IDENTIFICAZIONE DI NUOVE VIE DI SEGNALE E MARCATORI PREDITTIVI DELLA MALATTIA DEL TRAPIANTO CONTRO L'OSPITE DOPO TRAPIANTO ALLOGENICO DI CELLULE STAMINALI EMATOPOIETICHE

IDENTIFYING NEW MOLECULAR PATHWAYS AND PREDICTOR BIOMARKERS OF GVHD AFTER ALLOGENEIC HSCT Background and rationale Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative option for patients with hematological malignancies. However, its success is limited by life-threatening graft-versus-host disease (GVHD). Strategies to control GVHD are often associated with suppression of the immune system leading to the impairment of the beneficial graft versus tumor (GVT) effect. The ideal approach to prevent and treat GVHD would limit alloantigen-specific reactivity while preserving immunity against malignant cells and pathogens. Chemokines are well known inducers of leukocyte trafficking and activation. Stimulation of the chemokine receptor signaling pathway leads to initiation of the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway that contributes to the pathogenesis of GVHD. The key role of JAK signaling in normal and abnormal lymphocyte development and function prompted us to hypotesize that the inhibition of the JAK/STAT signaling could prevent GVHD from developing. We therefore evaluated the efficacy of two distinct JAK inhibitors, CP-690,550 and INC-424, in the allogeneic setting. Moreover, we tested whether the treatment with JAK inhibitors preserved the GVT effect in a mouse model of aGVHD. However, it would be a great advantage to predict aGVHD before its onset in order to tailor the immunosuppressive therapy in a patient-specific manner. To date, noninvasive, diagnostic and prognostic tests for aGVHD are lacking but necessary to predict aGVHD and improve the safety of allo-HSCT. We therefore performed a prospective analysis of miRNA expression profile in plasma of allografted patients to detect specific miRNAs with predictive role for aGVHD. Methods A major histocompatibility complex (MHC) mismatched HSCT mouse model was set up. Lethally irradiated BALB/c mice received spleen (SC) and bone marrow (BM) cells from donors C57BL/6 (B6) mice, and were treated with CP690,550 or INCB18424 for 14 days at 15mg/Kg/day or 45mg/Kg/day respectively. Syngeneic transplants (B6-B6) and BALB/c recipients treated with B6 BM cells only were also used. To determine the GVT activity, allo-HSCT recipients were co-injected with either a B-cell lymphoma cell line (A20) or a myeloid leukemia cell line (RMB-1) and treated or not with INC-424 at the dose of 45mg/kg/day. Mice were monitored for overall survival (OS) and weight loss. GVHD was histologically scored in tissues harvested on day 14, 30 and 60 and cytokine production by ELISA. Immune reconstitution and tumor cells were monitored by flow cytometry. For biomarker discovery, plasma samples from 24 patients who received unmanipulated allo-HSCT were collected weekly. MicroRNAs were isolated from plasma and miRNA expression profile examined using quantitative Real Time PCR. Results JAK/STAT inhibition, especially by INC-424 treatment, caused significantly less GVHD when compared to untreated animals, as determined by survival, weight loss, and histopathology of GVHD target organs. Plasma levels of inflammatory cytokines (IL-6 and IL-12) were significantly reduced in all treated animals when compared to controls. Daily administration of INC-424 post allo-HSCT did not alter hematologic parameters and did not impair immune reconstitution but shifted the CD4+ Treg to CD8+ effector cell ratio because of a relative decrease in the latter population and a relative increase in Treg in GVHD treated mice. A reduction in the Th17/Treg ratio was also observed. When allo-HSCT recipients were challenged with tumor cells, the GVT activity in INCB-424 treated mice was intact in the absence of GVHD, resulting in significantly improved survival compared to untreated animals (p<0.001) and reduced the presence of tumor cells. On the other hand, circulating miRNA profiling before aGVHD onset was able to identify patients at high risk of developing the disease. Two unique miRNAs (miR-194 and miR-518f) were upregulated in samples of patients that would lately develop aGVHD. Pathway prediction analysis indicated that these miRNAs are critical in GVHD pathogenesis. Conclusions The inhibition of JAK/STAT signaling using the sensitive and specific inhibitor of JAK1/JAK2 INC-424 at the optimal dose of 45mg/kg/day, conferred effective protection from lethal acute GVHD in a MHC mismatched HSCT mouse model. Mice retain the ability to mount aggressive graft-versus-tumor (GVT) effects and to generate complete donor T-cell reconstitution. Taken together, these data suggest that INC-424, recently approved for the treatment of myelofibrosis, might also be tested for GVHD prophylaxis and treatment in clinical trials. In addition, considering the non-invasive characteristics of plasma sampling and the feasibility of detecting miRNAs after allo-HSCT, our results indicate that circulating miRNAs represent a promising tool for the early diagnosis of aGVHD. However, inconsistencies with prior published data highlight the need to develop standards for circulating miRNA studies to move their discovery from the molecular biology laboratory to the clinic.