PRE-CLINICAL EFFICACY OF A NOVEL COMBINED PHARMACOLOGICAL APPROACH TARGETING JAK AND ERK IN A MYELOFIBROSIS MOUSE MODEL Matteo Bertesi Haematologica, 2026 Introduction: Myelofibrosis (MF) is the most severe form of Philadelphia–negative myeloproliferative neoplasms (MPNs), characterized by high risk of leukemic transformation and reduced survival. Neoplastic transformation is linked to somatic mutations in JAK/STAT pathway arising in the hematopoietic stem cell compartment, while MF pathology is defined by clonal myeloproliferation, extramedullary hematopoiesis with splenomegaly, chronic inflammation and extensive bone marrow fibrosis. Currently, the molecular basis of abnormal cytokine production and the altered interaction between malignant hematopoietic cells and the bone marrow microenvironment in MF pathogenesis remains unclear, and JAK1/2 inhibitors, while effective in reducing splenomegaly and inflammation-related symptoms, fail to address BM fibrosis and disease burden in most patients. A key mediator in bone marrow fibrosis development is Osteopontin (OPN), a pro-fibrotic molecule induced by MAPK signalling and JAK/STAT pathway hyperactivation driving MF pathogenesis. This study aimed at identifying a rapidly translatable and effective therapeutic strategy to counteract OPN production and provide a new potential druggable approach to restore bone marrow microenvironment in MF patients. Methods: With the aim of defining a pharmacological strategy to restore bone marrow microenvironment in MF, we selected Cobimetinib - a MEK1 inhibitor of MAPK pathway already approved for clinical use - to test a combined treatment against OPN overproduction and JAK2 hyperactivation. Thrombopoietin receptor agonist-treated mice developing thrombocytosis, bone marrow fibrosis and splenomegaly were used to test a dual JAK/ERK inhibition strategy comprising Ruxolitinib and Cobimetinib, and its efficacy was evaluated in terms of symptoms improvement.Results: The efficacy study in this murine model of myeloproliferation showed the ability of Cobimetinib monotherapy to reduce OPN plasma levels and to mitigate fibrosis to a greater extent than Ruxolitinib treatment alone. Strikingly, combined therapy Cobimetinib plus Ruxolitinib showed a synergistic effect in reducing MF-associated symptoms. Specifically, combined treatment has demonstrated greater efficacy in reducing spleen index if compared to Ruxolitinib treatment alone and in reducing bone marrow fibrosis when compared to Cobimetinib monotherapy. Conclusions: These data demonstrate the translational relevance of the combined treatment of Cobimetinib and Ruxolitinib in counteracting MF hallmark of pathology in a preclinical model of myelofibrosis, defining a rationale for the development of novel combinatory therapy able to target both clonal myeloproliferation and fibrotic microenvironment in MF patients.
SINGLE-CELL MULTI-OMICS UNRAVELS MOLECULAR AND CELLULAR PROGRAMS UNDERLYING RUXOLITINIB RESPONSE IN MYELOFIBROSIS Chiara Carretta Haematologica, 2026 Introduction: Myelofibrosis (MF) is a clonal hematological disease originating by the sequential acquisition of somatic mutations in hematopoietic stem and progenitor cells (HSPC). Alongside so-called “driver mutations” triggering the constitutive activation of JAK-STAT pathway and inducing myeloproliferation, MF may present with additional mutations often affecting epigenetic regulators. The JAK-inhibitor Ruxolitinib (Ruxo) effectively relieves MF symptoms but rarely eradicates the malignant clone, with highly variable responses among patients. Understanding the molecular mechanisms underlying Ruxo heterogeneous efficacy is essential to improve therapeutic strategies.Methods: To elucidate the clonal dynamics associated with Ruxo response, we conducted a longitudinal single-cell (SC) proteogenomic study on 12 MF patients (6 responders and 6 non-responders, as defined by clinical features) at diagnosis and after at least 6 months of Ruxo therapy. Cryopreserved peripheral blood mononuclear cells and CD34+ HSPC from each time point were analyzed through Tapestri platform. Next, longitudinal samples from a Responder and a Non-responder patient form the same cohort were subjected to SC-RNA+protein analysis by means of 10X genomics platform.Results: Ruxo responders showed a marked reduction in circulating CD34+ cells and a decrease in JAK2V617F allele frequency in granulocytes. SC-genomic analysis revealed that the mutation acquisition order determines Ruxo sensitivity: patients in whom the driver mutation either occurred alone or preceded mutations in TET2 or ASXL1 generally responded to therapy, while those in whom epigenetic mutations arose first exhibited limited therapeutic benefit. Using SC-proteomics we identified 14 HSPC and differentiated cell clusters and differences in the clonal dynamics of CD34+ and CD34- cells were observed. Non-responders displayed post-treatment expansion of highly mutated monocytes, suggesting myeloid lineage–driven resistance, whereas responders exhibited an increase in wild-type monocytes. Conversely, in CD34+ cells from responders, we observed both a reduction in driver-homozygous cells and an expansion of heterozygous co-mutated clones despite Ruxo treatment. Notably, despite MF being a primarily myeloid disorder, a fraction of mutated T and B cells was also detected, particularly in non-responders. SC-RNA+protein profiling recapitulated the cellular heterogeneity observed in the SC-proteogenomic dataset, revealing persistent activation of JAK-mediated interferon-response signaling in non-responder monocytes upon treatment, while this pathway was suppressed in responders.Conclusions: Our data demonstrate that the order of mutation acquisition impacts Ruxo response in MF patients. Ruxo primarily affects JAK2-only mutated clones. As a result, co-mutated clones may evade this treatment and outcompete other neoplastic cell populations, thus contributing to disease persistence.
CD9 DEFINES THE DISEASE-PROPAGATING HEMATOPOIETIC STEM CELL POPULATION IN MYELOFIBROSIS Ruggiero Norfo Haematologica, 2026 Introduction. Myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders, among which myelofibrosis (MF) represents the most severe form. MF is marked by progressive bone marrow fibrosis, worsening cytopenia, and a median overall survival of approximately five years. The disease originates from the expansion of a single hematopoietic stem and progenitor cell (HSPC) that has acquired somatic driver mutations in JAK2, CALR, or MPL, followed by additional cooperating mutations that promote clonal dominance over wild-type HSPCs. Although current therapies, including JAK inhibitors, provide symptomatic benefit, they fail to eradicate the malignant HSPC pool or induce molecular remission. Therefore, identifying and characterizing the stem cell population that sustains the MF clone is essential for developing curative strategies.Methods. HSPCs were purified from peripheral blood samples, collected from MF patients with JAK2, CALR, or MPL driver mutations and healthy donors (HDs). We performed RNA sequencing on purified HSPCs from 13 MF samples and 5 HDs, focusing on surface molecules with higher expression in MF. Candidate markers were then assessed by flow cytometry in an expanded cohort (30 MF, 15 HDs).To explore whether CD9 expression identified the neoplastic stem cell population, we performed single-cell mutational profiling combined with surface protein analysis using the Tapestri platform (Mission Bio) in 8 MF patients. CD9+ and CD9- HSCs from both MF and HD samples were compared in clonogenic and differentiation assays in vitro. Finally, the disease-propagating potential of each population was tested through patient-derived xenografts, generated by transplanting sorted cells into sub-lethally irradiated NSGS mice.Results. Transcriptomic profiling highlighted CD9 as one of the most upregulated surface molecules in MF HSPCs. Flow cytometry confirmed a clear expansion of the CD9+ HSPC compartment in patient samples. Single-cell proteogenomics data then revealed that the majority of mutated HSPCs clustered within the CD9+ fraction, supporting CD9 expression as a phenotypic marker of the malignant stem cell population.In functional assays performed in vitro, MF CD9+ HSPCs exhibited a more primitive phenotype and enhanced clonogenic potential unlike healthy and MF CD9- HSPCs. Our in vivo data further indicate that CD9+ MF HSPCs displayed higher engraftment levels relative to their CD9- counterparts, supporting their role as key disease-propagating cells.Conclusions. Across MF patients with distinct driver mutations, CD9 marks an expanded, mutation-enriched HSPC population with enhanced clonogenic activity and disease-propagating potential. These findings support CD9 as a promising surface marker of MF HSPCs, with potential implications for future therapeutic targeting aimed at eradicating the malignant clone.
Combined MEK and JAK inhibition reduces osteopontin plasma level and bone marrow fibrosis in a myelofibrosis mouse model Lara Tavernari, Matteo Bertesi, Anita Neroni, Elisa Papa, Sebastiano Rontauroli, Elisa Bianchi, Margherita Mirabile, Ruggiero Norfo, Sandra Parenti, Camilla Tombari, Chiara Carretta, Marica Malerba, Luca Fabbiani, Paola Guglielmelli, Lorena Losi, Enrico Tagliafico, Alessandro Maria Vannucchi, Rossella Manfredini, and Blood Cancer Journal, 2025 Myelofibrosis (MF), either primary or secondary, is the most aggressive among Philadelphia-negative myeloproliferative neoplasms (MPNs), since it is characterized by an increased risk of leukemic progression and an inferior life expectancy [ 1 ]. Key features of MF include clonal myeloproliferation, extramedullary hematopoiesis, extensive bone marrow (BM) fibrosis deposition, and chronic inflammation [ 2 ]. MF progression is primarily driven by neoplastic clone hyperproliferation and the excessive production of proinflammatory and profibrotic mediators [ 3 ]. While considerable progress has been made in identifying the genomic alterations responsible for clonal myeloproliferation, particularly driver mutations in JAK2 , MPL , and CALR that activate the JAK/STAT pathway [ 4 ], the molecular mechanisms underlying the abnormal mediator production and the disrupted interaction between malignant hematopoietic cells and the BM microenvironment are still not fully understood. Currently, with the exception of hematopoietic stem cell transplantation, no medical or pharmacologic treatment cures MF. Targeted JAK1/2 inhibitors, while effective in reducing splenomegaly and inflammation-related symptoms, fail to address BM fibrosis and disease burden in most patients [ 5 , 6 ]. Therefore, the development of novel therapeutic strategies aimed at restoring BM architecture and targeting both clonal myeloproliferation and fibrosis remains a critical unmet need in MF management.
CD44 Participates to Extramedullary Haematopoiesis Onset by Mediating the Interplay Between Monocytes and Haematopoietic Stem Cells in Myelofibrosis Margherita Mirabile, Camilla Tombari, Anita Neroni, Lara Tavernari, Ruggiero Norfo, et al. Journal of Cellular and Molecular Medicine, 2025 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.
Targeting exhausted cytotoxic T cells through CTLA-4 inhibition promotes elimination of neoplastic cells in human myelofibrosis xenografts Lara Tavernari, Sebastiano Rontauroli, Ruggiero Norfo, Margherita Mirabile, Monica Maccaferri, Barbara Mora, Elena Genovese, Sandra Parenti, Chiara Carretta, Elisa Bianchi, Matteo Bertesi, Francesca Pedrazzi, Elena Tenedini, Silvia Martinelli, Maria Teresa Bochicchio, Paola Guglielmelli, Leonardo Potenza, Alessandro Lucchesi, Francesco Passamonti, Enrico Tagliafico, Mario Luppi, Alessandro Maria Vannucchi, Rossella Manfredini, and American Journal of Hematology, 2024 Myeloproliferative neoplasms represent a group of clonal hematopoietic disorders of which myelofibrosis (MF) is the most aggressive. In the context of myeloid neoplasms, there is a growing recognition of the dysregulation of immune response and T‐cell function as significant contributors to disease progression and immune evasion. We investigated cytotoxic T‐cell exhaustion in MF to restore immune response against malignant cells. Increased expression of inhibitory receptors like CTLA‐4 was observed on cytotoxic T cells from MF patients together with a reduced secretion of IFNɣ and TNFɑ. CTLA‐4 ligands CD80 and CD86 were increased on MF granulocytes and monocytes highlighting a possible role for myeloid cells in suppressing T‐cell activation in MF patients. Unlike healthy donors, the activation of cytotoxic T cells from MF patients was attenuated in the presence of myeloid cells and restored when T cells were cultured alone or treated with anti‐CTLA‐4. Moreover, anti‐CTLA‐4 treatment promoted elimination of neoplastic monocytes and granulocytes in a co‐culture system with cytotoxic T cells. To test CTLA‐4 inhibition in vivo, patient‐derived xenografts were generated by transplanting MF CD34+ cells and by infusing homologous T cells in NSGS mice. CTLA‐4 blockade reduced human myeloid chimerism and led to T‐cell expansion in spleen and bone marrow. Overall, these findings shed light on T‐cell dysfunction in MF and suggest that CTLA‐4 blockade can boost the cytotoxic T cell‐mediated immune response against tumor cells.
Chromosome 9p trisomy increases stem cells clonogenic potential and fosters T-cell exhaustion in JAK2-mutant myeloproliferative neoplasms Chiara Carretta, Sandra Parenti, Matteo Bertesi, Sebastiano Rontauroli, Filippo Badii, Lara Tavernari, Elena Genovese, Marica Malerba, Elisa Papa, Samantha Sperduti, Elena Enzo, Margherita Mirabile, Francesca Pedrazzi, Anita Neroni, Camilla Tombari, Barbara Mora, Margherita Maffioli, Marco Mondini, Marco Brociner, Monica Maccaferri, Elena Tenedini, Silvia Martinelli, Niccolò Bartalucci, Elisa Bianchi, Livio Casarini, Leonardo Potenza, Mario Luppi, Enrico Tagliafico, Paola Guglielmelli, Manuela Simoni, Francesco Passamonti, Ruggiero Norfo, Alessandro Maria Vannucchi, Rossella Manfredini, and Leukemia, 2024 JAK2V617F is the most recurrent genetic mutation in Philadelphia-negative chronic Myeloproliferative Neoplasms (MPNs). Since the JAK2 locus is located on Chromosome 9, we hypothesized that Chromosome 9 copy number abnormalities may be a disease modifier in JAK2V617F-mutant MPN patients. In this study, we identified a subset of MPN patients with partial or complete Chromosome 9 trisomy (+9p patients), who differ from JAK2V617F-homozygous MPN patients as they carry three JAK2 alleles as well as three copies of all neighboring gene loci, including CD274, encoding immunosuppressive Programmed death-ligand 1 (PD-L1) protein. Investigation of the clonal hierarchy revealed that the JAK2V617F occurs first, followed by +9p. Functionally, CD34+ cells from +9p MPN patients demonstrated increased clonogenicity, generating a greater number of primitive colonies, due to high OCT4 and NANOG expression, with knock-down of these genes leading to a genotype-specific decrease in colony numbers. Moreover, our analysis revealed increased PD-L1 surface expression in malignant monocytes from +9p patients, while analysis of the T cell compartment unveiled elevated levels of exhausted cytotoxic T cells. Overall, here we identify a distinct novel subgroup of MPN patients, who feature a synergistic interplay between +9p and JAK2V617F that shapes immune escape characteristics and increased stemness in CD34+ cells.
Clonal dynamics and copy number variants by single-cell analysis in leukemic evolution of myeloproliferative neoplasms Laura Calabresi, Chiara Carretta, Simone Romagnoli, Giada Rotunno, Sandra Parenti, Matteo Bertesi, Niccolò Bartalucci, Sebastiano Rontauroli, Chiara Chiereghin, Sara Castellano, Giulia Gentili, Chiara Maccari, Fiorenza Vanderwert, Francesco Mannelli, Matteo Della Porta, Rossella Manfredini, Alessandro Maria Vannucchi, Paola Guglielmelli American Journal of Hematology, 2023 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 Elisa Bianchi, Sebastiano Rontauroli, Lara Tavernari, Margherita Mirabile, Francesca Pedrazzi, Elena Genovese, Stefano Sartini, Massimiliano Dall’Ora, Giulia Grisendi, Luca Fabbiani, Monica Maccaferri, Chiara Carretta, Sandra Parenti, Sebastian Fantini, Niccolò Bartalucci, Laura Calabresi, Manjola Balliu, Paola Guglielmelli, Leonardo Potenza, Enrico Tagliafico, Lorena Losi, Massimo Dominici, Mario Luppi, Alessandro Maria Vannucchi, Rossella Manfredini Leukemia, 2023 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 Sebastiano Rontauroli, Chiara Carretta, Sandra Parenti, Matteo Bertesi, Rossella Manfredini International Journal of Molecular Sciences, 2022 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 Elena Genovese, Margherita Mirabile, Sebastiano Rontauroli, Stefano Sartini, Sebastian Fantini, Lara Tavernari, Monica Maccaferri, Paola Guglielmelli, Elisa Bianchi, Sandra Parenti, Chiara Carretta, Selene Mallia, Sara Castellano, Corrado Colasante, Manjola Balliu, Niccolò Bartalucci, Raffaele Palmieri, Tiziana Ottone, Barbara Mora, Leonardo Potenza, Francesco Passamonti, Maria Teresa Voso, Mario Luppi, Alessandro Maria Vannucchi, Enrico Tagliafico, Rossella Manfredini, and Antioxidants, 2022
Gene expression profile correlates with molecular and clinical features in patients with myelofibrosis Sebastiano Rontauroli, Sara Castellano, Paola Guglielmelli, Roberta Zini, Elisa Bianchi, Elena Genovese, Chiara Carretta, Sandra Parenti, Sebastian Fantini, Selene Mallia, Lara Tavernari, Stefano Sartini, Margherita Mirabile, Carmela Mannarelli, Francesca Gesullo, Annalisa Pacilli, Daniela Pietra, Elisa Rumi, Silvia Salmoiraghi, Barbara Mora, Laura Villani, Andrea Grilli, Vittorio Rosti, Giovanni Barosi, Francesco Passamonti, Alessandro Rambaldi, Luca Malcovati, Mario Cazzola, Silvio Bicciato, Enrico Tagliafico, Alessandro M. Vannucchi, Rossella Manfredini Blood Advances, 2021
Characterization of new ATM deletion associated with hereditary breast cancer Sandra Parenti, Claudio Rabacchi, Marco Marino, Elena Tenedini, Lucia Artuso, Sara Castellano, Chiara Carretta, Selene Mallia, Laura Cortesi, Angela Toss, Elena Barbieri, Rossella Manfredini, Mario Luppi, Tommaso Trenti, Enrico Tagliafico Genes, 2021
KLF4 mediates the effect of 5-ASA on the b-catenin pathway in colon cancer cells Sandra Parenti, Lucia Montorsi, Sebastian Fantini, Fabiana Mammoli, Claudia Gemelli, Claudio Giacinto Atene, Lorena Losi, Chiara Frassineti, Bruno Calabretta, Enrico Tagliafico, Sergio Ferrari, Tommaso Zanocco-Marani, Alexis Grande Cancer Prevention Research, 2018
