Signal integration and coincidence detection in the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) cascade: concomitant activation of receptor tyrosine kinases and of LRP-1 leads to sustained ERK phosphorylation via down-regulation of dual specificity phosphatases (DUSP1 and -6).

Diverse stimuli can feed into the MAPK/ERK cascade; this includes receptor tyrosine kinases, G protein-coupled receptors, integrins, and scavenger receptors (LDL receptor-related protein (LRP)). Here, we investigated the consequence of concomitant occupancy of the receptor tyrosine kinases (by EGF, basic FGF, VEGF, etc.) and of LRP family members (by LDL or lactoferrin). The simultaneous stimulation of a receptor tyrosine kinase by its cognate ligand and of LRP-1 (by lactoferrin or LDL) resulted in sustained activation of ERK, which was redirected to the cytoplasm. Accordingly, elevated levels of active cytosolic ERK were translated into accelerated adhesion to vitronectin. The sustained ERK response was seen in several cell types, but it was absent in cells deficient in LRP-1 (but not in cells lacking the LDL receptor). This response was also contingent on the presence of urokinase (uPA) and its receptor (uPAR), because it was absent in uPA(-/-) and uPAR(-/-) fibroblasts. Combined stimulation of the EGF receptor and of LRP-1 delayed nuclear accumulation of phosphorylated ERK. This shift in favor of cytosolic accumulation of phospho-ERK was accounted for by enhanced proteasomal degradation of dual specificity phosphatases DUSP1 and DUSP6, which precluded dephosphorylation of cytosolic ERK. These observations demonstrate that the ERK cascade can act as a coincidence detector to decode the simultaneous engagement of a receptor tyrosine kinase and of LRP-1 and as a signal integrator that encodes this information in a spatially and temporally distinct biological signal. In addition, the findings provide an explanation of why chronic elevation of LRP-1 ligands (e.g. PAI-1) can predispose to cancer.

Control of Metastases via Myeloid CD39 and NK Cell Effector Function.

Natural killer (NK) cell protection from tumor metastases is a critical feature of the host immune response to cancer, but various immunosuppression mechanisms limit NK cell effector function. The ectoenzyme, CD39, expressed on tumor-infiltrating myeloid cells, granulocytes, and lymphocytes, including NK cells, converts extracellular ATP (eATP) into AMP and, thus, potentially suppresses eATP-mediated proinflammatory responses. A CD39-targeting monoclonal antibody (mAb) that inhibits the mouse ectoenzyme CD39 suppressed experimental and spontaneous metastases in a number of different tumor models and displayed superior antimetastatic activity compared with the CD39 inhibitor POM1 and inhibitors and mAbs that block other members of the adenosinergic family (e.g., A2AR and CD73). The antimetastatic activity of anti-CD39 was NK cell and IFNγ dependent, and anti-CD39 enhanced the percentage and quantity of IFNγ produced and CD107a expression in lung-infiltrating NK cells following tumor challenge and anti-CD39 therapy. Using conditional Cd39 gene-targeted mouse strains and adoptive NK cell transfers, we showed that CD39 expressed on bone marrow-derived myeloid cells was essential for anti-CD39's antimetastatic activity, but NK cell expression of CD39 was not critical. The eATP receptor P2X7 and the NALP3 inflammasome, including downstream IL18, were critical in the mechanism of action of anti-CD39, and the frequency of P2X7 and CD39 coexpressing lung alveolar macrophages was specifically reduced 1 day after anti-CD39 therapy. The data provide a mechanism of action involving NK cells and myeloid cells, and anti-CD39 combined with anti-PD-1, NK cell-activating cytokines IL15 or IL2, or an inhibitor of A2AR to effectively suppress tumor metastases.

Immunogenicity and protective efficacy of "Mycobacterium w" against Mycobacterium tuberculosis in mice immunized with live versus heat-killed M. w by the aerosol or parenteral route.

As the disease caused by Mycobacterium tuberculosis continues to be a burden, there is a concerted effort to find new vaccines to combat this problem. One of the important vaccine strategies is whole bacterial vaccines. This approach relies on multiple antigens and built-in adjuvanticity. Other mycobacterial strains which share cross-reactive antigens with M. tuberculosis have been considered as alternatives to M. bovis for vaccine use. One such strain, "Mycobacterium w", had been evaluated for its immunomodulatory properties in leprosy. A vaccine against leprosy based on killed M. w is approved for human use, where it has resulted in clinical improvement, accelerated bacterial clearance, and increased immune responses to Mycobacterium leprae antigens. M. w shares antigens not only with M. leprae but also with M. tuberculosis, and initial studies have shown that vaccination with killed M. w induces protection against tuberculosis in Mycobacterium bovis BCG responder, as well as BCG nonresponder, strains of mice. Hence, we further studied the protective potential of M. w and the underlying immune responses in the mouse model of tuberculosis. We analyzed the protective efficacy of M. w immunization in both live and killed forms through the parenteral route and by aerosol immunization, compared with that of BCG. Our findings provide evidence that M. w has potential protective efficacy against M. tuberculosis. M. w activates macrophage activity, as well as lymphocytes. M. w immunization by both the parenteral route and aerosol administration gives higher protection than BCG given by the parenteral route in the mouse model of tuberculosis.

The role of NK cells and CD39 in the immunological control of tumor metastases.

Tumor metastases are responsible for death in the majority of cancer patients. Here we have explored the role of the ectonucleotidase CD39 in select models of tumor metastases and further tested the therapeutic anticancer activity of the NTPDase inhibitor sodium polyoxotungstate (POM-1). CD39 was expressed on tumor-infiltrating regulatory T cells (Treg), myeloid cells and some NK cells, and it was upregulated on these cells within tumors early after inoculation in vivo. NK cell numbers and effector functions were increased in globally CD39-deficient mice and also in WT mice treated with POM-1. Dosing with POM-1 suppressed experimental and spontaneous metastases in four different tumor models and was well tolerated. This anti-metastatic activity was completely abrogated in mice, that were depleted of NK cells, had IFNγ neutralized or were deficient in CD39 expression in bone marrow-derived cells. POM-1 was highly effective in suppressing metastases when used in combination with BRAFi/MEKi or anti-PD-1/anti-CTLA-4 or IL-2. These data highlight the importance of the CD39 pathway in suppressing NK cell-mediated anti-tumor immunity and validate further the development of CD39-based therapies in the clinic.

Targeting CD39 in Cancer Reveals an Extracellular ATP- and Inflammasome-Driven Tumor Immunity.

We explored the mechanism of action of CD39 antibodies that inhibit ectoenzyme CD39 conversion of extracellular ATP (eATP) to AMP and thus potentially augment eATP-P2-mediated proinflammatory responses. Using syngeneic and humanized tumor models, we contrast the potency and mechanism of anti-CD39 mAbs with other agents targeting the adenosinergic pathway. We demonstrate the critical importance of an eATP-P2X7-ASC-NALP3-inflammasome-IL18 pathway in the antitumor activity mediated by CD39 enzyme blockade, rather than simply reducing adenosine as mechanism of action. Efficacy of anti-CD39 activity was underpinned by CD39 and P2X7 coexpression on intratumor myeloid subsets, an early signature of macrophage depletion, and active IL18 release that facilitated the significant expansion of intratumor effector T cells. More importantly, anti-CD39 facilitated infiltration into T cell-poor tumors and rescued anti-PD-1 resistance. Anti-human CD39 enhanced human T-cell proliferation and Th1 cytokine production and suppressed human B-cell lymphoma in the context of autologous Epstein-Barr virus-specific T-cell transfer. SIGNIFICANCE: Overall, these data describe a potent and novel mechanism of action of antibodies that block mouse or human CD39, triggering an eATP-P2X7-inflammasome-IL18 axis that reduces intratumor macrophage number, enhances intratumor T-cell effector function, overcomes anti-PD-1 resistance, and potentially enhances the efficacy of adoptive T-cell transfer.This article is highlighted in the In This Issue feature, p. 1631.