Correcting T-cell immunosuppression may unleash powerful antitumor responses; however, knowledge about the mechanisms and modifiers that may be targeted to improve therapy remains incomplete. Here, we report that polyamine elevation in cancer, a common metabolic aberration in aggressive lesions, contributes significantly to tumor immunosuppression and that a polyamine depletion strategy can exert antitumor effects that may also promote immunity. A polyamine-blocking therapy (PBT) that combines the well-characterized ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO) with AMXT 1501, a novel inhibitor of the polyamine transport system, blocked tumor growth in immunocompetent mice but not in athymic nude mice lacking T cells. PBT had little effect on the proliferation of epithelial tumor cells, but it increased the number of apoptotic cells. Analysis of CD45⁺ tumor immune infiltrates revealed that PBT decreased levels of Gr-1⁺CD11b⁺ myeloid suppressor cells and increased CD3⁺ T cells. Strikingly, in a model of neoadjuvant therapy, mice administered with PBT one week before surgical resection of engrafted mammary tumors exhibited resistance to subsequent tumor rechallenge. Collectively, our results indicate that therapies targeting polyamine metabolism do not act exclusively as antiproliferative agents, but also act strongly to prevent immune escape by the tumor. PBT may offer a general approach to heighten immune responses in cancer. Cancer Immunol Res; 2(3); 274–85. ©2013 AACR.

A novel polyamine blockade therapy activates an anti-tumor immune response A novel polyamine blockade therapy activates an anti-tumor immune response

Ornithine decarboxylase (ODC) is aberrantly regulated in tumor cells and results in high basal levels of ODC and polyamines in many epithelial tumors. To determine if elevated ODC/polyamine levels can co-operate with a mutant Ha-ras gene in mouse skin tumorigenesis, double transgenic mice were generated by breeding K6/ODC transgenic mice with TG.AC v-Ha-ras transgenic mice. A K6 keratin promoter drives the ODC transgene in K6/ ODC transgenic mice, which results in elevated ODC/ polyamine levels directed to the outer root sheath cells of hair follicles. TG.AC transgenic mice carry a v-Ha-ras transgene while still retaining two normal c-Ha-ras alleles. Transgenic mice that possess only the K6/ODC or the v-Ha-ras transgene did not develop tumors unless treated with either a carcinogen or a tumor promoter, respectively. However, a high percentage of double transgenic mice possessing both the K6/ODC and v-Ha-ras transgenes developed spontaneous tumors. All tumors were well-differentiated keratoacanthomas, some of which progressed to carcinomas within 2 months. The development and the maintenance of these ODC/ras tumors was ODC-dependent since alpha-difluoromethylornithine (DFMO), a specific ODC inhibitor, prevented the formation and caused the regression of these tumors. These findings indicate that ODC overexpression and an activated Ha-ras are sufficient to produce a high rate of malignant transformation in an animal model. The ODC/ras double transgenic mouse provides a simple in vivo model without the use of chemical carcinogens or tumor promoters in which to test downstream effectors that play a key role in mediating the development of epithelial tumors resulting from the cooperation between ODC and v-Ha-ras.

Despite unprecedented advances in the treatment of cancer through the use of immune checkpoint blockade (ICB), responses are not universal and alternative strategies are needed to enhance responses to ICB. We have shown previously that a novel polyamine blocking therapy (PBT), consisting of cotreatment with α-difluoromethylornithine (DFMO) to block polyamine biosynthesis and a Trimer polyamine transport inhibitor, decreases myeloid-derived suppressor cells (MDSC) and M2-like tumor-associated macrophages (TAM). Both MDSCs and TAMs promote tumor progression, inhibit antitumor immunity, and limit the efficacy of ICB. In this study, we investigated the use of PBT to heighten therapeutic responses to PD-1 blockade in mice bearing 4T1 mammary carcinoma and B16F10 melanoma tumors. Whereas PBT inhibited primary tumor growth in both tumor models, 4T1 lung metastases were also dramatically decreased in mice treated with PBT. Reductions in MDSC and TAM subpopulations in 4T1 tumors from PBT-treated mice were accompanied by reduced cytoprotective autophagy only in tumor-infiltrating MDSC and macrophage subpopulations but not in the lung or spleen. PBT treatment blunted M2-like alternative activation of bone marrow–derived macrophages and reduced STAT3 activation in MDSC cultures while increasing the differentiation of CD80+, CD11c+ macrophages. PBT significantly enhanced the antitumor efficacy of PD-1 blockade in both 4T1 and B16F10 tumors resistant to anti–PD-1 monotherapy, increasing tumor-specific cytotoxic T cells and survival of tumor-bearing animals beyond that with PBT or PD-1 blockade alone. Our results suggest that cotreatment with DFMO and the Trimer polyamine transport inhibitor may improve the therapeutic efficacy of immunotherapies in patients with cancer with resistant tumors.

Coagulation proteases and the generation of thrombin are increased in breast tumor epithelial and stromal cells. Since thrombin can modify tumor cell behavior directly through the activation of pro...