The CUT&RUN greenlist: genomic regions of consistent noise are effective normalizing factors for quantitative epigenome mapping.

Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a recent development for epigenome mapping, but its unique methodology can hamper proper quantitative analyses. As traditional normalization approaches have been shown to be inaccurate, we sought to determine endogenous normalization factors based on the human genome regions of constant nonspecific signal. This constancy was determined by applying Shannon's information entropy, and the set of normalizer regions, which we named the 'Greenlist', was extensively validated using publicly available datasets. We demonstrate here that the greenlist normalization outperforms the current top standards, and remains consistent across different experimental setups, cell lines and antibodies; the approach can even be applied to different species or to CUT&Tag. Requiring no additional experimental steps and no added cost, this approach can be universally applied to CUT&RUN experiments to greatly minimize the interference of technical variation over the biological epigenome changes of interest.

MLL2/KMT2D and MLL3/KMT2C expression correlates with disease progression and response to imatinib mesylate in chronic myeloid leukemia.

BACKGROUND: Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm whose pathogenesis is linked to the Philadelphia chromosome presence that generates the BCR-ABL1 fusion oncogene. Tyrosine kinase inhibitors (TKI) such as imatinib mesylate (IM) dramatically improved the treatment efficiency and survival of CML patients by targeting BCR-ABL tyrosine kinase. The disease shows three distinct clinical-laboratory stages: chronic phase, accelerated phase and blast crisis. Although patients in the chronic phase respond well to treatment, patients in the accelerated phase or blast crisis usually show therapy resistance and CML relapse. It is crucial, therefore, to identify biomarkers to predict CML genetic evolution and resistance to TKI therapy, considering not only the effects of genetic aberrations but also the role of epigenetic alterations during the disease. Although dysregulations in epigenetic modulators such as histone methyltrasnferases have already been described for some hematologic malignancies, to date very limited data is available for CML, especially when considering the lysine methyltransferase MLL2/KMT2D and MLL3/KMT2C. METHODS: Here we investigated the expression profile of both genes in CML patients in different stages of the disease, in patients showing different responses to therapy with IM and in non-neoplastic control samples. Imatinib sensitive and resistant CML cell lines were also used to investigate whether treatment with other tyrosine kinase inhibitors interfered in their expression. RESULTS: In patients, both methyltransferases were either upregulated or with basal expression level during the chronic phase compared to controls. Interestingly, MLL3/KMT2C and specially MLL2/KMT2D levels decreased during disease progression correlating with distinct clinical stages. Furthermore, MLL2/KMT2D was decreased in patients resistant to IM treatment. A rescue in the expression of both MLL genes was observed in KCL22S, a CML cell line sensitive to IM, after treatment with dasatinib or nilotinib which was associated with a higher rate of apoptosis, an enhanced expression of p21 (CDKN1A) and a concomitant decrease in the expression of CDK2, CDK4 and Cyclin B1 (CCNB1) in comparison to untreated KCL22S control or IM resistant KCL22R cell line, which suggests involvement of p53 regulated pathway. CONCLUSION: Our results established a new association between MLL2/KMT2D and MLL3/KMT2C genes with CML and suggest that MLL2/KMT2D is associated with disease evolution and may be a potential marker to predict the development of therapy resistance.

The expression of Death Inducer-Obliterator (DIDO) variants in Myeloproliferative Neoplasms.

Chronic Myeloid Leukemia (CML), Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation without cell maturation impairment. CML pathogenesis is associated with the Ph chromosome leading to BCR-ABL tyrosine-kinase constitutive expression. The Ph negative MPN (PV, ET and PMF) are characterized by the mutation JAK2(V617F) of the JAK2 protein in the auto-inhibitory JH2 domain, which is found in most PV patients and in approximately half of ET and PMF patients. Considerable effort is being made to understand the role of JAK2(V617F) at the MPN initiation and to clarify the pathogenesis and apoptosis resistance in CML, PV, ET and PMF patients. In the present investigation, we evaluated the Death Inducer-Obliterator (DIDO) (variants DIDO 1, 2 and 3) levels in CML, PV, ET and PMF patients. Our data reported the DIDO 1, 2 and 3 differential expressions in Myeloproliferative Neoplasms.

Hippo pathway-related genes expression is deregulated in myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are hematological disorders characterized by increased proliferation of precursor and mature myeloid cells. MPN patients may present driver mutations in JAK2, MPL, and CALR genes, which are essential to describe the molecular mechanisms of MPN pathogenesis. Despite all the new knowledge on MPN pathogenesis, many questions remain to be answered to develop effective therapies to cure MPN or impair its progression to acute myeloid leukemia. The present study examined the expression levels of the Hippo signaling pathway members in patients with polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), as well as the role that they play in disease pathogenesis. The Hippo pathway is a tumor suppressor pathway that participates in the regulation of cell proliferation, differentiation, and death. Our main finding was that the expression of tumor suppressor genes from Hippo pathway were downregulated and seemed to be associated with cell resistance to apoptosis and increased proliferation rate. Therefore, the decreased expression of Hippo pathway-related genes may contribute to the malignant phenotype, apoptosis resistance, and cell proliferation in MPN pathogenesis.

Bone Marrow Soluble Mediator Signatures of Patients With Philadelphia Chromosome-Negative Myeloproliferative Neoplasms.

BACKGROUND: Essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) are clonal hematological diseases classified as Philadelphia chromosome-negative myeloproliferative neoplasms (MPN). MPN pathogenesis is associated with the presence of somatic driver mutations, bone marrow (BM) niche alterations, and tumor inflammatory status. The relevance of soluble mediators in the pathogenesis of MPN led us to analyze the levels of cytokines, chemokines, and growth factors related to inflammation, angiogenesis and hematopoiesis regulation in the BM niche of MPN patients. METHODS: Soluble mediator levels in BM plasma samples from 17 healthy subjects, 28 ET, 19 PV, and 16 PMF patients were determined using a multiplex assay. Soluble mediator signatures were created from categorical analyses of high mediator producers. Soluble mediator connections and the correlation between plasma levels and clinic-laboratory parameters were also analyzed. RESULTS: The soluble mediator signatures of the BM niche of PV patients revealed a highly inflammatory and pro-angiogenic milieu, with increased levels of chemokines (CCL2, CCL5, CXCL8, CXCL12, CXCL10), and growth factors (GM-CSF M-CSF, HGF, IFN-γ, IL-1ß, IL-6Ra, IL-12, IL-17, IL-18, TNF-α, VEGF, and VEGF-R2). ET and PMF patients presented intermediate inflammatory and pro-angiogenic profiles. Deregulation of soluble mediators was associated with some clinic-laboratory parameters of MPN patients, including vascular events, treatment status, risk stratification of disease, hemoglobin concentration, hematocrit, and red blood cell count. CONCLUSIONS: Each MPN subtype exhibits a distinct soluble mediator signature. Deregulated production of BM soluble mediators may contribute to MPN pathogenesis and BM niche modification, provides pro-tumor stimuli, and is a potential target for future therapies.

Differential cytokine network profile in polycythemia vera and secondary polycythemia.

Polycythemia vera (PV) is a clonal disorder resulting from neoplastic transformation of hematopoietic stem cells, while secondary polycythemia (SP) is a disease characterized by increased absolute red blood cell mass caused by stimulation of red blood cell production. Although the physiopathology of SP and PV is distinct, patients with these diseases share similar symptoms. The early differential diagnosis may improve the quality of life and decrease the disease burden in PV patients, as well as enable curative treatment for SP patients. PV is considered an oncoinflammatory disease because PV patients exhibit augmented levels of several pro-inflammatory cytokines. In this sense, we examined whether analysis of the cytokine production profile of SP and PV patients would help to distinguish them, despite their clinical similarities. Here we reported that SP patients exhibited decreased plasma levels of, IL-17A, IFN-γ, IL-12p70 and TNF-α when compared with PV patients, suggesting that analysis of the cytokine production profile may be an useful diagnostic biomarker to distinguish PV from SP patients.

Epigenetic Silencing of CDR1as Drives IGF2BP3-Mediated Melanoma Invasion and Metastasis.

Metastasis is the primary cause of death of cancer patients. Dissecting mechanisms governing metastatic spread may uncover important tumor biology and/or yield promising therapeutic insights. Here, we investigated the role of circular RNAs (circRNA) in metastasis, using melanoma as a model aggressive tumor. We identified silencing of cerebellar degeneration-related 1 antisense (CDR1as), a regulator of miR-7, as a hallmark of melanoma progression. CDR1as depletion results from epigenetic silencing of LINC00632, its originating long non-coding RNA (lncRNA) and promotes invasion in vitro and metastasis in vivo through a miR-7-independent, IGF2BP3-mediated mechanism. Moreover, CDR1as levels reflect cellular states associated with distinct therapeutic responses. Our study reveals functional, prognostic, and predictive roles for CDR1as and expose circRNAs as key players in metastasis.

Expression of Hippo signaling pathway and Aurora kinase genes in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm resulting from clonal expansion of hematopoietic stem cells positive for the Philadelphia chromosome. The CML pathogenesis is associated with expression of the BCR-ABL1 oncogene, which encodes the Bcr-Abl protein with tyrosine kinase activity, promoting the leukemic cell exacerbated myeloproliferation and resistance to apoptosis. CML patients are usually treated with tyrosine kinase inhibitors (TKI), but some of them acquire resistance or are refractory to TKI. Thus, it is still relevant to elucidate the CML pathogenesis and seek new therapeutic targets, such as the Hippo signaling pathway and cell cycle regulatory genes from the Aurora kinase family. The present study quantified the expression level of genes encoding components of the Hippo signaling pathway (LATS1, LATS2, YAP, and TAZ), AURKA and AURKB in CML patients at different stages of the disease, who were resistant or sensitive to imatinib mesylate therapy, and in healthy individuals. The expression levels of the target genes were correlated with the CML Sokal's prognostic score. The most striking results were the LATS2 and AURKA overexpression in CML patients, the overexpression of TAZ and AURKB in CML patients at advanced phases and TAZ in CML IM-resistant. The development of drugs and/or identification of tumor markers for the Hippo signaling pathway and the Aurora kinase family, either alone or in combination, can optimize CML treatment by enhancing the susceptibility of leukemic cells to apoptosis and leading to a better disease prognosis.

Crosstalk between BCR-ABL and protease-activated receptor 1 (PAR1) suggests a novel target in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome, which generates the oncogene BCR-ABL1. Protease-activated receptor 1 (PAR1) is involved in tumor progression and angiogenesis. We have previously reported that PAR1 expression is elevated in human leukemias that display a more aggressive clinical behavior, including the blast crisis of CML. In this study, we analyzed the crosstalk between the oncoprotein BCR-ABL and PAR1 in CML. Leukemic cell lines transfected with the BCR-ABL1 oncogene showed significantly higher expression levels of PAR1 compared with that of wild-type counterparts. This phenomenon was reversed by treatment with tyrosine kinase inhibitors (TKIs). Conversely, treatment with the PAR1 antagonist SCH79797 inhibited BCR-ABL expression. The PAR1 antagonist induced apoptosis in a dose- and time-dependent manner. Higher vascular endothelial growth factor (VEGF) levels were observed in cells transfected with BCR-ABL1 than in their wild-type counterparts. VEGF expression was strongly inhibited after treatment with either TKIs or the PAR1 antagonist. Finally, we evaluated PAR1 expression in CML patients who were either in the blast or chronic phases and had either received TKI treatment or no treatment. A significant decrease in PAR1 expression was observed in treatment-responsive patients, as opposed to a significant increase in PAR1 expression levels in treatment-resistant patients. Patients classified as high risk according to the Sokal index showed higher PAR1 expression levels. Our results demonstrate the crosstalk between BCR-ABL and PAR1. These data may offer important insight into the development of new therapeutic strategies for CML.

Hippo pathway deregulation: implications in the pathogenesis of haematological malignancies.

The Hippo pathway participates in the regulation of cell proliferation, differentiation and apoptosis. It is composed by a large array of proteins whose deregulation has been associated with pro-oncogenic and antioncogenic processes. The present review focuses on the Hippo pathway signalling network and discusses its dual role in oncogenesis, particularly in haematological malignancies.

The L-amino acid oxidase from Calloselasma rhodostoma snake venom modulates apoptomiRs expression in Bcr-Abl-positive cell lines.

Anti-apoptotic genes and apoptomiRs deregulated expression contribute to apoptosis resistance in chronic myeloid leukemia (CML) Bcr-Abl(+) cells. Here, the L-amino acid oxidase from Calloselasma rhodostoma (CR-LAAO) venom altered the apoptotic machinery regulation by modulating the expression of the miR-145, miR-26a, miR-142-3p, miR-21, miR-130a, and miR-146a, and of the apoptosis-related proteins Bid, Bim, Bcl-2, Ciap-2, c-Flip, and Mcl-1 in Bcr-Abl(+) cells. CR-LAAO is a potential tool to instigate apoptomiRs regulation that contributes to drive CML therapy.