Somatic mutations in calreticulin (CALR) are present in approximately 40% of patients with myeloproliferative neoplasms (MPN) but the mechanism by which mutant CALR is oncogenic remains unclear. Here, we demonstrate that expression of mutant CALR alone is sufficient to engender MPN in mice and recapitulates the disease phenotype of CALR-mutant MPN patients. We further show that the thrombopoietin receptor, MPL is required for mutant CALR-driven transformation through JAK-STAT pathway activation, thus rendering mutant CALR-transformed hematopoietic cells sensitive to JAK2 inhibition. Finally, we demonstrate that the oncogenicity of mutant CALR is dependent on the positive electrostatic charge of the C-terminus of the mutant protein, which is necessary for physical interaction between mutant CALR and MPL. Together, our findings elucidate a novel paradigm of cancer pathogenesis and reveal how CALR mutations induce MPN.

Cell transplantation into adult zebrafish has lagged behind mouse due to the lack of immune compromised models. Here, we have created homozygous rag2E450fs mutant zebrafish that have reduced numbers of functional T and B cells but are viable and fecund. Mutant fish engraft zebrafish muscle, blood stem cells, and cancers. rag2E450fs mutant zebrafish are the first immune compromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer.

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.

T cell recognition of specific antigens mediates protection from pathogens and controls neoplasias, but can also cause autoimmunity. Our knowledge of T cell antigens and their implications for human health is limited by the technical limitations of T cell profiling technologies. Here, we present T-Scan, a high-throughput platform for identification of antigens productively recognized by T cells. T-Scan uses lentiviral delivery of antigen libraries into cells for endogenous processing and presentation on major histocompatibility complex (MHC) molecules. Target cells functionally recognized by T cells are isolated using a reporter for granzyme B activity, and the antigens mediating recognition are identified by next-generation sequencing. We show T-Scan correctly identifies cognate antigens of T cell receptors (TCRs) from viral and human genome-wide libraries. We apply T-Scan to discover new viral antigens, perform high-resolution mapping of TCR specificity, and characterize the reactivity of a tumor-derived TCR. T-Scan is a powerful approach for studying T cell responses.

T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive malignancy of thymocytes. Using a transgenic screen in zebrafish, thymocyte selection-associated high mobility box protein (TOX) was uncovered as a collaborating oncogenic driver that accelerated T-ALL onset by expanding the initiating pool of transformed clones and elevating genomic instability. TOX is highly expressed in a majority of human T-ALL and is required for proliferation and continued xenograft growth in mice. Using a wide array of functional analyses, we uncovered that TOX binds directly to KU70/80 and suppresses recruitment of this complex to DNA breaks to inhibit Non-Homologous End Joining repair (NHEJ). Impaired NHEJ is well known to cause genomic instability, including development of T cell malignancies in Ku70 and Ku80 deficient mice. Collectively, our work has uncovered important roles for TOX in regulating NHEJ by elevating genomic instability during leukemia initiation and sustaining leukemic cell proliferation following transformation.

Somatic mutations in calreticulin (CALR), an endoplasmic reticulum (ER) chaperone protein, are found in up to 40% of patients with myeloproliferative neoplasms (MPN). All pathologic CALR mutations are out-of-frame insertion and/or deletions (indels) in exon 9, generating a 1 base-pair (bp) frame shift and a common mutant-specific C-terminus, with the most common mutation being a 52 bp deletion (del52). The observation that CALR mutations are mutually exclusive with other MPN-initiating mutations such as JAK2V617F suggests a key pathogenic role for mutant CALR. To determine if mutant CALR alone is sufficient to induce MPN we began by over-expressing CALR-del52 in a retroviral bone marrow transplant (BMT) mouse model. We found that CALR-del52-expressing mice develop thrombocytosis and megakaryocytic hyperplasia, recapitulating the megakaryocyte-specific phenotype of CALR-mutant MPN patients. These findings suggest that the thrombopoietin receptor, MPL plays a key role in the pathogenesis of mutant CALR-driven MPN. To evaluate the role of MPL in mutant CALR driven oncogenesis, we over-expressed CALR-del52 in interleukin-3 (IL-3)-dependent Ba/F3 hematopoietic cells. We found that CALR-del52 over-expression results in transformation to IL3-independent growth only in Ba/F3 cells co-expressing MPL, but not in parental Ba/F3 cells or Ba/F3 cells co-expressing the EPO receptor (EPOR) or the G-CSF receptor (GCSFR). We found similar results in human cytokine-dependent UT-7 cells. We also introduced +1 frameshift mutations into the endogenous Calr locus in Ba/F3-MPL cells using CRISPR/Cas9 gene editing and successfully engendered IL-3 independent growth, indicating that endogenous levels of mutant Calr expression are sufficient for transformation. Together, these data indicate that MPL is specifically required for the transforming capacity of mutant CALR. Using RNA-sequencing followed by gene set enrichment analysis (GSEA), we confirmed that mutant CALR transformed Ba/F3-MPL cells display strong enrichment of Stat5 and Stat3 gene expression signatures. Concordantly, we also saw differential phosphorylation of Stat5 and Stat3 in these cells. Furthermore, we found that the IL-3 independent proliferation of mutant CALR expressing Ba/F3-MPL cells is decreased upon shRNA-mediated knockdown of Jak2, and that differential activation of Stat5 and Stat3 is abrogated by the JAK2 inhibitor, ruxolitinib. Together, these data demonstrate that mutant CALR signals through the JAK/STAT axis downstream of MPL. We next sought to define the specific domains within mutant CALR required for oncogenic transformation. We found that neither expression of the mutant C-terminus alone nor expression of CALR lacking the C-terminus leads to cytokine-independent growth, suggesting that the novel C-terminus is necessary (but not sufficient) for transformation. We therefore generated an extensive series of truncation, domain deletion and point mutations within the C-terminus and assessed their respective transforming capabilities. Surprisingly, we found that the oncogenic activity of mutant CALR is not encoded within a specific sequence or domain of the mutant C-terminus. Rather, we found that the positive electrostatic charge of the mutant C-terminus is critical for its transforming capacity. Mutagenizing all 18 lysine/arginine residues (positively charged) within the C-terminus to a neutral glycine residue abrogates CALR-del52 transformation activity. In contrast, mutagenizing the 18 non-lysine/arginine residues within the C-terminus to glycine does not affect transforming activity, a remarkable finding considering that, in this mutant, 50% of the amino acids have been modified. Finally, using co-immunoprecipitation assays we found that mutant CALR, but not wild-type CALR, physically interacts with MPL, and that neither the mutant C-terminus alone nor mutant CALR lacking the C-terminus can bind to MPL. This suggests that the tertiary structure of mutant CALR is required for binding to MPL. Moreover, we found that the ability of our engineered CALR mutants to bind MPL perfectly correlates with their ability to mediate transformation, suggesting that the interaction with MPL is critical for mutant CALR-mediated transformation. Together, our findings elucidate a novel mechanism of pathogenesis in MPN and provide insights into how CALR mutations drive the development of MPN. Disclosures: No relevant conflicts of interest to declare.

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

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