Targeting the CALR interactome in myeloproliferative neoplasms
Elodie Pronier, Paolo Cifani, Tiffany R. Merlinsky, Katharine Barr Berman, Amritha Varshini Hanasoge Somasundara, Raajit K. Rampal, John LaCava, Karen E. Wei, Friederike Pastore, Jesper L.V. Maag, Jane Park, Richard Koche, Alex Kentsis, Ross L. Levine
Elodie Pronier, Paolo Cifani, Tiffany R. Merlinsky, Katharine Barr Berman, Amritha Varshini Hanasoge Somasundara, Raajit K. Rampal, John LaCava, Karen E. Wei, Friederike Pastore, Jesper L.V. Maag, Jane Park, Richard Koche, Alex Kentsis, Ross L. Levine
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Research Article Hematology Oncology

Targeting the CALR interactome in myeloproliferative neoplasms

Abstract

Mutations in the ER chaperone calreticulin (CALR) are common in myeloproliferative neoplasm (MPN) patients, activate the thrombopoietin receptor (MPL), and mediate constitutive JAK/STAT signaling. The mechanisms by which CALR mutations cause myeloid transformation are incompletely defined. We used mass spectrometry proteomics to identify CALR-mutant interacting proteins. Mutant CALR caused mislocalization of binding partners and increased recruitment of FLI1, ERP57, and CALR to the MPL promoter to enhance transcription. Consistent with a critical role for CALR-mediated JAK/STAT activation, we confirmed the efficacy of JAK2 inhibition on CALR-mutant cells in vitro and in vivo. Due to the altered interactome induced by CALR mutations, we hypothesized that CALR-mutant MPNs may be vulnerable to disruption of aberrant CALR protein complexes. A synthetic peptide designed to competitively inhibit the carboxy terminal of CALR specifically abrogated MPL/JAK/STAT signaling in cell lines and primary samples and improved the efficacy of JAK kinase inhibitors. These findings reveal what to our knowledge is a novel potential therapeutic approach for patients with CALR-mutant MPN.

Authors

Elodie Pronier, Paolo Cifani, Tiffany R. Merlinsky, Katharine Barr Berman, Amritha Varshini Hanasoge Somasundara, Raajit K. Rampal, John LaCava, Karen E. Wei, Friederike Pastore, Jesper L.V. Maag, Jane Park, Richard Koche, Alex Kentsis, Ross L. Levine

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Figure 3

CALR-mutated cells are sensitive to JAK1/2 inhibitors in vitro and in vivo.

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CALR-mutated cells are sensitive to JAK1/2 inhibitors in vitro and in v...
(A) IC50 values for MPL-WT-Ba/F3 cells expressing EV or WT, DEL, or INS CALR cultured for 48 hours with increased concentration of CHZ868 (CHZ, nM) (n = 3 in triplicate). (B) Percentage (%) of cleaved caspase-3–positive MPL-WT-Ba/F3 cells expressing different CALR mutants 24 hours after treatment with increasing concentrations of CHZ868 (CHZ, nM) (n = 3 in triplicate). (C) JAK/STAT axis analysis by Western blot analysis on total protein extracts from MPL-WT-Ba/F3 cells expressing different CALR constructs cultured with increasing concentrations of CHZ868 (CHZ, μM) as compared with negative controls (EV and WT). (D) Kaplan-Meier survival curve, at death, of BALB/c mice injected (arrows) with MPL-WT-Ba/F3 cells expressing DEL CALR or INS CALR and treated with CHZ868. (E) Box-and-whisker plots of spleen weights (mg), liver weights (g), and WBC (M/μl) of DEL CALR– or INS CALR–expressing cells treated with CHZ868. In box-and-whisker plots, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate 10th and 90th percentiles. Dots outside of box plots represent outliers (F) Mutant allele burden, as reflected by the percentage of GFP+ cells, in the BM and the spleen, at death of BALB/c mice injected with MPL-WT-Ba/F3 cells expressing DEL CALR or INS CALR and treated with CHZ868 (30 mg/kg). CHZ, CHZ868; veh, vehicle (n =10/11 animals per condition). In A, B, and D–F, mean values ± SEM are represented. Statistical significance was assessed using (A, B, E, and F) 1-way ANOVA or (D) Mantel-Cox; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Blots are representative of 3 independent experiments.