Introduction: Myelofibrosis (MF) is characterized by clonal hemopoiesis, inflammatory microenvironment and mutations in JAK2, MPL, or CALR genes. Around 10% of patients (pts) do not carry the 3 mutations (Triple negative, TN). Microparticles (MPs; 0.1 - 1 µm) are small vesicles deriving from the cell plasma membrane with a role in intercellular signalling. Circulating MPs are increased in inflammation and cancer including MF. MicroRNAs (miRs) contribute to MF pathogenesis. However, the miRs profile of MPs has never been investigated in MF. Here we studied miR expression of circulating MPs from JAK2(V617F) mutated and TN pts. Methods: Peripheral blood was collected from 6 MF pts (at diagnosis or out of cytotoxic treatment for at least 3 months) and 3 age/sex-matched healthy donors (HD). MF pts were JAK2(V617F) mutated (n=3) and TN (n=3). MPs were purified from platelet poor plasma by ultracentrifugation and quantified using the Nanosight technology. MiR expression of isolated pts/HD-MPs (10e9) was investigated using miRNeasy Mini Kit and TaqMan™ Array Human MicroRNA A Cards (ThermoFisher). RT-PCR validation assays are under way. Results: As compared with the HD counterparts, many miRs were significantly upregulated in MPs from both JAK2(V617F) mutated and TN pts. Among them miR-21, known to be the most commonly upregulated miR in haematological tumours, is an anti-apoptotic factor with oncogenic potential and able to inhibit megakaryocyto-erithropoiesis. MiR-155, 222, 24 were upregulated in TN-MPs only. MiR-155 is upregulated in response to inflammation and, targeting p53 pathway, inhibits apoptosis. Its overexpression stimulates granulo-monocytopoiesis while impairing megakaryocytopoiesis. MiR-221/222, markers of poor prognosis in aggressive tumours and both increased in TN-MPs, show anti-apoptotic effect acting downstream of the oncogenic RAS-RAF-MEK pathway. MiR-423-5p, a pro-apoptic miR, shows high expression in JAK2(V617F)/TN MPs but is almost absent in the HD counterparts. MiR-34a-5p, shown to be associated with MF, is upregulated in pts-MPs; however, its expression is lower in TN-MPs. Interestingly, many miRs regulating inflammation (miR-21, 146a, 223, 19a) and proliferation (miR-199a-3p, 21, 99b) were overexpressed in MPs-pts. Finally, miR-155, 146a, 19a and 194 overexpression affects the JAK-STAT pathway by blocking SOCS3, a negative regulator of the JAK-STAT signalling, and promoting cell survival. Comparing TN vs JAK2(V617F) MPs, the expression of 6 miRs (miR-122, 27a, 744, 584c, 365, 483-5p) was increased, whereas 2 onco-suppressors miRs (let-7b and miR-361) were less expressed. Of note, 6 out of the above mentioned 8 miRs have SRSF1 gene as a common target which is both a protooncogene and a splicing regulator. Conclusions: Circulating TN-MPs show distinct miR signature as compared with the JAK2(V617F) mutated counterparts. This study has the potential to identify disease-related biomarker(s).

TOWARD THE IDENTIFICATION OF A MICRORNA-BASED SIGNATURE OF CIRCULATING MICROPARTICLES FROM TRIPLE NEGATIVE AND JAK2(V617F) MUTATED PATIENTS WITH MYELOFIBROSIS

M. Barone;C. Morsiani;D. Sollazzo;D. Forte;S. Carloni;G. Auteri;M. Romano;M. Cavo;N. Vianelli;C. Franceschi;M. Capri;F. Palandri;L. Catani
2018

Abstract

Introduction: Myelofibrosis (MF) is characterized by clonal hemopoiesis, inflammatory microenvironment and mutations in JAK2, MPL, or CALR genes. Around 10% of patients (pts) do not carry the 3 mutations (Triple negative, TN). Microparticles (MPs; 0.1 - 1 µm) are small vesicles deriving from the cell plasma membrane with a role in intercellular signalling. Circulating MPs are increased in inflammation and cancer including MF. MicroRNAs (miRs) contribute to MF pathogenesis. However, the miRs profile of MPs has never been investigated in MF. Here we studied miR expression of circulating MPs from JAK2(V617F) mutated and TN pts. Methods: Peripheral blood was collected from 6 MF pts (at diagnosis or out of cytotoxic treatment for at least 3 months) and 3 age/sex-matched healthy donors (HD). MF pts were JAK2(V617F) mutated (n=3) and TN (n=3). MPs were purified from platelet poor plasma by ultracentrifugation and quantified using the Nanosight technology. MiR expression of isolated pts/HD-MPs (10e9) was investigated using miRNeasy Mini Kit and TaqMan™ Array Human MicroRNA A Cards (ThermoFisher). RT-PCR validation assays are under way. Results: As compared with the HD counterparts, many miRs were significantly upregulated in MPs from both JAK2(V617F) mutated and TN pts. Among them miR-21, known to be the most commonly upregulated miR in haematological tumours, is an anti-apoptotic factor with oncogenic potential and able to inhibit megakaryocyto-erithropoiesis. MiR-155, 222, 24 were upregulated in TN-MPs only. MiR-155 is upregulated in response to inflammation and, targeting p53 pathway, inhibits apoptosis. Its overexpression stimulates granulo-monocytopoiesis while impairing megakaryocytopoiesis. MiR-221/222, markers of poor prognosis in aggressive tumours and both increased in TN-MPs, show anti-apoptotic effect acting downstream of the oncogenic RAS-RAF-MEK pathway. MiR-423-5p, a pro-apoptic miR, shows high expression in JAK2(V617F)/TN MPs but is almost absent in the HD counterparts. MiR-34a-5p, shown to be associated with MF, is upregulated in pts-MPs; however, its expression is lower in TN-MPs. Interestingly, many miRs regulating inflammation (miR-21, 146a, 223, 19a) and proliferation (miR-199a-3p, 21, 99b) were overexpressed in MPs-pts. Finally, miR-155, 146a, 19a and 194 overexpression affects the JAK-STAT pathway by blocking SOCS3, a negative regulator of the JAK-STAT signalling, and promoting cell survival. Comparing TN vs JAK2(V617F) MPs, the expression of 6 miRs (miR-122, 27a, 744, 584c, 365, 483-5p) was increased, whereas 2 onco-suppressors miRs (let-7b and miR-361) were less expressed. Of note, 6 out of the above mentioned 8 miRs have SRSF1 gene as a common target which is both a protooncogene and a splicing regulator. Conclusions: Circulating TN-MPs show distinct miR signature as compared with the JAK2(V617F) mutated counterparts. This study has the potential to identify disease-related biomarker(s).
Haematologica XV Congress of the Italian Society of Experimental Hematology Rimini, Italy, October 18-20, 2018 ABSTRACT BOOK
M. Barone, C. Morsiani, D. Sollazzo, D. Forte, S. Carloni, F. Fabbri, G. Auteri, M. Romano, M. Ottaviani, M. Cavo, M. Martinelli, N. Vianelli, C. Franceschi, M. Capri, F. Palandri, L. Catani
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/662922
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