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 2

CALR mutants’ binding to a subset of partners affects their cellular localization and influences MPL transcription.

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CALR mutants’ binding to a subset of partners affects their cellular loc...
(A) Cytoplasm/membrane extracts and (B) nucleus/chromatin extracts from EV or WT CALR–, DEL CALR–, and INS CALR–expressing MPL-WT-Ba/F3 cells were used to detect, by Western blot analysis, cellular localization of CALR (MBP) and its partners: BiP, Myl9, Fli1, and Erp57. L, light exposure time; D, dark exposure time. (C) Cytoplasm (Cyto) and nuclear (Nuc) extracts from mononuclear cells isolated from healthy donors (CTRL) or patients with CALR-mutated MPN. (D) Quantitative reverse transcription PCR (qRT-PCR) analysis of MPL expression in MPL-WT-Ba/F3 cells expressing WT, DEL, or INS CALR compared with EV control. qRT-PCR analysis of ChIP assays showing (E) Fli1, (F) Erp57, and (G) CALR binding to the Mpl promoter in MPL-WT-Ba/F3 cells expressing WT, DEL, or INS CALR. Data are expressed as the percentage of pre-immunoprecipitation input for each sample and are representative of at least 3 independent immunoprecipitations. (H) qPCR analysis of Mpl in MPL-WT-Ba/F3 cells expressing EV or WT, DEL, or INS CALR transduced by Fli1 or Erp57 shRNA compared with Renilla controls (CTRL). (I) Signal distribution of Fli1 ChIP-seq peaks’ intensity in EV or WT CALR–, DEL CALR–, or INS CALR–expressing cells. (J) Mean peak intensity of Fli1 peaks per the following genomic regions: 3′-UTR, 5′-UTR, promoter TSSs, and TSSs. TSS, transcription start site for MPL-WT-Ba/F3 cells expressing EV or WT, DEL, or INS CALR. In D–H, mean values ± SEM are represented. Statistical significance was assessed using 1-way ANOVA. *P < 0.05, **P < 0.01, and ****P < 0.0001. Blots are representative of 3 independent experiments. Gapdh or histone H3 was used as a loading control. Gapdh was used as a housekeeping gene (n = 3 in triplicate).