Combinatorial inhibition of Tec kinases BTK and ITK is beneficial in ameliorating murine sclerodermatous chronic graft versus host disease

Abstract

Graft-versus-host disease (GVHD) is the major factor limiting the widespread use of potentially curative allogeneic hematopoietic stem cell transplant (allo-HSCT). Chronic GVHD is characterized by the activation of alloreactive donor immune cells, especially B- and T-cells, leading to tissue damage and pathogenic fibrosis. In this study, we used highly specific next-generation inhibitors of ITK (PCYC-274), BTK (PCYC-804), and ibrutinib-like BTK/ITK inhibitors (PCYC-914 and PCYC-401) in the B10.D2 → BALB/C model of murine sclerodermatous cGVHD. From the third week onward, allogeneic recipients in each group of respective Tec kinase inhibitors were treated three times weekly with inhibitors at doses of 10 and 30 mg/kg or with saline control via oral gavage. Overall, we found that selective BTK inhibition was less effective than combined ITK/BTK or ITK inhibition in lengthening survival and reducing symptoms of cGVHD. ITK inhibition was most efficacious, with PCYC-274 and PCYC-401 demonstrating a nearly 50 percent reduction in GVHD scoring even at the 10 mg/kg dose, while 30 mg/kg of these compounds almost completely ameliorated GVHD symptomology. BTK/ITK and ITK-treated mice showed significant reductions in overall pathology. Significant reductions in dermal thickness and fibrosis were shown for all treatment groups. There was evidence of mixed Th1 and Th2 cytokine profiles in the skin of mice with dermal cGVHD, as both IFN-gamma and IL-4 were upregulated in the allogeneic control group, while kinase inhibition significantly reduced levels of these cytokines. Using an in vitro model of T-cell polarization, Th1 cell production of TNF-alpha and IFN-gamma were partially blocked by ITK. Th2 cell production of IL-4 was almost completely blocked synergistically by ITK and BTK inhibition. BTK-specific inhibition was unable to block either Th1 or Th2 cytokine production. Taken together, these results confirm previous reports that ITK-focused inhibition inhibits Th1 and Th2 cells. Additionally, the compound's effects on T-cell proliferation were tested by CFSE assay. Pure ITK inhibition was most effective at blocking T-cell proliferation, with no proliferation in PCYC-274-treated cells even at 0.1uM. PCYC-401 and PCYC-914 showed some inhibition at lower doses, with complete inhibition evident at 10uM. PCYC-804 was only partially able to block proliferation even at 10uM. In conclusion, we observed substantial benefit for differential inhibition of Tec kinases in GVHD, with ITK being most efficacious and Th1 cells being more resistant to inhibition, matching the previously reported findings of a Th2 to Th1 selective pressure in cells treated with ibrutinib. Our data warrants the further development of ITK and ITK/BTK inhibitors with specific inhibitory ratios to improve the treatment of GVHD and other T-cell mediated diseases.