Courtney Nicholas

Cross-species identification of genomic drivers of squamous cell carcinoma development across preneoplastic intermediates

Cutaneous squamous cell carcinoma (cuSCC) comprises 15–20% of all skin cancers, accounting for over 700,000 cases in USA annually. Most cuSCC arise in association with a distinct precancerous lesion, the actinic keratosis (AK). To identify potential targets for molecularly targeted chemoprevention, here we perform integrated cross-species genomic analysis of cuSCC development through the preneoplastic AK stage using matched human samples and a solar ultraviolet radiation-driven Hairless mouse model. We identify the major transcriptional drivers of this progression sequence, showing that the key genomic changes in cuSCC development occur in the normal skin to AK transition. Our data validate the use of this ultraviolet radiation-driven mouse cuSCC model for cross-species analysis and demonstrate that cuSCC bears deep molecular similarities to multiple carcinogen-driven SCCs from diverse sites, suggesting that cuSCC may serve as an effective, accessible model for multiple SCC types and that common treatment and prevention strategies may be feasible.

Intratumoral STING Activation with T-cell Checkpoint Modulation Generates Systemic Antitumor Immunity

Intratumoral administration of myeloid agonists combined with a cocktail of T-cell checkpoint–modulating antibodies elicited systemic antitumor immunity against bilateral TRAMP-C2 prostate tumors. Limited agonist dosing coupled with intratumoral antibody injection reduced toxicity but preserved abscopal immunity. Coordinated manipulation of independent immune regulatory pathways in the tumor microenvironment—including blockade of T-cell checkpoint receptors and reversal of suppressive myeloid programs—can render aggressive cancers susceptible to immune rejection. Elevated toxicity associated with combination immunotherapy, however, prevents translation of the most efficacious regimens. We evaluated T-cell checkpoint–modulating antibodies targeting CTLA-4, PD-1, and 4-1BB together with myeloid agonists targeting either STING or Flt3 in the TRAMP-C2 model of prostate cancer to determine whether low-dose intratumoral delivery of these agents could elicit systemic control of multifocal disease. Intratumoral administration of the STING agonist cyclic di-GMP (CDG) or Flt3 Ligand (Flt3L) augmented the therapeutic effect of systemic triple checkpoint modulation and promoted the cure of 75% of mice with bilateral TRAMP-C2; however, when all agents were administered locally, only CDG mobilized abscopal immunity. Combination efficacy correlated with globally enhanced ratios of CD8+ T cells to regulatory T cells (Treg), macrophages, and myeloid-derived suppressor cells, and downregulation of the M2 marker CD206 on tumor-associated macrophages. Flt3L improved CD8+ T-cell and dendritic cell infiltration of tumors, but was diminished in efficacy by concomitant Treg expansion. Although intratumoral CDG/checkpoint therapy invokes substantial ulceration at the injection site, reduced CDG dosing can preserve tissue integrity without sacrificing therapeutic benefit. For high-order combinations of T-cell checkpoint antibodies and local myeloid agonists, systemic antibody administration provides the greatest efficacy; however, local administration of CDG and antibody provides substantial systemic benefit while minimizing the potential for immune-related adverse events. Cancer Immunol Res; 5(8); 676–84. ©2017 AACR.

Debio 0617B Inhibits Growth of STAT3-Driven Solid Tumors through Combined Inhibition of JAK, SRC, and Class III/V Receptor Tyrosine Kinases.

Tumor survival, metastases, chemoresistance, and escape from immune responses have been associated with inappropriate activation of STAT3 and/or STAT5 in various cancers, including solid tumors. Debio 0617B has been developed as a first-in-class kinase inhibitor with a unique profile targeting phospho-STAT3 (pSTAT3) and/or pSTAT5 in tumors through combined inhibition of JAK, SRC, ABL, and class III/V receptor tyrosine kinases (RTK). Debio 0617B showed dose-dependent inhibition of pSTAT3 in STAT3-activated carcinoma cell lines; Debio 0617B also showed potent antiproliferative activity in a panel of cancer cell lines and in patient-derived tumor xenografts tested in an in vitro clonogenic assay. Debio 0617B showed in vivo efficacy by inhibiting tumor growth in several mouse xenograft models. To increase in vivo efficacy and STAT3 inhibition, Debio 0617B was tested in combination with the EGFR inhibitor erlotinib in a non–small cell lung cancer xenograft model. To evaluate the impact of in vivo STAT3 blockade on metastases, Debio 0617B was tested in an orthotopic tumor model. Measurement of primary tumor weight and metastatic counts in lung tissue demonstrated therapeutic efficacy of Debio 0617B in this model. These data show potent activity of Debio 0617B on a broad spectrum of STAT3-driven solid tumors and synergistic activity in combination with EGFR inhibition. Mol Cancer Ther; 15(10); 2334–43. ©2016 AACR.

Drug-induced RAF dimerization is independent of RAS mutation status and does not lead to universal MEK dependence for cell survival in head and neck cancers

Treatments for recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) have limited efficacy. One potential therapeutic target for HNSCC is the RAS/RAF/MEK/ERK cascade, which is one of the major signaling pathways for HNSCC cell survival. In HNSCC, RAS can be activated either by HRAS mutation or by upstream signaling. The ABL inhibitor nilotinib acts as a weak RAF inhibitor that induces RAF dimerization and subsequent activation of MEK/ERK in other cancer cell lines with activated RAS, leading to an unexpected dependence on MEK/ERK for cell survival. We hypothesized that nilotinib and the MEK inhibitor MEK162 would be synergistic in HNSCC cell lines owing to the frequent activation of RAS. We treated HNSCC cell lines with nilotinib and performed immunoblotting and cell-viability experiments. We used an orthotopic mouse model to assess synergistic effects in vivo. Nilotinib induced significant BRAF-CRAF heterodimerization and ERK activation irrespective of RAS mutation status. In cell-viability assays, nilotinib synergized with MEK162. MEK162 alone induced G1 arrest that was minimally enhanced by nilotinib. In the mouse model, treatment with MEK162 alone or combined with nilotinib led to tumor growth inhibition. In HNSCC, nilotinib-induced RAF dimerization is independent of RAS mutation status, but this dimerization does not lead to MEK dependence for cell survival in all HNSCC cell lines. MEK inhibition alone leads to decreased proliferation both in vitro and in vivo. Although nilotinib has some synergistic effects with MEK162, other agents may be more effective against HNSCC when combined with MEK162.

Targeted hypoxia reduction restores T cell infiltration and sensitizes prostate cancer to immunotherapy

Despite the success of immune checkpoint blockade against melanoma, many “cold” tumors like prostate cancer remain unresponsive. We found that hypoxic zones were prevalent across preclinical prostate cancer and resisted T cell infiltration even in the context of CTLA-4 and PD-1 blockade. We demonstrated that the hypoxia-activated prodrug TH-302 reduces or eliminates hypoxia in these tumors. Combination therapy with this hypoxia-prodrug and checkpoint blockade cooperated to cure more than 80% of tumors in the transgenic adenocarcinoma of the mouse prostate–derived (TRAMP-derived) TRAMP-C2 model. Immunofluorescence imaging showed that TH-302 drives an influx of T cells into hypoxic zones, which were expanded by checkpoint blockade. Further, combination therapy reduced myeloid-derived suppressor cell density by more than 50%, and durably reduced the capacity of the tumor to replenish the granulocytic subset. Spontaneous prostate tumors in TRAMP transgenic mice, which completely resist checkpoint blockade, showed minimal adenocarcinoma tumor burden at 36 weeks of age and no evidence of neuroendocrine tumors with combination therapy. Survival of Pb-Cre4, Ptenpc–/–Smad4pc–/– mice with aggressive prostate adenocarcinoma was also significantly extended by this combination of hypoxia-prodrug and checkpoint blockade. Hypoxia disruption and T cell checkpoint blockade may sensitize some of the most therapeutically resistant cancers to immunotherapy.

MEK Is a Therapeutic and Chemopreventative Target in Squamous Cell Carcinoma

ACKNOWLEDGMENTS The Italian Melanoma Intergroup (IMI) includes the following additional members who participated as investigators in this study and should be considered as co-authors: Nicola Mozzillo (Istituto Nazionale Tumori, Napoli, Italy), Panagiotis Paliogiannis (Chirurgia, Microchirurgia e Scienze Mediche, Universita di Sassari, Italy), PaolaQueirolo (Oncologia, Azienda Ospedaliera, Genoa, Italy), Corrado Rubino (Chirurgia Plastica, Universita di Salerno, Italy), MariaCristina Sini (IstitutodiChimicaBiomolecolare,CNR, Sassari, Italy), Ignazio Stanganelli (Dermatologia, Universita di Parma—Skin Cancer Unit, IRST, Meldola, Italy), Francesco Tanda (Anatomia Patologica, Universita di Sassari, Italy). The authors are grateful to patients for their important contribution to this study. The work was supported by Ricerca Finalizzata Ministero Salute andSardinianRegionalGovernment (Regione Autonoma della Sardegna).

Mucosal HPV E6/E7 Peptide Vaccination in Combination with Immune Checkpoint Modulation Induces Regression of HPV+ Oral Cancers

High-risk human papillomavirus (HPV)-associated squamous cell carcinomas of the oropharynx (SCCOP) are among the fastest growing cancers. After standard-of-care treatment, however, patients with HPV+ SCCOP have better overall and disease-specific survival than patients with HPV- SCCOP, suggesting the importance of HPV-specific immunity. We reasoned that therapeutic vaccination targeting the HPV-16 E6 and E7 oncogenes could elicit high-affinity, high-frequency tumor antigen-specific T-cell responses, which could then be augmented and shielded from suppression in the tumor microenvironment by immune checkpoint modulation. In this study, we used a preclinical syngeneic mouse model of oral cancer comprised of mouse tonsil-derived epithelial cells stably expressing HPV-16 E6 and E7 genes along with H-ras oncogene (mEER) to identify combinations of vaccination and checkpoint antibodies capable of promoting tumor regression. Intranasal HPV E6/E7 peptide vaccination and single checkpoint antibodies failed to elicit responses in more than half of animals; however, 4-1BB agonist antibody along with either CD40 agonist antibody or CTLA-4 blockade eliminated the majority of established mEER tumors. The combination of intranasal HPV peptide vaccine and α4-1BB and αCTLA-4 antibodies produced curative efficacy and a better safety profile against orally implanted mEER tumors. Correlates of protective immunity included enhanced intratumoral levels of CD8 T cells relative to immunosuppressive regulatory T cells and myeloid-derived suppressor cells. Overall, our results demonstrate combination vaccine-immunotherapy modalities as novel treatment options for HPV+ SCCOP.Significance: Combinations of vaccine and checkpoint modulation are effective and safe treatment options for HPV+ oral cancers. Cancer Res; 78(18); 5327-39. ©2018 AACR.

Abstract 406: A proteome-transcriptome-miRnome integrated analysis identifies similarity between UV-exposed skin and wounding skin

Cutaneous squamous cell carcinoma (cuSCC) is the second most common skin cancer, for which long term UV exposure and chronic wounding are the dominant risk factors. Despite these clinically established connections, little is understood about the early molecular response of human skin to UV exposure and its connection to acute wounding and cuSCC. Thus, our goal is to find common and specific signatures driven by UV-exposure and wounding as a means of developing new approaches for treating and preventing cuSCC. Here, we perform integrated analyses of proteomic, RNA-seq and miR-seq on 3 datasets: (1) UV-unexposed and acute UV-exposed human skin, (2) public dataset on acute wound healing and (3) our previously published dataset on normal skin and cuSCC from humans. We find that biological signatures and processes regulated by acute UV exposure and wounding has profound similarity. miR-seq data shows that miR-223, miR-132 and miR-142 are significantly upregulated in both acute events. Combined gene set enrichment analysis shows that G-protein-coupled-receptors (GPCRs) pathways are upregulated, possibly through Gαi activation. While ECM remodeling is significantly enriched in all three datasets, gene expression regulated by PPARα is suppressed. Interestingly, upregulation of matrisome components is observed among all three datasets. This suggests that these changes are important early events that regulated by both UV-exposure and wounding which eventually can promote cuSCC initiation. Thus, our findings suggest that these common signatures can be potentially validated as chemopreventive targets for cuSCC. Citation Format: Tran N. Nguyen, Kimal Rajapakshe, Stanislav Avdieiev, Courtney Nicholas, Vida Chitsazzadeh, Eric Welsh, Bin Fang, John Koomen, Cristian Coarfa, Janine Einspahr, Kenneth Y. Tsai. A proteome-transcriptome-miRnome integrated analysis identifies similarity between UV-exposed skin and wounding skin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 406.

Abstract 2613: MEK is a therapeutic and chemopreventative target in squamous cell carcinoma

Purpose: While the prognosis of cutaneous squamous cell carcinoma (cuSCC) is excellent overall, advanced metastatic disease represents a substantial mortality burden for which no standard targeted therapy exists. Findings from genomic and proteomic studies and the observed induction of cuSCC by BRAF inhibitor driven MEK/ERK pathway engagement suggest that MEK/ERK activation is essential for cuSCC tumorigenesis and tumor proliferation. Most cuSCC arise from a clinically and histopathologically defined preneoplastic precursor, the actinic keratosis (AK). Molecular studies of early events in cuSCC pathogenesis strongly implicate MEK/ERK signaling at the proteomic and transcriptional level. This occurs at the earliest recognizable transitions from chronically UV-exposed skin to AK, with sharp elevation of ETS2/ELK1 transcriptional target expression. With these compelling rationales in mind, we tested whether MEK inhibitors (MEKi) are a clinically actionable treatment and chemoprevention approach for cuSCC. Given that several MEKi are approved, this is a readily translated strategy. Experimental Design: Preclinical testing was performed in 10 cuSCC cell lines and two mouse models of cuSCC using two distinct MEK inhibitors, trametinib and cobimetinib. We show that two MEKi, trametinib and cobimetinib are highly effective against cuSCC cell lines in culture, effectively engage MEK/ERK signaling in cells and in vivo, and potently induce cell cycle arrest and senescence. Both established and new tumors are powerfully inhibited in both xenograft and UV-driven autochthonous mouse models. This model, which utilizes immunocompetent SKH-1E mice exposed to chronic, low-dose, solar simulated UV light (12.5 kJ/m2 UVB weekly administered across three doses) more faithfully recapitulates human cuSCC molecularly than chemical carcinogenesis models. Lesions in this model exhibit heterogeneity in latency and responses to therapy, as do human tumors. Results: MEK inhibitor treatment of cuSCC lines strongly reduces proliferation and induces senescence markers in cells, including p21 and beta-galactosidase. This response was universal, but highly heterogeneous. Sensitivity to MEKi was determined, in part, by modulation of AKT activity, and combination MEKi and AKTi. In-vivo, an SRB1 xenograft model was exquisitely sensitive to oral trametinib treatment, and in our spontaneous UV-driven Hairless mouse model of cuSCC, treatment with the MEK inhibitors trametinib and cobimetinib strongly reduced tumor growth and almost completely abrogated tumor induction. We confirmed target engagement in-vivo showing that ERK signaling was significantly suppressed. Conclusions: Overall, we conclude MEK signaling is critical for cuSCC tumor induction and maintenance, and that MEK inhibition is an attractive approach for both advanced cuSCC treatment as well as chemoprevention. Citation Format: Charles H. Adelmann, Kimberly Truong, Roger Liang, Varun Bansal, Rachael Saporito, Woojin Lee, Lili Du, Courtney Nicholas, Marco Napoli, Elsa R. Flores, Kenneth Y. Tsai. MEK is a therapeutic and chemopreventative target in squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2613.

Abstract 5578: SOCS2 (suppressor of cytokine signaling protein 2) is a prognostic indicator of progression-free survival in head and neck squamous cell carcinoma (HNSCC) patients.

HNSCC is a common, deadly, and disfiguring disease. While combinations of radiotherapy, surgery, and chemotherapy are highly effective in HNSCC, there is significant morbidity associated with the disease and recurrence is common. There is great interest in identifying molecular events and pathways which drive HNSCC progression in order that targeted therapies may be developed. The loss of the tumor suppressor function such as p53 and NOTCH1, as well as activation of the STAT3 and STAT5 pathways, has been implicated in HNSCC progression. We have identified interactions between STAT3, STAT5, and the novel tumor suppressor SOCS2 to be important in HNSCC. To extend these findings to humans, we performed experiments to identify prognostic markers in HNSCC and to investigate the expression of STAT3, STAT5, and SOCS2 in HNSCC tissue. Immunohistochemistry was performed on tissue microarrays from resected tumor specimens of 123 stage I-IVB oral cavity SCC patients (treated with surgery +/- adjuvant radiation therapy) with annotated clinical outcome information from a median follow up of 76 months collected at the UT/MD Anderson Cancer Center. The array was screened with antibodies against STAT5, STAT3, and SOCS2, and scored in a blinded fashion by a pathologist. SOCS2 expression (present vs. absent) correlated significantly with recurrence-free survival in both the univariate (hazard ratio 0.24, p=0.0003; Cox proportional hazards model) and multivariate (hazard ratio 0.24, p=0.0004) analyses. Notably all patients who lacked SOCS2 tumor expression recurred within 45 months. There was a trend towards a correlation of SOCS2 expression with overall survival (HR 0.50, p=0.11) and disease-specific survival (HR 0.52, p=0.28). SOCS2 expression did not significantly correlate with tumor stage and extracapsular extension emphasizing that its absence is an independent marker of poor prognosis. Our prior published work demonstrates that STAT5 drives SOCS2 expression. The expression of SOCS2 in these patient samples positively correlated with total and phosphoSTAT5 (r=0.29 and 0.21 respectively; p This work was supported The University of Texas SPORE in Head and Neck Cancer (P50 CA097007), The Cancer Center Support Grant, and the ASCO Cancer Foundation Young Investigator Award (WW). Citation Format: Courtney Nicholas, Maria I. Nunez, Nusrat Harun, J. Jack Lee, Jeffrey Myers, Ignacio I. Wistuba, Banibrata Sen, Adel K. El-Naggar, Stephen Y. Lai, Faye M. Johnson, William N. William. SOCS2 (suppressor of cytokine signaling protein 2) is a prognostic indicator of progression-free survival in head and neck squamous cell carcinoma (HNSCC) patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5578. doi:10.1158/1538-7445.AM2013-5578