MPL Overexpression Induces a High Level of Mutant-Calr/MPL Complex: A Novel Mechanism of Ruxolitinib Resistance in Myeloproliferative Neoplasms with Calr 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, causing poor prognosis of MPN patients. Until now, various studies have elucidated the mechanisms of RUX-resistance in JAK2V617F-mutant MPN cells, including 1) increased JAK2 heterodimerization leading to sustained JAK2 activation, 2) JAK2 kinase domain mutations, and 3) JAK1/JAK3 activations. However, mechanisms of RUX-resistance in MPN cells with CALR mutations have not been fully characterized to date. In this study, we have clarified a mechanism of RUX-resistance in MPN tumor cells with CALR mutations. 【Materials and Methods】 At first, we have created several human cell lines with exogenous MPL expressions (exMPL) and CALR +1 frameshift mutations (CALR-fs) by introductions of V5-tagged MPL and/or FLAG-tagged CALR Del52/Ins5 expressing vectors or the CRISPR/Cas9 technology. We have confirmed that these cell lines had increased JAK/STAT signaling and respond to RUX-treatment. 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. 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. 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. Three weeks after injections of the tumor cells, the mice were euthanized, and the subcutaneous tumors pathologically examined. MPL knockdown experiments showed that high levels of MPL were indispensable in the resistant cell lines. Co-immunoprecipitation assaies showed the interactions of mutant CALR and MPL proteins in RUX-resistant cells. To examine reversibility of RUX-resistance, RUX-resistant cells were cultured without RUX for three months. Finally, we examined pathological features of bone marrow samples of MPN patients with CALR mutations by immunohistochemical staining. 【Results】We have found that RUX-resistant cells had high MPL transcripts, overexpression of both immature and mature MPL, and JAK2. We also found that RUX-resistant cells had increased phosphorylations of JAK1, JAK2, JAK3 STAT5, MEK and ERK. In vivo assay using immunocompromised mice showed the immuno