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Abstract The treatment of non-small cell lung cancer (NSCLC) patients has significantly improved with recent therapeutic strategies; however, many patients still do not benefit from them. As a result, new treatment approaches are urgently needed. In this study, we evaluated the antitumor efficacy of co-targeting G9a and DNMT1 enzymes and its potential as a cancer drug sensitizer. We observed co-expression and overexpression of G9a and DNMT1 in NSCLC, which were associated with poor prognosis. Co-targeting G9a/DNMT1 with the drug CM-272 reduced proliferation and induced cell death in a panel of human and murine NSCLC cell lines. Additionally, the transcriptomes of these cells were reprogrammed to become highly responsive to chemotherapy (cisplatin), targeted therapy (trametinib), and epigenetic therapy (vorinostat). In vivo, CM-272 reduced tumor volume in human and murine cell-derived cancer models, and this effect was synergistically enhanced by cisplatin. The expression of SCARA5 and AOX1 was induced by CM-272, and both proteins were found to be essential for the antiproliferative response, as gene silencing decreased cytotoxicity. Furthermore, the expression of SCARA5 and AOX1 was positively correlated with each other and inversely correlated with G9a and DNMT1 expression in NSCLC patients. SCARA5 and AOX1 DNA promoters were hypermethylated in NSCLC, and SCARA5 methylation was identified as an epigenetic biomarker in tumors and liquid biopsies from NSCLC patients. Thus, we demonstrate that co-targeting G9a/DNMT1 is a promising strategy to enhance the efficacy of cancer drugs, and SCARA5 methylation could serve as a non-invasive biomarker to monitor tumor progression.

Abstract The development of computational tools for the systematic prediction of metabolic vulnerabilities of cancer cells constitutes a central question in systems biology. Here, we present gmctool, a freely accessible online tool that allows us to accomplish this task in a simple, efficient and intuitive environment. gmctool exploits the concept of genetic Minimal Cut Sets (gMCSs), a theoretical approach to synthetic lethality based on genome-scale metabolic networks, including a unique database of synthetic lethals computed from Human1, the most recent metabolic reconstruction of human cells. gmctool introduces qualitative and quantitative improvements over our previously developed algorithms to predict, visualize and analyze metabolic vulnerabilities in cancer, demonstrating a superior performance than competing algorithms. A detailed illustration of gmctool is presented for multiple myeloma (MM), an incurable hematological malignancy. We provide in vitro experimental evidence for the essentiality of CTPS1 (CTPS synthase) and UAP1 (UDP-N-Acetylglucosamine Pyrophosphorylase 1) in specific MM patient subgroups.

Abstract Despite the development of novel therapies for acute myeloid leukemia, outcomes remain poor for most patients, and therapeutic improvements are an urgent unmet need. Although treatment regimens promoting differentiation have succeeded in the treatment of acute promyelocytic leukemia, their role in other acute myeloid leukemia subtypes needs to be explored. Here we identify and characterize two lysine deacetylase inhibitors, CM-444 and CM-1758, exhibiting the capacity to promote myeloid differentiation in all acute myeloid leukemia subtypes at low non-cytotoxic doses, unlike other commercial histone deacetylase inhibitors. Analyzing the acetylome after CM-444 and CM-1758 treatment reveals modulation of non-histone proteins involved in the enhancer–promoter chromatin regulatory complex, including bromodomain proteins. This acetylation is essential for enhancing the expression of key transcription factors directly involved in the differentiation therapy induced by CM-444/CM-1758 in acute myeloid leukemia. In summary, these compounds may represent effective differentiation-based therapeutic agents across acute myeloid leukemia subtypes with a potential mechanism for the treatment of acute myeloid leukemia.

Not available.

Aims: Rotator cuff (RC) injuries are characterized by tendon rupture, muscle atrophy, retraction, and fatty infiltration, which increase injury severity and jeopardize adequate tendon repair. Epigenetic drugs, such as histone deacetylase inhibitors (HDACis), possess the capacity to redefine the molecular signature of cells, and they may have the potential to inhibit the transformation of the fibro-adipogenic progenitors (FAPs) within the skeletal muscle into adipocyte-like cells, concurrently enhancing the myogenic potential of the satellite cells. Methods: HDACis were added to FAPs and satellite cell cultures isolated from mice. The HDACi vorinostat was additionally administered into a RC injury animal model. Histological analysis was carried out on the isolated supra- and infraspinatus muscles to assess vorinostat anti-muscle degeneration potential. Results: Vorinostat, a HDACi compound, blocked the adipogenic transformation of muscle-associated FAPs in culture, promoting myogenic progression of the satellite cells. Furthermore, it protected muscle from degeneration after acute RC in mice in the earlier muscle degenerative stage after tenotomy. Conclusion: The HDACi vorinostat may be a candidate to prevent early muscular degeneration after RC injury. Cite this article: Bone Joint Res 2024;13(4):169–183.

Abstract Alterations in the epigenetic machinery in both tumor and immune cells contribute to bladder cancer (BC) development, constituting a promising target as an alternative therapeutic option. Here, we have explored the effects of a novel histone deacetylase (HDAC) inhibitor CM-1758, alone or in combination with immune checkpoint inhibitors (ICI) in BC. We determined the antitumor effects of CM-1758 in various BC cell lines together with the induction of broad transcriptional changes, with focus on the epigenetic regulation of PD-L1. Using an immunocompetent syngeneic mouse model of metastatic BC, we studied the effects of CM-1758 alone or in combination with anti-PD-L1 not only on tumor cells, but also in the tumor microenvironment. In vitro, we found that CM-1758 has cytotoxic and cytostatic effects either by inducing apoptosis or cell cycle arrest in BC cells at low micromolar levels. PD-L1 is epigenetically regulated by histone acetylation marks and is induced after treatment with CM-1758. We also observed that treatment with CM-1758 led to an important delay in tumor growth and a higher CD8 + T cell tumor infiltration. Moreover, anti-PD-L1 alone or in combination with CM-1758 reprogramed macrophage differentiation towards a M1-like polarization state and increased of pro-inflammatory cytokines systemically, yielding potential further antitumor effects. Our results suggest the possibility of combining HDAC inhibitors with immunotherapies for the management of advanced metastatic BC.

Non-syndromic orofacial cleft (OFC) is the most common facial developmental defect in the global population. The etiology of these birth defects is complex and multifactorial, involving both genetic and environmental factors. This study aimed to determine if SNPs in the WNT gene family (rs1533767, rs708111, rs3809857, rs7207916, rs12452064) are associated with OFCs in a Polish population. The study included 627 individuals: 209 children with OFCs and 418 healthy controls. DNA was extracted from saliva for the study group and from umbilical cord blood for the control group. Polymorphism genotyping was conducted using quantitative PCR. No statistically significant association was found between four variants and clefts, with odds ratios for rs708111 being 1.13 (CC genotype) and 0.99 (CT genotype), for rs3809857 being 1.05 (GT genotype) and 0.95 (TT genotype), for rs7207916 being 0.86 (AA genotype) and 1.29 (AG genotype) and for rs12452064 being 0.97 (AA genotype) and 1.24 (AG genotype). However, the rs1533767 polymorphism in WNT showed a statistically significant increase in OFC risk for the GG genotype (OR = 1.76, p < 0.001). This research shows that the rs1533767 polymorphism in the WNT gene is an important risk marker for OFC in the Polish population.

Summary: Naive human pluripotent stem cells (hPSCs) are defined as the in vitro counterpart of the human preimplantation embryo’s epiblast and are used as a model system to study developmental processes. In this study, we report the discovery and characterization of distinct cell populations coexisting with epiblast-like cells in 5iLAF naive human induced PSC (hiPSC) cultures. It is noteworthy that these populations closely resemble different cell types of the human embryo at early developmental stages. While epiblast-like cells represent the main cell population, interestingly we detect a cell population with gene and transposable element expression profile closely resembling the totipotent eight-cell (8C)-stage human embryo, and three cell populations analogous to trophectoderm cells at different stages of their maturation process: transition, early, and mature stages. Moreover, we reveal the presence of cells resembling primitive endoderm. Thus, 5iLAF naive hiPSC cultures provide an excellent opportunity to model the earliest events of human embryogenesis, from the 8C stage to the peri-implantation period.

Acute myeloid leukemia (AML) in the elderly remains a clinical challenge, with a five-year overall survival rate below 10%. The current ELN 2017 genetic risk classification considers cytogenetic and mutational characteristics to stratify fit AML patients into different prognostic groups. However, this classification is not validated for elderly patients treated with a non-intensive approach, and its performance may be suboptimal in this context. Indeed, the transcriptomic landscape of AML in the elderly has been less explored and it might help stratify this group of patients. In the current study, we analyzed the transcriptome of 224 AML patients > 65 years-old at diagnosis treated in the Spanish PETHEMA-FLUGAZA clinical trial in order to identify new prognostic biomarkers in this population. We identified a specific transcriptomic signature for high-risk patients with mutated TP53 or complex karyotype, revealing that low expression of B7H3 gene with high expression of BANP gene identifies a subset of high-risk AML patients surviving more than 12 months. This result was further validated in the BEAT AML cohort. This unique signature highlights the potential of transcriptomics to identify prognostic biomarkers in in elderly AML.

Artificial intelligence (AI) can unveil novel personalized treatments based on drug screening and whole-exome sequencing experiments (WES). However, the concept of “black box” in AI limits the potential of this approach to be translated into the clinical practice. In contrast, explainable AI (XAI) focuses on making AI results understandable to humans. Here, we present a novel XAI method -called multi-dimensional module optimization (MOM)- that associates drug screening with genetic events, while guaranteeing that predictions are interpretable and robust. We applied MOM to an acute myeloid leukemia (AML) cohort of 319 ex-vivo tumor samples with 122 screened drugs and WES. MOM returned a therapeutic strategy based on the FLT3, CBFβ-MYH11, and NRAS status, which predicted AML patient response to Quizartinib, Trametinib, Selumetinib, and Crizotinib. We successfully validated the results in three different large-scale screening experiments. We believe that XAI will help healthcare providers and drug regulators better understand AI medical decisions.

Castration-resistant prostate cancer (CRPC) is an incurable form of prostate cancer (PCa), with DNMT1 and G9a being reported as overexpressed, rendering them highly attractive targets for precision medicine. CM-272 is a dual inhibitor of both methyltransferases’ activity. Herein, we assessed the response of different PCa cell lines to CM-272, in both 2D and 3D models, and explored the molecular mechanisms underlying CM-272 inhibitory effects.CRPC tissues displayed significantly higher DNMT1, G9a and H3K9me2 expression than localized PCa. In vitro, CM-272 caused a significant decrease in PCa cell viability and proliferation alongside with increased apoptotic levels. We disclose that, under the evaluated dose, CM-272 led to G9a activity inhibition, while not significantly affecting DNMT1 activity. Upon G9a knockdown, DU145 and PC3 showed decreased cell viability. Remarkably, DU145 cells treated with CM-272 or with G9a knockdown displayed no differences in viability, suggesting a SET-dependent mechanism. Contrarily, PC3 cell viability impact was higher in G9a knockdown, compared with CM-272 treatment, suggesting an additional G9a function. Moreover, DU145 cells overexpressing catalytically functional G9a disclosed higher resistance to CM-272 treatment, reinforcing that the drug mechanism of action is dependent on G9a catalytic function.Importantly, we successfully assembled spheroids from several prostate cell lines. Our results showed that CM-272 retained its anti-tumoral effects in 3D PCa models, leading to a clear reduction in cancer cell survival.We concluded that inhibition of G9a methyltransferase activity by CM-272 has anti-tumor effect in PCa cells, holding therapeutic potential against CRPC.

The dynamics of genome architecture during human cell differentiation and upon neoplastic transformation remain poorly characterized. Here, the authors integrate in situ Hi-C and nine additional omic layers to characterize the dynamic changes in 3D genome architecture during normal B cell differentiation and in neoplastic cells from chronic lymphocytic leukemia and mantle cell lymphoma patients.

Mesenchymal stromal cells (MSCs) have been shown to support multiple myeloma (MM) development. Here, MSCs isolated from the bone marrow of MM patients are shown to have altered DNA methylation patterns and a methyltransferase inhibitor reverts MM-associated bone loss and reduces tumour burden in MM murine models.

With the frenetic growth of high-dimensional datasets in different biomedical domains, there is an urgent need to develop predictive methods able to deal with this complexity. Feature selection is a relevant strategy in machine learning to address this challenge. We introduce a novel feature selection algorithm for linear regression called BOSO (Bilevel Optimization Selector Operator). We conducted a benchmark of BOSO with key algorithms in the literature, finding a superior accuracy for feature selection in high-dimensional datasets. Proof-of-concept of BOSO for predicting drug sensitivity in cancer is presented. A detailed analysis is carried out for methotrexate, a well-studied drug targeting cancer metabolism.

Immunotherapy has improved the treatment of malignant skin cancer of the melanoma type, yet overall clinical response rates remain low. Combination therapies could be key to meet this cogent medical need. Because epigenetic hallmarks represent promising combination therapy targets, we studied the immunogenic potential of a dual inhibitor of histone methyltransferase G9a and DNA methyltransferases (DNMTs) in the preclinical B16-OVA melanoma model. Making use of tumor transcriptomic and functional analyses, methylation-targeted epigenetic reprogramming was shown to induce tumor cell cycle arrest and apoptosis in vitro coinciding with transient tumor growth delay and an IFN-I response in immune-competent mice. In consideration of a potential impact on immune cells, the drug was shown not to interfere with dendritic cell maturation or T-cell activation in vitro. Notably, the drug promoted dendritic cell and, to a lesser extent, T-cell infiltration in vivo, yet failed to sensitize tumor cells to programmed cell death-1 inhibition. Instead, it increased therapeutic efficacy of TCR-redirected T cell and dendritic cell vaccination, jointly increasing overall survival of B16-OVA tumor-bearing mice. The reported data confirm the prospect of methylation-targeted epigenetic reprogramming in melanoma and sustain dual G9a and DNMT inhibition as a strategy to tip the cancer-immune set-point towards responsiveness to active and adoptive vaccination against melanoma.

Synthetic Lethality (SL) is currently defined as a type of genetic interaction in which the loss of function of either of two genes individually has limited effect in cell viability but inactivation of both genes simultaneously leads to cell death. Given the profound genomic aberrations acquired by tumor cells, which can be systematically identified with -omics data, SL is a promising concept in cancer research. In particular, SL has received much attention in the area of cancer metabolism, due to the fact that relevant functional alterations concentrate on key metabolic pathways that promote cellular proliferation. With the extensive prior knowledge about human metabolic networks, a number of computational methods have been developed to predict SL in cancer metabolism, including the genetic Minimal Cut Sets (gMCSs) approach. A major challenge in the application of SL approaches to cancer metabolism is to systematically integrate tumor microenvironment, given that genetic interactions and nutritional availability are interconnected to support proliferation. Here, we propose a more general definition of SL for cancer metabolism that combines genetic and environmental interactions, namely loss of gene functions and absence of nutrients in the environment. We extend our gMCSs approach to determine this new family of metabolic synthetic lethal interactions. A computational and experimental proof-of-concept is presented for predicting the lethality of dihydrofolate reductase (DHFR) inhibition in different environments. Finally, our approach is applied to identify extracellular nutrient dependences of tumor cells, elucidating cholesterol and myo-inositol depletion as potential vulnerabilities in different malignancies.

Long non-coding RNAs (lncRNA) constitute a large fraction of human transcriptome, and are reported, individually, for context-specific regulatory functions. Here the authors expand our understanding by providing a systemic, unbiased annotation of lncRNA to establish an atlas of lncRNA landscape during the induction of human humoral immune responses.

Cancer cells are under the surveillance of the host immune system. Nevertheless, a number of immunosuppressive mechanisms allow tumors to escape protective responses and impose immune tolerance. Epigenetic alterations are central to cancer cell biology and cancer immune evasion. Accordingly, epigenetic modulating agents (EMAs) are being exploited as anti-neoplastic and immunomodulatory agents to restore immunological fitness. By simultaneously acting on cancer cells, e.g. by changing expression of tumor antigens, immune checkpoints, chemokines or innate defense pathways, and on immune cells, e.g. by remodeling the tumor stroma or enhancing effector cell functionality, EMAs can indeed overcome peripheral tolerance to transformed cells. Therefore, combinations of EMAs with chemo- or immunotherapy have become interesting strategies to fight cancer. Here we review several examples of epigenetic changes critical for immune cell functions and tumor-immune evasion and of the use of EMAs in promoting anti-tumor immunity. Finally, we provide our perspective on how EMAs could represent a game changer for combinatorial therapies and the clinical management of cancer.

Cell identity relies on the cross-talk between genetics and epigenetics and their impact on gene expression. Oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) is the first step of an active DNA demethylation process occurring mainly at enhancers and gene bodies and, as such, participates in processes governing cell identity in normal and pathological conditions. Although genetic alterations are well documented in multiple myeloma (MM), epigenetic alterations associated with this disease have not yet been thoroughly analyzed. To gain insight into the biology of MM, genome-wide 5hmC profiles were obtained and showed that regions enriched in this modified base overlap with MM enhancers and super enhancers and are close to highly expressed genes. Through the definition of a MM-specific 5hmC signature, we identified FAM72D as a poor prognostic gene located on 1q21, a region amplified in high risk myeloma. We further uncovered that FAM72D functions as part of the FOXM1 transcription factor network controlling cell proliferation and survival and we evidenced an increased sensitivity of cells expressing high levels of FOXM1 and FAM72 to epigenetic drugs targeting histone deacetylases and DNA methyltransferases.

Exploiting synthetic lethality is a promising approach for cancer therapy. Here, the authors present an approach to identifying such interactions by finding genetic minimal cut sets (gMCSs) that block cancer proliferation, and apply it to study the lethality of RRM1 inhibition in multiple myeloma.

Epigenetic drugs are emerging as a powerful therapeutic option for cancer treatment. Here, the authors synthesized selective chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity and demonstrate their anti-tumour activity usingin vitro and in vivomodels of haematological neoplasia.

In this study we interrogated the DNA methylome of myelofibrosis patients using high-density DNA methylation arrays. We detected 35,215 differentially methylated CpG, corresponding to 10,253 genes, between myelofibrosis patients and healthy controls. These changes were present both in primary and secondary myelofibrosis, which showed no differences between them. Remarkably, most differentially methylated CpG were located outside gene promoter regions and showed significant association with enhancer regions. This aberrant enhancer hypermethylation was negatively correlated with the expression of 27 genes in the myelofibrosis cohort. Of these, we focused on the ZFP36L1 gene and validated its decreased expression and enhancer DNA hypermethylation in an independent cohort of patients and myeloid cell-lines. In vitro reporter assay and 5’-azacitidine treatment confirmed the functional relevance of hyper-methylation of ZFP36L1 enhancer. Furthermore, in vitro rescue of ZFP36L1 expression had an impact on cell proliferation and induced apoptosis in SET-2 cell line indicating a possible role of ZFP36L1 as a tumor suppressor gene in myelofibrosis. Collectively, we describe the DNA methylation profile of myelofibrosis, identifying extensive changes in enhancer elements and revealing ZFP36L1 as a novel candidate tumor suppressor gene.

The homeobox NKX2 family of transcriptional factors has been shown to regulate fundamental developmental processes. Here, the authors show that NKX2-3 is a bona fideoncogenic driver in marginal-zone B-cell lymphoma and that it promotes lymphomagenesis by shaping lymphocyte dynamics and promoting BCR signalling.

The identification of therapeutic strategies exploiting the metabolic alterations of malignant cells is a relevant area in cancer research. Here, we discuss a novel computational method, based on the COBRA (COnstraint-Based Reconstruction and Analysis) framework for metabolic networks, to perform this task. Current and future steps are presented.

Epigenetic drug discovery is an emerging strategy for the treatment of cancer and other pathologies. Here, we discuss our recent discovery of first-in-class dual reversible inhibitors of the histone methyltransferase activity of G9a/EHMT2 and DNA methyltransferases showing in vivo efficacy in human tumors. Current and future investigation lines are presented.

The combination of defined factors with small molecules targeting epigenetic factors is a strategy that has been shown to enhance optimal derivation of iPSCs and could be used for disease modelling, high throughput screenings and/or regenerative medicine applications. In this study, we showed that a new first-in-class reversible dual G9a/DNMT1 inhibitor compound (CM272) improves the efficiency of human cell reprogramming and iPSC generation from primary cells of healthy donors and patient samples, using both integrative and non-integrative methods. Moreover, CM272 facilitates the generation of human iPSC with only two factors allowing the removal of the most potent oncogenic factor cMYC. Furthermore, we demonstrated that mechanistically, treatment with CM272 induces heterochromatin relaxation, facilitates the engagement of OCT4 and SOX2 transcription factors to OSKM refractory binding regions that are required for iPSC establishment, and enhances mesenchymal to epithelial transition during the early phase of cell reprogramming. Thus, the use of this new G9a/DNMT reversible dual inhibitor compound may represent an interesting alternative for improving cell reprogramming and human iPSC derivation for many different applications while providing interesting insights into reprogramming mechanisms.

El éxito de las investigaciones de crímenes internacionales (genocidio, crímenes de guerra y crímenes de lesa humanidad)depende en gran parte de poder establecer el patrón delictivo. El patrón delictivo es el conjunto de características que tienen en común varios incidentes con relación a las víctimas, los perpetradores y el modus operandi. Las pruebas y los análisis con base en patrones han sido utilizados con éxito principalmente en las investigaciones de masacres, destrucciones y desplazamiento, pero su utilización en imputaciones de violencia sexual ha sido notoriamente más limitada. Es necesario cubrir este vacío por medio del establecimiento de métodos apropiados de recolección y análisis de datos. A nivel de la recolección de pruebas, la carencia de denuncias presentadas por lasvíctimas se debe suplir por medio de encuestas a las víctimas o por medio del análisis de la información secundaria disponible en diferentes fuentes. A nivel de análisis, la evidencia disponible debe ser evaluada de manera imparcial, independientemente de las ideas preconcebidas de las partes en conflicto y de los grupos de defensa dederechos, cumpliendo con los estándares y métodos científicos para el análisis cuantitativo, cualitativo y de sistemas de información geográfica (GIS). Un análisis de varias experiencias investigativas y de la jurisprudencia permitirá establecer la metodología más apropiada y será la manera más eficiente de ayudar a poner fin a la impunidadrelacionada con los crímenes sexuales.

Interstitial deletion of chromosome 5q is the most common chromosomal abnormality in myelodysplastic syndromes. The catalogue of genes involved in the molecular pathogenesis of myelodysplastic syndromes is rapidly expanding and next-generation sequencing technology allows detection of these mutations at great depth. Here we describe the design, validation and application of a targeted next-generation sequencing approach to simultaneously screen 25 genes mutated in myeloid malignancies. We used this method alongside single nucleotide polymorphism-array technology to characterize the mutational and cytogenetic profile of 43 cases of early or advanced del(5q) myelodysplastic syndromes. A total of 29 mutations were detected in our cohort. Overall, 45% of early and 66.7% of advanced cases had at least one mutation. Genes with the highest mutation frequency among advanced cases were TP53 and ASXL1 (25% of patients each). These showed a lower mutation frequency in cases of 5q- syndrome (4.5% and 13.6%, respectively), suggesting a role in disease progression in del(5q) myelodysplastic syndromes. Fifty-two percent of mutations identified were in genes involved in epigenetic regulation (ASXL1, TET2, DNMT3A and JAK2). Six mutations had allele frequencies

Most DNA methylation studies in classic Philadelphia-negative myeloproliferative neoplasms have been performed on a gene-by-gene basis. Therefore, a more comprehensive methylation profiling is needed to study the implications of this epigenetic marker in myeloproliferative neoplasms. Here, we have analyzed 71 chronic (24 polycythemia vera, 23 essential thrombocythemia and 24 primary myelofibrosis) and 13 transformed myeloproliferative neoplasms using genome-wide DNA methylation arrays. The three types of chronic Philadelphia-negative myeloproliferative neoplasms showed a similar aberrant DNA methylation pattern when compared to control samples. Differentially methylated regions were enriched in a gene network centered on the NF-κB pathway, indicating that they may be involved in the pathogenesis of these diseases. In the case of transformed myeloproliferative neoplasms, we detected an increased number of differentially methylated regions with respect to chronic myeloproliferative neoplasms. Interestingly, these genes were enriched in a list of differentially methylated regions in primary acute myeloid leukemia and in a gene network centered around the IFN pathway. Our results suggest that alterations in the DNA methylation landscape play an important role in the pathogenesis and leukemic transformation of myeloproliferative neoplasms. The therapeutic modulation of epigenetically-deregulated pathways may allow us to design targeted therapies for these patients.

The disruption of RUNX1 function is one of the main mechanisms of disease observed in hematopoietic malignancies and the description of novel genetic events that lead to a RUNX1 loss of function has been accelerated with the development of genomic technologies. Here we describe the molecular characterization of a new t(4;21)(q21;q22) in a de novo myelodysplastic syndrome that resulted in the deletion of the RUNX1 gene. We demonstrated by quantitative real-time RT-PCR an almost complete depletion of the expression of the RUNX1 gene in our t(4;21) case compared with CD34+ cells that was independent of mutation or DNA methylation. More importantly, we explored and confirmed the possibility that this abrogation also prevented transactivation of RUNX1 target genes, perhaps confirming the genetic origin of the thrombocytopenia and the myelodysplastic features observed in our patient, and certainly mimicking what has been observed in the presence of the RUNX1/ETO fusion protein.

miRNAs are small RNA molecules (' 22nt) that interact with their corresponding target mRNAs inhibiting the translation of the mRNA into proteins and cleaving the target mRNA. This second effect diminishes the overall expression of the target mRNA. Several miRNA-mRNA relationship databases have been deployed, most of them based on sequence complementarities. However, the number of false positives in these databases is large and they do not overlap completely. Recently, it has been proposed to combine expression measurement from both miRNA and mRNA and sequence based predictions to achieve more accurate relationships. In our work, we use LASSO regression with non-positive constraints to integrate both sources of information. LASSO enforces the sparseness of the solution and the non-positive constraints restrict the search of miRNA targets to those with down-regulation effects on the mRNA expression. We named this method TaLasso (miRNA-Target LASSO).We used TaLasso on two public datasets that have paired expression levels of human miRNAs and mRNAs. The top ranked interactions recovered by TaLasso are especially enriched (more than using any other algorithm) in experimentally validated targets. The functions of the genes with mRNA transcripts in the top-ranked interactions are meaningful. This is not the case using other algorithms.TaLasso is available as Matlab or R code. There is also a web-based tool for human miRNAs at http://talasso.cnb.csic.es/.

Acute myeloid leukemia patients with normal cytogenetics (CN-AML) account for almost half of AML cases. We aimed to study the frequency and relationship of a wide range of genes previously reported as mutated in AML (ASXL1, NPM1, FLT3, TET2, IDH1/2, RUNX1, DNMT3A, NRAS, JAK2, WT1, CBL, SF3B1, TP53, KRAS and MPL) in a series of 84 CN-AML cases. The most frequently mutated genes in primary cases were NPM1 (60.8%) and FLT3 (50.0%), and in secondary cases ASXL1 (48.5%) and TET2 (30.3%). We showed that 85% of CN-AML patients have mutations in at least one of ASXL1, NPM1, FLT3, TET2, IDH1/2 and/or RUNX1. Serial samples from 19 MDS/CMML cases that progressed to AML were analyzed for ASXL1/TET2/IDH1/2 mutations; seventeen cases presented mutations of at least one of these genes. However, there was no consistent pattern in mutation acquisition during disease progression. This report concerns the analysis of the largest number of gene mutations in CN-AML studied to date, and provides insight into the mutational profile of CN-AML.

Chronic myelomonocytic leukemia (CMML) has recently been associated with a high incidence of diverse mutations in genes such as TET2 or EZH2 that are implicated in epigenetic mechanisms. We have performed genome-wide DNA methylation arrays and mutational analysis of TET2, IDH1, IDH2, EZH2 and JAK2 in a group of 24 patients with CMML. 249 genes were differentially methylated between CMML patients and controls. Using Ingenuity pathway analysis, we identified enrichment in a gene network centered around PLC, JNK and ERK suggesting that these pathways, whose deregulation has been recently described in CMML, are affected by epigenetic mechanisms. Mutations of TET2, JAK2 and EZH2 were found in 15 patients (65%), 4 patients (17%) and 1 patient (4%) respectively while no mutations in the IDH1 and IDH2 genes were identified. Interestingly, patients with wild type TET2 clustered separately from patients with TET2 mutations, showed a higher degree of hypermethylation and were associated with higher risk karyotypes. Our results demonstrate the presence of aberrant DNA methylation in CMML and identifies TET2 mutant CMML as a biologically distinct disease subtype with a different epigenetic profile.

Background The EVI1 gene (3q26) codes for a zinc finger transcription factor with important roles in both mammalian development and leukemogenesis. Over-expression of EVI1 through either 3q26 rearrangements, MLL fusions, or other unknown mechanisms confers a poor prognosis in acute myeloid leukemia.Design and Methods We analyzed the prevalence and prognostic impact of EVI1 over-expression in a series of 476 patients with acute myeloid leukemia, and investigated the epigenetic modifications of the EVI1 locus which could be involved in the transcriptional regulation of this gene.Results Our data provide further evidence that EVI1 over-expression is a poor prognostic marker in acute myeloid leukemia patients less than 65 years old. Moreover, we found that patients with no basal expression of EVI1 had a better prognosis than patients with expression/over-expression (P=0.036). We also showed that cell lines with over-expression of EVI1 had no DNA methylation in the promoter region of the EVI1 locus, and had marks of active histone modifications: H3 and H4 acetylation, and trimethylation of histone H3 lysine 4. Conversely, cell lines with no expression of EVI1 have DNA hypermethylation and are marked by repressive trimethylation of histone H3 lysine 27 at the EVI1 promoter.Conclusions Our results identify EVI1 over-expression as a poor prognostic marker in a large, independent cohort of acute myeloid leukemia patients less than 65 years old, and show that the total absence of EVI1 expression has a prognostic impact on the outcome of such patients. Furthermore, we demonstrated for the first time that an aberrant epigenetic pattern involving DNA methylation, H3 and H4 acetylation, and trimethylation of histone H3 lysine 4 and histone H3 lysine 27 might play a role in the transcriptional regulation of EVI1 in acute myeloid leukemia. This study opens new avenues for a better understanding of the regulation of EVI1 expression at a transcriptional level.

Background LMO2 is highly expressed at the most immature stages of lymphopoiesis. In T-lymphocytes, aberrant LMO2 expression beyond those stages leads to T-cell acute lymphoblastic leukemia, while in B cells LMO2 is also expressed in germinal center lymphocytes and diffuse large B-cell lymphomas, where it predicts better clinical outcome. The implication of LMO2 in B-cell acute lymphoblastic leukemia must still be explored.Design and Methods We measured LMO2 expression by real time RT-PCR in 247 acute lymphoblastic leukemia patient samples with cytogenetic data (144 of them also with survival and immunophenotypical data) and in normal hematopoietic and lymphoid cells.Results B-cell acute lymphoblastic leukemia cases expressed variable levels of LMO2 depending on immunophenotypical and cytogenetic features. Thus, the most immature subtype, pro-B cells, displayed three-fold higher LMO2 expression than pre-B cells, common-CD10+ or mature subtypes. Additionally, cases with TEL-AML1 or MLL rearrangements exhibited two-fold higher LMO2 expression compared to cases with BCR-ABL rearrangements or hyperdyploid karyotype. Clinically, high LMO2 expression correlated with better overall survival in adult patients (5-year survival rate 64.8% (42.5%–87.1%) vs. 25.8% (10.9%–40.7%), P= 0.001) and constituted a favorable independent prognostic factor in B-ALL with normal karyotype: 5-year survival rate 80.3% (66.4%–94.2%) vs. 63.0% (46.1%–79.9%) (P= 0.043).Conclusions Our data indicate that LMO2 expression depends on the molecular features and the differentiation stage of B-cell acute lymphoblastic leukemia cells. Furthermore, assessment of LMO2 expression in adult patients with a normal karyotype, a group which lacks molecular prognostic factors, could be of clinical relevance.

Aberrant DNA methylation is one of the most frequent alterations in patients with Acute Lymphoblastic Leukemia (ALL). Using methylation bead arrays we analyzed the methylation status of 807 genes implicated in cancer in a group of ALL samples at diagnosis (n = 48). We found that 154 genes were methylated in more than 10% of ALL samples. Interestingly, the expression of 13 genes implicated in the TP53 pathway was downregulated by hypermethylation. Direct or indirect activation of TP53 pathway with 5-aza-2'-deoxycitidine, Curcumin or Nutlin-3 induced an increase in apoptosis of ALL cells. The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients. Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (p = 0.001) and mortality (p

Recent studies have shown aberrant expression of SOX11 in various types of aggressive B-cell neoplasms. To elucidate the molecular mechanisms leading to such deregulation, we performed a comprehensive SOX11 gene expression and epigenetic study in stem cells, normal hematopoietic cells and different lymphoid neoplasms. We observed that SOX11 expression is associated with unmethylated DNA and presence of activating histone marks (H3K9/14Ac and H3K4me3) in embryonic stem cells and some aggressive B-cell neoplasms. In contrast, adult stem cells, normal hematopoietic cells and other lymphoid neoplasms do not express SOX11. Such repression was associated with silencing histone marks H3K9me2 and H3K27me3. The SOX11 promoter of non-malignant cells was consistently unmethylated whereas lymphoid neoplasms with silenced SOX11 tended to acquire DNA hypermethylation. SOX11 silencing in cell lines was reversed by the histone deacetylase inhibitor SAHA but not by the DNA methyltransferase inhibitor AZA. These data indicate that, although DNA hypermethylation of SOX11 is frequent in lymphoid neoplasms, it seems to be functionally inert, as SOX11 is already silenced in the hematopoietic system. In contrast, the pathogenic role of SOX11 is associated with its de novo expression in some aggressive lymphoid malignancies, which is mediated by a shift from inactivating to activating histone modifications.

Chronic myeloid leukemia (CML) is a malignant clonal disorder of the hematopoietic system caused by the expression of the BCR/ABL fusion oncogene. Although it is well known that CML cells are genetically unstable, the mechanisms accounting for this genomic instability are still poorly understood. Because the Fanconi anemia (FA) pathway is believed to control several mechanisms of DNA repair, we investigated whether this pathway was disrupted in CML cells. Our data show that CML cells have a defective capacity to generate FANCD2 nuclear foci, either in dividing cells or after DNA damage. Similarly, human cord blood CD34(+) cells transduced with BCR/ABL retroviral vectors showed impaired FANCD2 foci formation, whereas FANCD2 monoubiquitination in these cells was unaffected. Soon after the transduction of CD34(+) cells with BCR/ABL retroviral vectors a high proportion of cells with supernumerary centrosomes was observed. Similarly, BCR/ABL induced a high proportion of chromosomal abnormalities, while mediated a cell survival advantage after exposure to DNA cross-linking agents. Significantly, both the impaired formation of FANCD2 nuclear foci, and also the predisposition of BCR/ABL cells to develop centrosomal and chromosomal aberrations were reverted by the ectopic expression of BRCA1. Taken together, our data show for the first time a disruption of the FA/BRCA pathway in BCR/ABL cells, suggesting that this defective pathway should play an important role in the genomic instability of CML by the co-occurrence of centrosomal amplification and DNA repair deficiencies.

Aberrant promoter DNA methylation has been shown to play a role in acute myeloid leukemia (AML) pathophysiology. However, further studies to discuss the prognostic value and the relationship of the epigenetic signatures with defined genomic rearrangements in acute myeloid leukemia are required.We carried out high-throughput methylation profiling on 116 de novo AML cases and we validated the significant biomarkers in an independent cohort of 244 AML cases. Methylation signatures were associated with the presence of a specific cytogenetic status. In normal karyotype cases, aberrant methylation of the promoter of DBC1 was validated as a predictor of the disease-free and overall survival. Furthermore, DBC1 expression was significantly silenced in the aberrantly methylated samples. Patients with chromosome rearrangements showed distinct methylation signatures. To establish the role of fusion proteins in the epigenetic profiles, 20 additional samples of human hematopoietic stem/progenitor cells (HSPC) transduced with common fusion genes were studied and compared with patient samples carrying the same rearrangements. The presence of MLL rearrangements in HSPC induced the methylation profile observed in the MLL-positive primary samples. In contrast, fusion genes such as AML1/ETO or CBFB/MYH11 failed to reproduce the epigenetic signature observed in the patients.Our study provides a comprehensive epigenetic profiling of AML, identifies new clinical markers for cases with a normal karyotype, and reveals relevant biological information related to the role of fusion proteins on the methylation signature.

BackgroundThe therapeutic use of multipotent stem cells depends on their differentiation potential, which has been shown to be variable for different populations. These differences are likely to be the result of key changes in their epigenetic profiles.Methodology/principal findingsto address this issue, we have investigated the levels of epigenetic regulation in well characterized populations of pluripotent embryonic stem cells (ESC) and multipotent adult stem cells (ASC) at the trancriptome, methylome, histone modification and microRNA levels. Differences in gene expression profiles allowed classification of stem cells into three separate populations including ESC, multipotent adult progenitor cells (MAPC) and mesenchymal stromal cells (MSC). The analysis of the PcG repressive marks, histone modifications and gene promoter methylation of differentiation and pluripotency genes demonstrated that stem cell populations with a wider differentiation potential (ESC and MAPC) showed stronger representation of epigenetic repressive marks in differentiation genes and that this epigenetic signature was progressively lost with restriction of stem cell potential. Our analysis of microRNA established specific microRNA signatures suggesting specific microRNAs involved in regulation of pluripotent and differentiation genes.Conclusions/significanceOur study leads us to propose a model where the level of epigenetic regulation, as a combination of DNA methylation and histone modification marks, at differentiation genes defines degrees of differentiation potential from progenitor and multipotent stem cells to pluripotent stem cells.

Alterations in the DNA methylation pattern are a hallmark of leukemias and lymphomas. However, most epigenetic studies in hematologic neoplasms (HNs) have focused either on the analysis of few candidate genes or many genes and few HN entities, and comprehensive studies are required.Here, we report for the first time a microarray-based DNA methylation study of 767 genes in 367 HNs diagnosed with 16 of the most representative B-cell (n = 203), T-cell (n = 30), and myeloid (n = 134) neoplasias, as well as 37 samples from different cell types of the hematopoietic system. Using appropriate controls of B-, T-, or myeloid cellular origin, we identified a total of 220 genes hypermethylated in at least one HN entity. In general, promoter hypermethylation was more frequent in lymphoid malignancies than in myeloid malignancies, being germinal center mature B-cell lymphomas as well as B and T precursor lymphoid neoplasias those entities with highest frequency of gene-associated DNA hypermethylation. We also observed a significant correlation between the number of hypermethylated and hypomethylated genes in several mature B-cell neoplasias, but not in precursor B- and T-cell leukemias. Most of the genes becoming hypermethylated contained promoters with high CpG content, and a significant fraction of them are targets of the polycomb repressor complex. Interestingly, T-cell prolymphocytic leukemias show low levels of DNA hypermethylation and a comparatively large number of hypomethylated genes, many of them showing an increased gene expression.We have characterized the DNA methylation profile of a wide range of different HNs entities. As well as identifying genes showing aberrant DNA methylation in certain HN subtypes, we also detected six genes--DBC1, DIO3, FZD9, HS3ST2, MOS, and MYOD1--that were significantly hypermethylated in B-cell, T-cell, and myeloid malignancies. These might therefore play an important role in the development of different HNs.

Background and Objectives Cancer testis antigens (CTA) provide attractive targets for cancer-specific immunotherapy. Although CTA genes are expressed in some normal tissues, such as the testis, this immunologically protected site lacks MHC I expression and as such, does not present self antigens to T cells. To date, CTA genes have been shown to be expressed in a range of solid tumors via demethylation of their promoter CpG islands, but rarely in chronic myeloid leukemia (CML) or other hematologic malignancies.Design and Methods In this study, the methylation status of the HAGE CTA gene promoter was analyzed by quantitative methylation-specific polymerase chain reaction (MSP) and sequencing in four Philadelphia-positive cell lines (TCC-S, K562, KU812 and KYO-1) and in CML samples taken from patients in chronic phase (CP n=215) or blast crisis (BC n=47). HAGE expression was assessed by quantitative reverse transcriptase-polymerase chain reaction.Results The TCC-S cell line showed demethylation of HAGE that was associated with over-expression of this gene. HAGE hypomethylation was significantly more frequent in BC (46%) than in CP (22%) (p=0.01) and was correlated with high expression levels of HAGE transcripts (p