Role of CALR Mutations on MPL/JAK Signaling: New Road for Novel Therapies in MPN

Undergraduate #6
Discipline: Biological Sciences
Subcategory: Cancer Research

Tiffany Merlinksy - Hunter College
Co-Author(s): Justin Whitfield Memorial Sloan Kettering Cancer Center, NY Elodie Pronier PhD, Memorial Sloan Kettering Cancer Center, NY Ross Levine MD, Memorial Sloan Kettering Cancer Center, NY

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Introduction Myeloproliferative Neoplasms (MPN) are clonal hematopoietic disorders characterized by the overproduction of mature blood cells of the myeloid lineage. In a substantive subset of MPN patients, especially those with essential thrombocythemia, two major frameshift mutations in the calreticulin (CALR) gene have been discovered: a 52-bp deletion (52DEL) and a 5-bp insertion (5INS) are observed. Both result in the expression of a novel C-terminal sequence. CALR mutants bind to a crucial receptor involved in platelet differentiation: the thrombopoietin receptor, MPL. CALR binding to MPL is thought to lead to constitutive activation of the downstream JAK-STAT signaling pathway. Hypothesis In this project we aim to improve understanding of the molecular pathogenesis of CALR mutations, and propose novel treatment options for MPN patients. Methods Human cDNA of WT CALR (WT) and mutants (52DEL and 5INS) were fused to an N-terminal maltose binding protein (MBP) cDNA and cloned into a MSCV-GFP expression vector. Retrovirus particles were used to infect the murine Ba/F3 leukemia cell line. The IC50 of Ruxolitinib, a specific JAK2 inhibitor (Rux) was determined for all conditions using increasing drug concentrations (10nM to 1µM) for 48 hours. Cells were treated with a synthetic peptide corresponding to the WT C-terminal part of CALR (New England Peptide Company) and cell growth was monitored for 4 days. Whole cell lysates from treated cells were immunoprecipitated with a MBP antibody (Abcam) and blotted with MPL antibody (Milipore) to determine the impact of treatment on CALR/MPL interaction. Flow Cytometry was used to determine cell surface level of MPL expression after treatment. Results We observed a decreased IC50 in CALR mutant cells treated with the JAK1/2 inhibitor ruxolitinib compared to controls, consistent with increased drug sensitivity. This was associated with a lower phosphorylation rate of both JAK2 and STAT3 upon JAK inhibitor treatment. These results demonstrate that CALR mutated cells are more sensitive to a JAK2 inhibitor than WT cells. We also found that WT CALR outcompetes mutant CALR in MPL binding assays, and used this as a novel therapeutic approach. We demonstrated that expression of a WT C-term CALR peptide treatment disrupts MPL binding by CALR mutants, leading to a decrease in cell surface MPL expression. This was associated with a lower phosphorylation of JAK2 and STAT3 and abrogation of cytokine independent growth . Conclusion Our data demonstrate JAK inhibitor therapy can be repurposed for MPN patients harboring CALR mutations. We have also shown that WT C-term CALR peptide could be used as a novel treatment option for these patients.

Funder Acknowledgement(s): Memorial Sloan Kettering Cancer Center

Faculty Advisor: Elodie Pronier, proniere@mskcc.org

Role: I performed all the technical experiments presented in this project. As well as outlined the hypothesis and questions to be answered and decided which scientific approaches should be used.