Palbociclib Prevents CDK4/6 Dependent-Myeloproliferation and Myelofibrosis

Abstract

Introduction Primary and secondary myelofibrosis (MF) present the worst prognosis in the group of BCR-ABL-negative myeloproliferative neoplasms. Presently there is no really efficient therapy of myelofibrosis, except allogenic bone marrow transplantation. In PMF patients, the JAK inhibitors ruxolitinib and fedratinib do not significantly reduce the MF itself or the natural progression of the disease. This could be due to the absence of direct link between JAK2 signaling level and the development of MF. However, ruxolitinib only partially inhibits JAK2 signaling and a reactivation of JAK/STAT signaling through heterodimerization of JAK2 with other JAK may occur (Koppikar et al, Nature, 2012). Complete inhibition of JAK2 signaling would likely induce a profound anemia and thrombocytopenia and at long-term an aplastic anemia. It therefore remains of great interest to identify and inhibit specific downstream targets dysregulated by JAK2 activation that may be responsible of the MF to circumvent the absence of a specific JAK2V617F inhibitor. JAK2V617F mutation leads to the autonomous activation of homodimeric type 1 cytokine receptors and their downstream signaling that induces G1/S cell cycle transition by activating the cyclin-dependent kinases CDK4/6. Cdk6 loss in Jak2V617F KI mice delays the development of the MPN and increases mouse survival (Uras et al, Blood, 2019). We hypothesized that intensifying the JAK2V617F-mediated CDK4/6 increased activity could potentially accelerate the MF development, which is a late event in the Jak2V617F KI model. Our study aims to explore the role of CDK4/6 overactivation or inhibition in the disease and MF development. Methods Since p19INK4D is a cyclin dependent kinase inhibitor, which inhibits CDK4 and to a lesser extent CDK6 and which specifically regulate megakaryocyte (MK) cell cycle and the arrest of endomitosis, we established a pre-clinical MF model by crossing p19Ink4d knock-out (KO) mice with heterozygous inducible Jak2V617F KI mice to obtain p19Ink4d-/-/Jak2FLEXV617F/+SCL-Cre+/-mice called here KOKI mice. With this model, we studied the development of the myeloproliferative disorder. To prevent a MF development in this model, a CDK4/6 inhibitor Palbociclib was administered from 2 to 4 weeks post-Jak2V617F induction. Results In contrast to Jak2V617F (KI) mice that developed a severe MF, in both bone marrow and spleen, at 24 weeks post-Jak2V617F induction, all KOKI mice developed a grade I MF as early as 3 weeks after Jak2V617F induction that progressed to severe fibrosis at 8 weeks (grade III). KOKI mice were characterized by the rapid onset of an extramedullary hematopoiesis (EMH) and megakaryocytic hyperplasia in bone marrow and spleen. As MK are known to be a major source of TGF-β1 in the development of MF, we further focused on the MK lineage. At 1 week post-Jak2V617F induction, prior to MF development, the numbers of MK progenitors (MK-P) were markedly increased in the spleen of KOKI mice (11-fold) while only 3.9-fold increase was observed for KI mice as compared to WT littermates. A significant 2.6-fold increase in the number of MK in the spleen was only visible in KOKI mice, in accordance with splenomegaly and EMH. The analysis of mean ploidy level (>4N) revealed that it increased to 34.8N in KI, 43.7N in KO p19 and 59.4N in KOKI mice as compared to 19N in WT mice. Total TGF-β1 bone marrow levels were moderately elevated in KOKI mice, with a higher proportion of activated TGF-β1. Treatment of KOKI mice with a CDK4/6 inhibitor, Palbociclib, prevented the development of MF and markedly decreased the EMH, the abnormalities of the megakaryocyte differentiation including the ploidy level and the total and activated TGF-β1 levels. Conclusions Altogether, using a KOKI mouse model we show that the deregulation of CDK4/6 mainly in megakaryocytes is sufficient to induce a severe MF strongly suggesting that CDK4/6 inhibitors could be a very promising therapeutic issue for MF patients (Figure). Disclosure of Prior Presentation/Publication : The abstract will not have been presented and published between January 1, 2020 and December 5, 2020. Figure 1 Disclosures No relevant conflicts of interest to declare.