Preclinical Efficacy of TGF-Beta Receptor I Kinase Inhibitor, Galunisertib, in Myelofibrosis

Abstract

Background: Bone marrow fibrosis is associated with poor prognosis in myeloproliferative neoplasms (MPN). Both polycythemia vera (PV) and essential thrombocythemia (ET) patients are at high risk for transformation into myelofibrosis. Although JAK2 inhibitors are effective treatment for MPNs, they are unable to alleviate the fibrotic phenotype. Since TGFβ has been implicated in the development of fibrosis in this disease, we determined the expression of TGF-β pathway in human MPN samples and also evaluated the anti-fibrotic effect of a clinically relevant TGF-β receptor inhibitor (Galunisertib, LY2157299) in MPN mouse models.Results: To determine the pathobiology of TGF-β signaling in MPNs, we analyzed gene expression profiles of 93 MPN patient derived neutrophils and 11 controls. We determined that the TGF-β1 isoform was the dominant ligand expressed in these samples and was significantly increased in primary myelofibrosis(PMF) samples (p=0.001). Examination of downstream SMAD pathways revealed that SMAD2 was the dominant expressed effector in MPN samples and was also significantly increased in PMF samples (p=0.005).Galunisertib(LY2157299) is a specific inhibitor of TGF-β receptor I kinase and abrogates the phosphorylation of SMAD2. Next, we determined preclinical efficacy of LY2157299 in a mouse model generated by transgenic expression of JAK2V617F using the vav-1 promoter. These mice display a PV-like phenotype with elevated blood cell counts at the age of 6 weeks and develop bone marrow and spleen fibrosis at 25-30 weeks. Microarray analysis of gene expression in JAK2V617F and control total bone marrow revealed significant increase in TGF-β1 expression in JAK2V617F animals and immunohistochemistry demonstrated increased SMAD2 activation consistent with primary human data. Thirty-week old mice with established myelofibrosis were treated with LY2157299 daily for 4 weeks. A significant decrease in fibrosis was assessed quantitatively by total hydroxyproline content in bone marrow after treatment (mean hydroxyproline content 5ug/ml in control mice vs 2.1ug/ml in treated mice, p=0.005). To validate the efficacy of inhibition of this pathway, we next treated 50 week-old JAK2V617F transgenic mice with a murine antibody against TGFβ1 (IMC-TR1, LY3022859) at 40mg/kg 3 times per week for 4 weeks. This treatment also resulted in significant decrease in fibrosis (p=0.02).For further verification, we used the MPLW515L transplantation mouse model that develops rapid thrombocytosis, leukocytosis, splenomegaly and myelofibrosis. For in vivo studies, LY2157299 was orally administered daily from day 12 after transplantation for 14 consecutive days. Through in vitro studies of MSCs in transplant recipients with WT-MPL, we found that LY2157299 effectively antagonizes the stimulatory effect of TGFβ on collagen I (p<0.05) and collagen III (p<0.01) production in a dose-dependent manner. Treatment of MPLW515L mice with LY2157299 for 14 days significantly decreased reticulin fiber formation compared with those treated with vehicle control (p<0.05). MSCs derived from LY2157299-treated mice also showed significantly less collagen production compared to MSCs from vehicle-treated MPLW515L mice (p<0.05). Although no reduction in spleen size was observed in MPLW515L-transplanted mice, a significant reduction in the peripheral WBC count was observed after treatment with LY2157299 at 150mg/kg (p<0.05).Conclusions: These results indicate that TGFβ-receptor blockade with Galunisertib inhibits TGFβ-induced collagen production and attenuates the fibrotic phenotype in MPN mouse models. DisclosuresLevine:CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.