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Despite increased understanding of the genomic landscape of Myeloproliferative Neoplasms (MPNs), the pathological mechanisms underlying abnormal megakaryocyte (Mk)-stromal crosstalk and fibrotic progression in MPNs remain unclear. We conducted mass spectrometry-based proteomics on mice with Romiplostim-dependent myelofibrosis to reveal alterations in signaling pathways and protein changes in Mks, platelets, and bone marrow (BM) cells. The chemokine Platelet Factor 4 (PF4)/Cxcl4 was up-regulated in all proteomes and increased in plasma and BM fluids of fibrotic mice. High TPO concentrations sustained in vitro PF4 synthesis and secretion in cultured Mks, while Ruxolitinib restrains the abnormal PF4 expression in vivo. We discovered that PF4 is rapidly internalized by stromal cells through surface glycosaminoglycans (GAGs) to promote myofibroblast differentiation. Cxcl4 gene silencing in Mks mitigated the profibrotic phenotype of stromal cells in TPO-saturated co-culture conditions. Consistently, extensive stromal PF4 uptake and altered GAGs deposition were detected in Romiplostim-treated, JAK2 mice and BM biopsies of MPN patients. BM PF4 levels and Mk/platelet CXCL4 expression were elevated in patients, exclusively in overt fibrosis. Finally, pharmacological inhibition of GAGs ameliorated in vivo fibrosis in Romiplostim-treated mice. Thus, our findings highlight the critical role of PF4 in the fibrosis progression of MPNs and substantiate the potential therapeutic strategy of neutralizing PF4-GAGs interaction.

MinION and GridION X5 from Oxford Nanopore Technologies are devices for real-time DNA and RNA sequencing. On the one hand, MinION is the only real-time, low cost and portable sequencing device and, thanks to its unique properties, is becoming more and more popular among biologists; on the other, GridION X5, mainly for its costs, is less widespread but highly suitable for researchers with large sequencing projects. Despite the fact that Oxford Nanopore Technologies' devices have been increasingly used in the last few years, there is a lack of high-performing and user-friendly tools to handle the data outputted by both MinION and GridION X5 platforms. Here we present NanoR, a cross-platform R package designed with the purpose to simplify and improve nanopore data visualization. Indeed, NanoR is built on few functions but overcomes the capabilities of existing tools to extract meaningful informations from MinION sequencing data; in addition, as exclusive features, NanoR can deal with GridION X5 sequencing outputs and allows comparison of both MinION and GridION X5 sequencing data in one command. NanoR is released as free package for R at https://github.com/davidebolo1993/NanoR.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by an excessive production of pro‐inflammatory cytokines resulting in chronic inflammation and genomic instability. Besides the driver mutations in JAK2, MPL, and CALR genes, the deregulation of miRNA expression may also contribute to the pathogenesis of PMF. To this end, we recently reported the upregulation of miR‐382‐5p in PMF CD34+ cells. In order to unveil the mechanistic details of the role of miR‐382‐5p in pathogenesis of PMF, we performed gene expression profiling of CD34+ cells overexpressing miR‐382‐5p. Among the downregulated genes, we identified superoxide dismutase 2 (SOD2), which is a predicted target of miR‐382‐5p. Subsequently, we confirmed miR‐382‐5p/SOD2 interaction by luciferase assay and we showed that miR‐382‐5p overexpression in CD34+ cells causes the decrease in SOD2 activity leading to reactive oxygen species (ROS) accumulation and oxidative DNA damage. In addition, our data indicate that inhibition of miR‐382‐5p in PMF CD34+ cells restores SOD2 function, induces ROS disposal, and reduces DNA oxidation. Since the pro‐inflammatory cytokine transforming growth factor‐β1 (TGF‐β1) is a key player in PMF pathogenesis, we further investigated the effect of TGF‐β1 on ROS and miR‐382‐5p levels. Our data showed that TGF‐β1 treatment enhances miR‐382‐5p expression and reduces SOD2 activity leading to ROS accumulation. Finally, inhibition of TGF‐β1 signaling in PMF CD34+ cells by galunisertib significantly reduced miR‐382‐5p expression and ROS accumulation and restored SOD2 activity. As a whole, this study reports that TGF‐β1/miR‐382‐5p/SOD2 axis deregulation in PMF cells is linked to ROS overproduction that may contribute to enhanced oxidative stress and inflammation. Our results suggest that galunisertib may represent an effective drug reducing abnormal oxidative stress induced by TGF‐β1 in PMF patients. Database linking GEO: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE103464. TGF‐β1/miR‐382‐5p/SOD2 axis is involved in the pathogenesis of primary myelofibrosis (PMF). TGF‐β1/miR‐382‐5p/SOD2 axis deregulation induces reactive oxygen species (ROS) overproduction in PMF stem/progenitor cells. Increased levels of TGF‐β1 in plasma enhance miR‐382‐5p expression, which in turn leads to ROS accumulation by targeting SOD2. The TGF‐β‐receptor I kinase inhibitor galunisertib decreases ROS production by reducing the expression level of miR‐382‐5p and restoring SOD2 activity.

Somatic mutations of calreticulin (CALR) have been described in approximately 60–80% of JAK2 and MPL unmutated Essential Thrombocythemia and Primary Myelofibrosis patients. CALR is an endoplasmic reticulum (ER) chaperone responsible for proper protein folding and calcium retention. Recent data demonstrated that the TPO receptor (MPL) is essential for the development of CALR mutant-driven Myeloproliferative Neoplasms (MPNs). However, the precise mechanism of action of CALR mutants haven’t been fully unraveled. In this study, we showed that CALR mutants impair the ability to respond to the ER stress and reduce the activation of the pro-apoptotic pathway of the unfolded protein response (UPR). Moreover, our data demonstrated that CALR mutations induce increased sensitivity to oxidative stress, leading to increase oxidative DNA damage. We finally demonstrated that the downmodulation of OXR1 in CALR-mutated cells could be one of the molecular mechanisms responsible for the increased sensitivity to oxidative stress mediated by mutant CALR. Altogether, our data identify novel mechanisms collaborating with MPL activation in CALR-mediated cellular transformation. CALR mutants negatively impact on the capability of cells to respond to oxidative stress leading to genomic instability and on the ability to react to ER stress, causing resistance to UPR-induced apoptosis.

AbstractInternational collaborations over the years have produced a series of prognostic models for primary myelofibrosis (PMF), including the recently unveiled mutation-enhanced international prognostic scoring systems for transplant-age patients (MIPSS70 and MIPSS70-plus). In the current study, we considered the feasibility of a genetically inspired prognostic scoring system (GIPSS) that is exclusively based on genetic markers. Among 641 cytogenetically annotated patients with PMF and informative for previously recognized adverse mutations, multivariable analysis identified “VHR” karyotype, “unfavorable” karyotype, absence of type 1/like CALR mutation and presence of ASXL1, SRSF2, or U2AF1Q157 mutation, as inter-independent predictors of inferior survival; the respective HRs (95% CI) were 3.1 (2.1–4.3), 2.1 (1.6–2.7), 2.1 (1.6–2.9), 1.8 (1.5–2.3), 2.4 (1.9–3.2), and 2.4 (1.7–3.3). Based on HR-weighted risk points, a four-tiered GIPSS model was devised: low (zero points; n = 58), intermediate-1 (1 point; n = 260), intermediate-2 (2 points; n = 192), and high (≥3 points; n = 131); the respective median (5-year) survivals were 26.4 (94%), 8.0 (73%), 4.2 (40%), and 2 (14%) years; the model was internally validated by bootstrapping and its predictive accuracy was shown to be comparable to that of MIPSS70-plus. GIPPS offers a low-complexity prognostic tool for PMF that is solely dependent on genetic risk factors and, thus, forward-looking in its essence.

One of the key parameters of an artificial biosensor is a high signal‐to‐noise ratio. This is achieved by limiting non‐specific interactions while simultaneously maximizing the targeted specific interaction. Here, it is combined non‐fouling characteristics of poly(2‐methyl‐2‐oxazoline) (PMOXA) coatings with an abundance of azide groups to create a multi‐azide containing poly(2‐methyl‐2‐oxazoline‐co‐2‐(3‐azidopropyl)‐2‐oxazoline) (PMCA) that can participate in bioorthogonal strain‐promoted azide‐alkyne cycloaddition (SPAAC) for functionalization. This functional polymer is made surface‐active using the PAcrAm™ technology to obtain well‐defined spontaneously adsorbed monolayers on gold surfaces. The resistance to non‐specific interactions is tested against full human serum (HS), analyzed via variable angle spectroscopic ellipsometry (VASE), and compared to equivalent coatings based on PMOXA and azido‐poly(ethylene glycol) (PEG‐N3). The specific interactions are investigated via VASE and quartz crystal microbalance with dissipation (QCM‐D) by immobilization of dibenzocyclooctyne‐PEG4‐biotin conjugate (DBCO‐biotin) and streptavidin. The new PMCA‐based coating shows superior resistance to non‐specific protein adhesion than equivalent coatings based on commercially available PEG‐N3 and significantly increases capacity for SPAAC. A proof of principle assay (biotin‐streptavidin/biotin‐BSA/anti‐BSA) shows improved binding for the new PMCA polymer compared with single azide PEG. To improve the signal of current biosensors a novel multi‐azide containing polyoxazoline is created. This polymer can impart increased number of functionalization's to a surface via SPAAC that is superior to a mono‐functionalized polymer. At the same time, the surfaces significantly suppress the non‐specific adhesion of proteins.

Dysregulated signaling of the JAK/STAT pathway is a common feature of chronic myeloproliferative neoplasms (MPN), usually associated with JAK2V617F mutation. Recent clinical trials with JAK2 inhibitors showed significant improvements in splenomegaly and constitutional symptoms in patients with myelofibrosis but meaningful molecular responses were not documented. Accordingly, there remains a need for exploring new treatment strategies of MPN. A potential additional target for treatment is represented by the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway that has been found constitutively activated in MPN cells; proof-of-evidence of efficacy of the mTOR inhibitor RAD001 has been obtained recently in a Phase I/II trial in patients with myelofibrosis. The aim of the study was to characterize the effects in vitro of mTOR inhibitors, used alone and in combination with JAK2 inhibitors, against MPN cells. Mouse and human JAK2V617F mutated cell lines and primary hematopoietic progenitors from MPN patients were challenged with an allosteric (RAD001) and an ATP-competitive (PP242) mTOR inhibitor and two JAK2 inhibitors (AZD1480 and ruxolitinib). mTOR inhibitors effectively reduced proliferation and colony formation of cell lines through a slowed cell division mediated by changes in cell cycle transition to the S-phase. mTOR inhibitors also impaired the proliferation and prevented colony formation from MPN hematopoietic progenitors at doses significantly lower than healthy controls. JAK2 inhibitors produced similar antiproliferative effects in MPN cell lines and primary cells but were more potent inducers of apoptosis, as also supported by differential effects on cyclinD1, PIM1 and BcLxL expression levels. Co-treatment of mTOR inhibitor with JAK2 inhibitor resulted in synergistic activity against the proliferation of JAK2V617F mutated cell lines and significantly reduced erythropoietin-independent colony growth in patients with polycythemia vera. These findings support mTOR inhibitors as novel potential drugs for the treatment of MPN and advocate for clinical trials exploiting the combination of mTOR and JAK2 inhibitor.

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.

Extramedullary haematopoiesis (EMH) refers to blood generation outside of the bone marrow (BM). In Myelofibrosis (MF), a myeloproliferative neoplasm, the disruption of BM microenvironment promotes haematopoietic stem and progenitor cells (HSPCs) mobilisation, resulting in the onset of EMH in the spleen, and then in splenomegaly. Although JAK2 inhibitors have a good efficacy in reducing splenomegaly, the presence of a significant proportion of non‐responder patients underlines the need to explore the cellular mechanisms responsible for the EMH onset. In a MF mouse model, Ruxolitinib induces a reduction in spleen volume but does not affect EMH. CD44 inhibition successfully reduces monocyte and HSPC migration in an in vitro extravasation model. Strikingly, MF monocytes are more effective in promoting HSPC migration through the production of hyaluronic acid. Collectively, our results demonstrate that CD44 regulates the migration of monocytes that are crucial for the onset of EMH in MF patients, as they produce CD44 ligands recruiting HSPCs from the BM.

Cytogenetic and genomic profiling of acute myeloid leukemia (AML) guides personalized treatment according to ELN2022 recommendations. However, marked outcome variability persists among cytogenetically normal (CN-) patients, representing an unmet clinical need. We used long-read whole-genome sequencing to interrogate the prognostic significance of structural variations (SVs) in a prospective cohort of 162 intensively treated CN-AML patients. After stringent filtering, we identified 5 somatic SVs associated with shorter overall survival (OS) (HR:4.18, p < 0.001) and event-free survival (EFS) (HR:3.59, p < 0.001) in 13% of the patients. Results were validated in a real-world cohort of 149 CN-AML, using target assays. These high-risk SVs (HRVs) operationally defined a “very high-risk” category in the framework of ELN2022, overall resulting in more accurate OS prediction. HRVs were independent of most frequent mutations, particularly FLT3 ITD and NPM1 mut . Among the latter patients, HRVs independently predicted shorter OS (8.2 months versus not-reached; p < 0.001), EFS (3.5 versus 25.7 months; p < 0.001), and lower complete response rates (66.7% versus 90.1%; p < 0.005). Finally, we provided evidence of transcriptional deregulation of SV-related genes in primary samples and engineered cell models. Current findings support the value of SVs for refining risk stratification in CN-AML, by identifying patients at exceedingly dismal outcome who might benefit from personalized approaches.