Biology subjects

Kandarpa, M.

Publications and source records attributed to Kandarpa, M..

4 recordsLinked to original sources

bioRxiv2025

Effects of Calreticulin Mutations on HLA Class I Expression in Myeloproliferative Neoplasms

Calreticulin (CRT) is important for human leukocyte antigen (HLA) class I assembly. Somatic mutations of the CRT gene (CALR) in hematopoietic lineage cells cause myeloproliferative neoplasms (MPNs). Typically, MPN patient cells have one copy each of the wild-type and mutant CALR allele. We find that heterozygous knock-in of a MPN CALR mutation into human cell lines maintains or slightly induces surface expression of HLA class I allotypes. However, full deficiency of wild-type CRT variably reduces the surface expression of HLA class I allotypes, and MPN CRT mutants fail to restore expression for all tested allotypes. Consistent with the largely heterozygous nature of CALR mutations in MPN, surface HLA class I expression in platelets and monocytes from MPN patients with CALR mutations generally falls within the normal range, with higher average expression measured in monocytes from patients treated with interferon alpha compared with other treatments. Overall, the studies indicate that loss of HLA class I expression in cells deficient in wild-type CRT (the CRT dependency) is allele-dependent and correlates with known effects of the assembly factor tapasin. Furthermore, heterozygous knock-in of a MPN-linked CALR mutation has no effect on some allotypes and slightly induces HLA class I expression for CRT-dependent allotypes.

immunology↗

bioRxiv2025

Identification of Omaveloxolone as An Endoplasmic Reticulum Associated Degradation Inhibitor That Induces Early Apoptotic Signaling in Multiple Myeloma

Endoplasmic reticulum-associated degradation (ERAD) is essential for maintaining protein homeostasis, yet its regulatory mechanisms remain poorly understood. A major challenge in studying ERAD is the lack of specific inhibitors targeting the ERAD complex. To address this, we conducted a cell-based high-throughput screen using the FDA-repurposing library and identified omaveloxolone (RTA408) as a potent ERAD inhibitor that selectively impairs the degradation of ER luminal and membrane substrates. Beyond its utility in identifying ERAD substrates, RTA408 exhibits strong cytotoxic effects in multiple myeloma (MM), an incurable plasma cell malignancy. RTA408 inhibits ERAD activity and rapidly induces apoptotic signaling via caspase 8 and the death-inducing signaling complex (DISC). Notably, RTA408 is cytotoxic to malignant plasma cells, including those resistant to proteasome inhibitors, and demonstrates in vivo anti-myeloma activity. Our findings establish ERAD inhibitors as valuable tools for dissecting ERAD regulation while also highlighting their potential as therapeutic agents for MM. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/646787v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@96254aorg.highwire.dtl.DTLVardef@16a3f6dorg.highwire.dtl.DTLVardef@6a1503org.highwire.dtl.DTLVardef@897c45_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIWe developed a cell-based screening approach to identify novel modulators of endoplasmic reticulum associated degradation (ERAD), with implications in studying ERAD biology and targeting plasma cell neoplasms. C_LIO_LIScreening of the FDA-repurposing library identified Omaveloxolone (RTA408) as an inhibitor of luminal and membrane ERAD substrate degradation, which can be leveraged to identify ERAD substrates. C_LIO_LImaveloxolone treatment rapidly induces the unfolded protein response and apoptosis that is dependent on caspase 8 and death-inducing signaling complex (DISC) in multiple myeloma cells. C_LIO_LImaveloxolone exhibits cytotoxic effects against multiple myeloma cells in vitro and in vivo and induces apoptosis in primary plasma cells from patients with relapsed/refractory myeloma C_LI

cell biology↗

bioRxiv2023

Lysosomal Degradation Pathways Target Mutant Calreticulin and the Thrombopoietin Receptor in Myeloproliferative Neoplasms

Somatic mutants of calreticulin (CRT) drive myeloproliferative neoplasms (MPNs) via binding to the thrombopoietin receptor (MPL) and aberrant activation of the JAK/STAT pathway. Compared with healthy donors, platelets from MPN patients with CRT mutations display low cell surface MPL. Co-expression of MPL with an MPN-linked CRT mutant (CRTDel52) reduces cell surface MPL expression, indicating the involvement of induced protein degradation, a better understanding of which could lead to new therapies. We show that lysosomal degradation is relevant to the turnover of both CRTDel52 and MPL. Drug-mediated activation of lysosomal degradation reduces CRTDel52 and MPL expression, with parallel inhibition of CRTDel52-induced cell proliferation and stem cell colony formation. Thus, reduced surface MPL, a marker of platelets from MPN patients with CRT mutations, results from mutant CRT-induced lysosomal degradation of MPL. Drug-induced activation of lysosomal degradation compromises the pathogenic effects of CRTDel52, which can be further exploited for therapeutic interventions.

cancer biology↗

bioRxiv2020

Mpl is activated by dimers of MPN-linked calreticulin mutants stabilized by disulfide bonds and ionic interactions

Myeloproliferative neoplasms (MPNs) are frequently driven by insertions and deletions within the gene encoding calreticulin (CRT). CRTDel52 and CRTIns5 are recurrent mutations. Although oncogenic transformation requires both mutated CRT and the myeloproliferative leukemia protein (Mpl), the molecular mechanism of CRT-mediated constitutive activation of Mpl is unknown. Our studies reveal that the novel C-domain of CRTDel52 encodes specificity both for Mpl binding and for disulfide-mediated CRT dimerization. Disulfide-stabilized CRTDel52 dimers and multimers are observed in MPN patient-derived platelet lysates and in transfected mammalian cells. Cysteine mutations within both the novel C-domain (C400A and C404A) and the conserved N-domain (C163A) of CRTDel52 are required to reduce disulfide-mediated dimers and multimers of CRTDel52. Based on these data and published structures of crystalized CRT oligomers, we tested the relevance of ionic interactions between charged residues proximal to C163 at the N-domain dimerization interface. Charge alteration at these residues affected dimerization and multimerization of both wild type and CRTDel52. Elimination of intermolecular disulfides and disruption of ionic interactions at both proposed dimerization interfaces was required to abrogate the ability of CRTDel52 to induce cytokine-independent cell proliferation via Mpl. Based on these findings, we propose a structural model of the Mpl-activating CRTDel52 unit as a covalently-linked dimer that is stabilized by disulfides and ionic interactions at both the C-domain and N-domain. MPNs exploit a natural dimerization interface of CRT combined with C-domain gain-of-functions to achieve cell transformation.

cancer biology↗