CALR Human

What is CALR and what is its role in normal human cells?

CALR (Calreticulin) is a multifunctional protein primarily located in the endoplasmic reticulum (ER) of human cells. It serves several critical functions including acting as a calcium-binding chaperone protein in the ER, facilitating proper protein folding, maintaining calcium homeostasis, participating in quality control of newly synthesized proteins, and functioning in immune response regulation. In normal human tissues, CALR is expressed ubiquitously but at varying levels. According to GeneCards database analysis, CALR shows particularly high expression in tissues with active protein synthesis and secretion .

How are CALR mutations associated with human diseases?

CALR mutations are predominantly associated with myeloproliferative neoplasms (MPNs), particularly in JAK2 wildtype cases. These mutations present as the main oncogenic drivers in essential thrombocythemia and myelofibrosis . The association between CALR mutations and MPNs follows specific patterns:

• CALR mutations are found in approximately 25-35% of essential thrombocythemia and primary myelofibrosis cases

• CALR mutations are mutually exclusive with JAK2 and MPL mutations in most cases

• Two major types of CALR mutations exist: type 1 (52-bp deletion) and type 2 (5-bp insertion), with type 1 being more common

• The mutations typically occur in exon 9 and result in a frameshift that generates a novel C-terminal peptide sequence

The mutated CALR protein interacts abnormally with the thrombopoietin receptor (MPL), leading to constitutive activation of JAK-STAT signaling, which drives excessive megakaryocyte proliferation and platelet production .

What are the main types of CALR mutations observed in human cancers?

Based on COSMIC database analysis, CALR mutations are found in various cancer types but are most frequently observed in myeloid neoplasms . The main types include:

• Type 1 mutations: 52-bp deletion (p.L367fs*46) - most common in myelofibrosis

• Type 2 mutations: 5-bp insertion (p.K385fs*47) - more common in essential thrombocythemia

These mutations share key characteristics:

• They occur in exon 9 of the CALR gene

• They cause a frameshift that alters the C-terminal domain

• The mutant protein loses its ER retention signal (KDEL)

• The novel C-terminus contains positively charged amino acids instead of the normal negatively charged ones

• Both types lead to constitutive activation of the thrombopoietin receptor (MPL)

In addition to these common types, other insertions/deletions in exon 9 have been identified, collectively referred to as "type 2-like" or "other" mutations.

How is CALR expression regulated in human tissues?

CALR expression is regulated through multiple mechanisms at the transcriptional, post-transcriptional, and post-translational levels. Based on Regulome Explorer analysis , CALR expression correlates with several regulatory factors:

• Transcription factors: Several have been identified that bind to the CALR promoter region

• Endoplasmic reticulum stress: CALR expression increases during ER stress as part of the unfolded protein response (UPR)

• Calcium depletion: Induces CALR upregulation

• Tissue-specific regulatory elements: Different tissues show varying levels of CALR expression

Interestingly, in CALR mutant cells, total CALR protein levels remain relatively unchanged despite the presence of the mutant protein, suggesting intact cell-intrinsic regulation of gene expression . This indicates sophisticated autoregulatory mechanisms that warrant further investigation.

What are the differences in CALR expression between normal and cancerous tissues?

CALR expression shows distinct patterns between normal and cancerous tissues. According to UALCAN database analysis :

• CALR is overexpressed in several cancer types compared to matched normal tissues

• Expression levels correlate with cancer stage in some malignancies

• Some cancers show decreased CALR expression

A comprehensive analysis of CALR expression across various cancer types using GEPIA and UALCAN databases revealed significant upregulation in breast cancer, lung adenocarcinoma, and hepatocellular carcinoma, no significant change in prostate cancer and lung squamous cell carcinoma, and correlation with advanced pathological stages in certain cancers, suggesting involvement in disease progression . These differences in expression patterns suggest cancer-specific roles of CALR that may be exploited for diagnostic or therapeutic purposes.

What mechanisms drive CALR mutation-induced myeloproliferative neoplasms?

CALR mutations drive myeloproliferative neoplasms through several interconnected mechanisms:

• Abnormal MPL activation: Mutant CALR proteins, through their novel positively charged C-terminal domain, abnormally bind to the thrombopoietin receptor (MPL) and induce its dimerization and constitutive activation.

• JAK-STAT pathway hyperactivation: The activated MPL receptor triggers hyperactivation of the JAK-STAT signaling pathway, particularly STAT5, leading to increased transcription of genes involved in megakaryocyte differentiation and proliferation.

• Thrombopoietin independence: CALR-mutated cells demonstrate thrombopoietin-independent megakaryopoiesis, a hallmark of the disease .

• Early HSPC reprogramming: Introduction of CALR mutations enforces early reprogramming of human hematopoietic stem and progenitor cells (HSPCs), altering their differentiation trajectory .

• Endoplasmic reticulum stress induction: CALR mutations trigger an endoplasmic reticulum stress response in HSPCs, which may contribute to disease pathogenesis .

• Chaperone upregulation: Compensatory upregulation of chaperones in response to ER stress creates mutation-specific vulnerabilities, including sensitivity to BiP chaperone inhibition and proteasome inhibition .

These findings highlight the complex nature of CALR mutation-driven pathogenesis and suggest multiple potential intervention points for therapeutic development.

How do heterozygous CALR mutations affect human hematopoietic stem and progenitor cells (HSPCs)?

Heterozygous CALR mutations induce multiple phenotypic and functional changes in human HSPCs:

• Lineage bias: CALR mutations induce myeloid-lineage skewing, particularly enhancing megakaryocyte differentiation while relatively preserving other lineages .

• Altered signaling: Enhanced JAK-STAT signaling in response to thrombopoietin, with constitutive STAT5 activation even in the absence of cytokine stimulation.

• Transcriptional reprogramming: Early reprogramming of HSPCs with altered gene expression profiles related to megakaryopoiesis, inflammation, and cellular stress .

• ER stress response: Induction of endoplasmic reticulum stress response pathways, including upregulation of chaperones and components of the unfolded protein response .

• Thrombopoietin independence: Development of thrombopoietin-independent megakaryocyte colony formation and differentiation .

• CD41+ progenitor expansion: Specific expansion of CD41+ megakaryocyte-primed progenitor cells, which may serve as disease-propagating cells .

• In vivo disease recapitulation: When xenografted into immunodeficient mice, heterozygous CALR-mutated HSPCs recapitulate multiple MPN hallmarks, including splenomegaly and bone marrow fibrosis .

These findings demonstrate that CALR mutations fundamentally alter HSPC behavior from the earliest stages of hematopoiesis, providing insight into disease initiation mechanisms.

What are the limitations of current mouse models in studying human CALR mutations?

Current mouse models of CALR mutations have several significant limitations that hamper translation to human disease:

• Lack of full disease phenotype: Mouse models expressing human CALR mutants show thrombocytosis and megakaryocyte hyperplasia but often fail to develop robust reticulin fibrosis in the bone marrow .

• No competitive advantage: Unlike in human disease, CALR-mutated HSPCs in mice do not demonstrate a strong competitive advantage upon transplantation .

• Species-specific differences: Important biological characteristics differ between species, most notably the binding capacity of mutant CALR to the thrombopoietin receptor (MPL), which affects downstream signaling intensity .

• Ectopic overexpression issues: Many mouse models use retroviral overexpression systems that may not accurately reflect physiological expression levels .

• Cross-species protein interactions: Human CALR mutants may interact differently with mouse cellular components compared to human counterparts.

These limitations underscore the need for developing humanized models that more accurately recapitulate the human disease, such as the CRISPR/Cas9 and adeno-associated virus (AAV)-mediated gene editing approach described in the research .

How does CALR mutation induce endoplasmic reticulum stress response?

CALR mutations induce endoplasmic reticulum (ER) stress response through several potential mechanisms:

• Altered chaperone function: Mutant CALR has impaired calcium-binding capacity and chaperone function, potentially leading to accumulation of misfolded proteins in the ER.

• Disrupted calcium homeostasis: As CALR is a major calcium-binding protein in the ER, mutations may alter calcium levels and signaling, triggering stress responses.

• Early HSPC reprogramming: Introduction of CALR mutations enforces early reprogramming of human HSPCs, with induction of an endoplasmic reticulum stress response as part of this process .

• Compensatory chaperone upregulation: In response to ER stress, CALR mutant cells show upregulation of other chaperones like BiP, creating a potential vulnerability to chaperone inhibition .

The observation that CALR mutant cells are particularly sensitive to inhibition of the BiP chaperone and the proteasome suggests that these cells exist in a state of elevated ER stress and are dependent on compensatory mechanisms for survival . This represents a potential therapeutic vulnerability that could be exploited for targeted treatment approaches.

What novel therapeutic targets emerge from studying CALR mutations in human cells?

Research on CALR mutations in human cells has revealed several promising therapeutic targets:

• BiP chaperone inhibition: CALR mutant cells show preferential sensitivity to inhibition of the BiP (GRP78) chaperone, which is upregulated as part of the compensatory response to ER stress .

• Proteasome inhibition: CALR mutant cells demonstrate increased sensitivity to proteasome inhibitors, suggesting dependency on protein degradation pathways .

• MPL receptor targeting: Since mutant CALR abnormally activates the MPL receptor, therapies that disrupt this interaction could be effective.

• JAK-STAT pathway inhibition: As downstream effectors of mutant CALR signaling, components of the JAK-STAT pathway represent established therapeutic targets.

• ER stress modulation: Compounds that modulate specific branches of the ER stress response might selectively affect CALR mutant cells.

The gene-engineered human HSPC models provide an excellent platform for testing these therapeutic approaches in a physiologically relevant context before advancing to clinical trials .

What gene engineering approaches are most effective for studying CALR mutations in human cells?

The most effective gene engineering approach for studying CALR mutations involves CRISPR/Cas9 and rAAV6-mediated knock-in strategy. This approach enables precise introduction of CALR mutations at their endogenous loci in primary human HSPCs while preserving natural gene regulation .

Key components of this system include:

• CRISPR/Cas9 ribonucleoprotein (RNP) targeting intron 7 of CALR

• Recombinant adeno-associated virus serotype 6 (rAAV6) carrying a CALR mutant or wildtype cDNA donor template flanked with homology arms

• Fluorescent reporter (GFP or BFP) driven by an SFFV promoter for cell tracking

• Homology-directed repair to ensure proper integration

This system achieves:

• Stable on-target integration

• Gene modification efficiency of approximately 25-35%

• Proper expression of both wildtype and mutant CALR transcripts

• Heterozygous genotype mimicking patient mutations

• Preserved cell intrinsic regulation of gene expression

Alternative approaches include lentiviral overexpression systems and transposon-based systems, but these have limitations such as non-physiological expression levels and random integration.

How can bioinformatics tools be utilized to analyze CALR in human cancers?

Several bioinformatics tools and databases are particularly useful for analyzing CALR in human cancers:

• GeneCards (www.genecards.org):

  • Provides comprehensive gene annotation data

  • Offers CALR mRNA expression information in normal human tissues

  • Integrates data from GTEx, BioGPS, and SAGE databases

• UALCAN (http://ualcan.path.uab.edu/analysis.html):

  • Enables comparison of CALR expression between normal and cancerous tissues

  • Provides data based on The Cancer Genome Atlas (TCGA) and MET500 cohort

  • Allows analysis of expression patterns across cancer types and stages

• GEPIA (http://gepia.cancer-pku.cn/index.html):

  • Facilitates exploration of CALR expression in different pathological stages

  • Integrates TCGA and GTEX databases (9,736 tumor tissues and 8,587 normal samples)

  • Provides standardized RNA-seq data analysis

• Kaplan-Meier Plotter (http://www.kmplot.com/analysis/):

  • Analyzes the effect of CALR expression on survival outcomes, particularly relapse-free survival (RFS)

  • Based on GEO, EGA, and TCGA databases

  • Calculates 95% confidence intervals and p-values for survival differences

• COSMIC (https://cancer.sanger.ac.uk/cosmic/):

  • Catalogs CALR mutation types across different cancers

  • Provides comprehensive mutation data including coding mutations, gene fusions, and expression variants

These tools collectively enable comprehensive analysis of CALR expression, mutation patterns, prognostic significance, and functional relationships across various cancer types.

What assays are most informative for assessing CALR mutation effects on hematopoiesis?

Several assays provide valuable insights into the effects of CALR mutations on hematopoiesis:

Table 1: Assays for Evaluating CALR Mutation Effects on Hematopoiesis

These assays, particularly when used in combination, provide comprehensive assessment of the complex effects of CALR mutations on hematopoietic stem and progenitor cell biology, lineage commitment, and disease development .

How can humanized models of CALR mutations be developed to overcome limitations of mouse models?

Developing humanized models of CALR mutations involves several strategies to overcome the limitations of traditional mouse models:

• Gene-engineered primary human HSPCs:

  • Utilize CRISPR/Cas9 and rAAV6-mediated knock-in to introduce CALR mutations into human CD34+ HSPCs

  • Maintain heterozygous mutation state, preserving endogenous expression regulation

  • Track modified cells using fluorescent reporters

• Xenotransplantation approaches:

  • Transplant gene-modified human HSPCs into immunodeficient mice (e.g., NSG or NSGS strains)

  • Create chimeric animals with human hematopoietic system bearing CALR mutations

  • Monitor disease development in vivo, including thrombocytosis, splenomegaly, and fibrosis

• Patient-derived xenografts (PDX):

  • Directly transplant primary cells from CALR-mutated MPN patients into immunodeficient mice

  • Maintain the complex cellular heterogeneity of patient samples

  • Study disease progression and drug responses

The humanized model using CRISPR/Cas9 and rAAV6-mediated gene editing in primary human HSPCs has successfully recapitulated many disease hallmarks, including thrombopoietin-independent megakaryopoiesis, myeloid-lineage skewing, splenomegaly, bone marrow fibrosis, and expansion of megakaryocyte-primed CD41+ progenitors . This approach overcomes the cross-species differences, ectopic overexpression issues, and lack of disease penetrance that hamper traditional mouse models.

How to resolve contradictions in CALR expression data across different cancer types?

Researchers may encounter contradictory data regarding CALR expression across different cancer types. These contradictions can be methodically addressed through:

• Meta-analysis approach:

  • Integrate data from multiple databases (UALCAN, GEPIA, TCGA)

  • Weight studies by sample size and methodological quality

  • Apply statistical methods to resolve discrepancies

• Experimental validation:

  • Verify expression using multiple techniques (qPCR, Western blot, immunohistochemistry)

  • Include appropriate controls and reference genes

  • Test multiple cell lines or patient samples of the same cancer type

• Contextual analysis:

  • Consider cancer subtypes, stages, and patient demographics

  • Examine molecular signatures rather than isolated gene expression

  • Account for tumor heterogeneity and microenvironment effects

• Functional significance assessment:

  • Determine whether expression differences translate to functional outcomes

  • Correlate with clinical parameters and survival data

  • Assess relationship with known cancer pathways

• Technical considerations:

  • Evaluate platform-specific biases (microarray vs. RNA-seq)

  • Consider sample preparation methods and quality control measures

  • Assess data normalization procedures

By systematically addressing these aspects, researchers can reconcile contradictory findings and develop a more nuanced understanding of CALR's role across cancer types.