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Microbes have been coevolving with their host for millions of years, exploiting host resources to their own benefit. We show that viral and bacterial pathogens convergently evolved to hijack cellular mitogen-activated protein kinase (MAPK) p90-ribosomal S6-kinases (RSKs). Theiler's virus leader (L) protein binds RSKs and prevents their dephosphorylation, thus maintaining the kinases active. Recruitment of RSKs enables L-protein-mediated inhibition of eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2 or PKR) and stress granule formation. Strikingly, ORF45 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) and YopM protein of use the same peptide motif as L to recruit and activate RSKs. All three proteins interact with a conserved surface-located loop of RSKs, likely acting as an allosteric regulation site. Some unrelated viruses and bacteria thus evolved to harness RSKs in a common fashion, yet to target distinct aspects of innate immunity. As documented for Varicella zoster virus ORF11, additional pathogens likely evolved to hijack RSKs, using a similar short linear motif.

Frameshifting mutations (−1/+2) of the calreticulin (CALR) gene are responsible for the development of essential thrombocythemia (ET) and primary myelofibrosis (PMF). The mutant CALR proteins activate the thrombopoietin receptor (TpoR) inducing cytokine-independent megakaryocyte progenitor proliferation. Here, we generated via CRISPR/Cas9 technology two knock-in mouse models that are heterozygous for a type-I murine Calr mutation. These mice exhibit an ET phenotype with elevated circulating platelets compared with wild-type controls, consistent with our previous results showing that murine CALR mutants activate TpoR. We also show that the mutant CALR proteins can be detected in plasma. The phenotype of Calr del52 is transplantable, and the Calr mutated hematopoietic cells have a slow-rising advantage over wild-type hematopoiesis. Importantly, a homozygous state of a type-1 Calr mutation is lethal at a late embryonic development stage, showing narrowed ventricular myocardium walls, similar to the murine Calr knockout phenotype, pointing to the C terminus of CALR as crucial for heart development.

Calreticulin (CALR) +1 frameshift mutations in exon 9 are prevalent in myeloproliferative neoplasms. Mutant CALRs possess a new C-terminal sequence rich in positively charged amino acids, leading to activation of the thrombopoietin receptor (TpoR/MPL). We show that the new sequence endows the mutant CALR with rogue chaperone activity, stabilizing a dimeric state and transporting TpoR and mutants thereof to the cell-surface in states that would not pass quality control, and this function is absolutely required for oncogenic transformation. Mutant CALRs determine traffic via the secretory pathway of partially immature TpoR, as they protect N117-linked glycans from further processing in the Golgi. A number of engineered or disease-associated TpoRs, such as TpoR/MPL R102P, which causes congenital thrombocytopenia are rescued for traffic and function by mutant CALRs, which can also overcome endoplasmic reticulum retention signals on TpoR. Besides requiring N-glycosylation of TpoR, mutant CALRs require a hydrophobic patch located in the extracellular domain of TpoR to induce TpoR thermal stability and initial intracellular activation, while full activation requires cell surface localization of TpoR. Thus, mutant CALRs are rogue chaperones for TpoR and traffic-defective TpoR mutants, a function required for the oncogenic effects

Cancer therapy is experiencing a paradigm shift due to the clinical success of immune checkpoint inhibitors (ICI), yet efficacy remains limited to a few tumor types and a minority of patients. In an increasing number of cancers, tumor populations called cancer stem cells (CSCs) or tumor-initiating cells (TIC) have been defined. Strong evidence is supporting the immunoresistance feature of CSCs. CSCs can also assist immunosuppression in the Tumor microenvironment (TME) through interaction with other tumor stroma cells. Understanding the mechanisms leading to the immunotolerance of CSCs will be essential for treatment of immune-resistant and metastatic tumors. The identification of such mechanisms faces challenges when applying the widely used transplanted tumor models since they fail to recapitulate the tumor microenvironment as it develops during the progression of human tumors. We previously generated a transgenic model of autochthonous melanoma, named TiRP, which express murine MAGE-type antigen P1A, and can be used as a more faithful preclinical model for immunotherapy. In this model, the CSCs and CTCs are immune resistant. They are devoid of surface MHC I expression and are resistant to both antigen-specific T-cells-and NK-cells-mediated killing. In contrast to non-CSC tumor clones from the TiRP tumor (T429.11), CSCs/CTCs, when transplanted into recipient mice, generate a highly immunosuppressive TME with high numbers of PMN-and Monocytic-MDSCs, and are resistant to immune therapy.

DNA vaccination against cancer has become a promising strategy for inducing a specific and long-lasting antitumor immunity. However, DNA vaccines fail to generate potent immune responses when used as a single therapy. To enhance their activity into the tumor, a DNA vaccine against murine P815 mastocytoma was combined with antibodies directed against the immune checkpoints CTLA4 and PD1. The combination of these two strategies delayed tumor growth and enhanced specific antitumor immune cell infiltration in comparison to the corresponding single therapies. The combination also promoted IFNg, IL12 and granzyme B production in the tumor microenvironment and decreased the formation of liver metastasis in a very early phase of tumor development, enabling 90% survival. These results underline the complementarity of DNA vaccination and immune checkpoint blockers in inducing a potent immune response, by exploiting the generation of antigen-specific T cells by the vaccine and the ability of immune checkpoint blockers to enhance T cell activity and infiltration in the tumor. These findings suggest how and why a rational combination therapy can overcome the limits of DNA vaccination but could also allow responses to immune checkpoint blockers in a larger proportion of subjects.