Mesothelin\u27s minimal MUC16 binding moiety converts TR3 into a potent cancer therapeutic via hierarchical binding events at the plasma membrane

TRAIL has been extensively explored as a cancer drug based on its tumor-selective activity profile but it is incapable per se of discriminating between death receptors expressed by normal host cells and transformed cancer cells. Furthermore, it is well documented that surface tethering substantially increases its biologic activity. We have previously reported on Meso-TR3, a constitutive TRAIL trimer targeted to the biomarker MUC16 (CA125), in which the entire ectodomain of human mesothelin was genetically fused to the TR3 platform, facilitating attachment to the cancer cells via the MUC16 receptor. Here, we designed a truncation variant, in which the minimal 64 amino acid MUC16 binding domain of mesothelin was incorporated into TR3. It turned out that the dual-domain biologic Meso64-TR3 retained its high MUC16 affinity and bound to the cancer cells quickly, independent of the TR3/death receptor interaction. Furthermore, it was substantially more potent than Meso-TR3 and TR3 in vitro and in a preclinical xenograft model of MUC16-dependent ovarian cancer. Phenotypically, Meso64-TR3 is more closely related to non-targeted TR3, evident by indistinguishable activity profiles on MUC16-deficient cancers and similar thermal stability characteristics. Overall, Meso64-TR3 represents a fully human, MUC16-targetd TRAIL-based biologic, ideally suited for exploring preclinical and clinical evaluation studies in MUC16-dependent malignancies

Novel treatment option for MUC16-positive malignancies with the targeted TRAIL-based fusion protein Meso-TR3

BACKGROUND: The targeted delivery of cancer therapeutics represents an ongoing challenge in the field of drug development. TRAIL is a promising cancer drug but its activity profile could benefit from a cancer-selective delivery mechanism, which would reduce potential side effects and increase treatment efficiencies. We recently developed the novel TRAIL-based drug platform TR3, a genetically fused trimer with the capacity for further molecular modifications such as the addition of tumor-directed targeting moieties. MUC16 (CA125) is a well characterized biomarker in several human malignancies including ovarian, pancreatic and breast cancer. Mesothelin is known to interact with MUC16 with high affinity. In order to deliver TR3 selectively to MUC16-expressing cancers, we investigated the possibility of targeted TR3 delivery employing the high affinity mesothelin/MUC16 ligand/receptor interaction. METHODS: Using genetic engineering, we designed the novel cancer drug Meso-TR3, a fusion protein between native mesothelin and TR3. The recombinant proteins were produced with mammalian HEK293T cells. Meso-TR3 was characterized for binding selectivity and killing efficacy against MUC16-positive cancer cells and controls that lack MUC16 expression. Drug efficacy experiments were performed in vitro and in vivo employing an intraperitoneal xenograft mouse model of ovarian cancer. RESULTS: Similar to soluble mesothelin itself, the strong MUC16 binding property was retained in the Meso-TR3 fusion protein. The high affinity ligand/receptor interaction was associated with a selective accumulation of the cancer drug on MUC16-expressing cancer targets and directly correlated with increased killing activity in vitro and in a xenograft mouse model of ovarian cancer. The relevance of the mesothelin/MUC16 interaction for attaching Meso-TR3 to the cancer cells was verified by competitive blocking experiments using soluble mesothelin. Mechanistic studies using soluble DR5-Fc and caspase blocking assays confirmed engagement of the extrinsic death receptor pathway. Compared to non-targeted TR3, Meso-TR3 displayed a much reduced killing potency on cells that lack MUC16. CONCLUSIONS: Soluble Meso-TR3 targets the cancer biomarker MUC16 in vitro and in vivo. Following attachment to the tumor via surface bound MUC16, Meso-TR3 acquires full activation with superior killing profiles compared to non-targeted TR3, while its bioactivity is substantially reduced on cells that lack the tumor marker. This prodrug phenomenon represents a highly desirable property because it has the potential to enhance cancer killing with fewer side-effects than non-targeted TRAIL-based therapeutics. Thus, further exploration of this novel fusion protein is warranted as a possible therapeutic for patients with MUC16-positive malignancies

Stromal senescence establishes an immunosuppressive microenvironment that drives tumorigenesis

Age is a significant risk factor for the development of cancer. However, the mechanisms that drive age-related increases in cancer remain poorly understood. To determine if senescent stromal cells influence tumorigenesis, we develop a mouse model that mimics the aged skin microenvironment. Using this model, here we find that senescent stromal cells are sufficient to drive localized increases in suppressive myeloid cells that contributed to tumour promotion. Further, we find that the stromal-derived senescence-associated secretory phenotype factor interleukin-6 orchestrates both increases in suppressive myeloid cells and their ability to inhibit anti-tumour T-cell responses. Significantly, in aged, cancer-free individuals, we find similar increases in immune cells that also localize near senescent stromal cells. This work provides evidence that the accumulation of senescent stromal cells is sufficient to establish a tumour-permissive, chronic inflammatory microenvironment that can shelter incipient tumour cells, thus allowing them to proliferate and progress unabated by the immune system

Tumor-induced STAT3 activation in monocytic myeloid-derived suppressor cells enhances stemness and mesenchymal properties in human pancreatic cancer

Pancreatic cancer (PC) mobilizes myeloid cells from the bone marrow to the tumor where they promote tumor growth and proliferation. Cancer stem cells (CSCs) are a population of tumor cells that are responsible for tumor initiation. Aldehyde dehydrogenase-1 activity in PC identifies CSCs, and its activity has been correlated with poor overall prognosis in human PC. Myeloid cells have been shown to impact tumor stemness, but the impact of immunosuppressive tumor-infiltrating granulocytic and monocytic myeloid-derived suppressor cells (Mo-MDSC) on ALDH1(Bright) CSCs and epithelial to mesenchymal transition is not well understood. In this study, we demonstrate that Mo-MDSC (CD11b(+)/Gr1(+)/Ly6G(−)/Ly6C(hi)) significantly increase the frequency of ALDH1(Bright) CSCs in a mouse model of PC. Additionally, there was significant upregulation of genes associated with epithelial to mesenchymal transition. We also found that human PC converts CD14(+) peripheral blood monocytes into Mo-MDSC (CD14(+)/HLA-DR(low/−)) in vitro, and this transformation is dependent on the activation of the STAT3 pathway. In turn, these Mo-MDSC increase the frequency of ALDH1(Bright) CSCs and promote mesenchymal features of tumor cells. Finally, blockade of STAT3 activation reversed the increase in ALDH1(Bright) CSCs. These data suggest that the PC tumor microenvironment transforms monocytes to Mo-MDSC by STAT3 activation, and these cells increase the frequency of ALDH1(Bright) CSCs. Therefore, targeting STAT3 activation may be an effective therapeutic strategy in targeting CSCs in PC. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00262-014-1527-x) contains supplementary material, which is available to authorized users

The Genome Sequence of the Leaf-Cutter Ant Atta cephalotes Reveals Insights into Its Obligate Symbiotic Lifestyle

Leaf-cutter ants are one of the most important herbivorous insects in the Neotropics, harvesting vast quantities of fresh leaf material. The ants use leaves to cultivate a fungus that serves as the colony's primary food source. This obligate ant-fungus mutualism is one of the few occurrences of farming by non-humans and likely facilitated the formation of their massive colonies. Mature leaf-cutter ant colonies contain millions of workers ranging in size from small garden tenders to large soldiers, resulting in one of the most complex polymorphic caste systems within ants. To begin uncovering the genomic underpinnings of this system, we sequenced the genome of Atta cephalotes using 454 pyrosequencing. One prediction from this ant's lifestyle is that it has undergone genetic modifications that reflect its obligate dependence on the fungus for nutrients. Analysis of this genome sequence is consistent with this hypothesis, as we find evidence for reductions in genes related to nutrient acquisition. These include extensive reductions in serine proteases (which are likely unnecessary because proteolysis is not a primary mechanism used to process nutrients obtained from the fungus), a loss of genes involved in arginine biosynthesis (suggesting that this amino acid is obtained from the fungus), and the absence of a hexamerin (which sequesters amino acids during larval development in other insects). Following recent reports of genome sequences from other insects that engage in symbioses with beneficial microbes, the A. cephalotes genome provides new insights into the symbiotic lifestyle of this ant and advances our understanding of host–microbe symbioses

CD73 and PD-L1 dual blockade amplifies antitumor efficacy of SBRT in murine PDAC models

Background Stereotactic body radiotherapy (SBRT) induces immunogenic cell death, leading to subsequent antitumor immune response that is in part counterbalanced by activation of immune evasive processes, for example, upregulation of programmed cell death-ligand 1 (PD-L1) and adenosine generating enzyme, CD73. CD73 is upregulated in pancreatic ductal adenocarcinoma (PDAC) compared with normal pancreatic tissue and high expression of CD73 in PDACs is associated with increased tumor size, advanced stage, lymph node involvement, metastasis, PD-L1 expression and poor prognosis. Therefore, we hypothesized that blockade of both CD73 and PD-L1 in combination with SBRT might improve antitumor efficacy in an orthotopic murine PDAC model.Methods We assessed the combination of systemic blockade of CD73/PD-L1 and local SBRT on tumor growth in primary pancreatic tumors, and investigated systemic antitumor immunity using a metastatic murine model bearing both orthotopic primary pancreatic tumor and distal hepatic metastases. Immune response was quantified by flow cytometric and Luminex analyses.Results We demonstrated that blockade of both CD73 and PD-L1 significantly amplified the antitumor effect of SBRT, leading to superior survival. The triple therapy (SBRT+anti-CD73+anti-PD-L1) modulated tumor-infiltrating immune cells with increases of interferon-γ+CD8+ T cells. Additionally, triple therapy reprogramed the profile of cytokines/chemokines in the tumor microenvironment toward a more immunostimulatory phenotype. The beneficial effects of triple therapy are completely abrogated by depletion of CD8+ T cells, and partially reversed by depletion of CD4+ T cells. Triple therapy promoted systemic antitumor responses illustrated by: (1) potent long-term antitumor memory and (2) enhanced both primary and liver metastases control along with prolonged survival