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 5

The C-Term peptide represents a tractable approach to target CALR-mutated MPN cells and improve JAK1/2 inhibitors’ potency in vitro.

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The C-Term peptide represents a tractable approach to target CALR-mutate...
(A) Quantification of STAT3/5 phosphorylation levels, by Western blot analysis and flow cytometry, in CD34+ cells isolated from patients with MPN harboring CALR mutations, treated with the C-Term peptide for 1 hour. (B) Quantification of change in MPL-positive cells (%) among CD34+ cells isolated from patients with CALR-mutated MPN treated with the C-Term peptide for 1 hour. JAK/STAT axis analysis by Western blot of MPL-WT-Ba/F3-cells expressing DEL or INS CALR treated with increasing concentrations (0–1 μM) of (C) Rux or (D) CHZ868 (CHZ) alone or in combination with the C-Term peptide for 4 hours. L, light exposure time; D, dark exposure time. IC50 values for MPL-WT-Ba/F3 cells expressing DEL or INS CALR cultured for 48 hours with increasing concentrations of (E) Rux (μM) or (F) CHZ (μM) alone or in combination with the C-Term peptide (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. In A, B, E, and F, mean values ± SEM are represented. Statistical significance was assessed using 2-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Blots are representative of 3 independent experiments. Histone H3 was used as a loading control.