Clonal dynamics and copy number variants by single-cell analysis in leukemic evolution of myeloproliferative neoplasms.

Transformation from chronic (CP) to blast phase (BP) in myeloproliferative neoplasm (MPN) remains poorly characterized, and no specific mutation pattern has been highlighted. BP-MPN represents an unmet need, due to its refractoriness to treatment and dismal outcome. Taking advantage of the granularity provided by single-cell sequencing (SCS), we analyzed paired samples of CP and BP in 10 patients to map clonal trajectories and interrogate target copy number variants (CNVs). Already at diagnosis, MPN present as oligoclonal diseases with varying ratio of mutated and wild-type cells, including cases where normal hematopoiesis was entirely surmised by mutated clones. BP originated from increasing clonal complexity, either on top or independent of a driver mutation, through acquisition of novel mutations as well as accumulation of clones harboring multiple mutations, that were detected at CP by SCS but were missed by bulk sequencing. There were progressive copy-number imbalances from CP to BP, that configured distinct clonal profiles and identified recurrences in genes including NF1, TET2, and BCOR, suggesting an additional level of complexity and contribution to leukemic transformation. EZH2 emerged as the gene most frequently affected by single nucleotide and CNVs, that might result in EZH2/PRC2-mediated transcriptional deregulation, as supported by combined scATAC-seq and snRNA-seq analysis of the leukemic clone in a representative case. Overall, findings provided insights into the pathogenesis of MPN-BP, identified CNVs as a hitherto poorly characterized mechanism and point to EZH2 dysregulation as target. Serial assessment of clonal dynamics might potentially allow early detection of impending disease transformation, with therapeutic implications.

Inhibition of ERK1/2 signaling prevents bone marrow fibrosis by reducing osteopontin plasma levels in a myelofibrosis mouse model.

Clonal myeloproliferation and development of bone marrow (BM) fibrosis are the major pathogenetic events in myelofibrosis (MF). The identification of novel antifibrotic strategies is of utmost importance since the effectiveness of current therapies in reverting BM fibrosis is debated. We previously demonstrated that osteopontin (OPN) has a profibrotic role in MF by promoting mesenchymal stromal cells proliferation and collagen production. Moreover, increased plasma OPN correlated with higher BM fibrosis grade and inferior overall survival in MF patients. To understand whether OPN is a druggable target in MF, we assessed putative inhibitors of OPN expression in vitro and identified ERK1/2 as a major regulator of OPN production. Increased OPN plasma levels were associated with BM fibrosis development in the Romiplostim-induced MF mouse model. Moreover, ERK1/2 inhibition led to a remarkable reduction of OPN production and BM fibrosis in Romiplostim-treated mice. Strikingly, the antifibrotic effect of ERK1/2 inhibition can be mainly ascribed to the reduced OPN production since it could be recapitulated through the administration of anti-OPN neutralizing antibody. Our results demonstrate that OPN is a novel druggable target in MF and pave the way to antifibrotic therapies based on the inhibition of ERK1/2-driven OPN production or the neutralization of OPN activity.

Novel Molecular Insights into Leukemic Evolution of Myeloproliferative Neoplasms: A Single Cell Perspective.

Myeloproliferative neoplasms (MPNs) are clonal disorders originated by the serial acquisition of somatic mutations in hematopoietic stem/progenitor cells. The major clinical entities are represented by polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), that are caused by driver mutations affecting JAK2, MPL or CALR. Disease progression is related to molecular and clonal evolution. PV and ET can progress to secondary myelofibrosis (sMF) but can also evolve to secondary acute myeloid leukemia (sAML). PMF is associated with the highest frequency of leukemic transformation, which represents the main cause of death. sAML is associated with a dismal prognosis and clinical features that differ from those of de novo AML. The molecular landscape distinguishes sAML from de novo AML, since the most frequent hits involve TP53, epigenetic regulators, spliceosome modulators or signal transduction genes. Single cell genomic studies provide novel and accurate information about clonal architecture and mutation acquisition order, allowing the reconstruction of clonal dynamics and molecular events that accompany leukemic transformation. In this review, we examine our current understanding of the genomic heterogeneity in MPNs and how it affects disease progression and leukemic transformation. We focus on molecular events elicited by somatic mutations acquisition and discuss the emerging findings coming from single cell studies.

The Response to Oxidative Damage Correlates with Driver Mutations and Clinical Outcome in Patients with Myelofibrosis.

Myelofibrosis (MF) is the Philadelphia-negative myeloproliferative neoplasm characterized by the worst prognosis and no response to conventional therapy. Driver mutations in JAK2 and CALR impact on JAK-STAT pathway activation but also on the production of reactive oxygen species (ROS). ROS play a pivotal role in inflammation-induced oxidative damage to cellular components including DNA, therefore leading to greater genomic instability and promoting cell transformation. In order to unveil the role of driver mutations in oxidative stress, we assessed ROS levels in CD34+ hematopoietic stem/progenitor cells of MF patients. Our results demonstrated that ROS production in CD34+ cells from CALR-mutated MF patients is far greater compared with patients harboring JAK2 mutation, and this leads to increased oxidative DNA damage. Moreover, CALR-mutant cells show less superoxide dismutase (SOD) antioxidant activity than JAK2-mutated ones. Here, we show that high plasma levels of total antioxidant capacity (TAC) correlate with detrimental clinical features, such as high levels of lactate dehydrogenase (LDH) and circulating CD34+ cells. Moreover, in JAK2-mutated patients, high plasma level of TAC is also associated with a poor overall survival (OS), and multivariate analysis demonstrated that high TAC classification is an independent prognostic factor allowing the identification of patients with inferior OS in both DIPSS lowest and highest categories. Altogether, our data suggest that a different capability to respond to oxidative stress can be one of the mechanisms underlying disease progression of myelofibrosis.

Magnesium favors the capacity of vitamin D3 to induce the monocyte differentiation of U937 cells.

The hematopoietic U937 cells are able to differentiate into monocytes, macrophages, or osteoclasts when stimulated, respectively, with vitamin D3 (VD3), phorbol 12-myristate 13-acetate (PMA) or PMA plus VD3. We have previously demonstrated that magnesium (Mg) strongly potentiates the osteoclastic differentiation of U937 cells. In this study, we investigated whether such an effect may be ascribed to a capacity of Mg to modulate the monocyte differentiation of U937 cells and/or to an ability of Mg and VD3 to act directly and independently on the early phases of the osteoclastic differentiation. To address this issue, we subjected U937 cells to an individual and combined treatment with Mg and VD3 and then we analyzed, by flow cytometry and quantitative real-time polymerase chain reaction, the expression of a number of genes related to the early phases of the differentiation pathways under consideration. The results obtained indicated that Mg favors the monocyte differentiation of U937 cells induced by VD3 and at the same time, Mg contrasts the inhibitory effect that VD3 exerts on the osteoclastic differentiation in the absence of PMA. The crucial and articulated role played by Mg in diverse pathways of the osteoclastic differentiation of U973 cells is emphasized.

Increased Plasma Levels of lncRNAs LINC01268, GAS5 and MALAT1 Correlate with Negative Prognostic Factors in Myelofibrosis.

Long non-coding RNAs (lncRNAs) have been recently described as key mediators in the development of hematological malignancies. In the last years, circulating lncRNAs have been proposed as a new class of non-invasive biomarkers for cancer diagnosis and prognosis and to predict treatment response. The present study is aimed to investigate the potential of circulating lncRNAs as non-invasive prognostic biomarkers in myelofibrosis (MF), the most severe among Philadelphia-negative myeloproliferative neoplasms. We detected increased levels of seven circulating lncRNAs in plasma samples of MF patients (n = 143), compared to healthy controls (n = 65). Among these, high levels of LINC01268, MALAT1 or GAS5 correlate with detrimental clinical variables, such as high count of leukocytes and CD34+ cells, severe grade of bone marrow fibrosis and presence of splenomegaly. Strikingly, high plasma levels of LINC01268 (p = 0.0018), GAS5 (p = 0.0008) or MALAT1 (p = 0.0348) are also associated with a poor overall-survival while high levels of LINC01268 correlate with a shorter leukemia-free-survival. Finally, multivariate analysis demonstrated that the plasma level of LINC01268 is an independent prognostic variable, suggesting that, if confirmed in future in an independent patients' cohort, it could be used for further studies to design an updated classification model for MF patients.

Gene expression profile correlates with molecular and clinical features in patients with myelofibrosis.

Myelofibrosis (MF) belongs to the family of classic Philadelphia-negative myeloproliferative neoplasms (MPNs). It can be primary myelofibrosis (PMF) or secondary myelofibrosis (SMF) evolving from polycythemia vera (PV) or essential thrombocythemia (ET). Despite the differences, PMF and SMF patients are currently managed in the same way, and prediction of survival is based on the same clinical and genetic features. In the last few years, interest has grown concerning the ability of gene expression profiles (GEPs) to provide valuable prognostic information. Here, we studied the GEPs of granulocytes from 114 patients with MF, using a microarray platform to identify correlations with patient characteristics and outcomes. Cox regression analysis led to the identification of 201 survival-related transcripts characterizing patients who are at high risk for death. High-risk patients identified by this gene signature displayed an inferior overall survival and leukemia-free survival, together with clinical and molecular detrimental features included in contemporary prognostic models, such as the presence of high molecular risk mutations. The high-risk group was enriched in post-PV and post-ET MF and JAK2V617F homozygous patients, whereas pre-PMF was more frequent in the low-risk group. These results demonstrate that GEPs in MF patients correlate with their molecular and clinical features, particularly their survival, and represent the proof of concept that GEPs might provide complementary prognostic information to be applied in clinical decision making.

Mutated clones driving leukemic transformation are already detectable at the single-cell level in CD34-positive cells in the chronic phase of primary myelofibrosis.

Disease progression of myeloproliferative neoplasms is the result of increased genomic complexity. Since the ability to predict disease evolution is crucial for clinical decisions, we studied single-cell genomics and transcriptomics of CD34-positive cells from a primary myelofibrosis (PMF) patient who progressed to acute myeloid leukemia (AML) while receiving Ruxolitinib. Single-cell genomics allowed the reconstruction of clonal hierarchy and demonstrated that TET2 was the first mutated gene while FLT3 was the last one. Disease evolution was accompanied by increased clonal heterogeneity and mutational rate, but clones carrying TP53 and FLT3 mutations were already present in the chronic phase. Single-cell transcriptomics unraveled repression of interferon signaling suggesting an immunosuppressive effect exerted by Ruxolitinib. Moreover, AML transformation was associated with a differentiative block and immune escape. These results suggest that single-cell analysis can unmask tumor heterogeneity and provide meaningful insights about PMF progression that might guide personalized therapy.

Characterization of New ATM Deletion Associated with Hereditary Breast Cancer.

Next-generation sequencing (NGS)-based cancer risk screening with multigene panels has become the most successful method for programming cancer prevention strategies. ATM germ-line heterozygosity has been described to increase tumor susceptibility. In particular, families carrying heterozygous germ-line variants of ATM gene have a 5- to 9-fold risk of developing breast cancer. Recent studies identified ATM as the second most mutated gene after CHEK2 in BRCA-negative patients. Nowadays, more than 170 missense variants and several truncating mutations have been identified in ATM gene. Here, we present the molecular characterization of a new ATM deletion, identified thanks to the CNV algorithm implemented in the NGS analysis pipeline. An automated workflow implementing the SOPHiA Genetics' Hereditary Cancer Solution (HCS) protocol was used to generate NGS libraries that were sequenced on Illumina MiSeq Platform. NGS data analysis allowed us to identify a new inactivating deletion of exons 19-27 of ATM gene. The deletion was characterized both at the DNA and RNA level.

Genomic Analysis of Hematopoietic Stem Cell at the Single-Cell Level: Optimization of Cell Fixation and Whole Genome Amplification (WGA) Protocol.

Single-cell genomics has become the method of choice for the study of heterogeneous cell populations and represents an elective application in defining the architecture and clonal evolution in hematological neoplasms. Reconstructing the clonal evolution of a neoplastic population therefore represents the main way to understand more deeply the pathogenesis of the neoplasm, but it is also a potential tool to understand the evolution of the tumor population with respect to its response to therapy. Pre-analytical phase for single-cell genomics analysis is crucial to obtain a cell population suitable for single-cell sorting, and whole genome amplification is required to obtain the necessary amount of DNA from a single cell in order to proceed with sequencing. Here, we evaluated the impact of different methods of cellular immunostaining, fixation and whole genome amplification on the efficiency and yield of single-cell sequencing.

Promoter Methylation Leads to Decreased ZFP36 Expression and Deregulated NLRP3 Inflammasome Activation in Psoriatic Fibroblasts.

The mRNA-destabilizing protein tristetraprolin (TTP), encoded by the ZFP36 gene, is known to be able to end inflammatory responses by directly targeting and destabilizing mRNAs encoding pro-inflammatory cytokines. We analyzed its role in psoriasis, a disease characterized by chronic inflammation. We observed that TTP is downregulated in fibroblasts deriving from psoriasis patients compared to those deriving from healthy individuals and that psoriatic fibroblasts exhibit abnormal inflammasome activity compared to their physiological counterpart. This phenomenon depends on TTP downregulation. In fact, following restoration, TTP is capable of directly targeting for degradation NLRP3 mRNA, thereby drastically decreasing inflammasome activation. Moreover, we provide evidence that ZFP36 undergoes methylation in psoriasis, by virtue of the presence of long stretches of CpG dinucleotides both in the promoter and the coding region. Besides confirming that a perturbation of TTP expression might underlie the pathogenesis of psoriasis, we suggest that deregulated inflammasome activity might play a role in the disease alongside deregulated cytokine expression.

Physiological expression of miR-130a during differentiation of CD34+ human hematopoietic stem cells results in the inhibition of monocyte differentiation.

MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression by targeting mRNAs in a sequence specific manner, thereby determining their degradation or inhibiting translation. They are involved in processes such as proliferation, differentiation and apoptosis by fine-tuning the expression of genes underlying such events. The expression of specific miRNAs is involved in hematopoietic differentiation and their deregulation contributes to the development of hematopoietic malignancies such as acute myeloid leukemia (AML). miR-130a is over-expressed in AML. Here we show that miR-130a is physiologically expressed in myeloblasts and down-regulated during monocyte differentiation. Gain- and loss-of-function experiments performed on CD34+ human hematopoietic stem cells confirmed that expression of miR-130a inhibits monocyte differentiation by interfering with the expression of key transcription factors HOXA10, IRF8, KLF4, MAFB and PU-1. The data obtained in this study highlight that the correct modulation of miR-130a is necessary for normal differentiation to occur and confirming that deregulation of this miRNA might underlie the differentiation block occurring in AML.

Gene expression profiles of human granulosa cells treated with bioequivalent doses of corifollitropin alfa (CFA) or recombinant human follicle-stimulating hormone (recFSH).

Using recombinant DNA technologies, a chimeric gene containing the coding sequences of follicle stimulating hormone (FSH) ß-subunit and C-terminal peptide of the human chorionic gonadotrophin (hCG) ß-subunit have been designed to generate a new gonadotrophin named corifollitropin alfa (CFA). CFA has longer elimination half-life and slower rate of absorption compared with FSH, which makes CFA a long-acting hormone employed as a substitute of the recombinant FSH (recFSH) in the controlled ovarian stimulation (COS). The purpose of this study is to compare the gene expression profiles elicited by bioequivalent doses of CFA or recFSH in primary cultures of human granulosa cells (hGCs). Gonadotrophins exert their functions by binding FSH receptors (FSHRs), activating signaling pathways that increase the cyclic adenosine monophosphate (cAMP) intracellular content. Bioequivalence has been defined as the dose/duration of gonadotrophin treatment able to promote the same amount of intracellular cAMP. hGCs were treated with different doses of either gonadotrophin and the cAMP was measured after different incubation times to establish the bioequivalence. Results obtained by comparing the bioequivalent treatments, showed that CFA is more effective than recFSH in inducing aromatase gene expression after 6 and 24 h from the initial stimulation in agreement with its long-acting characteristic.

Magnesium Is a Key Regulator of the Balance between Osteoclast and Osteoblast Differentiation in the Presence of Vitamin D3.

Magnesium (Mg) is crucial for bone health. Low concentrations of Mg inhibit the activity of osteoblasts while promoting that of osteoclasts, with the final result of inducing osteopenia. Conversely, little is known about the effects of high concentrations of extracellular Mg on osteoclasts and osteoblasts. Since the differentiation and activation of these cells is coordinated by vitamin D3 (VD3), we investigated the effects of high extracellular Mg, as well as its impact on VD3 activity, in these cells. U937 cells were induced to osteoclastic differentiation by VD3 in the presence of supra-physiological concentrations (>1 mM) of extracellular Mg. The effect of high Mg concentrations was also studied in human bone-marrow-derived mesenchymal stem cells (bMSCs) induced to differentiate into osteoblasts by VD3. We demonstrate that high extra-cellular Mg levels potentiate VD3-induced osteoclastic differentiation, while decreasing osteoblastogenesis. We hypothesize that Mg might reprogram VD3 activity on bone remodeling, causing an unbalanced activation of osteoclasts and osteoblasts.

Loss of expression of µ-protocadherin and protocadherin-24 in sporadic and hereditary nonpolyposis colorectal cancers.

Colorectal cancer (CRC) is a neoplastic disease in which normal mucosa undergoes a process of malignant transformation due to the progressive accumulation of molecular alterations affecting proto-oncogenes and oncosuppressor genes. Some of these modifications exert their carcinogenic potential by promoting a constitutive activation of the ß-catenin signaling proliferation pathway, and when present, loss of cadherin expression also significantly contributes to the same effect. Using a combined approach of molecular and immunohistochemical analysis, we have previously demonstrated that most sporadic CRCs exhibit a down-regulated expression of a cadherin, named µ-protocadherin, that is generally observed in association with a higher proliferation rate and a worse prognosis. The aim of this report was to perform a comparative immunohistochemical assessment of µ-protocadherin and a similar cadherin, named protocadherin-24, in sporadic CRC and hereditary nonpolyposis colorectal cancer. The data obtained put in evidence that double-negative CRCs, lacking both the analyzed protocadherins, are more represented among sporadic tumors, whereas double-positive CRCs, maintaining their expression, exhibit an opposite trend. As expected, loss of protocadherin expression was accompanied by nuclear localization of ß-catenin and increased positivity of the Ki-67 proliferation marker. This finding is consistent with the different clinical evolution of the 2 considered CRC sets according to which patients with hereditary nonpolyposis colorectal cancer experience a better prognosis as compared with those affected by a sporadic CRC.

Genomic alterations at the basis of treatment resistance in metastatic breast cancer: clinical applications.

The standard of care for breast cancer has gradually evolved from empirical treatments based on clinical-pathological characteristics to the use of targeted approaches based on the molecular profile of the tumor. Consequently, an increasing number of molecularly targeted drugs have been developed. These drugs target specific alterations, called driver mutations, which confer a survival advantage to cancer cells. To date, the main challenge remains the identification of predictive biomarkers for the selection of the optimal treatment. On this basis, we evaluated a panel of 25 genes involved in the mechanisms of targeted treatment resistance, in 16 primary breast cancers and their matched recurrences, developed during treatment. Overall, we found a detection rate of mutations higher than that described in the literature. In particular, the most frequently mutated genes were ERBB2 and those involved in the PI3K/AKT/mTOR and the MAPK signaling pathways. The study revealed substantial discordances between primary tumors and metastases, stressing the need for analysis of metastatic tissues at recurrence. We observed that 85.7% of patients with an early-stage or locally advanced primary tumor showed at least one mutation in the primary tumor. This finding could explain the subsequent relapse and might therefore justify more targeted adjuvant treatments. Finally, the mutations detected in 50% of relapsed tissues could have guided subsequent treatment choices in a different way. This study demonstrates that mutation events may be present at diagnosis or arise during cancer treatment. As a result, profiling primary and metastatic tumor tissues may be a major step in defining optimal treatments.

KLF4 Mediates the Effect of 5-ASA on the ß-Catenin Pathway in Colon Cancer Cells.

Mesalazine (5-ASA) is an aminosalicylate anti-inflammatory drug capable of inducing µ-protocadherin, a protein expressed by colorectal epithelial cells that is downregulated upon malignant transformation. Treatment with 5-ASA restores µ-protocadherin expression and promotes the sequestration of ß-catenin to the plasma membrane. Here, we show that 5-ASA-induced µ-protocadherin expression is directly regulated by the KLF4 transcription factor. In addition, we suggest the existence of a dual mechanism whereby 5-ASA-mediated ß-catenin inhibition is caused by µ-protocadherin-dependent sequestration of ß-catenin to the plasma membrane and by the direct binding of KLF4 to ß-catenin. In addition, we found that 5-ASA treatment suppresses the expression of miR-130a and miR-135b, which target KLF4 mRNA, raising the possibility that this mechanism is involved in the increased expression of KLF4 induced by 5-ASA. Cancer Prev Res; 11(8); 503-10. ©2018 AACR.

Loss of ZFP36 expression in colorectal cancer correlates to wnt/ ß-catenin activity and enhances epithelial-to-mesenchymal transition through upregulation of ZEB1, SOX9 and MACC1.

The mRNA-destabilizing protein ZFP36 has been previously described as a tumor suppressor whose expression is lost during colorectal cancer development. In order to evaluate its role in this disease, we restored ZFP36 expression in different cell contexts, showing that the presence of this protein impairs the epithelial-to-mesenchymal transition (EMT) and induces a higher susceptibility to anoikis. Consistently, we found that ZFP36 inhibits the expression of three key transcription factors involved in EMT: ZEB1, MACC1 and SOX9. Finally, we observed for the first time that its expression negatively correlates with the activity of Wnt/ß-catenin pathway, which is constitutively activated in colorectal cancer. This evidence provides a clue on the mechanism leading to the loss of ZFP36 in CRC.

Monocyte-macrophage differentiation of acute myeloid leukemia cell lines by small molecules identified through interrogation of the Connectivity Map database.

The transcription factor C/EBPα is required for granulocytic differentiation of normal myeloid progenitors and is frequently inactivated in acute myeloid leukemia (AML) cells. Ectopic expression of C/EBPα in AML cells suppresses proliferation and induces differentiation suggesting that restoring C/EBPα expression/activity in AML cells could be therapeutically useful. Unfortunately, current approaches of gene or protein delivery in leukemic cells are unsatisfactory. However, "drug repurposing" is becoming a very attractive strategy to identify potential new uses for existing drugs. In this study, we assessed the biological effects of candidate C/EBPα-mimetics identified by interrogation of the Connectivity Map database. We found that amantadine, an antiviral and anti-Parkinson agent, induced a monocyte-macrophage-like differentiation of HL60, U937, Kasumi-1 myeloid leukemia cell lines, as indicated by morphology and differentiation antigen expression, when used in combination with suboptimal concentration of all trans retinoic acid (ATRA) or Vit D3. The effect of amantadine depends, in part, on increased activity of the vitamin D receptor (VDR), since it induced VDR expression and amantadine-dependent monocyte-macrophage differentiation of HL60 cells was blocked by expression of dominant-negative VDR. These results reveal a new function for amantadine and support the concept that screening of the Connectivity Map database can identify small molecules that mimic the effect of transcription factors required for myelo-monocytic differentiation.

ZFP36 stabilizes RIP1 via degradation of XIAP and cIAP2 thereby promoting ripoptosome assembly.

BACKGROUND: ZFP36 is an mRNA binding protein that exerts anti-tumor activity in glioblastoma by triggering cell death, associated to an increase in the stability of the kinase RIP1. METHODS: We used cell death assays, size exclusion chromatography, Co-Immunoprecipitation, shRNA lentivectors and glioma neural stem cells to determine the effects of ZFP36 on the assembly of a death complex containing RIP1 and on the induction of necroptosis. RESULTS: Here we demonstrate that ZFP36 promotes the assembly of the death complex called Ripoptosome and induces RIP1-dependent death. This involves the depletion of the ubiquitine ligases cIAP2 and XIAP and leads to the association of RIP1 to caspase-8 and FADD. Moreover, we show that ZFP36 controls RIP1 levels in glioma neural stem cell lines. CONCLUSIONS: We provide a molecular mechanism for the tumor suppressor role of ZFP36, and the first evidence for Ripoptosome assembly following ZFP36 expression. These findings suggest that ZFP36 plays an important role in RIP1-dependent cell death in conditions where IAPs are depleted.