Overexpression of MPL Causes Ruxolitinib-Resistance in Myeloproliferative Neoplasms with Calreticulin Frame-Shift Mutations

【Introduction】 Although Ruxolitinib (Rux), a JAK1/2-inhibitor, is an effective treatment option for primary myelofibrosis, tumor cells become resistant to this drug in many MPN patients. Currently, Rux-resistance is a major problem and contributes to poor prognosis of MPN patients. Mechanisms of Rux-resistance in MPN cells with JAK2V617F mutations have already been reported. On the other hand, MPN cells with CALR mutations resistant to Rux-treatment have not been fully characterized yet. In this study, we have clarified a mechanism of Rux-resistance in MPN tumor cells with CALR mutations in both cell lines and clinical samples.　

【Materials and Methods】To establish human cell lines with CALR mutations, we have introduced deletion of 46-nucleotides generating the CALR frame-shift mutation (del46), one of the type1-like mutations, to UT-7/EPO by the CRISPR/Cas9 system. Then, we introduced exogenous MPL to UT-7/EPO with the CALR mutations by transfection. We confirmed that UT-7/EPO with CALR frame-shift mutations (CALR-fs) and exogenous MPL (exMPL) activate STAT5 and showed cytokine-independent cell growth. To establish Rux-resistant cell lines, we have cultured these cell lines with low-dose Rux (0.2μM), and gradually increased the concentrations of Rux by 0.1μM every week. Cells were considered as resistant when the half-maximal inhibitory concentrations (IC₅₀) of the cells was double the IC₅₀ of the parental cells. We have successfully established Rux-resistant cells that proliferated

in the presence of Rux at 0.8μM. Then, we characterized the Rux-resistant cells with CALR-fs/exMPL. Next, reversibility of Rux-resistance, the Rux-resistant cells was examined.

To examine whether Rux-resistant cells shows the resistant-phenotype in vivo, we have subcutaneously implanted Rux-resistant cells as well as Rux-sensitive cells into immunocompromised mice and treated the animals with Rux. Three weeks after injections of the tumor cells, the mice were euthanized, and the subcutaneous tumors were excised and weighted. Finally, we examined pathological features of bone marrow samples of MPN patients with CALR mutations by immunohistochemical staining.

【Results】We found that Rux-resistant cells had overexpressions of both MPL and JAK2, and increased phosphorylations of both JAK2 and STAT5. We found that high levels of MPL transcripts in the resistant cells. Cycloheximide treatment assay showed that mature MPL proteins were more stable in Rux-resistant cells. Then, we examined mechanisms of stability of mature MPL proteins in Rux-resistant cells. Our data suggested that the proteasome/system degrading mature MPL was attenuated in Rux-resistant cells. When Rux-resistant cells were cultured in the absence of Rux, Rux-resistance was reversed with reduction of mature MPL and JAK2. In vivo assay using immunocompromised mice showed the Rux resistant cells were not sufficiently suppressed by Rux-treatment. Since our data using the cell lines suggested that high MPL expression causes resistance to Rux in MPN cells with CALR mutations, we examined levels of MPL proteins in bone marrow samples of MPN patients with CALR mutations by immunohistochemical staining. Immunohistochemical staining of MPL showed that the megakaryocytes of MPN patients with high-risk and/or resistant to ruxolitinib treatment expressed higher MPL.

【Discussion】 MPL overexpression is one of mechanisms of MPN cells with CALR mutations to gain resistance to Rux. This mechanism may be a key element to overcome Rux-resistance in this fatal disease.