Pharmacologic blockade of JAK1/JAK2 reduces GvHD and preserves the graft-versus-leukemia effect

Abstract

We have recently reported that interferon gamma receptor deficient (IFNγR-/-) allogeneic donor T cells result in significantly less graft-versus-host disease (GvHD) than wild-type (WT) T cells, while maintaining an anti-leukemia or graft-versus-leukemia (GvL) effect after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We demonstrated that IFNγR signaling regulates alloreactive T cell trafficking to GvHD target organs through expression of the chemokine receptor CXCR3 in alloreactive T cells. Since IFNγR signaling is mediated via JAK1/JAK2, we tested the effect of JAK1/JAK2 inhibition on GvHD. While we demonstrated that pharmacologic blockade of JAK1/JAK2 in WT T cells using the JAK1/JAK2 inhibitor, INCB018424 (Ruxolitinib), resulted in a similar effect to IFNγR-/- T cells both in vitro (reduction of CXCR3 expression in T cells) and in vivo (mitigation of GvHD after allo-HSCT), it remains to be determined if in vivo administration of INCB018424 will result in preservation of GvL while reducing GvHD. Here, we report that INCB018424 reduces GvHD and preserves the beneficial GvL effect in two different murine MHC-mismatched allo-HSCT models and using two different murine leukemia models (lymphoid leukemia and myeloid leukemia). In addition, prolonged administration of INCB018424 further improves survival after allo-HSCT and is superior to other JAK1/JAK2 inhibitors, such as TG101348 or AZD1480. These data suggest that pharmacologic inhibition of JAK1/JAK2 might be a promising therapeutic approach to achieve the beneficial anti-leukemia effect and overcome HLA-barriers in allo-HSCT. It might also be exploited in other diseases besides GvHD, such as organ transplant rejection, chronic inflammatory diseases and autoimmune diseases.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. In vivo administration of INCB018424 maintains a beneficial GvL effect and improves survival after allo-HSCT.

Allo-HSCT was performed as described in the Materials and Method. CBRluc-expressing A20 B cell lymphoma cells were injected either i.v. (1×104 cells) or s.c. (1×105 cells in 100 ul PBS). In vivo BLI imaging technique was used to measure tumor burden once every week starting day 1 to day 43 after allo-HSCT. INCB018424 was injected for three weeks (days 3–23) twice a day (100 µg/injection, i.p.). (A) Systemic leukemia (left panel) and solid tumor (right panel) models. Photon flux was measured with a region of interest drawn over the entire body of each mouse. (B) Actual images of 1 representative mouse from each group are shown. Data represents the pool of three independent experiments. Systemic A20 lymphoid leukemia model is shown in upper panels and solid tumor A20 lymphoid leukemia model shown in bottom panel. (C) Survival of mice shown in Kaplan-Meier plots of both a systemic (left panel) and a solid tumor model (right panel).

Figure 2. Prolonged administration of INCB018424 further improves survival after allo-HSCT.

(A) Administration of INCB018424 for one month (days +1–+31) twice a day (100 µg/injection, s.c.) significantly improves survival after allo-HSCT (p = 0.0003). (B–C) Prolonged administration of INCB018424 improves survival while preserving GvL (B). 1×105 A20 cells (i.v) were transplanted along with allogeneic TCD BM and pan T cells at day 0. Clinical GvHD scores according to Cooke et al (C). (D–E) B6 mice were lethally irradiated (1,200 cGy) at day -1 and injected (i.p.) with 50 µg of anti-NK1.1 mAb (PK136). BM cells (5×106) and whole splenocytes (20×106) harvested from Balb/c were transplanted into lethally irradiated B6 at day 0, followed by INCB018424 for 31 days (days +1–+31). Clinical GvHD scores according to Cooke et al (D) and weight change (E). (F–G) B6 mice were lethally irradiated (1,200 cGy) at day -1 and injected (i.p.) with 50 µg of anti-NK1.1 mAb (PK136). TCD BM (5×106) and pan T cells (5×106) isolated from Balb/c were transplanted into the lethally irradiated B6 at day 0. Banked primary APL cells (5×105) were injected (i.v.) along with TCD BM and pan T cells at day 0. INCB018424 was administered s.c. twice a day for 31 days (days +1–+31). (F) Percentage of APL cells in the PB was measured by flow cytometry after staining PB cells starting at day +16 once every week until day +41. Anti-H-2Kb and anti-CD45.2 antibodies were used to identify the APL cells. H-2b+ CD45.1+ B6 recipients and H-2d+ CD45.2+ Balb/c donors were used to discriminate APL from all other cells. (G) Shown is a Kaplan-Meier survival curve. (H) INCB018424 is superior to other JAK inhibitors, such as TG101348 and AZD1480, in blocking IFNγR signaling. As an indicator of IFNγR signaling, we assessed STAT1 phosphorylation by intracellular pSTAT1 staining and FACS. MFI: geometric mean fluorescence intensity. (I) Lethally irradiated (900 cGy) Balb/c mice were transplanted with TCD BM (5×106) obtained from B6 WT mice and pan T cells (5×105) isolated from B6 WT or IFNγR−/− mice at day 0. INCB018424 was administered s.c. twice a day for 31 days (days +1–+31). INCB018424 treatment for 31 days results in free of GvHD in 90% of recipients and 100% survival when mice are transplanted with IFNγR−/− T cells.