Hematoxylin binds to mutant calreticulin and disrupts its abnormal interaction with thrombopoietin receptor

Abstract

Somatic mutations of calreticulin (CALR)have been identified as one of the main disease drivers of myeloproliferative neoplasms (MPNs), suggesting that developing drugs targeting mutant CALR is of great significance. Site-directed mutagenesis in the N-glycan binding domain (GBD)abolishes the ability of mutant CALRto oncogenically activate the thrombopoietin receptor (MPL).We thus hypothesized that a small molecule targeting the GBD might inhibit the oncogenicity of the mutant CALR. Using an in-silico molecular docking study, we identified candidate binders to the GBD of CALR. Further experimental validation of the hits identified a group of catechols inducing selective growth inhibitory effect on cells that depend on oncogenic CALRs for survival and proliferation. Apoptosis-inducing effects by the compound were significantly higher in the CALR mutated cells than in CALR wild type cells. Additionally, knockout or C-terminal truncation of CALR abolished the drug hypersensitivity in CALR mutated cells. We experimentally confirmed the direct binding of the selected compound to CALR, the disruption of the mutant CALR-MPL interaction, the inhibition of the JAK2-STAT5 pathway, and reduction of intracellular level of mutant CALR upon the drug treatment. Our data conclude that small molecules targeting the GBD of CALR can selectively kill CALR mutated cells by disrupting the CALR-MPL interaction and inhibiting the oncogenic signaling.

Functional Consequences of Mutations in Myeloproliferative Neoplasms

“… 22 , 77 , 78 The interaction between the mutant CALR proteins and MPL leads to thermal stabilization of MPL and can be interrupted by a small molecule that binds to the N-glycan binding domain of CALR. 79 While intracellular complexes of MPL and CALR mutants do induce JAK-STAT signaling, this is not sufficient to transform to autonomous growth cytokine-dependent hematopoietic cells. For this, cell-surface localization of the complex is required and full activation of JAK2-STAT5/STAT3, mitogen-activated protein kinase, and phosphoinositide 3-kinase pathways appears to require cell-surface localization.…”

High-throughput drug screening identifies the ATR-CHK1 pathway as a therapeutic vulnerability of CALR mutated hematopoietic cells

“…Endogenous CALR mutations were generated using CRISPR-Cas9 in UT-7/Tpo and Ba/F3-MPL cell lines. The mutated cell lines were characterized and cultured as previously described [ 16 , 21 ]. In both cases, gRNAs were designed to cut at the site where deletion 52 bp mutation starts.…”

“…The two competing cell lines can be distinguished by FACS, as one expresses mCherry. The establishment of the mCherry expressing cell lines was described previously [ 21 ]. Three biological replicates were performed for each experiment.…”

“…UT-7/Tpo CALR WT, CALR del61/WT cells, and primary CD34+ cells were treated with the indicated drugs for 24 h. Cells were then washed twice with PBS (Gibco, USA). The cell pellets were used for protein extraction and Western blot analysis, as previously described [ 21 ]. All the antibodies used in this assay were listed in Supplementary Table 1 .…”

“…However, none of these features have been exploited sufficiently to develop novel drug treatments. In a recent study, our group demonstrated the possibility of targeting the glycan-binding pocket of mutant CALR protein by small molecules as a chemotherapeutic approach [ 21 ].…”