Mutations in calreticulin () are phenotypic drivers in the pathogenesis of myeloproliferative neoplasms. Mechanistic studies have demonstrated that mutant CALR binds to the thrombopoietin receptor MPL, and that the positive electrostatic charge of the mutant CALR C terminus is required for mutant CALR-mediated activation of JAK-STAT signaling. Here we demonstrate that although binding between mutant CALR and MPL is required for mutant CALR to transform hematopoietic cells; binding alone is insufficient for cytokine independent growth. We further show that the threshold of positive charge in the mutant CALR C terminus influences both binding of mutant CALR to MPL and activation of MPL signaling. We find that mutant CALR binds to the extracellular domain of MPL and that 3 tyrosine residues within the intracellular domain of MPL are required to activate signaling. With respect to mutant CALR function, we show that its lectin-dependent function is required for binding to MPL and for cytokine independent growth, whereas its chaperone and polypeptide-binding functionalities are dispensable. Together, our findings provide additional insights into the mechanism of the pathogenic mutant CALR-MPL interaction in myeloproliferative neoplasms.
The cohesin complex plays an essential role in chromosome maintenance and transcriptional regulation. Recurrent somatic mutations in the cohesin complex are frequent genetic drivers in cancer, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Here, using genetic dependency screens of stromal antigen 2–mutant ( STAG2 -mutant) AML, we identified DNA damage repair and replication as genetic dependencies in cohesin-mutant cells. We demonstrated increased levels of DNA damage and sensitivity of cohesin-mutant cells to poly(ADP-ribose) polymerase (PARP) inhibition. We developed a mouse model of MDS in which Stag2 mutations arose as clonal secondary lesions in the background of clonal hematopoiesis driven by tet methylcytosine dioxygenase 2 ( Tet2 ) mutations and demonstrated selective depletion of cohesin-mutant cells with PARP inhibition in vivo. Finally, we demonstrated a shift from STAG2- to STAG1-containing cohesin complexes in cohesin-mutant cells, which was associated with longer DNA loop extrusion, more intermixing of chromatin compartments, and increased interaction with PARP and replication protein A complex. Our findings inform the biology and therapeutic opportunities for cohesin-mutant malignancies.
Calreticulin (CALR) is mutated in the majority of JAK2/MPL-unmutated myeloproliferative neoplasms (MPNs). Mutant CALR (CALRdel52) exerts its effect by binding to the thrombopoietin receptor MPL to cause constitutive activation of JAK-STAT signaling. In this study, we performed an extensive mutagenesis screen of the CALR globular N-domain and revealed 2 motifs critical for CALRdel52 oncogenic activity: (1) the glycan-binding lectin motif and (2) the zinc-binding domain. Further analysis demonstrated that the zinc-binding domain was essential for formation of CALRdel52 multimers, which was a co-requisite for MPL binding. CALRdel52 variants incapable of binding zinc were unable to homomultimerize, form CALRdel52-MPL heteromeric complexes, or stimulate JAK-STAT signaling. Finally, treatment with zinc chelation disrupted CALRdel52-MPL complexes in hematopoietic cells in conjunction with preferential eradication of cells expressing CALRdel52 relative to cells expressing other MPN oncogenes. In addition, zinc chelators exhibited a therapeutic effect in preferentially impairing growth of CALRdel52-mutant erythroblasts relative to unmutated erythroblasts in primary cultures of MPN patients. Together, our data implicate zinc as an essential cofactor for CALRdel52 oncogenic activity by enabling CALRdel52 multimerization and interaction with MPL, and suggests that perturbation of intracellular zinc levels may represent a new approach to abrogate the oncogenic activity of CALRdel52 in the treatment of MPNs.
Recurrent mutations in the ER chaperone calreticulin (CALR) are found in ~30% of MPNs. All known CALR mutations result in a +1-frameshift of the CALR reading frame that retains the N-domain and P-arm of wild-type CALR and generates a novel mutant-specific C-terminal domain encoded by an alternative reading frame. Multiple groups have previously demonstrated that mutant CALR (CALRMUT) physically binds to and activates the thrombopoietin receptor MPL and causes inappropriate receptor activation and downstream JAK-STAT signaling in a ligand-independent manner. These oncogenic activities of CALRMUT have been shown to be dependent on a threshold of basic amino acid residues in the mutant-specific C-terminus and on the lectin motif within the N-domain. In this study, we extended the analysis of the role of the N-domain in CALRMUT activity by performing a comprehensive alanine mutagenesis screen of all 177 non-alanine residues within the N-domain and assessed the ability of alanine-mutants to confer cytokine independent growth in Ba/F3-MPL cells. We identified two functional groups of residues as being important for CALRMUT activity: (i) Residues associated with CALR lectin motif. Consistent with previous work which had shown that Asp135 and Asp317 was essential for CALRMUT activity, we observed that loss of any of 4 residues within the lectin motif (Cys105, Lys111, Gly133 and Asp135) led to total impairment in the ability of CALRMUT to confer cytokine independence, bind MPL or elicit JAK-STAT signaling. Surprisingly, other residues implicated in lectin activity (Tyr109, Tyr128, Met131) had either only a partial effect or no effect on CALRMUT oncogenic activity, suggesting that different regions within the lectin motif of CALR have differential effects in facilitating MPL binding. (ii) Residues associated with zinc binding. We also identified a group of zinc-binding histidine residues (His99, His145 and His170) as being important for modulating CALRMUT activity. We observed that loss of any single histidine residue (1xHis) led to partial abrogation of cytokine independence in Ba/F3-MPL cells. Strikingly, combined loss of two (or more) histidines (2xHis) led to complete impairment in the capacity to confer cytokine independent growth or elicit JAK-STAT signaling, in conjunction with impaired ability to physically associate with Zn2+ by affinity chromatography. In addition, 2xHis-CALRMUT failed to bind MPL in pulldown assays or colocalize with MPL in FRET-based immunocytochemical analysis. We next investigated in greater detail how the zinc binding capacity of CALRMUT regulates its oncogenic activity. Previous studies have shown that CALRMUT forms homomultimers (Araki, Leukemia, 33:122), and that zinc is able to increase homomultimerization of recombinant wild-type CALR in vitro (Li, Biochemistry, 40:11193). We therefore tested the multimerization capacity of lectin-deficient or zinc binding-deficient CALRMUT by assessing the ability of heterologously-expressed FLAG-tagged CALRMUT species to complex and co-immunoprecipitate with a V5-tagged CALRMUT species in 293T cells. We observed that lectin-deficient CALRMUT and 1xHis-CALRMUT retained the capacity to form homomultimers, but the 2xHis- CALRMUT could not, suggesting that zinc binding was required for homomultimer formation. Treatment with ZnCl2 which increases free intracellular Zn2+ resulted in increased CALRMUT homomultimerization in a dose-dependent manner, as well as increased binding to MPL within heteromeric complexes. Finally, treatment with the zinc chelator TPEN led to decreased CALRMUT homomultimer formation, decreased CALRMUT-MPL heteromultimeric complexes and attenuated JAK-STAT signaling. Cumulatively, our data suggest that zinc is required for homomultimerization of CALRMUT, and is a necessary prerequisite event to facilitate binding to and activation of MPL in CALR-mutated MPNs. Disclosures Mullally: Janssen: Research Funding.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.