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Background: The National Cancer Institute (NCI) PREVENT Cancer Program (PREVENT) is a peer-reviewed R&D pipeline with the core emphasis on preclinical development and clinical translation of novel cancer preventive interventions. One of the latest PREVENT-supported projects include cGMP production and IND enabling studies of a broad spectrum experimental human papillomavirus (HPV) vaccine- HPV16 RG1-VLP. This monovalent chimeric virus-like particle (VLP) displays 360 copies of the highly conserved epitope RG1 (aa 17-36 of minor capsid protein HPV16 L2) in the DE loop of HPV16L1 VLP backbone, and is capable of eliciting broadly neutralizing antibodies that target several clinically relevant HPV genotypes. RG-1 induced cross-neutralizing titers are typically lower than anti-L1 antibodies generated by currently licensed HPV vaccines. Hence, durability of protection has been cited as a cause of concern. Here, using engineering-run cGMP grade HPV16-RG1VLPs formulated with alhdyrogel®, the durability of protection 6 month post-vaccination of HPV16 RG1-VLPs against Gardasil-9®, a licensed HPV vaccine was evaluated using an established papillomavirus disease model. Methods: New Zealand white rabbits (n=15 per treatment group) were administered three intra-muscular vaccinations of HPV16-RG1 (80 µg), human doses of Gardasil-9®, or no vaccine (adjuvant only). Following vaccination, in vivo protection was assessed against 8 high-risk oncogenic HPVs utilizing methods from an established cottontail rabbit papillomavirus (CRPV) disease model. Within each treatment group, 5 rabbits were challenged two weeks post-final vaccination (at peak serum ELISA titer), while another 5 were challenged six months post-final vaccination to assess durability of protection. The remainder 5 rabbits will be challenged one year post final vaccination. Results: During the peak-titer period, rabbits vaccinated with monovalent HPV16-RG1VLP were protected from disease development, which was comparable to the protection afforded by Gardasil-9®. Six months after final vaccination, despite lower serum titers, HPV16-RG1VLP immunized rabbits were still protected from disease development with vaccine efficacy comparable to that of Gardasil-9®. And, in some instances, HPV16-RG1VLP vaccine demonstrated a superior cross-protection. Conclusions: Even as a monovalent formulation, HPV16 RG1 VLP vaccination was able to provide comparable protection against a number of high-risk oncogenic HPV types, including types not covered by Gardasil-9®, even after six months post-vaccination. As a monovalent VLP, HPV16 RG1 VLP holds promise as a broad-spectrum preventative vaccine candidate that could potentially provide broader protection at lower production costs. Studies evaluating protection one-year-post vaccination is currently in progress. Citation Format: Jiafen Hu, Karla Balogh, Ken Matsui, Huimin Tan, Pola Olczak, George Buchman, Brian Howard, Jonathan White, Michelle Kennedy, Shizuko Sei, Elizabeth Glaze, Sarah Brendle, Christina Schellenbacher, Reinhard Kirnbauer, Richard Roden, Robert Shoemaker, Neil Christensen, Joshua Weiyuan Wang. A cGMP-grade chimeric papillomavirus candidate vaccine (HPV16 RG1-VLP) confers long term cross-protection compared to a nonavalent hpv vaccine in a pre-clinical papillomavirus animal model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-200.

Multiple classes of pharmacologic agents have the potential to induce the expression and release of proinflammatory factors from dying tumor cells. As a result, these cells can in theory elicit an immune response through various defined mechanisms to permanently eradicate disseminated cancer. However, the impact of chemotherapy on the tumor-specific immune response in the context of the tumor microenvironment is largely unknown. Within the tumor microenvironment, the immune response promoted by chemotherapy is antagonized by an immune-suppressive milieu, and the balance of these opposing forces dictates the clinical course of disease. Here, we report that high antigen exposure within the tumor microenvironment following chemotherapy is sufficient to skew this balance in favor of a productive immune response. In elevating antigen exposure, chemotherapy can achieve long-term control of tumor progression without the need of an additional adjuvant. We found that chemotherapy initiated this phenomenon in the tumor microenvironment through an accumulation of dendritic cells, which stimulated CD8+ T cells and the type I IFN pathway. From this conceptual base, we developed a simple approach to cancer therapy combining chemotherapy and vaccination that may be widely applicable. Cancer Res; 73(8); 2493–504. ©2013 AACR.

Oncogenic human papillomavirus (HPV) infection is a necessary cause of cervical cancer. Therefore, vaccination to prevent or eliminate HPV infection could reduce the incidence of cervical cancer. A fusion protein comprising HPV16 L2, E6, and E7 is a candidate combination preventive and therapeutic HPV vaccine. The L1- and L2-specific and neutralizing serum antibody titers and peripheral blood mononucleocyte antigen–specific proliferative responses generated by vaccination thrice at monthly intervals with HPV16 L2E7E6 were compared in two studies: a phase I randomized double-blind placebo controlled dose escalation trial in 40 healthy volunteers and a phase II trial of HPV16 L2E7E6 at the maximum dose in 29 women with high-grade anogenital intraepithelial neoplasia (AGIN). Vaccination of healthy volunteers induced L2-specific serum antibodies that were detected 1 month after the final vaccination (Pbinomial < 0.001). There was a significant trend to seroconversion for HPV16 and HPV18 neutralizing antibodies with increasing vaccine dose (P = 0.006 and P = 0.03, respectively). Seroconversion for HPV18 neutralizing antibodies showed a significant positive trend with increasing dose (P = 0.03) and was associated with seroconversion for HPV16 neutralizing antibodies (Pexact = 0.04). The antigen-specific proliferative response of vaccinated healthy volunteers also showed a significant trend with increasing vaccine dose (P = 0.04). However, AGIN patients responded less effectively to vaccination than healthy patients for induction of HPV16 L2–specific antibody (P < 0.001) and proliferative responses (P < 0.001). Vaccination of healthy volunteers thrice with 533-μg HPV16 L2E7E6 at monthly intervals induced L2-specific serum antibodies that neutralized across papillomavirus species. Responses in AGIN patients were infrequent. (Cancer Res 2006; 66(23): 11120-4)

An elevated number of Gr-1+CD11b+ myeloid cells has been described in mice bearing transplantable tumors, and has been associated with immune suppression. We examined the role of such myeloid suppressor cells in mice bearing the spontaneously transformed syngeneic mouse ovarian surface epithelial cell line, 1D8. We observed high levels of CD80 expression by Gr-1+CD11b+ cells from spleen, ascites, and tumor tissue of mice bearing 1D8 ovarian carcinoma, whereas CD40 and CD86 were absent. CD80 expression was not detected on Gr-1+CD11b+ cells from naïve mice. However, the expression of CD80 by Gr-1+CD11b+ cells from naïve mice was promoted by coculture with 1D8 cells. Because irradiated 1D8 cells, but not 1D8-conditioned medium, up-regulate CD80 expression by Gr-1+CD11b+ cells, this phenomenon likely requires direct interaction. Gr-1+CD11b+ cells derived from 1D8 tumor–bearing mice provided significant suppression of antigen-specific immune responses, but Gr-1+CD11b+ cells from naïve mice did not. Both short interfering RNA–mediated knockdown and genetic knockout of CD80 expression by Gr-1+CD11b+ cells of 1D8 tumor–bearing mice alleviated the suppression of antigen-specific immune responses. Suppression via CD80 on Gr-1+ CD11b+ myeloid cells was mediated by CD4+CD25+ T regulatory cells and required CD152. CD80 knockout or antibody blockade of either CD80 or CD152 retarded the growth of 1D8 tumor in mice, suggesting that expression of CD80 on Gr-1+CD11b+ myeloid cells triggered by 1D8 ovarian carcinoma suppresses antigen-specific immunity via CD152 signaling and CD4+CD25+ T regulatory cells. Thus, CD80-dependent responses to myeloid suppressor cells may contribute to tumor tolerance and the progression of ovarian carcinoma.(Cancer Res 2006; 66(13): 6807-15)

Biomarkers for early detection of epithelial ovarian cancer (EOC) are urgently needed. Patients can generate antibodies to tumor-associated antigens (TAAs). We tested multiplex detection of antibodies to candidate ovarian TAAs and statistical modeling for discrimination of sera of EOC patients and controls. Binding of serum antibody of women with EOC or healthy controls to candidate TAA-coated microspheres was assayed in parallel. A Bayesian model/variable selection approach using Markov Chain Monte Carlo computations was applied to these data, and serum CA125 values, to determine the best predictive model. The selected model was subjected to area under the receiver-operator curve (AUC) analysis. The best model generated an AUC of 0.86 [95% confidence interval (95% CI), 0.78-0.90] for discrimination between sera of EOC patients and healthy patients using antibody specific to p53, NY-CO-8, and HOXB7. Inclusion of CA125 in the model provided an AUC of 0.89 (95% CI, 0.84-0.92) compared with an AUC of 0.83 (95% CI, 0.81-0.85) using CA125 alone. However, using TAA responses alone, the model discriminated between independent sera of women with nonmalignant gynecologic conditions and those with advanced-stage or early-stage EOC with AUCs of 0.71 (95% CI, 0.67-0.76) and 0.70 (95% CI, 0.48-0.75), respectively. Serum antibody to p53 and HOXB7 is positively associated with EOC, whereas NY-CO-8-specific antibody shows negative association. Bayesian modeling of these TAA-specific serum antibody responses exhibits similar discrimination of patients with early-stage and advanced-stage EOC from women with nonmalignant gynecologic conditions and may be complementary to CA125. (Cancer Res 2006; 66(3): 1792-8)

Background: Bone loss has been consistently reported among long-term older breast cancer (BC) survivors. However, it remains unclear how early and to what degree BC survivors experience bone loss compared to their cancer-free peers and to what extent this can be attributed to age and premature menopause. Methods: We studied 796 women (210 BC survivors, 586 cancer-free) with familial risk of breast and/or ovarian cancer in the Breast and Ovarian Surveillance Service (BOSS) cohort at Johns Hopkins Hospital. Survivors were diagnosed with stage I-III BC Results: BC survivors were slightly older (mean age 48 vs. 45 years), more likely to be post-menopausal (51% vs. 29%) and had higher current vitamin D use (21% vs. 8%) compared to cancer-free women. During a mean follow-up time of 6.2 years, 77% of BC survivors and 60% of cancer-free women reported having ≥1 bone density exam and 115 incident osteopenia and/or osteoporosis cases were identified (75% osteopenia only). In MV-adjusted models, BC survivors had 66% higher risk of being diagnosed with osteopenia and/or osteoporosis compared to cancer-free women (HR=1.66, 95% CI=1.11-2.47). BC survivors with no current vitamin D or calcium use had 76% and 93% higher osteopenia and/or osteoporosis risk compared to cancer-free women, respectively (MV HR=1.76, 95% CI=1.14-2.72; MV HR=1.93, 95% CI=1.15-3.24). BC survivors ever treated with chemotherapy or hormone therapy had higher osteopenia and/or osteoporosis risk compared to cancer-free women (MV HR=2.00, 95% CI=1.14-3.50; MV HR=1.79, 95% CI=1.14-2.82, respectively). Analyses restricted to women with no change in menopause status during follow-up were only slightly attenuated (MV HR=1.58, 95% CI=0.96-2.59). Conclusion: Our results demonstrate that bone loss is significantly greater than and occurs early in young BC survivors compared to cancer-free women even after taking into consideration increasing age and premature menopause. Our findings also provide support for a baseline evaluation of bone density at diagnosis with subsequent monitoring in young BC survivors with familial risk. Further analyses are ongoing to evaluate the effect of specific BC treatments on bone health in this cohort. Citation Format: Cody A. Ramin, Betty J. May, Richard B. Roden, Dana Petry, Deborah K. Armstrong, Kala Visvanathan. Evaluation of bone health in breast cancer survivors compared to cancer-free women: A prospective study within a young familial risk cohort [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 3271. doi:10.1158/1538-7445.AM2017-3271

Human papilloma virus (HPV) high risk (hr) types cause several cancers, including cervical, vaginal, penile, and oropharyngeal, whereas low risk types cause benign diseases such as skin warts, anogenital warts and recurrent respiratory papillomatosis. Although hrHPV infection is one of the most common sexually transmitted diseases most infections remain asymptomatic and are cleared while a sub-population develops persistent HPV-related pathologies including cancer. Further, immune-compromised populations, notably organ transplant and HIV-positive patients, are particularly vulnerable to HPV-related pathologies. Here, we describe the first establishment of mouse papillomavirus-1 (MusPV1) disease in outbred and immune competent SKH1-Elite mice (Crl:SKH1-Hrhr). Challenge of SKH1 mice with MusPV1 resulted in three clinical outcomes: 1) persistent (>2 months) papillomas (∼15%), 2) transient papillomas that spontaneously regress typically within 2 months (∼10%), 3) no visible papillomas and viral clearance (∼75%). SKH1 mice with persisitent papillomas were treated using a potentially preventive and therapeutic DNA vaccine composed of human calreticulin (hCRT) linked to MusPV1 early proteins, mE6 and mE7 and the late protein mL2 (hCRTmE6/mE7/mL2). Three intramuscular DNA vaccinations (15 μg in PBS) via in vivo electroporation were administered weekly and immune responses were measured, including mE6/mE7/mL2 serum antibody response and T-cell epitope mapping studies. Previously persistent papillomas disappeared within 2 months after final vaccination. Clearance of MusPV1 was confirmed by loss of mE6/mE7 transcript staining using RNA-CISH on formalin-fixed, paraffin-embedded tail sections. Vaccination induced a strong mE6 and mE7 CD8+ T cell response in all mice. Interestingly, mL2 antibody responses were faster and stronger in asymptomatic challenged mice and in those that spontaneously cleared their papillomas after challenge as compared to mice with persistent papillomas. In sum, MusPV1 challenge of outbred, immune-competent SKH1 mice represents a promising model to further study potential immunotherapies for HPV-related disease and the relationship between spontaneous clearance of papillomavirus and host genetics. Citation Format: Rosie T. Jiang, Joshua W. Wang, Shiwen Peng, Chien-Fu Hung, Richard B.S Roden. Spontaneous and vaccine-induced clearance of Mus Musculus Papillomavirus1 (MusPV1) infection. [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 2364.

The NCI Division of Cancer Prevention's PREVENT Cancer Preclinical Drug Development Program supports preclinical development and clinical translation of novel cancer preventive interventions. One of the projects currently supported came from principal investigators, Drs. Reinhard Kirnbauer and Richard Roden, who requested cGMP production of a novel chimeric virus-like particle (VLP)-based human papillomavirus (HPV) vaccine, comprising HPV16 L1 VLP with HPV16 L2-RG1 epitope repetitively displayed on the capsid surface (RG1-VLP). The vaccine is designed to prevent infection by a broad range of HPV types, including mucosal and cutaneous types not targeted by current vaccines (J Invest Dermatol 2013, 133:2706), and thus has the potential to become a broadly effective next generation HPV vaccine. The cGMP process is currently being developed and validated by Paragon Bioservices in Baltimore, MD, under NCI contract with MRIGlobal. RG1-VLP is expressed in Sf9 insect cells infected with a triple-plaque-purified recombinant baculovirus. After cell culture clarification and capsid maturation steps, VLPs are purified by a series of chromatography steps to remove host cell derived impurities. The purified VLPs are analyzed by ELISA and Western blot immunoassays. Removal of host cell protein and DNA is confirmed by ELISA and qPCR, respectively. Assembly of a chimeric RG1-VLP protein into full size VLP structures (approximately 50-60 nm in diameter) is verified by transmission electron microscopy. cGMP-produced VLPs should be available during the first half of 2016. The PREVENT Program will perform the required toxicology studies and will assist with IND filing, for a first-in-human Phase I study of RG1-VLP vaccine. While the clinical protocol is still under development, it is anticipated that 15-20 healthy adult volunteers will be enrolled into each of three to four escalating dose groups. While the primary objective of the Phase I study will be to establish the safety of RG1-VLP, preliminary immunogenicity studies will be included. Immunogenicity of RG1-VLP will be evaluated by serum anti-L1 and anti-RG1 antibody levels as well as protective and cross-neutralizing antibodies against HPV16 and a broad panel of HPV types including at least one type not covered by marketed vaccines. Citation Format: George W. Buchman, Brian P. Howard, Nadia Abdallah, Bala Medicherla, Mary Anne Fisher, Jonathan M. White, Michelle L. Kennedy, Shizuko Sei, Richard B.S. Roden, Christina Schellenbacher, Reinhard Kirnbauer, Robert H. Shoemaker. cGMP production of a chimeric virus-like particle vaccine for prevention of HPV-associated cancers. [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 2367.

The ubiquitin-proteasome system (UPS) mediates targeted protein degradation. Notably, the UPS determines levels of key checkpoint proteins controlling apoptosis and proliferation by controlling protein half-life. Herein, we show that ovarian carcinoma manifests an overstressed UPS by comparison with normal tissues by accumulation of ubiquitinated proteins despite elevated proteasome levels. Elevated levels of total ubiquitinated proteins and 19S and 20S proteasome subunits are evident in both low-grade and high-grade ovarian carcinoma tissues relative to benign ovarian tumors and in ovarian carcinoma cell lines relative to immortalized surface epithelium. We find that ovarian carcinoma cell lines exhibit greater sensitivity to apoptosis in response to proteasome inhibitors than immortalized ovarian surface epithelial cells. This sensitivity correlates with increased cellular proliferation rate and UPS stress rather than absolute proteasome levels. Proteasomal inhibition in vitro induces cell cycle arrest and the accumulation of p21 and p27 and triggers apoptosis via activation of caspase-3. Furthermore, treatment with the licensed proteasome inhibitor PS-341 slows the growth of ES-2 ovarian carcinoma xenograft in immunodeficient mice. In sum, elevated proliferation and metabolic rate resulting from malignant transformation of the epithelium stresses the UPS and renders ovarian carcinoma more sensitive to apoptosis in response to proteasomal inhibition. (Cancer Res 2006; 66(7): 3754-63)

The interactions between neoplastic cells and their tumor microenvironment (TME) have become increasingly evident. Cancer-associated fibroblasts (CAFs) and M2 macrophages (M2 Mθ) are important components of the TME that have been recognized to orchestrate tumor-promoting inflammation and thereby tumor growth and progression. Here, we aim to determine the presence and role of CAFs and M2 Mθ in high grade cervical intraepithelial neoplasia (CIN 2/3) and invasive cervical carcinomas (SCCx). We optimized immunohistochemistry (IHC) markers for M2 Mθ (CD163, ARG1), CAFs (αSMA) and Th1 T cells (TBX21) in paraffin-embedded tissue sections from CIN2/3 (n=6) and SCCx cases (n=4). We found an increased infiltration of TBX21+ cells in the epithelium and stroma of CIN2/3 and SCCx cases compared to their normal adjacent stroma. This suggests that despite increased Th1 T cells, the progression from normal to CIN2/3 to SCCx might be facilitated by M2 Mθ and CAFs. Moreover, by performing double staining Immunofluorescence (IF) we found that M2 Mθ (CD163+) were the cells responsible for ARG1 production, an immunesuppressive mechanism utilized by these cells. We recently stained by IHC a cervical tissue microarray (TMA) of normal, CIN2/3 and SCCx cases (n=63) with CD163, αSMA and TBX21, and we are currently analyzing the correlation of these markers with clinicopathological data available for these cases (HPV status, tumor stage, therapy response, overall survival). With the hypothesis in mind that CAFs induce the polarization of macrophages into a M2 Mθ phenotype, we isolated fibroblasts from normal (n=2), CIN2/3 (n=1) and SCCx (n=2) fresh tissue explants obtained from surgery. We performed Immunocytochemistry (ICC) with αSMA, and observed a progressive increase in αSMA+ fibroblasts from normal to CIN2/3 and from CIN2/3 to SCCx fibroblasts, suggesting that CAFs might play an important role in the transformation of normal cervix into CIN2/3 and SCCx. Furthermore, we are currently performing co-culture experiments with normal, CIN2/3 and SCCx isolated fibroblasts and peripheral blood monocytes obtained from healthy donors. We will perform triple staining IF with vimentin, αSMA and CD163 in the slides generated from the co-culture, to evaluate if monocytes acquired a M2 Mθ phenotype with the presence of any of these 3 subsets of fibroblasts. Moreover, we will perform a cytokine analysis in the supernatants of these co-cultures to evaluate for potential cytokines produced by CAFs, implicated in M2 Mθ polarization. In this study we provide evidence regarding the role of CAFs in SCCx initiation and progression. Although the mechanisms that regulate fibroblast activation have not been elucidated, it is possible that CAFs might serve as novel therapeutic targets in cancer, either alone or in combination with existing anti-cancer therapies. Citation Format: Leonel Maldonado, Teresa Diaz-Montes, Abdulrahman Sinno, Edward J. Tanner, Christopher VandenBussche, Richard Roden, Iveta Yotova, Benjamin Tycko, Cornelia L. Trimble. Role and presence of cancer associated fibroblasts and M2 macrophages in high grade cervical intraepithelial neoplasia and invasive cervical carcinomas. [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 3672. doi:10.1158/1538-7445.AM2014-3672

Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) is a rare and highly aggressive malignancy that affects children and young women at a mean age of 24 (range 14 months - 58 years). SCCOHT is refractory to standard of care therapy for ovarian cancer, with ∼75% mortality within 18 months of diagnosis. The early age of onset of SCCOHT and reports of familial occurrence in some cases, strongly suggest an underlying hereditary etiology. To understand the molecular pathogenesis of SCCOHT, we performed next-generation genomic sequencing on a series of tumor and germline samples from SCCOHT patients. This analysis revealed germline and somatic inactivating mutations in SMARCA4, a subunit of the SWI/SNF chromatin-remodeling complex, in 75% (9/12) of SCCOHT patients. Moreover, immunohistochemical (IHC) analysis of 15 tumors revealed that 87% (13/15) of tumors lacked SMARCA4 protein. The high prevalence of SMARCA4 mutations in SCCOHT has not been previously reported in other, more common ovarian carcinomas. We therefore examined the expression of SMARCA4 protein in 300 ovarian carcinomas of different histologies by IHC and found SMARCA4 protein loss in only 6 tumors. In addition, the BIN-67 SCCOHT cell line, which harbors 2 splice site mutations in SMARCA4, showed complete absence of SMARCA4 protein by Western blot while representative cell lines from 4 other ovarian carcinoma subtypes as well as immortalized granulosa cells (SVOG) and adult granulosa tumor cells (KGN) all maintained SMARCA4 expression. The prevalence of germline and sporadic SMARCA4 mutations as well as frequent SMARCA4 protein loss in SCCOHTs implicates this gene as a tumor suppressor in this cancer and more broadly suggests a role for the SWI/SNF complex in its pathogenesis. In addition to providing evidence to the pathogenesis of SCCOHT, this finding provides the opportunity to develop treatment approaches for SCCOHT based on targeting vulnerabilities of SMARCA4-deficient cells. Citation Format: Pilar Ramos, Anthony Karnezis, David Craig, Aleksandar Sekulic, Megan Russell, William Hendricks, Michael Barrett, Karey Shumansky, Yidong Yang, Sohrab Shah, Leah Prentice, Marco Marra, Jeffrey Kiefer, Victoria Zismann, Troy McEachron, Bodour Salhia, Joseph Pressey, John Farley, Stephen Anthony, Richard Roden, Heather Cunliffe, David Huntsman, Jeffrey Trent. The rare, highly malignant small cell carcinoma of the ovary displays common inactivating germline and somatic mutations in the tumor suppressor SMARCA4. [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 LB-202. doi:10.1158/1538-7445.AM2014-LB-202

Title. Small-Molecule Inhibitors of de-Ubiquitinating Enzymes for Cervical Cancer Treatment Abstract: Human Papillomavirus (HPV) is the primary cause of cervical cancer and responsible for 5% of all cancers worldwide. While HPV vaccines can be an effective preventive measure against cervical cancer, there are currently no virus-specific therapies for it, and the efficacy of standard surgical and chemo/radiotherapies is limited for advanced disease. The E6 oncoprotein of HPV exerts its oncogenic activity by binding to the E3 ubiquitin ligase E6-AP and redirects its activity towards p53 and other tumor suppressor proteins for rapid ubiquitin-mediated proteasomal degradation. Therefore, stabilization of p53 via preventing its ubiquitin-mediated degradation represents a valid therapeutic approach for cervical cancer treatment. De-ubiquitinating enzymes are an attractive novel “druggable” target for cervical cancer as they are responsible for controlling the steady state levels of proteins (i.e. p53 and E6-AP) crucial for maintaining the transformed status of cervical cancer cells, upstream proteasome. Thus, their inhibition is potentially associated with less toxic effects as compared to inhibition of proteasomes. During a structure-based screening of chalcone-based small-molecule inhibitors of ubiquitin-mediated protein degradation we have recently identified a novel class of molecules capable of inhibiting the ubiquitin-mediated protein degradation upstream proteasomes. The lead compound of the series, contains the α-β unsaturated carbonyl system, as the molecular determinant for inhibiting de-ubiquitinating enzyme activities and: i) is capable of inducing acute perturbation of the ubiquitin-mediated protein degradation pathways accompanied by accumulation of poly-ubiquitinated species and reduction of mono-, di-, and tri-ubiquitin species, ii) has no effect on the catalytic activities of purified proteasomes or proteasome in living cells, iii) is capable of induce aggresome formation, iv) is capable of rescuing p53 levels/functions in cervical cancer cells which are accompanied by decrease in the levels of Cyclin D1 and failure for the cells to enter the S-phase of the cell cycle, v) is capable of preventing tumor-colony formation and induces onset of apoptosis via caspase-3 activation in cervical cancer cells without affecting the viability of normal cells. Therefore, we believe in the feasibility of using our lead compound in preclinical model of cervical cancer to test its efficacy as antineoplastic agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-258. doi:10.1158/1538-7445.AM2011-LB-258

In Response: In the literature, including our own ( [1][1]), diverse terms (such as Gr-1+CD11b+ myeloid cells, Gr-1+ myeloid cells, Gr-1+CD11b+ immature myeloid cells, CD11b+Gr1+CD31+myeloid progenitor, immature myeloid cells, myeloid suppressor cells, etc.) are used to describe murine Gr1+CD11b+