Targeting the CALR interactome in myeloproliferative neoplasms
Elodie Pronier, … , Alex Kentsis, Ross L. Levine
Elodie Pronier, … , Alex Kentsis, Ross L. Levine
Published November 15, 2018
Citation Information: JCI Insight. 2018;3(22):e122703. https://doi.org/10.1172/jci.insight.122703.
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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 4

CALR-mutant cells are sensitive to a peptide corresponding to the C-terminal sequence of the WT CALR.

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CALR-mutant cells are sensitive to a peptide corresponding to the C-ter...
(A) Proliferation assays of MPL-WT-Ba/F3 cells expressing CALR mutant constructs treated with the C-Term peptide in the absence of IL-3 compared with untreated controls (Unt). Growth curves are means (in total number of viable cells) ± SEM (n = 6 in triplicate). (B) JAK/STAT axis analysis by Western blot analysis of MPL-WT-Ba/F3-cells expressing DEL or INS CALR treated with the C-Term peptide for different incubation times (0–2 hours). (C) Percentage (%) of cleaved caspase-3–positive MPL-WT-Ba/F3 cells expressing CALR mutants after treatment with the C-Term peptide for 4 hours (n = 3 in triplicate). 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. (D) Quantification of change in MPL mean fluorescence index (MFI) in MPL-WT-Ba/F3 cells expressing DEL or INS CALR treated with the C-Term peptide for the indicated time points. (E) Co-immunoprecipitation analysis and (F) quantitative densitometry (AU) of MPL protein expression in 293T cells cotransfected with MPL-WT and MBP-tagged CALR-mutant constructs treated with the peptide for 15 minutes or 1 hour. (G) Western blot analysis of MBP expression in 293T cells transfected with MPL-WT and MBP-tagged CALR-mutant constructs and treated with the C-Term peptide for 1 hour. (H) Cytoplasm/membrane and nucleus/chromatin extracts from EV or WT, DEL, or INS CALR–expressing MPL-WT-Ba/F3 cells were used to detect, by Western blot, cellular localization of CALR partners BiP, Myl9, Fli1, and Erp57 after C-Term treatment for 1 hour. In A, C, D, and F, mean values ± SEM are represented. Statistical significance was assessed using 2-way ANOVA, ****P < 0.0001. Blots are representative of 3 independent experiments. Gapdh, histone H3, and vinculin were used as loading controls.