Falguni Parikh

TGF-β2 dictates disseminated tumour cell fate in target organs through TGF-β-RIII and p38α/β signalling

In patients, non-proliferative disseminated tumour cells (DTCs) can persist in the bone marrow (BM) while other organs (such as lung) present growing metastasis. This suggested that the BM might be a metastasis ‘restrictive soil’ by encoding dormancy-inducing cues in DTCs. Here we show in a head and neck squamous cell carcinoma (HNSCC) model that strong and specific transforming growth factor-β2 (TGF-β2) signalling in the BM activates the MAPK p38α/β, inducing an (ERK/p38)low signalling ratio. This results in induction of DEC2/SHARP1 and p27, downregulation of cyclin-dependent kinase 4 (CDK4) and dormancy of malignant DTCs. TGF-β2-induced dormancy required TGF-βxa0receptor-I (TGF-β-RI), TGF-β-RIII and SMAD1/5 activation to induce p27. In lungs, a metastasis ‘permissive soil’ with low TGF-β2 levels, DTC dormancy was short-lived and followed by metastatic growth. Importantly, systemic inhibition of TGF-β-RI or p38α/β activities awakened dormant DTCs, fuelling multi-organ metastasis. Our work reveals a ‘seed and soil’ mechanism where TGF-β2 and TGF-β-RIII signalling through p38α/β regulates DTC dormancy and defines restrictive (BM) and permissive (lung) microenvironments for HNSCC metastasis.

Tumor-Expressed Inducible Nitric Oxide Synthase Controls Induction of Functional Myeloid-Derived Suppressor Cells through Modulation of Vascular Endothelial Growth Factor Release

Inducible NO synthase (iNOS) is a hallmark of chronic inflammation that is also overexpressed in melanoma and other cancers. Whereas iNOS is a known effector of myeloid-derived suppressor cell (MDSC)-mediated immunosuppression, its pivotal position at the interface of inflammation and cancer also makes it an attractive candidate regulator of MDSC recruitment. We hypothesized that tumor-expressed iNOS controls MDSC accumulation and acquisition of suppressive activity in melanoma. CD11b+GR1+ MDSC derived from mouse bone marrow cells cultured in the presence of MT-RET-1 mouse melanoma cells or conditioned supernatants expressed STAT3 and reactive oxygen species (ROS) and efficiently suppressed T cell proliferation. Inhibition of tumor-expressed iNOS with the small molecule inhibitor L-NIL blocked accumulation of STAT3/ROS-expressing MDSC, and abolished their suppressive function. Experiments with vascular endothelial growth factor (VEGF)-depleting Ab and recombinant VEGF identified a key role for VEGF in the iNOS-dependent induction of MDSC. These findings were further validated in mice bearing transplantable MT-RET-1 melanoma, in which L-NIL normalized elevated serum VEGF levels; downregulated activated STAT3 and ROS production in MDSC; and reversed tumor-mediated immunosuppression. These beneficial effects were not observed in iNOS knockout mice, suggesting L-NIL acts primarily on tumor- rather than host-expressed iNOS to regulate MDSC function. A significant decrease in tumor growth and a trend toward increased tumor-infiltrating CD8+ T cells were also observed in MT-RET transgenic mice bearing spontaneous tumors. These data suggest a critical role for tumor-expressed iNOS in the recruitment and induction of functional MDSC by modulation of tumor VEGF secretion and upregulation of STAT3 and ROS in MDSC.

NR2F1 controls tumour cell dormancy via SOX9- and RARβ-driven quiescence programmes

Metastases can originate from disseminated tumor cells (DTCs), which may be dormant for years before reactivation. Here we find that the orphan nuclear receptor NR2F1 is epigenetically upregulated in experimental HNSCC dormancy models and in DTCs from prostate cancer patients carrying dormant disease for 7–18 years. NR2F1-dependent dormancy is recapitulated by a co-treatment with the DNA demethylating agent 5-Aza-C and retinoic acid across various cancer types. NR2F1-induced quiescence is dependent on SOX9, RARβ and CDK inhibitors. Intriguingly, NR2F1 induces global chromatin repression and the pluripotency gene NANOG, which contributes to dormancy of DTCs in the bone marrow. When NR2F1 is blocked in vivo, growth arrest or survival of dormant DTCs is interrupted in different organs. We conclude that NR2F1 is a critical node in dormancy induction and maintenance by integrating epigenetic programs of quiescence and survival in DTCs.

Chemoradiotherapy-Induced Upregulation of PD-1 Antagonizes Immunity to HPV-Related Oropharyngeal Cancer

While viral antigens in human papillomavirus (HPV)-related oropharyngeal cancer (HPVOPC) are attractive targets for immunotherapy, the effects of existing standard-of-care therapies on immune responses to HPV are poorly understood. We serially sampled blood from patients with stage III-IV oropharyngeal cancer undergoing concomitant chemoradiotherapy with or without induction chemotherapy. Circulating immunocytes including CD4(+) and CD8(+) T cells, regulatory T cells (Treg), and myeloid-derived suppressor cells (MDSC) were profiled by flow cytometry. Antigen-specific T-cell responses were measured in response to HPV16 E6 and E7 peptide pools. The role of PD-1 signaling in treatment-related immunosuppression was functionally defined by performing HPV-specific T-cell assays in the presence of blocking antibody. While HPV-specific T-cell responses were present in 13 of 18 patients before treatment, 10 of 13 patients lost these responses within 3 months after chemoradiotherapy. Chemoradiotherapy decreased circulating T cells and markedly elevated MDSCs. PD-1 expression on CD4(+) T cells increased by nearly 2.5-fold after chemoradiotherapy, and ex vivo culture with PD-1-blocking antibody enhanced HPV-specific T-cell responses in 8 of 18 samples tested. Chemoradiotherapy suppresses circulating immune responses in patients with HPVOPC by unfavorably altering effector:suppressor immunocyte ratios and upregulating PD-1 expression on CD4(+) T cells. These data strongly support testing of PD-1-blocking agents in combination with standard-of-care chemoradiotherapy for HPVOPC.

Inducible Nitric Oxide Synthase Drives mTOR Pathway Activation and Proliferation of Human Melanoma by Reversible Nitrosylation of TSC2

Melanoma is one of the cancers of fastest-rising incidence in the world. Inducible nitric oxide synthase (iNOS) is overexpressed in melanoma and other cancers, and previous data suggest that iNOS and nitric oxide (NO) drive survival and proliferation of human melanoma cells. However, specific mechanisms through which this occurs are poorly defined. One candidate is the PI3K-AKT-mTOR pathway, which plays a major role in proliferation, angiogenesis, and metastasis of melanoma and other cancers. We used the chick embryo chorioallantoic membrane (CAM) assay to test the hypothesis that melanoma growth is regulated by iNOS-dependent mTOR pathway activation. Both pharmacologic inhibition and siRNA-mediated gene silencing of iNOS suppressed melanoma proliferation and in vivo growth on the CAM in human melanoma models. This was associated with strong downregulation of mTOR pathway activation by Western blot analysis of p-mTOR, p70 ribosomal S6 kinase (p-P70S6K), p-S6RP, and p-4EBP1. iNOS expression and NO were associated with reversible nitrosylation of tuberous sclerosis complex (TSC) 2, and inhibited dimerization of TSC2 with its inhibitory partner TSC1, enhancing GTPase activity of its target Ras homolog enriched in brain (Rheb), a critical activator of mTOR signaling. Immunohistochemical analysis of tumor specimens from stage III melanoma patients showed a significant correlation between iNOS expression levels and expression of the mTOR pathway members. Exogenously supplied NO was also sufficient to reverse the mTOR pathway inhibition by the B-Raf inhibitor vemurafenib. In summary, covalent modification of TSC2 by iNOS-derived NO is associated with impaired TSC2/TSC1 dimerization, mTOR pathway activation, and proliferation of human melanoma. This model is consistent with the known association of iNOS overexpression and poor prognosis in melanoma and other cancers.

iNOS Expression in CD4+ T Cells Limits Treg Induction by Repressing TGFβ1: Combined iNOS Inhibition and Treg Depletion Unmask Endogenous Antitumor Immunity

Purpose: Expression of inducible nitric oxide synthase (iNOS) in different cellular compartments may have divergent effects on immune function. We used a syngeneic tumor model to functionally characterize the role of iNOS in regulation of CD4+FOXP3+ regulatory T cells (Treg), and optimize the beneficial effects of iNOS inhibition on antitumor immunity. Experimental Design: Wild-type (WT) or iNOS knockout mice bearing established MT-RET-1 melanoma were treated with the small-molecule iNOS inhibitor L-NIL and/or cyclophosphamide alone or in combination. The effect of iNOS inhibition or knockout on induction of Treg from mouse and human CD4+ T cells in ex vivo culture was determined in parallel in the presence or absence of TGFβ1-depleting antibodies, and TGFβ1 levels were assessed by ELISA. Results: Whereas intratumoral myeloid-derived suppressor cells (MDSC) were suppressed by iNOS inhibition or knockout, systemic and intratumoral FOXP3+ Treg levels increased in tumor-bearing mice. iNOS inhibition or knockout similarly enhanced induction of Treg from activated cultured mouse splenocytes or purified human or mouse CD4+ T cells in a TGFβ1-dependent manner. Although either iNOS inhibition or Treg depletion with low-dose cyclophosphamide alone had little effect on growth of established MT-RET1 melanoma, combination treatment potently inhibited MDSC and Treg, boosted tumor-infiltrating CD8+ T-cell levels, and arrested tumor growth in an immune-dependent fashion. Conclusions: iNOS expression in CD4+ T cells suppresses Treg induction by inhibiting TGFβ1 production. Our data suggest that iNOS expression has divergent effects on induction of myeloid and lymphoid-derived regulatory populations, and strongly support development of combinatorial treatment approaches that target these populations simultaneously. Clin Cancer Res; 20(24); 6439–51. ©2014 AACR.

Abstract 2211: Characterization of monoclonal antibodies specific for HPV-positive head and neck cancer

HPV-driven head and neck squamous cell carcinoma (HNSCC) are among the fastest growing cancers. Although, HPV-HNSCC patients have an overall favorable prognosis, a significant number of patients relapse post-treatment, and currently there is no specific therapeutic approach targeting the unique biology of HPV-driven HNSCC. Our ongoing efforts to target HPV-HNSCC focus on the identification of cell surface antigens that are upregulated by HPV infection. We have developed an antigen-agnostic approach for generating HPV-HNSCC-targeting monoclonal antibodies for cancer diagnosis and treatment that does not rely on prior identification of target antigens. We used HPV-HNSCC membrane fractions to immunize recipient mice and generated HPV-specific hybridomas. We then screened five thousand hybridoma colonies by flow cytometry to test the specificity of binding to HPV-positive cancer cell lines (2HNSCC and 2 Cervical Cancer) and HPV-negative cancer cell lines (4 HNSCC and 1 CC). After primary screening, we narrowed down to forty-four clones; among these hybridoma clones, 6D8 and 6B3 bound preferentially to HPV-positive cancer cell lines. The binding targets of 6D8 and 6B3 were identified by immunoprecipitation and mass spectrometry; their targets are integrin alpha6 (ITGA6) and tissue factor (F3) respectively. Future work will validate the biological function of these mAbs in in vitro and in vivo models, and continue identifying more binding target of mAbs. We propose mAbs specifically targeting membrane-expressed antigens on HPV-related cancer cells as a potential approach for early diagnosis and targeted therapy. Citation Format: Hsuan-Chen Liu, Falguni Parikh, Thomas Kraus, Thomas Moran, Andrew Sikora. Characterization of monoclonal antibodies specific for HPV-positive head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2211. doi:10.1158/1538-7445.AM2017-2211

Abstract 4559: Novel T-cell targets for HPV16-associated oropharyngeal cancer immune therapy

Background: Human papillomavirus subtype 16 (HPV16) is the primary cause of an increasing number of oropharyngeal cancers (HPVOPC) worldwide. Despite effective prophylactic vaccines, low vaccine uptake and high incidence of HPV16 infections provides a strong rationale for the development of novel targeted immune therapies against HPVOPC. We sought to 1) Identify novel CD8+ T-cell (CTL) epitopes from HPV16 antigens; 2) Understand mechanisms of immune resistance in HPVOPC to enhance T-cell based immune therapies. Methods: We developed a comprehensive bioinformatic strategy to predict potential CTL epitopes for 10 HLA class I alleles representing common HLA-supertypes (with >90% global coverage) against HPV16 antigens E2, E6 and E7. T-cell reactivity against candidate epitopes was assessed by short term ex vivo T-cell cultures from healthy donors (n=15) and HPVOPC patients (n=18). We data mined publicly available OPC transcriptomes (University of Michigan (UM-OPC) cohort, n=36) for gene expression analysis. Results: IFN-gamma Elispot assays using 58 candidate epitopes from HPV16 E2, E6 and E7 revealed that >70% of HPVOPC patients had either E2 or E6-specific CTL reactivity. E2-CTL reactivity was >2-fold higher in HPVOPC patients than in healthy controls (mean = 68.75 vs. 24.6 SFUs/106 cells, P = 0.027), while no difference in E6- CTL responses were observed between patients and controls. In contrast, E7-specific CTL responses were low to undetectable. We have further identified 7 novel E2 and 3 novel E6 CTL-epitopes, and confirmed immunogenicity of previously described E2, E6 and E7 epitopes. RNAseq analysis of the UM-OPC dataset indicated that 78% of HPV16+ OPCs expressed E2 (P Conclusion: HPV16-E2 represents a novel antigen target for HPVOPC immune therapy, as demonstrated by T-cell reactivity and tumor antigen expression. Several novel and previously defined E2, E6 and E7 CTL have been confirmed for T-cell reactivity in HPVOPC patients. Transcriptomic analysis of OPCs indicate extensive immune dysfunction in HPVOPCs that are distinct from non-HPV OPCs. The T-cell epitopes identified in this study in combination with blockade of HPVOPC-specific checkpoints may be useful for targeted immunotherapy. Citation Format: Sri Krishna, Falguni Parikh, Peaches Ulrich, Shanshan Yang, Amelia Clark, Seunghee Kim-Schulze, Marshall Posner, Jin Park, Andrew Sikora, Karen S. Anderson. Novel T-cell targets for HPV16-associated oropharyngeal cancer immune therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4559. doi:10.1158/1538-7445.AM2017-4559

Abstract 626: Cisplatin-based concurrent chemoradiotherapy antagonizes anti-tumor immunity in patients with HPV-positive oropharyngeal cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnPurpose/Objectives: While viral antigens in HPV-related oropharyngeal cancer (HPVOPC) make it an attractive target for immunotherapy, understanding the immune effects of existing therapeutic approaches is essential for integrating immunotherapy into HPVOPC treatment. Preclinical data in several cancer models support an overall immunostimulatory effect of chemotherapy and radiation; however their effect on HPV-specific immunity on HPVOPC patients is unknown. We tested the hypotheses that platinum-based concomitant chemoradiation, with or without taxane-platinum-5FU (TPF) induction chemotherapy, induces a favorable profile of circulating immunocytes ad enhanced HPV-specific T cell responses in HPVOPC patients.nnMaterials/Methods: Patients with stage II-IV HPVOPC treated with standard-of-care chemoradiation underwent serial blood sampling before, during, and after treatment for up to one year. Circulating immunocytes including effector CD4+ and CD8+ T cells and immunosuppressive regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) were profiled by flow cytometry. Expression levels of the negative costimulatory molecule PD-1 on T cells were also assessed by flow cytometry. HPV antigen-specific T cell responses in response to HPV16 E6 and E7 peptides were measured by luminex analysis of IFN-γ production.nnResults: Pre-treatment HPV-specific T cell responses were present in 12/18 patients, and an additional 4 patients acquired measureable responses following induction chemotherapy. Of the 16 patients who developed HPV-specific responses before completion of therapy, 10 lost these responses by 3 months post-treatment. The average level of IFN-g release by PBMC after stimulation with HPV peptides was non-significantly decreased at 3 weeks post-treatment, and significantly decreased at 3 and 6 months. Loss of pre-existing tumor-specific immune responses was associated with a striking (2-fold) decline in circulating CD4+ and CD8+ T cell numbers, a lesser decline in Treg (1.5-fold), marked elevation of MDSC (>2-fold), and decline in the CD8+:Treg and CD8+:MDSC ratios following chemoradiotherapy. PD-1 expression levels on total and CD45RO+ (memory) CD4+ T cells were elevated at 3 weeks after completion of chemoradiation, returning to baseline by 3 months.nnConclusions: Contrary to our starting hypothesis, we found an overall immunosuppressive effect of chemoradiotherapy on immune responses in HPVOPC patients. Upregulation of PD-1 on CD4 T cells is a potential immunosuppressive mechanism amenable to targeted therapy with clinically available anti-PD-1 and anti-PD-L1 antibodies. Taken together, our results suggest that chemoradiation has profound effects on circulating immunocyte populations and the immune response to HPV+ OPC, and highlight the importance of further studies of the immune effects of standard-of-care treatment for HPVOPC.nnCitation Format: Andrew Sikora, Marshall Posner, Falguni Parikh, Seunghee Kim-Schulze, Vishal Gupta, Krzysztof Misiukiewicz, Alexis Patsias, Amelia Clark, Sangkon Oh, Dorothee Duluc. Cisplatin-based concurrent chemoradiotherapy antagonizes anti-tumor immunity in patients with HPV-positive oropharyngeal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 626. doi:10.1158/1538-7445.AM2014-626

Abstract 1090: NR2F1 coordinates a dormancy program by inducing low self-renewal capacity and a repressive chromatin state.

Clinical studies have shown that solitary disseminated tumor cells (DTCs) in distant organs can be detected years after treatment of primary tumors. This has led to the notion that these solitary DTCs must enter a non-proliferative state or cellular dormancy. This may also hold true for residual tumor cells left behind in loco-regional sites after surgery. Unfortunately, the mechanisms regulating the timing of dormancy and the awakening of DTCs to fuel recurrences are unknown. Similarly, tools to stably induce dormancy of residual disease are unavailable. Here we show that transient differentiation signals propagated by treatment (3-4 days) with retinoic acid (atRA) along with the DNA demethylating agent 5-Aza-C reprogrammed malignant cells into dormancy for remarkably long periods of time (weeks). This reprogramming was associated with upregulation of the orphan nuclear receptor NR2F1/COUP-TF1 and other genes included in a gene signature previously linked to dormancy and late metastasis in breast patients. Importantly, the reprogramming was dependent on NR2F1, which is also silenced in breast and HNSCC tumors, cell lines and mouse cancer models. NR2F1-induced dormancy was associated with a global repressive chromatin state characterized by a H3K27-/K9-me3high/H3K4-me3/H3K27aclow profile. In both breast and HNSCC models NR2F1 blocked self-renewal extinguishing tumor initiating cell subpopulations. Tracking label retaining H2B-GFP cells, gain and loss of function studies showed that NR2F1-induced quiescence occurs via upregulation of SOX9, RARβ and CDK inhibitors. Importantly, inducible shRNAmir-mediated knock down of NR2F1 revealed that it mediates dormancy of occult loco-regional, spleen and lung DTCs, but not of dormant bone marrow DTCs. We conclude that the ability of NR2F1 to limit self-renewal and promote quiescence is key to dormancy induction. Manipulation of such mechanisms via epigenetic and differentiation therapies might represent a strategy to generate induced dormant cancer cells (iDCs) and maintain or eliminate asymptomatic residual disease. Citation Format: Maria S. Sosa, Yeriel Estrada, Falguni Parikh, Almudena Bosch Gutierrez, Yang Zheng, Paloma Bragado, Esther Ekpin, Ajish George, Eduardo Farias, Julio Aguirre-ghiso. NR2F1 coordinates a dormancy program by inducing low self-renewal capacity and a repressive chromatin state. [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 1090. doi:10.1158/1538-7445.AM2013-1090