p16 Represses DNA Damage Repair via a Novel Ubiquitin-Dependent Signaling Cascade.

Squamous cell carcinoma driven by human papillomavirus (HPV) is more sensitive to DNA-damaging therapies than its HPV-negative counterpart. Here, we show that p16, the clinically used surrogate for HPV positivity, renders cells more sensitive to radiotherapy via a ubiquitin-dependent signaling pathway, linking high levels of this protein to increased activity of the transcription factor SP1, increased HUWE1 transcription, and degradation of ubiquitin-specific protease 7 (USP7) and TRIP12. Activation of this pathway in HPV-positive disease led to decreased homologous recombination and improved response to radiotherapy, a phenomenon that can be recapitulated in HPV-negative disease using USP7 inhibitors in clinical development. This p16-driven axis induced sensitivity to PARP inhibition and potentially leads to "BRCAness" in head and neck squamous cell carcinoma (HNSCC) cells. Thus, these findings support a functional role for p16 in HPV-positive tumors in driving response to DNA damage, which can be exploited to improve outcomes in both patients with HPV-positive and HPV-negative HNSCC. SIGNIFICANCE: In HPV-positive tumors, a previously undiscovered pathway directly links p16 to DNA damage repair and sensitivity to radiotherapy via a clinically relevant and pharmacologically targetable ubiquitin-mediated degradation pathway.

IDO1 Inhibition Overcomes Radiation-Induced "Rebound Immune Suppression" by Reducing Numbers of IDO1-Expressing Myeloid-Derived Suppressor Cells in the Tumor Microenvironment.

PURPOSE: The limitation of hypofractionated radiation efficacy is due partly to the immunosuppressive tumor microenvironment. Indoleamine 2,3-dioxygenase 1 (IDO1) is an important regulator of tumor immune suppression. We evaluated the effects of IDO1 in hypofractionated radiation using a Lewis lung carcinoma (LLC) mouse model and tested whether IDO1 inhibition could sensitize those tumors to hypofractionated radiation. METHODS AND MATERIALS: Bilateral LLC tumors were established in C57BL/6 mice. Primary tumors were treated with 3 fractions of either 12 Gy or 6 Gy, and the IDO1 inhibitor INCB023843 was given starting on the first day of radiation. Plasma tryptophan and kynurenine levels were quantified by liquid chromatography and tandem mass spectrometry. Tumor-infiltrating immune cells were isolated from the tumors, stained, and quantified by flow cytometry. RESULTS: The combination of INCB023843 and three 12-Gy fractions led to better tumor control and survival than radiation alone; INCB023843 plus three 6-Gy fractions had no benefit. IDO1 expression by tumor-infiltrating immune cells was increased by three 12-Gy doses and inhibited by the addition of INCB023843. Nearly all IDO1+ immune cells were also F4/80+. Percentages of IDO1+F4/80+ immune cells were drastically increased by three 12-Gy fractions and by three 6-Gy fractions, but only INCB023843 combined with three 12-Gy fractions reduced those percentages. IDO1+F4/80+ immune cells were further found to be CD11b+, Gr1-intermediate-expressing, CD206-, and CD11c- (ie, myeloid-derived suppressor cells). Three 12-Gy fractions also increased the percentages of tumor-infiltrating T regulatory cells and CD8+ T cells, but adding INCB023843 did not affect those percentages. CONCLUSIONS: In addition to its immune activation effects, hypofractionated radiation induced "rebound immune suppression" in the tumor microenvironment by activating and recruiting IDO1-expressing myeloid-derived suppressor cells in a dose-dependent manner. Adding an IDO1 inhibitor to hypofractionated radiation reduced the percentages of these cells, overcame the immune suppression, and sensitized LLC tumors to hypofractionated radiation.

Indoleamine 2,3-dioxygenase 1 inhibition targets anti-PD1-resistant lung tumors by blocking myeloid-derived suppressor cells.

Indoleamine 2,3-dioxygenase 1 (IDO1), involved in the catabolism of tryptophan (Trp) to kynurenine (Kyn) is an important regulator of tumor-mediated immunosuppression implicated in resistance to anti-PD1 immunotherapy. We investigated the role of IDO1 in an anti-PD1-resistant lung cancer model (344SQ_R) compared to the parental 344SQ tumors (344SQ_P). IDO1 was overexpressed in tumor-infiltrating leukocytes, and plasma Kyn levels were increased, in 344SQ_R vs. 344SQ_P. The IDO1 inhibitor INCB023843 retarded tumor growth and reduced lung metastases in 344SQ_R. IDO1 was expressed at higher levels in F4/80+Gr1intCD11b+ myeloid-derived suppressor cells (MDSCs) that were prominent in 344SQ_R. The INCB023843 reduced IDO1 expression and percentages of these MDSCs while increasing CD8+ T cells infiltration, hence reactivating antitumor T-cell responses in 344SQ_R. Therefore, IDO1 inhibition holds promise for treating lung cancer that does not respond to anti-PD1 therapy.

Suppression of Type I IFN Signaling in Tumors Mediates Resistance to Anti-PD-1 Treatment That Can Be Overcome by Radiotherapy.

Immune checkpoint therapies exhibit impressive efficacy in some patients with melanoma or lung cancer, but the lack of response in most cases presses the question of how general efficacy can be improved. In addressing this question, we generated a preclinical tumor model to study anti-PD-1 resistance by in vivo passaging of Kras-mutated, p53-deficient murine lung cancer cells (p53R172HΔg/+K-rasLA1/+ ) in a syngeneic host exposed to repetitive dosing with anti-mouse PD-1 antibodies. PD-L1 (CD274) expression did not differ between the resistant and parental tumor cells. However, the expression of important molecules in the antigen presentation pathway, including MHC class I and II, as well as ß2-microglobulin, were significantly downregulated in the anti-PD-1-resistant tumors compared with parental tumors. Resistant tumors also contained fewer CD8+ (CD8α) and CD4+ tumor-infiltrating lymphocytes and reduced production of IFNγ. Localized radiotherapy induced IFNß production, thereby elevating MHC class I expression on both parental and resistant tumor cells and restoring the responsiveness of resistant tumors to anti-PD-1 therapy. Conversely, blockade of type I IFN signaling abolished the effect of radiosensitization in this setting. Collectively, these results identify a mechanism of PD-1 resistance and demonstrate that adjuvant radiotherapy can overcome resistance. These findings have immediate clinical implications for extending the efficacy of anti-PD-1 immune checkpoint therapy in patients. Cancer Res; 77(4); 839-50. ©2016 AACR.

Inhibition of EGFR or IGF-1R signaling enhances radiation response in head and neck cancer models but concurrent inhibition has no added benefit.

Interaction between the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGF-1R) has been well established in many cancer types. We investigated the effects of cetuximab (EGFR antibody) and IMC-A12 (IGF-1R antibody) on the response of head and neck squamous cell carcinoma (HNSCC) to radiation therapy (RT). The effects of cetuximab and IMC-A12 on cell viability and radiosensitivity were determined by clonogenic cell survival assay. Formation of nuclear γ-H2AX and 53BP1 foci was monitored by immunofluorescence. Alterations in target signaling were analyzed by Western blots. In vivo tumor growth delay assay was performed to determine the efficacy of triple therapy with IMC-A12, cetuximab, and RT. In vitro data showed that cetuximab differentially affected the survival and the radiosensitivity of HNSCC cells. Cetuximab suppressed DNA repair that was evident by the prolonged presence of nuclear γ-H2AX and 53BP1 foci. IMC-A12 did not have any effect on the cell survival. However, it increased the radiosensitivity of one of the cell lines. EGFR inhibition increased IGF-1R expression levels and also the association between EGFR and IGF-1R. Addition of IMC-A12 to cetuximab did not increase the radiosensitivity of these cells. Tumor xenografts exhibited enhanced response to RT in the presence of either cetuximab or IMC-A12. Concurrent treatment regimen failed to further enhance the tumor response to cetuximab and/or RT. Taken together our data suggest that concomitant inhibition of both EGFR and IGF-1R pathways did not yield additional therapeutic benefit in overcoming resistance to RT.

Noncovalent assembly of targeted carbon nanovectors enables synergistic drug and radiation cancer therapy in vivo.

Current chemotherapeutics are characterized by efficient tumor cell-killing and severe side effects mostly derived from off-target toxicity. Hence targeted delivery of these drugs to tumor cells is actively sought. In an in vitro system, we previously demonstrated that targeted drug delivery to cancer cells overexpressing epidermal growth factor receptor (EGFR+) can be achieved by poly(ethylene glycol)-functionalized carbon nanovectors simply mixed with a drug, paclitaxel, and an antibody that binds to the epidermal growth factor receptor, cetuximab. This construct is unusual in that all three components are assembled through noncovalent interactions. Here we show that this same construct is effective in vivo, enhancing radiotherapy of EGFR+ tumors. This targeted nanovector system has the potential to be a new therapy for head and neck squamous cell carcinomas, deserving of further preclinical development.

Vandetanib restores head and neck squamous cell carcinoma cells' sensitivity to cisplatin and radiation in vivo and in vitro.

PURPOSE: We investigated whether vandetanib, an inhibitor of the tyrosine kinase activities of vascular endothelial growth factor receptor-2 (VEGFR-2), epidermal growth factor receptor (EGFR), and rearranged during transfection (RET), could augment the antitumor activity of radiation with or without cisplatin in preclinical in vitro and in vivo models of human head and neck squamous cell carcinoma (HNSCC). EXPERIMENTAL DESIGN: OSC-19 and HN5 HNSCC cells that were cisplatin and radioresistant were treated with vandetanib, cisplatin, and radiation alone or in combination in vitro and in vivo using an orthotopic nude mouse model. Treatment effects were assessed using clonogenic survival assay, tumor volume, bioluminescence imaging, tumor growth delay, survival, microvessel density, tumor and endothelial cell apoptosis, and EGFR and Akt phosphorylation data. RESULTS: Vandetanib plus cisplatin radiosensitized HNSCC cells in vitro and in vivo. The combination treatment with vandetanib, cisplatin, and radiation was superior to the rest of treatments (including the double combinations) in antitumoral effects, prolonging survival, decreasing cervical lymph node metastases in vivo. It also increased both tumor and tumor-associated endothelial cell apoptosis and decreased microvessel density in vivo. An analysis of tumor growth delay data revealed that vandetanib plus cisplatin enhanced radioresponse in vivo. All vandetanib-containing treatments inhibited EGFR and Akt phosphorylation in vitro and in vivo. CONCLUSION: The addition of vandetanib to combination therapy with cisplatin and radiation was able to effectively overcome cisplatin and radioresistance in in vitro and in vivo models of HNSCC. Further study of this regimen in clinical trials may be warranted.