescent protein-complementation assay (PCA), we demonstrated that the N-terminal domains of the Ubn-1 and ZO-1 proteins triggered a functional interaction inside the cell. Indeed, expression of both complementary fragments of venus fused to the N-terminal parts of Ubn-1 and ZO-1 was able to reconstitute a fluorescent venus protein. Furthermore, nuclear expression of the chimeric Ubn-1 triggered nuclear localization of the chimeric ZO-1. We could localize this interaction to the PDZ2 domain of ZO-1 using an in vitro pull-down assay. More precisely, a 184-amino acid region (from amino acids 39 to 223) at the N-terminal region of Ubn-1 was responsible for the interaction with the PDZ2 domain of ZO-1. Co-imunoprecipitation and confocal microscopy experiments also revealed the tight junction protein cingulin as a new interacting partner of Ubn-1. A proteomic approach based on mass spectrometry analysis (MS) was then undertaken to identify further binding partners of GST-Ubn-1 fusion protein in different subcellular fractions of human epithelial HT29 cells. LYRIC (Lysine-rich CEACAM1-associated protein) and RACK-1 (receptor for activated C-kinase) proteins were validated as bona fide interacting partners of Ubn-1. Altogether, these results suggest that Ubn-1 is a scaffold protein influencing protein subcellular localization and is involved in several processes such as cell-cell contact signalling or modulation of gene activity.

Mutation analysis of ASXL1, CBL, DNMT3A, IDH1, IDH2, JAK2, MPL, NF1, SF3B1, SUZ12, and TET2 in myeloproliferative neoplasms.

Since the discovery of the JAK2V617F tyrosine kinase-activating mutation several genes have been found mutated in nonchronic myeloid leukemia (CML) myeloproliferative neoplasms (MPNs), which mainly comprise three subtypes of "classic" MPNs; polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF). We searched for mutations in ASXL1, CBL, DNMT3A, IDH1, IDH2, JAK2, MPL, NF1, SF3B1, SUZ12, and TET2 genes in 149 non-CML MPNs, including 127 "classic" MPNs cases. JAK2 was mutated in 100% PV, 66% ET and 68% MF. We found a high incidence of ASXL1 mutation in MF patients (20%) and a low incidence in PV (7%) and ET (4%) patients. Mutations in the other genes were rare (CBL, DNMT3A, IDH2, MPL, SF3B1, SUZ12, NF1) or absent (IDH1).

How outing conditions relate to the motivation of movement-restricted cattle to access an outdoor exercise yard.

Assessing animal motivation to access a given resource is one method available to evaluate what to provide in the living environments of captive animals. Providing increased opportunities for movement can be seen as an important source of enrichment, but we need to know the point of view of the animal. The objective of our study was to test a novel combination of behaviours in order to assess the motivation of cows to access an outdoor exercise paddock. Three trials were conducted, each enrolling 15-16 tie-stall-housed cows as a model for movement-restricted animals. Cows were provided with access to an outdoor exercise yard 5 days/week for the duration of the trial, each trial presenting different conditions such as paddock size, duration of access and animal handling. We recorded the trips' durations and cows' behaviours during the trips going to (go-out) and coming back (go-in) from the paddock. LMr comparisons on PCA were used to assess cow motivation profiles. The same two dimensions of speed and stop quality emerged from the PCA in all three trials, showing the method's robustness. Additionally, three motivation profiles were established, representing how the cows' motivation was affected by the conditions prevailing in each trial.

Erythroleukemia: Classification.

Acute erythroid leukemia (AEL) is a rare (2%-5%) form of acute myeloid leukemia (AML). Molecular alterations found in AEL resemble those of other AMLs. We report a classification of AELs in three major classes, with different prognosis and some specific features such as a tendency to mutual exclusion of mutations in epigenetic regulators and signaling genes.

The nuclear and adherent junction complex component protein ubinuclein negatively regulates the productive cycle of Epstein-Barr virus in epithelial cells.

The Epstein-Barr Virus (EBV) productive cycle is initiated by the expression of the viral trans-activator EB1 (also called Zebra, Zta, or BZLF1), which belongs to the basic leucine zipper transcription factor family. We have previously identified the cellular NACos (nuclear and adherent junction complex components) protein ubinuclein (Ubn-1) as a partner for EB1, but the function of this complex has never been studied. Here, we have evaluated the consequences of this interaction on the EBV productive cycle and find that Ubn-1 overexpression represses the EBV productive cycle whereas Ubn-1 downregulation by short hairpin RNA (shRNA) increases virus production. By a chromatin immunoprecipitation (ChIP) assay, we show that Ubn-1 blocks EB1-DNA interaction. We also show that in epithelial cells, relocalization and sequestration of Ubn-1 to the tight junctions of nondividing cells allow increased activation of the productive cycle. We propose a model in which Ubn-1 is a modulator of the EBV productive cycle: in proliferating epithelial cells, Ubn-1 is nuclear and inhibits activation of the productive cycle, whereas in differentiated cells, Ubn-1 is sequestrated to tight junctions, thereby allowing EB1 to fully function in the nucleus.

Myeloid malignancies: mutations, models and management.

Myeloid malignant diseases comprise chronic (including myelodysplastic syndromes, myeloproliferative neoplasms and chronic myelomonocytic leukemia) and acute (acute myeloid leukemia) stages. They are clonal diseases arising in hematopoietic stem or progenitor cells. Mutations responsible for these diseases occur in several genes whose encoded proteins belong principally to five classes: signaling pathways proteins (e.g. CBL, FLT3, JAK2, RAS), transcription factors (e.g. CEBPA, ETV6, RUNX1), epigenetic regulators (e.g. ASXL1, DNMT3A, EZH2, IDH1, IDH2, SUZ12, TET2, UTX), tumor suppressors (e.g. TP53), and components of the spliceosome (e.g. SF3B1, SRSF2). Large-scale sequencing efforts will soon lead to the establishment of a comprehensive repertoire of these mutations, allowing for a better definition and classification of myeloid malignancies, the identification of new prognostic markers and therapeutic targets, and the development of novel therapies. Given the importance of epigenetic deregulation in myeloid diseases, the use of drugs targeting epigenetic regulators appears as a most promising therapeutic approach.

Acute myeloid leukemia with myelodysplasia-related changes are characterized by a specific molecular pattern with high frequency of ASXL1 mutations.

To determine whether the distinct and heterogeneous WHO category called "AML with myelodysplasia-related changes" (MRC-AML), presents specific molecular alterations we searched for mutations in genes known to be mutated in malignant myeloid diseases. In 48 MRC-AML patients analyzed, we found 17 mutations in ASXL1 (35%), eight in RUNX1 (17%), seven in TET2 (15%), 12 in IDH (n = 2) or IDH2 (n = 10) (25%), four in DNMT3A (8%), four in NPM1 (8%), and one in FLT3 (2%). Mutations were more frequent in the intermediate cytogenetic (IC) subgroup of 36 patients than in the unfavorable karyotype subgroup, with an average ratio mutations/patients of 1.36 [0-3] vs. 0.33 [0-2] (P < 0.001). Then, we compared these 36 patients with IC MRC-AML with a control panel of 37 no-MRC-AML patients, who had both IC and no dysplasia. IC MRC-AMLs were associated with higher incidence of ASXL1 mutations (47% vs. 0%, P < 0.001) and lower incidence of DNMT3A (6% vs. 38%, P = 0.001), NPM1 (11% vs. 62%, P < 0.001) and FLT3 (3% vs. 49%, P < 0.001) mutations. No difference was found in the incidence of IDH1/2 or TET2 mutations according to the presence of dysplasia. Complete remission rate after intensive treatment was lower in the MRC-AML group than in the no-MRC-AML group (48% vs. 78%, P = 0.023) and in wild type NPM1 patients (50% vs. 84%, P = 0.009). Our study showed that MRC-AML as defined in the WHO 2008 classification presents a specific mutation pattern characterized by a high frequency of ASXL1 mutations and a low rate of NPM1, FLT3, and DNMT3A mutations.

Azacitidine Plus Venetoclax for the Treatment of Relapsed and Newly Diagnosed Acute Myeloid Leukemia Patients.

Venetoclax (VEN) belongs the BH3-mimetic class that selectively targets BCL-2, activating apoptosis. The combination of VEN and azacitidine (AZA) has changed the paradigm of treatment of newly diagnosed (ND) acute myeloid leukemia (AML) patients ineligible for intensive chemotherapy. There is scarce evidence for the use of VEN-AZA for relapsed or refractory (R/R) AML. We compared the outcome of 39 R/R AML and 38 ND AML patients treated between 01/20 and 12/21. The median age was 69 (22-86) and 73 (61-81) in the R/R and ND groups, respectively. Adverse cytogenetics were found in 36% of patients in the R/R group and 59% of patients in the ND group. Overall response rate was 37% in R/R AML, including 13% CR, 8% CRi, 3% PR and 13% MLFS, and 58% in the ND AML, including 32% CR, 13% CRi and 13% MLFS. Adverse cytogenetics was associated with treatment failure in the R/R group (Relative Risk = 0.13, p = 0.005). Median overall survival (OS) was 5.9 months in the R/R group and 9.4 months in the ND group. Median OS was 2.2 months in the adverse cytogenetics group versus 8.7 months in the intermediate cytogenetics group in the R/R group (p = 0.02). Median leukemia-free survival was not different between the two groups (9.4 months and 10.3 months), indicating that VEN-AZA can be an efficient salvage treatment for selected R/R AML patients. In conclusion, VEN-AZA is a promising treatment for ND AML and for selected R/R AML patients.