Pharmacologic Inhibition of JAK1/JAK2 Signaling Protects from Acute GvHD While Preserving Gvt in Mice

Background:

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents a potential curative strategy for patients with hematological malignancies. Despite recent improvements in transplantation procedurs and supportive care, acute graft versus host disease (aGvHD) remains the most significant barrier to the success of allo-HSCT. In this contest, strategies directed against GvHD adversely affect survival because they increase malignancy relapse and infections. Approaches aimed at separating graft-versus-tumor (GvT) effect from GvHD are warranted, but difficult to achieve because of their shared biology. Given that JAK signaling dictates T cell differentiation, we postulated that JAKs might be potential therapeutic targets in allo-HSCT through a pharmacological approach. The relative importance of JAK2 signaling in myeloid and lymphoid malignancies drew our attention to the usage of INCB18424 (Ruxolitinb), a JAK1/JAK2 specific inhibitor that was recently shown to be an effective treatment for patients with myelofibrosis. We tested our hypothesis in a mouse model of aGvHD in order to abrogate GvHD, while maintaining a GvT effect.

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 INCB18424 for 14 days at the dose of 90mg/kg/day (INC90), 45mg/kg/day (INC45) or 22.5mg/kg/day (INC22.5). 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 INCB18424 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.

Results:

Significantly less GvHD, as determined by survival (INC45, p<0.005; INC22.5 p<0.05; INC90 ns), weight loss, and histopathology of GvHD target organs (INC90 and INC45, p<0.0005; INC22.5 p<0.005) was observed compared to untreated animals indicating that INCB18424 treatment has a dose-dependent effect on acute GvHD reduction. In the INC90 cohort, liver examination revealed moderate centrilobular hepatocellular hypertrophy, compatible with the development of potential adverse effects. Post-transplant IL-6 concentration was significantly reduced with all three doses (p<0.05), but the reduction was significantly higher with 90 and 45 mg/Kg/day relative to 22,5 mg/Kg/day (11.7±0.1 vs 15.9±2.0 vs 32.0±1.0 pg/ml, p< 0.05) again indicating a dose-dependent effect. Daily administration of INCB18424 post allo-HSCT, did not alter hematologic parameters, did not impair donor T cell alloreactivity, did not increase T regulatory cell number, but significantly modulated the expression of CXCR3 both on spleen CD4+ and CD8+ cells compared to untreated animals and migration of T cells to GvHD target organs as highlighted by immune-histochemical analysis. Consistent with the persistent in-vivo alloreactivity of donor T cells, when allo-HSCT recipients were challenged with tumor cells (A20 B-cell lymphoma cells and RMB-1 myeloid leukemia cells), the GvT activity in INCB18424 treated mice remained intact in the absence of GvHD, resulting in significantly improved survival compared to untreated animals (A20, p<0.0001; RMB1, p<0.001) and reduced presence of tumor cells.

Conclusions:

The inhibition of Jak/STAT signalingusing the sensitive and specific inhibitor of Jak1/Jak2, INCB18424 conferred effective protection from lethal acute GvHD in a MHC mismatched HSCT mouse model while sparing the GvT effect. Our work provides novel evidences of the mechanisms implied in INCB18424-mediated prevention of acute GvHD, demonstrating a significant inhibition of T cell trafficking into GvHD target tissues associated with reduced expression of CXCR3. We expect that these experimental observations may be readily translated into a prospective clinical trial evaluating INCB18424 (Ruxolitinb) for aGvHD prophilaxis after allo-HSCT for patients with hematologic malignancies.