Carfilzomib Enhances the Suppressive Effect of Ruxolitinib in Myelofibrosis.

Simone Claudiani,Avirup Chowdhury,Michael Albert,Katya Mokretar,Andrea Bianchi,Alistair G Reid,Clinton C Mason,Michael W Deininger,Kanagaraju Ponnusamy,Mark Robinson,Kikkeri Naresh,Antinisca Di Marco,Jamshid S Khorashad,Dragana Milojkovic,Cristina Pellegrini,Carme R Fiol,Jane F Apperley

doi:10.3390/cancers13194863

Abstract

Simple SummaryMyelofibrosis (MF) is a progressive myeloproliferative neoplasm with tendency towards leukemic transformation and has the poorest prognosis amongst the Philadelphia-negative classical myeloproliferative neoplasms (MPN). Ruxolitinib, the first FDA-approved JAK1/2 tyrosine kinase inhibitor, is efficient in reducing spleen size and improving patient symptoms, but it has not been shown to eradicate the MF clone. In this study, using functional genomics techniques, we identified the proteasome family as an additional therapeutic target and demonstrated that inhibition of the proteasome family by carfilzomib in combination with ruxolitinib enhanced suppression of primary MF cells in vitro.As the first FDA-approved tyrosine kinase inhibitor for treatment of patients with myelofibrosis (MF), ruxolitinib improves clinical symptoms but does not lead to eradication of the disease or significant reduction of the mutated allele burden. The resistance of MF clones against the suppressive action of ruxolitinib may be due to intrinsic or extrinsic mechanisms leading to activity of additional pro-survival genes or signalling pathways that function independently of JAK2/STAT5. To identify alternative therapeutic targets, we applied a pooled-shRNA library targeting ~5000 genes to a JAK2V617F-positive cell line under a variety of conditions, including absence or presence of ruxolitinib and in the presence of a bone marrow microenvironment-like culture medium. We identified several proteasomal gene family members as essential to HEL cell survival. The importance of these genes was validated in MF cells using the proteasomal inhibitor carfilzomib, which also enhanced lethality in combination with ruxolitinib. We also showed that proteasome gene expression is reduced by ruxolitinib in MF CD34+ cells and that additional targeting of proteasomal activity by carfilzomib enhances the inhibitory action of ruxolitinib in vitro. Hence, this study suggests a potential role for proteasome inhibitors in combination with ruxolitinib for management of MF patients.

Highlights

Philadelphia-negative classical myeloproliferative neoplasms (MPN), comprising essential thrombocythemia (ET), polycythaemia vera (PV) and myelofibrosis (MF) are clonal disorders of the haematopoietic stem cell, characterised by abnormal proliferation of the myeloid compartment with maintained differentiation, decreased apoptosis and defective maturation of megakaryocytes
Cells harbouring an shRNA which targeted an essential gene for viability would be depleted following 9 days of culture
We applied a pooled-shRNA library screen to identify the mechanisms of resistance to ruxolitinib as we had previously described in chronic myeloid leukaemia [17], with modifications

Summary

Introduction

Philadelphia-negative classical myeloproliferative neoplasms (MPN), comprising essential thrombocythemia (ET), polycythaemia vera (PV) and myelofibrosis (MF) are clonal disorders of the haematopoietic stem cell, characterised by abnormal proliferation of the myeloid compartment with maintained differentiation, decreased apoptosis and defective maturation of megakaryocytes. Two clinical trials evaluating ruxolitinib in MF (COMFORT I and II) [5,6] showed efficacy in reducing spleen size and improving patient symptoms, with a marginal improvement in survival [7], but did not show a significant change in the natural history of the disease [8,9] nor eradication of the MF clone. This may be due to activation of alternative intrinsic or extrinsic pathways which provide an additional survival mechanism for the cells when the JAK2 pathway is inhibited. An extrinsic mechanism may activate parallel viable signals through physical contact with the cells and matrix of the microenvironment or secretion of cytokines from cellular components, such as mesenchymal stromal cells [11,12]

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