Myelofibrosis (MF) is the deadliest form of myeloproliferative neoplasms (MPN), characterized by deposition of fibrous tissues in the bone marrow, abnormal megakaryopoiesis, ineffective erythropoiesis and extramedullary hematopoiesis. The median survival for patients with MF is ~5 years. The JAK2V617F mutation, which results in constitutive activation of the JAK2 tyrosine kinase, has been found in 50-60% patients with MF. Additional mutations in the genes encoding thrombopoietin receptor (MPL) and calreticulin (CALR) have been found at a lower frequency than JAK2V617F in MF. The JAK1/JAK2 inhibitor ruxolitinib has been approved for treatment of MF. Although ruxolitinib can reduce splenomegaly and constitutional symptoms, it does not cure or prevent fibrosis in patients with MPN/MF. Additionally, ruxolitinib treatment does not improve survival in MF patients. So, there is a critical need to identify new therapeutic targets and develop novel therapies for MF.
We have found that expression of CDK6 is significantly elevated in mouse and human MPN hematopoietic cells/progenitors. We also observed that knockdown of CDK6 significantly inhibited proliferation of BA/F3-EpoR-JAK2V617F and HEL cells expressing JAK2V617F but not wild-type JAK2 expressing BA/F3-EpoR cells, suggesting that CDK6 is a bona fide target in MPN. In this study, we investigated the efficacy of CDK4/6 inhibitor Palbociclib (PD-0332991) in hematopoietic cells expressing JAK2V617F and MPLW515L and murine models of MF. Since Palbociclib is an FDA-approved drug for treatment of breast cancer, information on the dose, pharmacokinetics and toxicity of Palbociclib in human is already available. Thus, it would be easier to move this drug into clinic and repurpose it for treatment of MPN/MF.
We observed that Palbociclib treatment (0.25-0.5 uM) significantly reduced the proliferation of murine BA/F3 cells expressing JAK2V617F or MPLW515L. Palbociclib treatment also significantly inhibited human JAK2V617F-positive HEL, SET-2 and UKE-1 cells. However, Palbociclibonly modestly affected proliferation of wild type JAK2-expressing BA/F3 cells at higher concentrations. Palbociclib treatment also resulted in marked apoptosis in BA/F3-EpoR cells expressing JAK2V617F. Furthermore, Palbociclib and ruxolitinib synergistically induced apoptosis in JAK2V617F-expressing hematopoietic cells. Additionally, Palbociclib significantly inhibited hematopoietic progenitor colony formation in MPN/MF CD34+ cells.
We previously generated conditional JAK2V617F knock-in mice. Whereas expression of heterozygous JAK2V617F induces a PV-like disease, homozygous JAK2V617F expression promotes rapid progression to myelofibrosis. We have utilized the homozygous JAK2V617F mice to test the in vivo efficacy of Palbociclib alone and in combination with ruxolitinib against MF. Treatment of Palbociclib alone significantly reduced the WBC and neutrophil counts and decreased the spleen size in homozygous JAK2V617F mice. Combined treatment of Palbociclib and ruxolitinib almost completely normalized the WBC and neutrophil counts and the spleen size in homozygous JAK2V617F mice. Histopathologic analysis revealed marked reduction in fibrosis in the bone marrow of homozygous JAK2V617F mice treated with Palbociclib or Palbociclib and ruxolitinib combination.
We also have tested the in vivo efficacy of Palbociclib and Palbociclib/ruxolitinib combination in MPL mutant (MPLW515L) mouse model of MF. We observed similar decrease in WBC and neutrophil counts and reduction in splenomegaly in MPL mutant mice upon treatment of Palbociclib alone or in combination with ruxolitinib. Combined treatment of Palbociclib and ruxolitinib remarkably reduced fibrosis in the bone marrow of MPL mutant mice. Overall, our results suggest that palbociclib in combination with ruxolitinib may have therapeutic potential against MF and support the clinical investigation of palbociclib and ruxolitinib combination in patients with MF.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
