Jim Dimitroulakos

Customized Viral Immunotherapy for HPV-Associated Cancer

Oncolytic Maraba virus can selectively infect HPV+ human cancers as well as generate substantial antitumor immunity. This resulted in complete destruction of advanced HPV+ tumors in mice, providing a promising immunological approach to combat HPV-associated cancer. The viral-transforming proteins E6 and E7 make human papillomavirus–positive (HPV+) malignancies an attractive target for cancer immunotherapy. However, therapeutic vaccination exerts limited efficacy in the setting of advanced disease. We designed a strategy to induce substantial specific immune responses against multiple epitopes of E6 and E7 proteins based on an attenuated transgene from HPV serotypes 16 and 18 that is incorporated into MG1-Maraba virotherapy (MG1-E6E7). Mutations introduced to the transgene abrogate the ability of E6 and E7 to perturb p53 and retinoblastoma, respectively, while maintaining the ability to invoke tumor-specific, multifunctional CD8+ T-cell responses. Boosting with MG1-E6E7 significantly increased the magnitude of T-cell responses compared with mice treated with a priming vaccine alone (greater than 50 × 106 E7-specific CD8+ T cells per mouse was observed, representing a 39-fold mean increase in boosted animals). MG1-E6E7 vaccination in the HPV+ murine model TC1 clears large tumors in a CD8+-dependent manner and results in durable immunologic memory. MG1-Maraba can acutely alter the tumor microenvironment in vivo and exploit molecular hallmarks of HPV+ cancer, as demonstrated by marked infection of HPV+ patient tumor biopsies and is, therefore, ideally suited as an oncolytic treatment against clinical HPV+ cancer. This approach has the potential to be directly translatable to human clinical oncology to tackle a variety of HPV-associated neoplasms that cause significant morbidity and mortality globally. Cancer Immunol Res; 5(10); 847–59. ©2017 AACR.

Vulvar squamous cell carcinoma (VSCC) as two diseases: HPV status identifies distinct mutational profiles including oncogenic fibroblast growth factor receptor 3

Purpose: Patients with advanced or recurrent invasive vulvar squamous cell carcinoma (VSCC) have limited treatment options and a grave prognosis. Understanding the genomic landscape may facilitate the identification of new therapies and improve clinical outcomes. Experimental Design: A retrospective chart review and molecular analysis of patients with VSCC from 2000 to 2016 was performed at the Ottawa Hospital Research Institute. The presence of oncogenic human papillomavirus (HPV) was determined by nested PCR and amplified DNA was sequenced using the Ion AmpliSeq Cancer Hotspot v2 Panel. The patients were divided into two groups according to HPV status (HPV-positive versus HPV-negative) and clinical outcome correlated with mutation status using descriptive statistics. Results: In 43 VSCC patients, there was a high mutation rate in both HPV-positive (73%) and HPV-negative (90%) disease with the two subgroups expressing distinct genetic profiles. HPV-positive tumors were characterized by oncogenic mutations in PIK3CA (27%), FGFR3 (14%), and PTEN (9%), whereas HPV-negative tumors were found to have mutations in TP53 (57%), HRAS (24%), PI3KCA (19%), and CDKN2A (14%). Mutation S249C in FGFR3 occurred in 14% of HPV-positive tumors. While there were notable differences in the occurrence of TP53, HRAS, PTEN, and FGFR3 mutations according to HPV status, only the rate of TP53 mutations was statistically significant (P = 0.0004). No significant difference in prognosis was found between patients with HPV-positive and HPV-negative VSCC. Conclusions: HPV-positive VSCC is characterized by oncogenic FGFR3 mutations that helps classify this subtype as a separate disease. Inhibitors of FGFR3 merit consideration as a therapeutic strategy in this neglected cancer in women. Clin Cancer Res; 23(15); 4501–10. ©2017 AACR.

Induction of Activating Transcription Factor 3 Is Associated with Cisplatin Responsiveness in Non–Small Cell Lung Carcinoma Cells

Non–small cell lung carcinoma (NSCLC) is the most common cause of cancer deaths, with platin-based combination chemotherapy the most efficacious therapies. Gains in overall survival are modest, highlighting the need for novel therapeutic approaches including the development of next-generation platin combination regimens. The goal of this study was to identify novel regulators of platin-induced cytotoxicity as potential therapeutic targets to further enhance platin cytotoxicity. Employing RNA-seq transcriptome analysis comparing two parental NSCLC cell lines Calu6 and H23 to their cisplatin-resistant sublines, Calu6cisR1 and H23cisR1, activating transcription factor 3 (ATF3) was robustly induced in cisplatin-treated parental sensitive cell lines but not their resistant sublines, and in three of six tumors evaluated, but not in their corresponding normal adjacent lung tissue (0/6). Cisplatin-induced JNK activation was a key regulator of this ATF3 induction. Interestingly, in both resistant sublines, this JNK induction was abrogated, and the expression of an activated JNK construct in these cells enhanced both cisplatin-induced cytotoxicity and ATF3 induction. An FDA-approved drug compound screen was employed to identify enhancers of cisplatin cytotoxicity that were dependent on ATF3 gene expression. Vorinostat, a histone deacetylase inhibitor, was identified in this screen and demonstrated synergistic cytotoxicity with cisplatin in both the parental Calu6 and H23 cell lines and importantly in their resistant sublines as well that was dependent on ATF3 expression. Thus, we have identified ATF3 as an important regulator of cisplatin cytotoxicity and that ATF3 inducers in combination with platins are a potential novel therapeutic approach for NSCLC.

Correction to: HPV DNA in saliva from patients with SCC of the head and neck is specific for p16-positive oropharyngeal tumours

Following publication of the original article [1], the authors reported an error in one of the author names. In this Correction the incorrect and correct author names are listed.

Effect of statin use on oncologic outcomes in head and neck squamous cell carcinoma

Preclinical and early‐phase clinical studies have suggested an oncoprotective role of statins in head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to determine whether incidental statin use in patients with human papillomavirus (HPV)‐negative HNSCC is predictive of improved oncologic outcomes.

Abstract A16: Induction of activating transcription factor 3 is associated with cisplatin responsiveness in NSCLC: A potential predictive biomarker of response.

The goal of this study was to identify treatment induced biomarkers of platin activity that may predict response to this important class of chemotherapeutic agents. We employed RNAseq transcriptome analysis comparing two parental NSCLC cell lines Calu6 and H23 to their resistant sub-lines, Calu6cisR1 and H23cisR1, derived following exposure to high dose cisplatin. To this end, we identified a stress pathway consisting of GADD45α, ATF3 and DDIT3/CHOP that was induced specifically in cisplatin treated parental but not their resistant sub-lines. Furthermore, ATF3 in particular was not expressed in untreated parental or resistant clones but was robustly induced only in the parental sensitive cell lines following cisplatin treatment. Cisplatin-induced MAPKinase activation, particularly the JNK pathway was abrogated in Calu6cisR1 cells that regulates ATF3 induction in Calu6 cells. In ex-vivo NSCLC tumors, ATF3 was induced in 2/4 tumors evaluated but not in their corresponding normal adjacent lung tissue (0/4) following cisplatin treatment. Thus, the lack of significant expression in untreated NSCLC cells and normal lung tissue but a robust induction following cisplatin treatment in sensitive parental NSCLC cell lines and a cohort of ex-vivo tumor samples suggest potential utility as a predictive biomarker of platin response that requires further study. Citation Format: Jair Bar, David Stewart, Goss D. Glenwood, Patrick J. Villeneuve, Jim Dimitroulakos. Induction of activating transcription factor 3 is associated with cisplatin responsiveness in NSCLC: A potential predictive biomarker of response. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A16.

154PMIR PROFILING IDENTIFIES CDK6 DOWN-REGULATION AS A POTENTIAL MECHANISM OF ACQUIRED CISPLATIN RESISTANCE IN NON-SMALL CELL LUNG CARCINOMA

ABSTRACT Aim: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death. NSCLC systemic treatment usually includes platinum-based chemotherapeutic drugs. Resistance to these drugs is common and multi-factorial. We aimed to gain insight into the molecular mechanisms involved in platinum-resistance. Methods: A set of NSCLC platinum resistant cell-lines was created from Calu6 and NCI-H23 cell lines. Cell viability was quantified by MTT assay. Differentially expressed micro RNAs (miRs) in these lines were identified by Affymetrix miR array. Potential genes targeted by these miRs were searched by TargetScan algorithm. miRs and mRNA levels were tested by real-time PCR. Results: miR-145 was reproducibly elevated in all the resistant sub-lines tested within one of the experimental sets; however, modulation of miR-145 levels alone in these cells did not affect their response to cisplatin. A potential target of miR-145 is cyclin dependent kinase 6 (CDK6), an important regulator of cell proliferation. Both mRNA and protein levels of CDK6 are down regulated in the resistant sub-lines. An inhibitor of CDK4/6 (PD0332991) protected cells from cisplatin cytotoxicity. Conclusions: We have identified a number of miRs differentially expressed in cisplatin-resistant cell lines including miR-145. A potential target of miR-145 is CDK6. Expression of CDK6 is down regulated in the resistant sub-lines, likely through multiple mechanisms that may include targeting by miRs. Inhibition of CDK6 antagonizes cisplatin-induced NSCLC cell cytotoxicity, suggesting that agents that inhibit CDK6 should be avoided during cisplatin therapy. Disclosure: All authors have declared no conflicts of interest.

Abstract 895: Activating transcription factor 3 (ATF3) down-regulation correlates with platinum resistance in non-small cell lung cancer (NSCLC).

Background: NSCLC is the most common cause of cancer-related death. Platinum-based chemotherapy is the mainstay of treatment, but a variety of mechanisms lead to platinum-resistance. ATF3 is a transcription factor, activated in response to a wide variety of stress signals including DNA damage and hypoxia. We recently demonstrated a role for ATF3 as an important regulator of platinum-induced cytotoxicity. In this study, we hypothesize that ATF3 expression correlates with platinum-sensitivity/resistance in NSCLC. Methods: ATF3 induction was examined by Western blots and real-time RT-PCR in isogenic sets of platinum- sensitive (S) or induced resistant (R) NSCLC cell lines. A 1200 compound library was screened to identify platinum-sensitizers in both sets of cell lines. Complete RNA sequencing (RNA-seq) was performed in parental (S) and resistant (R) derived cell lines comparing expression patterns of either untreated or platinum treated cells. Similarly, platinum R and S tumors were identified by screening NSCLC patients’ clinical records and expression patterns compared with RNA-seq analysis. Results: Both mRNA and potein levels of ATF3 were induced 35-120 fold (mRNA) by cisplatin treatment in S cell lines, but only 1-12 fold (mRNA) in the corresponding R lines. The 1200 compound library screen of FDA approved compounds identified several commonly used chemotherapeutic agents as sensitizers of platinum cytotoxicity, as well as vorinostat, a histone deacetylase inhibitor that sensitized only S but not R cell lines. An analogue of this agent, M344, enhanced ATF3 induction and cisplatin cytotoxicity in the S lines but not in the R lines. Comparing RNA-seq results of the S and R lines revealed up-regulation of GADD45A, ATF3 and DDIT3/CHOP in the S but not R cell lines. Comparing samples of S and R NSCLC tumors by RNA-seq demonstrated ATF3 to be among the five genes significantly up-regulated in the S tumors compared to the R tumors. Conclusions: Stress-induced ATF3 is correlated with platinum-sensitivity in NSCLC cells. Tumor ATF3 levels represent a potential predictive marker of response to platinum-based treatment in NSCLC. Revealing the mechanism of ATF3 induction by platinum may lead to the identification of novel platinum-sensitizing therapeutic targets. Citation Format: Jair Bar, Ivan Gorn-Hondermann, Reid Stephanie, Patricia Moretto, Iris Shiran, Shlomit Jessel, Marina Perelman, Eyal Heller, Iris Kamer, Inbal Daniel-Meshulam, Glenwood D. Goss, Jim Dimitroulakos. Activating transcription factor 3 (ATF3) down-regulation correlates with platinum resistance in non-small cell lung cancer (NSCLC). [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 895. doi:10.1158/1538-7445.AM2013-895

Abstract 4949: microRNA (miR) analysis as a tool for discovering platinum resistance mechanisms in non-small cell lung cancer (NSCLC)

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnBackground: NSCLC is the most common cause of cancer-related death in the world. Platinum-based chemotherapy is the mainstay of treatment both in the adjuvant and the metastatic settings, but platinum resistance is common. A variety of mechanisms have been reported to underlie platinum-resistance. miRs are short non-coding RNA molecules, each regulating a large number of genes. We hypothesized that in platinum-resistant NSCLC cells, a small number of differentially expressed miRs modulating an abundance of gene products, activate in concert a large number of platinum-resistance mechanisms.nnAim: To uncover mechanisms of platinum-resistance in NSCLC, by indentifying miRs differentially expressed in platinum resistant cells.nnMethods: Platinum-resistant cell lines were created by cisplatin selection of Calu6 and NCI-H23, NSCLC cell lines. RNA was hybridized to Affymetrix miR arrays, followed by TargetScan algorithm analysis. Levels of specific miRs were quantified by RT-PCR (ABI) and were modulated by miR inhibitors (Ambion).nnResults: Two sets of sensitive (S) and platinum-resistant (R) cell lines were created, each set consisting of a parental S line and several R sub-lines. miR array analysis was performed on the S and R lines. miRs that were significantly more abundant in each of four R lines compared to the corresponding S lines were compiled. Genes predicted to be targeted by each of those miRs with a p value < .05 were tabulated. Surprisingly, only 16 genes were common to those four gene lists, including CDK6, an important G1-S cell cycle regulator. mRNA and protein levels of CDK6 were higher in S compared to R lines, and accordingly, growth rate of R lines was slower. miR145, one of the miRs upregulated in the R lines, is predicted to target CDK6. Transfection of a miR145 inhibitor resulted in a mild increase in mRNA levels of CDK6, supporting its role as a regulator of CDK6.nnConclusions: Utilizing a number of biological systems (sets of cells lines) that model the same phenotype (platinum resistance) allows for a robust analysis of the involved molecules. miR profiling of multiple systems can identify genes that are commonly regulated in different systems by different miRs. Using this approach we discovered upregulation of miRs, including miR145, resulting in downregulation of CDK6, as a potential mechanism of platinum resistance in NSCLC.nnCitation 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 4949. doi:10.1158/1538-7445.AM2011-4949

Abstract A53: Lovastatin inhibits EGFR dimerization and AKT activation in squamous cell carcinoma cells: Potential regulation through targeting rho proteins

We recently demonstrated the ability of lovastatin to inhibit the function of the epidermal growth factor receptor (EGFR) and its downstream signaling of the PI3K/AKT pathway. Combining lovastatin with gefitinib, a potent EGFR inhibitor, induced synergistic cytotoxicity in various tumor-derived cell lines. In this study, lovastatin treatment inhibits ligand-induced EGFR dimerization in squamous cell carcinoma (SCC) cells and its activation of AKT and its downstream targets 4EBP1 and S6K1. This inhibition was associated with global protein translational inhibition demonstrated by a decrease in RNA-associated polysome fractions. The effects of lovastatin on EGFR function were reversed by the addition of the geranylgeranyl pyrophosphate that acts as a protein membrane anchor. Lovastatin treatment induced actin cytoskeletal disorganization and the expression of the geranylgeranylated rho family proteins that regulate the actin cytoskeleton, including rhoA. Lovastatin-induced rhoA was inactive as EGF stimulation failed to activate rhoA and inhibition of the rho-associated kinase, a target and mediator of rhoA function, with Y-27632 also showed inhibitory effects on EGFR dimerization. The ability of lovastatin to inhibit EGFR dimerization is a novel exploitable mechanism regulating this therapeutically relevant target. To assess the potential of this approach, we evaluated the effect of statin use in patients enrolled in the BR21 erlotinib phase III study in non-small cell carcinoma (NSCLC) patients. In the erlotinib arm of this trial, although not statistically significant due to the limited number of patients on statins, in general, patients that were on statins performed better than patients without statins. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A53.