Kindlin-1 regulates IL-6 secretion and modulates the immune environment in breast cancer models.

The adhesion protein Kindlin-1 is over-expressed in breast cancer where it is associated with metastasis-free survival; however, the mechanisms involved are poorly understood. Here, we report that Kindlin-1 promotes anti-tumor immune evasion in mouse models of breast cancer. Deletion of Kindlin-1 in Met-1 mammary tumor cells led to tumor regression following injection into immunocompetent hosts. This was associated with a reduction in tumor infiltrating Tregs. Similar changes in T cell populations were seen following depletion of Kindlin-1 in the polyomavirus middle T antigen (PyV MT)-driven mouse model of spontaneous mammary tumorigenesis. There was a significant increase in IL-6 secretion from Met-1 cells when Kindlin-1 was depleted and conditioned media from Kindlin-1-depleted cells led to a decrease in the ability of Tregs to suppress the proliferation of CD8+ T cells, which was dependent on IL-6. In addition, deletion of tumor-derived IL-6 in the Kindlin-1-depleted tumors reversed the reduction of tumor-infiltrating Tregs. Overall, these data identify a novel function for Kindlin-1 in regulation of anti-tumor immunity, and that Kindlin-1 dependent cytokine secretion can impact the tumor immune environment.

Cyclophosphamide depletes tumor infiltrating T regulatory cells and combined with anti-PD-1 therapy improves survival in murine neuroblastoma.

The outcome for children with high-risk neuroblastoma is poor despite intensive multi-modal treatment protocols. Toxicity from current treatments is significant, and novel approaches are needed to improve outcome. Cyclophosphamide (CPM) is a key component of current chemotherapy regimens and is known to have immunomodulatory effects. However, this has not been investigated in the context of tumor infiltrating lymphocytes in neuroblastoma. Using murine models of neuroblastoma, the immunomodulatory effects of low-dose CPM were investigated using detailed immunophenotyping. We demonstrated that CPM resulted in a specific depletion of intratumoral T regulatory cells by apoptosis, and when combined with anti-PD-1 antibody therapy, this resulted in improved therapeutic efficacy. CPM combined with anti-PD-1 therapy was demonstrated to be an effective combinational therapy, with metronomic CPM found to be more effective than single dosing in more resistant tumor models. Overall, this pre-clinical data strongly support clinical evaluation of such combination strategies in neuroblastoma.

A Conformation Selective Mode of Inhibiting SRC Improves Drug Efficacy and Tolerability.

Despite the approval of several multikinase inhibitors that target SRC and the overwhelming evidence of the role of SRC in the progression and resistance mechanisms of many solid malignancies, inhibition of its kinase activity has thus far failed to improve patient outcomes. Here we report the small molecule eCF506 locks SRC in its native inactive conformation, thereby inhibiting both enzymatic and scaffolding functions that prevent phosphorylation and complex formation with its partner FAK. This mechanism of action resulted in highly potent and selective pathway inhibition in culture and in vivo. Treatment with eCF506 resulted in increased antitumor efficacy and tolerability in syngeneic murine cancer models, demonstrating significant therapeutic advantages over existing SRC/ABL inhibitors. Therefore, this mode of inhibiting SRC could lead to improved treatment of SRC-associated disorders. SIGNIFICANCE: Small molecule-mediated inhibition of SRC impairing both catalytic and scaffolding functions confers increased anticancer properties and tolerability compared with other SRC/ABL inhibitors.

Immune characterization of pre-clinical murine models of neuroblastoma.

Immunotherapy offers a potentially less toxic, more tumor-specific treatment for neuroblastoma than conventional cytotoxic therapies. Accurate and reproducible immune competent preclinical models are key to understanding mechanisms of action, interactions with other therapies and mechanisms of resistance to immunotherapy. Here we characterized the tumor and splenic microenvironment of two syngeneic subcutaneous (NXS2 and 9464D), and a spontaneous transgenic (TH-MYCN) murine model of neuroblastoma, comparing histological features and immune infiltrates to previously published data on human neuroblastoma. Histological sections of frozen tissues were stained by immunohistochemistry and immunofluorescence for immune cell markers and tumor architecture. Tissues were dissociated by enzymatic digestion, stained with panels of antibodies to detect and quantify cancer cells, along with lymphocytic and myeloid infiltration by flow cytometry. Finally, we tested TH-MYCN mice as a feasible model for immunotherapy, using prior treatment with cyclophosphamide to create a therapeutic window of minimal residual disease to favor host immune development. Immune infiltration differed significantly between all the models. TH-MYCN tumors were found to resemble immune infiltration in human tumors more closely than the subcutaneous models, alongside similar GD2 and MHC class I expression. Finally, TH-MYCN transgenic mice were administered cyclophosphamide alone or in combination with an anti-GD2 or anti-4-1BB monoclonal antibody, which resulted in increase in survival in both combination therapies. The TH-MYCN transgenic mouse is a promising in vivo model for testing immunotherapy compounds and combination therapy in a preclinical setting.