Dorian Forte1,2, Martina Barone3,4, Cristina Morsiani5, Giorgia Simonetti6, Francesco Fabbri6, Samantha Bruno3,4, Erika Bandini6, Daria Sollazzo4, Salvatore Collura5, Maria Chiara Deregibus7, Giuseppe Auteri3,4, Emanuela Ottaviani3, Nicola Vianelli3, Giovanni Camussi7, Claudio Franceschi8, Miriam Capri5, Francesca Palandri3, Michele Cavo3,4, Lucia Catani3,4. 1. Azienda Ospedaliero-Universitaria di Bologna, via Albertoni 15, Bologna, Italy. dorian.forte2@unibo.it. 2. Istituto di Ematologia "Seràgnoli", Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università degli Studi, Bologna, Italy. dorian.forte2@unibo.it. 3. Azienda Ospedaliero-Universitaria di Bologna, via Albertoni 15, Bologna, Italy. 4. Istituto di Ematologia "Seràgnoli", Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università degli Studi, Bologna, Italy. 5. Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Bologna, Italy. 6. Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy. 7. Department of Internal Medicine, Centre for Molecular Biotechnology and Centre for Research in Experimental Medicine, Torino, Italy. 8. Laboratory of Systems Medicine of Healthy Aging and Department of Applied Mathematics, Lobachevsky University, Nizhny Novgorod, Russia.
Abstract
BACKGROUND: Myelofibrosis (MF) is a clonal disorder of hemopoietic stem/progenitor cells (HSPCs) with high prevalence in elderly patients and mutations in three driver genes (JAK2, MPL, or CALR). Around 10-15% of patients are triple-negative (TN) for the three driver mutations and display significantly worse survival. Circulating extracellular vesicles (EVs) play a role in intercellular signaling and are increased in inflammation and cancer. To identify a biomolecular signature of TN patients, we comparatively evaluated the circulating HSPCs and their functional interplay with the microenvironment focusing on EV analysis. METHODS: Peripheral blood was collected from MF patients (n = 29; JAK2V617F mutation, n = 23; TN, n = 6) and healthy donors (HD, n = 10). Immunomagnetically isolated CD34+ cells were characterized by gene expression profiling analysis (GEP), survival, migration, and clonogenic ability. EVs were purified from platelet-poor plasma by ultracentrifugation, quantified using the Nanosight technology and phenotypically characterized by flow cytometry together with microRNA expression. Migration and survival of CD34+ cells from patients were also analyzed after in vitro treatments with selected inflammatory factors, i.e. (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, IL6) or after co-culture with EVs from MF patients/HD. RESULTS: The absolute numbers of circulating CD34+ cells were massively increased in TN patients. We found that TN CD34+ cells show in vitro defective functions and are unresponsive to the inflammatory microenvironment. Of note, the plasma levels of crucial inflammatory cytokines are mostly within the normal range in TN patients. Compared to JAK2V617F-mutated patients, the GEP of TN CD34+ cells revealed distinct signatures in key pathways such as survival, cell adhesion, and inflammation. Importantly, we observed the presence of mitochondrial components within plasma EVs and a distinct phenotype in TN-derived EVs compared to the JAK2V617F-mutated MF patients and HD counterparts. Notably, TN EVs promoted the survival of TN CD34+ cells. Along with a specific microRNA signature, the circulating EVs from TN patients are enriched with miR-361-5p. CONCLUSIONS: Distinct EV-driven signals from the microenvironment are capable to promote the TN malignant hemopoiesis and their further investigation paves the way toward novel therapeutic approaches for rare MF.
BACKGROUND:Myelofibrosis (MF) is a clonal disorder of hemopoietic stem/progenitor cells (HSPCs) with high prevalence in elderly patients and mutations in three driver genes (JAK2, MPL, or CALR). Around 10-15% of patients are triple-negative (TN) for the three driver mutations and display significantly worse survival. Circulating extracellular vesicles (EVs) play a role in intercellular signaling and are increased in inflammation and cancer. To identify a biomolecular signature of TNpatients, we comparatively evaluated the circulating HSPCs and their functional interplay with the microenvironment focusing on EV analysis. METHODS: Peripheral blood was collected from MF patients (n = 29; JAK2V617F mutation, n = 23; TN, n = 6) and healthy donors (HD, n = 10). Immunomagnetically isolated CD34+ cells were characterized by gene expression profiling analysis (GEP), survival, migration, and clonogenic ability. EVs were purified from platelet-poor plasma by ultracentrifugation, quantified using the Nanosight technology and phenotypically characterized by flow cytometry together with microRNA expression. Migration and survival of CD34+ cells from patients were also analyzed after in vitro treatments with selected inflammatory factors, i.e. (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, IL6) or after co-culture with EVs from MF patients/HD. RESULTS: The absolute numbers of circulating CD34+ cells were massively increased in TNpatients. We found that TNCD34+ cells show in vitro defective functions and are unresponsive to the inflammatory microenvironment. Of note, the plasma levels of crucial inflammatory cytokines are mostly within the normal range in TNpatients. Compared to JAK2V617F-mutated patients, the GEP of TNCD34+ cells revealed distinct signatures in key pathways such as survival, cell adhesion, and inflammation. Importantly, we observed the presence of mitochondrial components within plasma EVs and a distinct phenotype in TN-derived EVs compared to the JAK2V617F-mutated MF patients and HD counterparts. Notably, TN EVs promoted the survival of TNCD34+ cells. Along with a specific microRNA signature, the circulating EVs from TNpatients are enriched with miR-361-5p. CONCLUSIONS: Distinct EV-driven signals from the microenvironment are capable to promote the TN malignant hemopoiesis and their further investigation paves the way toward novel therapeutic approaches for rare MF.
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