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Human papillomavirus (HPV) infection causes 600,000 new cancers worldwide each year. HPV-related cancers express the oncogenic proteins E6 and E7, which could serve as tumor-specific antigens. It is not known whether immunity to E6 and E7 evolves during chemoradiotherapy or affects survival. Using T cells from 2 HPV16+ patients, we conducted functional T-cell assays to identify candidate HPV-specific T cells and common T-cell receptor motifs, which we then analyzed across 86 patients with HPV-related cancers. The HPV-specific clones and E7-related T-cell receptor motifs expanded in the tumor microenvironment over the course of treatment, whereas non–HPV-specific T cells did not. In HPV16+ patients, improved recurrence-free survival was associated with HPV-responsive T-cell expansion during chemoradiotherapy.

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Supplemental TablesTables S1-S13

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PDF file, 400K, MLN4924 and cisplatin enhance apoptosis induction in ovarian cancer cells.

Cell-free circulating linear DNA is being explored for noninvasive diagnosis and management of tumors and fetuses, the so-called liquid biopsy. Previously, we observed the presence of small extrachromosomal circular DNA (eccDNA), called microDNA, in the nuclei of mammalian tissues and cell lines. Now, we demonstrate that cell-free microDNA derived from uniquely mapping regions of the genome is detectable in plasma and serum from both mice and humans and that they are significantly longer (30%–60% >250 bases) than cell-free circulating linear DNA (∼150 bases). Tumor-derived human microDNA is detected in the mouse circulation in a mouse xenograft model of human ovarian cancer. Comparing the microDNA from paired tumor and normal lung tissue specimens reveals that the tumors contain longer microDNA. Consistent with human cancers releasing microDNA into the circulation, serum and plasma samples (12 lung and 11 ovarian cancer) collected prior to surgery are enriched for longer cell-free microDNA compared with samples from the same patient obtained several weeks after surgical resection of the tumor. Thus, circular DNA in the circulation is a previously unexplored pool of nucleic acids that could complement miRNAs and linear DNA for diagnosis and for intercellular communication.Implications: eccDNA derived from chromosomal genomic sequence, first discovered in the nuclei of cells, are detected in the circulation, are longer than linear cell-free DNA, and are released from normal tissue and tumors into the circulation. Mol Cancer Res; 15(9); 1197–205. ©2017 AACR.

Supplementary Methods

PDF file, 340K, CDT1 levels in SKOV3 and ES2 cells.

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Table S1: Length and GC content of plasma microDNA isolated from healthy volunteers.

Properties of mouse-derived microDNA in the serum of SKOV3 xenograft mice & Properties of microDNA in human matched tumor and normal lung tissues

Table S3: Summary of microDNA sequencing and mapping to human genomes in lung cancer patients.

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Purpose: The goal of this study was to determine whether distinct gene expression profiles are associated with intrinsic and/or acquired chemoresistance in epithelial ovarian carcinoma.Experimental Design: Gene expression profiles were generated from 21 primary chemosensitive tumors and 24 primary chemoresistant tumors using cDNA-based microarrays. Gene expression profiles of both groups of primary tumors were then compared with those of 15 ovarian carcinomas obtained following platinum-based chemotherapy (“postchemotherapy” tumors). A theme discovery tool was used to identify functional categories of genes involved in drug resistance.Results: Comparison of primary chemosensitive and chemoresistant tumors revealed differential expression of 85 genes (P < 0.001). Comparison of gene expression profiles of primary chemosensitive tumors and postchemotherapy tumors revealed more robust differences with 760 genes differentiating the two groups (P < 0.001). In contrast, only 230 genes were differentially expressed between primary chemoresistant and postchemotherapy groups (P < 0.001). Common to both gene lists were 178 genes representing transcripts differentially expressed between postchemotherapy tumors and all primary tumors irrespective of intrinsic chemosensitivity. The gene expression profile of postchemotherapy tumors compared with that of primary tumors revealed statistically significant overrepresentation of genes encoding extracellular matrix–related proteins.Conclusions: These data show that gene expression profiling can discriminate primary chemoresistant from primary chemosensitive ovarian cancers. Gene expression profiles were also identified that correlate with states of intrinsic and acquired chemoresistance and that represent targets for future investigation and potential therapeutic interventions.

Supplementary Figure S1 from Gene Expression Profiles Associated with Response to Chemotherapy in Epithelial Ovarian Cancers

Article Figures 1-4 from Gene Expression Profiles Associated with Response to Chemotherapy in Epithelial Ovarian Cancers

Supplementary Figures S1-2,4, describe cell viability curves of tested cell lines. Figure S3 and S5 show bioinformatic data for resistance upregulated gene sets (S3) and cisplatin-taxane comparison analysis(S5). Figure S6 shows ECAR and OCR experiments. Figure S7 is a heatmap of combination indices of cisplatin with JQ1. Figure S8 shows apoptosis rates of cell lines. S9-12 describe cell viability dynamics of SOX9 KOs.

Chemoresistance is driven by unique regulatory networks in the genome that are distinct from those necessary for cancer development. Here, we investigate the contribution of enhancer elements to cisplatin resistance in ovarian cancers. Epigenome profiling of multiple cellular models of chemoresistance identified unique sets of distal enhancers, super-enhancers (SE), and their gene targets that coordinate and maintain the transcriptional program of the platinum-resistant state in ovarian cancer. Pharmacologic inhibition of distal enhancers through small-molecule epigenetic inhibitors suppressed the expression of their target genes and restored cisplatin sensitivity in vitro and in vivo. In addition to known drivers of chemoresistance, our findings identified SOX9 as a critical SE-regulated transcription factor that plays a critical role in acquiring and maintaining the chemoresistant state in ovarian cancer. The approach and findings presented here suggest that integrative analysis of epigenome and transcriptional programs could identify targetable key drivers of chemoresistance in cancers.Significance:Integrative genome-wide epigenomic and transcriptomic analyses of platinum-sensitive and -resistant ovarian lines identify key distal regulatory regions and associated master regulator transcription factors that can be targeted by small-molecule epigenetic inhibitors.

Human papillomavirus (HPV) infection causes 600,000 new cancers worldwide each year. HPV-related cancers express the oncogenic proteins E6 and E7, which could serve as tumor-specific antigens. It is not known whether immunity to E6 and E7 evolves during chemoradiotherapy or affects survival. Using T cells from 2 HPV16+ patients, we conducted functional T-cell assays to identify candidate HPV-specific T cells and common T-cell receptor motifs, which we then analyzed across 86 patients with HPV-related cancers. The HPV-specific clones and E7-related T-cell receptor motifs expanded in the tumor microenvironment over the course of treatment, whereas non–HPV-specific T cells did not. In HPV16+ patients, improved recurrence-free survival was associated with HPV-responsive T-cell expansion during chemoradiotherapy.

Adoptive cell therapy (ACT) for cancer shows tremendous potential; however, several challenges preclude its widespread use. These include poor T-cell function in hostile tumor microenvironments, a lack of tumor-specific target antigens, and the high cost and poor scalability of cell therapy manufacturing. Creative genome-editing strategies are beginning to emerge to address each of these limitations, which has initiated the next generation of cell therapy products now entering clinical trials. CRISPR is at the forefront of this revolution, offering a simple and versatile platform for genetic engineering. This review provides a comprehensive overview of CRISPR applications that have advanced ACT. Significance: The clinical impact of ACT for cancer can be expanded by implementing specific genetic modifications that enhance the potency, safety, and scalability of cellular products. Here we provide a detailed description of such genetic modifications, highlighting avenues to enhance the therapeutic efficacy and accessibility of ACT for cancer. Furthermore, we review high-throughput CRISPR genetic screens that have unveiled novel targets for cell therapy enhancement.

Background: The potential of cytokines as cancer therapeutics has been limited by short half-life and severe adverse effects associated with high systemic exposure when delivered intravenously. Many strategies are being explored to overcome these limitations. A locoregional delivery approach to achieve high sustained local concentrations in the tumor microenvironment with minimal systemic exposure could widen the therapeutic window. Early experience with free recombinant human IL2 (rhIL-2) given intraperitoneally (IP) showed meaningful clinical activity in relapsed ovarian cancer. Still, the cumbersome delivery procedure requiring indwelling catheters and need for high volume IP infusions leading to frequent complications and poor patient compliance limited the utility of this approach. To overcome these shortcomings, we developed a clinically translatable localized delivery LOCOcyteTM platform composed of polymer encapsulated allogeneic epithelial cells engineered to produce immune effector molecules for local delivery with temporal regulation. The first product, AVB-001, produces native hIL-2, for the treatment of ovarian cancer and other peritoneal malignancies. IP AVB-001 inhibited tumor growth and improved survival in an in vivo ID8 ovarian cancer murine model. Sustained IL-2 production with well tolerated high IP concentrations were achieved, with >100x differential concentration vs. systemic blood levels in both mice and non-human primates. Strong local and systemic immune activating effects, optimized T cell profile and immune memory were observed without concomitant increase of T regs. The first in human study of AVB-001 in patients with advanced ovarian cancer (NCT05538624) is described here. Methods: Part 1 dose escalation exploring 4 ascending dose levels (capsules target production of 0.6, 1.2, 2.4, and 3.6 ug hIL2/kg/d) with a Bayesian Optimal Interval 3+3 design, in patients with recurrent high grade serous adenocarcinoma of the ovary, primary peritoneum, or fallopian tube. A minimum of 12 and up to 24 patients will be enrolled to receive one dose of AVB-001 administered IP. The primary objective is to evaluate safety (Incidence and severity of adverse events per NCI CTCAE v5.0), tolerability, and feasibility of delivering AVB-001 IP, and establish the RP2D. Secondary objectives include assessment of antitumor activity (RECIST v1.1, iRECIST), pharmacokinetics, and pharmacodynamic correlates of immune activation. Part 2 will enroll 20 patients at the RP2D with efficacy as the primary objective. This multicenter study will be conducted at 5 sites, currently one site is open. The first patient was dosed in December 2022. Recruitment in dose level 1 cohort continues. Citation Format: Shannon N. Westin, Travis Sims, Karen Andreas, Eric Soliman, Manish Jain, Claudio A. Dansky Ullmann, Amir A. Jazaeri. A phase 1/2 open-label, multicenter, dose escalation and expansion study of AVB-001, an intraperitoneally administered, cell-generated, human IL-2 immunotherapy in patients with platinum-resistant, high-grade, serous adenocarcinoma of the ovary, primary peritoneum, or fallopian tube [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT122.

Background: High-grade serous ovarian cancer (HGSC) is the most aggressive epithelial ovarian cancer, exhibiting tumor heterogeneity, molecular abnormality, and variable clinical outcomes. The heterogeneity and apparent adaptability of the HGSC genome under selective pressure by chemotherapy likely explain the high rates of drug resistance in HGSC. However, the underlying mechanisms of drug resistance are not well understood. Following our previous findings of significant distinct molecular and cellular characteristics in pre-treatment tumor samples from highly clinically annotated HGSC patients who received neoadjuvant chemotherapy (NACT), we examined post-treatment transcriptomic changes associated with excellent or poor responders to NACT in HGSC patients. Methods: Thirty tumor tissues (pre- and post-treatment) after 3 to 4 cycles of NACT were collected from HGSC patients at interval cytoreductive debulking surgery from the following groups: excellent response to NACT with carboplatin/paclitaxel (post-ER, n=8); poor response to NACT (post-PR, n=8). Primary or metastatic sites for each patient were obtained (n=13, post-ER; n=17, post-PR) and subjected to RNA sequencing. The downstream bioinformatics analysis was then performed and compared with the data from the matched pre-NACT samples from the same patients (Lee et al., Cell Reports, 2020). Results: We identified a total of 9,764 upregulated and 2,762 downregulated differentially expressed genes (DEGs, absolute log2FC >=2, adj-p Conclusions: Our transcriptomic analysis revealed distinct transcripts changes in post-NACT vs pre-NACT tumor tissues from HGSC patients with excellent or poor response to the NACT. These findings could enhance treatment decision-making and rationale for developing alternative therapies for patients with HGSC. Citation Format: Sanghoon Lee, Li Zhao, Nicole D. Fleming, Joseph Celestino, Richard A. Hajek, Margaret B. Morgan, Yan Liu, Shannon N. Westin, Amir A. Jazaeri, Jinsong Liu, Jianhua Zhang, P Andrew Futreal, Anil K. Sood. Transcriptomic profiles of response to neoadjuvant chemotherapy in patients with high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5738.

Introduction: Despite FDA approval of pembrolizumab in microsatellite instability-high (MSI-H)/mismatch repair deficient solid tumors, approximately half of patients with MSI-H endometrial cancer are treatment-refractory. We sought to evaluate pre-treatment MSI-H endometrial tumor samples to examine cell subpopulation differences in the tumor microenvironment (TME) associated with resistance to pembrolizumab. Methods: Archival tumor samples from MSI-H endometrial cancer patients treated with pembrolizumab at MD Anderson Cancer Center were obtained under an IRB-approved protocol. Twenty-one patients were identified, and pre-treatment archival tumor samples were collected and submitted for RNA-seq and imaging mass cytometry (IMC) with an optimized 38-antibody panel to identify predictive immuno-genomic signatures and cell subpopulations associated with treatment response. Results: Among the 21 patients treated with pembrolizumab, there were 14 responders and 7 non-responders. Based on transcriptomic signatures, TME heterogeneity was observed. The 14 responders consisted of samples with immunologically “hot” (5/5; 100%), “cold” (6/8; 75%), and “warm” TMEs (3/8; 37.5%) while the 7 non-responders consisted of only “cold” (2/8; 25%) and “warm” (5/8; 62.5%) TME samples. There was an enrichment of fibroblasts and endothelial cell transcriptomic signatures in the samples of the non-responders compared to responders (p=0.018) with a trend of increasing enrichment in those signatures as response strength decreased. IMC performed on archival tissue from 20 patients demonstrated similar trend of higher population of activated fibroblasts (SMA+, MFAP5+) and endothelial cells (CD31+) in non-responders. Furthermore, non-responders had significantly higher total regulatory T cells (CD4+FOXP3+) in the tumor (p=0.027) and stroma (p=0.0282) compared to responders. Additionally, significantly higher activated regulatory T cells (CD4+FOXP3+CD25+) were observed in the tumor (p=0.016) and stroma (p=0.008) of non-responders compared to responders. Similar abundance of total and subpopulations of CD8+ T cells were observed between responders and non-responders. Conclusion: The MSI-H endometrial TME is heterogeneous. Increased presence of fibroblasts, endothelial cells, and regulatory T-cells in the TME correlate with innate resistance to pembrolizumab. Treatment aimed toward the reduction of these cellular subpopulations may improve sensitivity to PD-1 inhibitors. Future studies are needed to validate these findings. Citation Format: Jeffrey A. How, Minghao Dang, Sammy Ferri-Borgogno, Elizabeth Euscher, Melinda S. Yates, Weiyi Peng, Shrina D. Patel, Jared J. Burks, Ivo Vletic, Javier Gomez, Karen Lu, Samuel C. Mok, Linghua Wang, Amir A. Jazaeri. Predictors of innate resistance to pembrolizumab in patients with microsatellite instability-high endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1248.

Background: Despite high rates of remission after frontline management, most patients with advanced stage ovarian cancer recur. Second look laparoscopy (SLL) can provide more sensitive detection of minimal residual disease (MRD) allowing for more individualized prognostication and possible therapeutic intervention. It may also assess tumor biology and microenvironment associated with undetectable chemoresistant MRD phase of ovarian cancer. The objective of this study was to determine SLL feasibility, and clinicopathologic and molecular characteristics of MRD after frontline therapy in ovarian cancer. Methods: SLL was offered to patients with stage III-IV high grade epithelial ovarian cancer who achieved complete response after frontline surgery and 6 cycles of carboplatin and paclitaxel chemotherapy. Patients were offered standard of care or investigational management options based on homologous recombination deficiency (HRD) and MRD status at SLL. Preliminary translational studies included RNAseq for comparison of serial, matched tumor from primary or interval TRS and SLL derived MRD+ biopsies at the time of SLL. In addition, Nanostring analyses of SLL surgical biopsies both with and without residual tumor were performed. Molecular profiling employed hierarchical clustering, principle component analysis, non-parametric testing, and pathway analyses. Results: Between 4/2017- 7/2021, 39 patients underwent SLL. The majority had stage III disease (74%), high grade serous histology (90%) and underwent neoadjuvant chemotherapy with interval tumor reductive surgery (TRS; 54%). MRD was present in 49% (n=19) of patients and was more frequent among neoadjuvant chemotherapy recipients (73% vs 26%, P=0.015) and patients with homologous recombination proficient tumors (81% vs 25%, P=0.01). Most patients without MRD were dispositioned to observation; 4 patients with HRD received a PARP inhibitor per standard of care. All other patients with MRD received bevacizumab as part of a clinical trial, except for one patient who received a PARP inhibitor. Median follow-up was 6.74 months (range 0.66 - 28.62). Presence of MRD was associated with worse PFS (HR 3.7, 95% CI 1.3- 10.9; 5.5 vs 24.6 months; P= 0.02). Based on transcriptional signature, MRD are immunologically distinct from untreated tumors or those collected at interval TRS (after 3 cycles of chemo) but also displayed heterogeneity with two distinct subclusters. Conclusions: SLL to assess for MRD has potential to further individualize post-frontline therapy, and identify patients at high risk for progression for early intervention clinical trials. Transcriptional profiling suggests that MRD phase of ovarian cancer is characterized by a distinct, heterogeneous, and evolving tumor and immune microenvironment. Citation Format: Roni Nitecki, Minghao Dang, Sanghoon Lee, Bryan Fellman, J Alejandro Rauh-hain, Jolyn Taylor, Lois Ramondetta, Michaela Grinsfelder, Lauren Cobb, David M. Boruta, Pamela T. Soliman, Aaron Shafer, Nicole D. Fleming, Shannon N. Westin, Anil K. Sood, Chris Tanguma, Pedro T. Ramrez, Karen H. Lu, Linghua Wang, Amir A. Jazaeri. Initial clinicopathologic and molecular characterization of minimal residual disease detected by second look laparoscopy after completion of frontline surgery and chemotherapy in patients with advanced stage ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4135.

Background: While high-grade serous ovarian cancers (HGSC) look alike at a histological level, there is marked variation in clinical outcomes; however, the underlying mechanisms are not well understood. We previously highlighted significant distinct molecular and cellular characteristics in highly clinically annotated HGSC tumor samples from patients who underwent complete gross resection (R0) or received neoadjuvant chemotherapy (NACT). Neurofibromatosis type 1 (NF1) is known to negatively regulate the Ras/MAPK signaling pathways and tumorigenesis. Here, we specifically validate the higher loss of NF1 copy number exhibited in the R0 compared to the NACT groups in patients with HGSC. Methods: Fresh-frozen tumor biopsies were collected from patients with HGSC treated under a systematic surgical algorithm: complete gross resection after primary surgery (R0, n=40); poor tumor response to NACT (NACT-PR, n=26); excellent tumor response to NACT with carboplatin and paclitaxel (NACT-ER, n=14). Primary or metastatic tumor sites for each patient were obtained pre-treatment and subjected to high-depth targeted DNA sequencing. Results: The average coverage of 568X for tumors and 291X for germline controls (matched blood samples) was obtained by targeted sequencing for each sample from the entire cohort. The median numbers of non-synonymous somatic mutations identified in the panel sequencing were 5 in R0, 3 in NACT-ER, and 5 in NACT-PR. Sixty-nine of the 80 samples (86.25%) had alterations in at least one of the ovarian cancer-related genes. The most frequently altered genes including somatic mutations and copy number variations were TP53 (68%), NF1 (65%) and ERBB2 (62%) in the R0 group whereas TP53 (50%), AKT1 (48%) and TERT (42%) in the NACT group. Gene alteration enrichment was evaluated by comparing between two groups. 17 genes were found to be enriched for alterations in the R0 group comparing to the NACT group; MLH3 (OR= 15.03, p Conclusions: Our findings, using an independent validation cohort of patients, verified the higher loss of NF1 copy number in the R0 versus the NACT group. These findings could have major implications for clinical decision making and developing novel therapies for patients with HGSC. Citation Format: Sanghoon Lee, Li Zhao, Joseph Celestino, Richard A. Hajek, Margaret B. Morgan, Mark S. Kim, Shannon N. Westin, Amir A. Jazaeri, Nicole D. Fleming, Jinsong Liu, Jianhua Zhang, P Andrew Futreal, Anil K. Sood. NF1 copy number alterations in clinically-defined subsets of high-grade serous ovarian cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2200.

Introduction: Despite FDA approval of pembrolizumab in microsatellite instability-high (MSI-H) / mismatch repair deficient solid tumors, approximately half of patients with MSI-H endometrial cancer are treatment-refractory. Our unpublished analysis of MSI-H endometrial cancer samples from the Cancer Genome Atlas (TCGA) suggests the possibility of immunologically “hot” and “cold” tumor microenvironments (TME); these differences may explain variable treatment responses. We sought to evaluate MSI-H endometrial tumor samples to examine differences in the TME and identify transcriptomic signatures associated with response/resistance to pembrolizumab. Methods: Archival tumor samples from MSI-H endometrial cancer patients treated at the University of Texas MD Anderson Cancer Center were obtained under an IRB-approved protocol. Tissue samples originating from patients who did not receive pembrolizumab treatment (“untreated cohort ”; n = 11) were submitted for RNA sequencing analysis (RNA-seq) to validate the TME heterogeneity observed in MSI-H samples of the TCGA dataset. Pre-treatment archival tumor samples (“treated cohort”; n = 23) from patients who were treated with pembrolizumab were collected and submitted for RNA-seq to identify predictive immuno-genomic signatures associated with treatment response. Results: In the untreated cohort, there were observable differences in the transcriptomic profiles of the samples. Four samples were immunologically “hot” as evidenced by an abundance of pro-inflammatory immune cell infiltrate (CD8+ T-cells, B-cells, monocytes, and dendritic cells). Three samples had a paucity of pro-inflammatory immune cell infiltrate (“cold”) while 4 samples had intermediate amounts (“warm”). In the treated cohort, there were 14 responders, 7 non-responders, and 2 patients with unknown response. The 14 responders consisted of samples with “hot” (5/5; 100%), “cold” (6/8; 75%), and “warm” TMEs (3/8; 37.5%) while the 7 non-responders consisted of only “cold” (2/8; 25%) and “warm” (5/8; 62.5%) TME samples. We observed an enrichment of fibroblasts and endothelial cell transcriptomic signatures in the samples of the non-responders compared to responders (p = 0.018). In particular, there was a significantly higher gene expression of PAMR1, HHIP, and MMRN1 in the non-responders. Subdividing samples into complete response, partial response, and no response to pembrolizumab, we observed a trend of increasing enrichment of fibroblast and endothelial cell transcriptomic signatures as response decreased. Specifically, there was increasing expression of TAGLN (fibroblast gene) and other endothelial cell genes (EMCN, KDR, MMRN1, MYCT1, PEAR1, PTPRB, and TEK). Conclusion: The TME composition appears to be heterogeneous among MSI-H endometrial cancer patients. Increased presence of fibroblasts and endothelial cells in the TME may contribute to innate resistance to pembrolizumab. Treatment aimed toward the reduction of these cellular subpopulations may improve sensitivity to PD-1 inhibitors. Future studies are needed to validate these findings. Citation Format: Jeffrey A. How, Minghao Dao, Elizabeth Euscher, Melinda Yates, Weiyi Peng, Shrina D. Patel, Karen H. Lu, Patrick Hwu, Linghua Wang, Amir A. Jazaeri. The impact of the tumor immune microenvironment on response to pembrolizumab in patients with microsatellite instability-high endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO015.

Objectives: To evaluate safety and efficacy of durvalumab (D) in combination with paclitaxel (P) and carboplatin (C) in newly diagnosed, advanced-stage, high grade, non-mucinous ovarian cancer (OC). To investigate informative pharmacodynamic changes including proteome changes and immune-related pathways by treatment with immuno-oncology therapy in combination with chemotherapy. Methods: In this single arm phase II study, patients (pts) with untreated, advanced stage (III/IV) OC dispositioned to neoadjuvant chemotherapy (NACT) received D (750mg) with weekly P (80mg/m2) and C (AUC6) after baseline tissue biopsy. Matched biopsies were obtained at tumor reductive surgery (TRS) after 3 cycles. Progression-free survival (PFS) and overall survival (OS) were estimated using Kaplan-Meier method. Reverse-phase protein arrays (RPPA) were performed on pre- and post-treatment tissues. Protein expression alteration and an enriched Reactome pathway analysis using differentially expressed proteins between pre- and post-treatments were assessed. Results: 19 pts were enrolled and 18 treated. Median age was 63 and 50% had stage III disease. No dose-limiting toxicities were observed. 94% of pts had an adverse event (AE). Most common AEs were anemia (94%), neutropenia (83%), fatigue (72%), hyperglycemia (73%), and thrombocytopenia (66%). Immune-related AEs observed included hyperthyroidism (22%), hypothyroidism (22%), diarrhea (6%), hypophysitis (6%). Eighty-three percent had optimal interval TRS, 70% had a pre-operative response by RECIST (PR=11, CR=1). At 13 months (mo) median follow up, 9 pts (50%) had disease progression and median PFS was 14.5 mo (95% CI: 9.2 - NA). Median OS was not reached; OS rate at 18 mo was 69% (95% CI 0.21 - 0.91). Fifty-five tumor specimens were available from 9 pts (31 pre- and 24 post-treatment). Thirteen significantly differentially expressed proteins among 211 proteins (adj. p Citation Format: Shannon N. Westin, Sanghoon Lee, Li Zhao, Ying Yuan, Jimin Wu, Richard Hajek, Sara Sharafi, Larissa A. Meyer, Nicole D. Fleming, Amir Jazaeri, Robert L. Coleman, Karen H. Lu, Gordon B. Mills, Jianhua Zhang, Andrew Futreal, Anil K. Sood. Pharmacodynamic changes by durvalumab in combination with chemotherapy in women with untreated, advanced stage ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT188.

Background: Prior molecular characterization efforts for high-grade serous ovarian cancer (HGSOC) were restricted to those who had upfront surgical debulking with variable treatment paradigms. Thus, we sought to examine molecular and cellular differences between clinically defined groups (tumor tissues from patients who had complete gross resection (CGR) versus those who were triaged to neoadjuvant chemotherapy (NACT) and experienced either excellent or poor response. Methods: Tumor biopsies were collected from three patient groups managed under a systematic surgical algorithm: CGR after primary surgery (R0, n=10); poor tumor response to NACT (NACT-PR, n=10); excellent tumor response to NACT (NACT-ER, n=10). Primary and multiple metastatic tumor sites per each patient were obtained pre-treatment and subjected to comprehensive omics analyses including high-pass whole-genome (WGS) and targeted deep DNA sequencing. Results: An average of 71 nonsynonymous somatic mutations from each sample for 75 samples with high-purity tumors (≥75%) by WGS were identified from each sample for the entire cohort. Fourteen ovarian-cancer-associated genes were found mutated in our patient cohort and, as expected, the most frequently mutated gene was TP53 in both primary and metastatic sites in all three groups. TP53 nonsense mutations were exclusively identified in the NACT-ER (36.0%) and NACT-PR groups (15.4%), while in the R0 group most TP53 mutations were missense mutations (62.5%). Nonsense mutations in CSMD3 and PIK3CA were exclusively identified in both primary and metastatic sites in the NACT-PR group. The most frequent copy number variations (CNVs) in the R0 were copy number gain/loss of CSMD3 (67%) and copy number loss of NF1 (54%) and CDK12 (50%) in both primary and metastatic sites. Interestingly, copy number losses of NF1 were significantly lower in the NACT groups (18%, p=0.002), especially in the NACT-PR (8%, p=0.0004), when compared to the R0 group (54%). We also identified significant less observation of chromothripsis-like patterns, and a significantly higher level of strong-binding neoantigens in the R0 than in the NACT groups. Conclusions: Our findings using HGSOC samples obtained from patients treated on a prospective algorithm identified distinct molecular abnormalities, and could have prognostic and therapeutic implications for patients with HGSOC. Citation Format: Sanghoon Lee, Li Zhao, Shannon N. Westin, Nicholas W. Bateman, Amir A. Jazaeri, Nicole D. Fleming, Karen H. Lu, Robert L. Coleman, Gordon B. Mills, Jianhua Zhang, Thomas P. Conrads, George L. Maxwell, P. Andrew Futreal, Anil K. Sood. High-depth whole genome sequencing of clinically-annotated high-grade serous ovarian cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2510.

Background: High-grade serous ovarian cancer (HGSOC) remains the leading cause of death from gynecologic malignancies. Here, we examined tumoral proteome changes following neoadjuvant chemotherapy (NACT) to identify potential predictive and prognostic biomarkers of response to primary chemotherapy. Methods: A total of 65 tissue specimens from 10 patients with advanced-stage HGSOC were collected from matched pre- and post-NACT (3 cycles of dose-dense paclitaxel and carboplatin). Protein expression was assessed using reverse-phase protein arrays (RPPA). All relative protein levels using 297 antibodies were normalized by the expression level of pre-treatment samples, and then protein expression alterations and functional analyses were performed by Reactome pathway analysis followed by statistical analysis. Results: The protein expression patterns of samples tended to cluster according to the time point (pre- and post-treatment) by a non-supervised clustering analysis using all the proteins assessed in the RPPA panel. Five differentially expressed proteins among 239 proteins (adj. p Conclusion: Our findings identified significant proteomic alterations following NACT, and could provide insights into interval proteomic alterations following induction chemotherapy in advanced-stage ovarian cancer patients. These data present information to optimize future clinical trial designs for patients with ovarian cancer. Citation Format: Sanghoon Lee, Li Zhao, Joseph Celestino, Kelly M. Rangel, Richard A. Hajek, Mark S. Kim, Sara E. Sharafi, Jinsong Liu, Nicole D. Fleming, Karen H. Lu, Jianhua Zhang, P. Andrew Futreal, Gordon B. Mills, Shannon N. Westin, Anil K. Sood, Amir A. Jazaeri, Robert L. Coleman. Functional proteomic aberrations post-chemotherapy with paclitaxel and carboplatin in patients with advanced ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2941.

Purpose: There is limited success with chemotherapeutic agents in women with advanced or recurrent endometrial cancer. Dysregulation of the PI3K/RAS signaling pathways in endometrial cancer have been well documented. However, responses with mTOR inhibitor such as temsirolimus have been modest. Previously we have shown that metformin prevents temsirolimus-induced AKT activation. Therefore, we enrolled patients with advanced endometrial carcinoma in the expansion cohort of a phase I study of temsirolimus in combination with metformin. Methods: Patients with advanced endometrial cancer refractory to or relapse after standard therapies, ECOG performance status 0 or 1, and with significant organ function reserve were enrolled. Patients were administered intravenous temsirolimus 25mg weekly and oral metformin 2000 mg daily in 28-day cycles. Response was assessed every 2 cycles by clinical evaluation, tumor markers, and imaging per RECIST 1.1. All toxicities were graded using NCI CTCAE, version 4.0. Results: Forty patients were treated. Median age is 67 years (range, 33-78). Drug-related adverse events of any grade were reported in 34 patients. The most common toxicities were mucositis (n=13), AST increase (n=13), anorexia (n=12), diarrhea (n=12) and anemia (n=10). Eleven grade 3 drug-related adverse events were reported. They were anemia (n=2), thrombocytopenia (n=2), mucositis, fatigue, weight loss, hypokalemia, hypophosphatemia, AST increase and ALT increase (n=1 each). Of the 33 patients evaluable for response, objective response was seen in 2 (6%) patients. Both had partial response (PR) and were on the study for 8.7 and 18.2 months respectively. In addition, 13 (39%) patients had stable disease (SD), including 11 with SD ≥4 months, representing a clinical benefit rate of 39%. Molecular characterization of tumor was available for 35 patients. Thirty of 35 patients had molecular alterations in the PI3K and/or RAS pathway. Of the 30 patients, 1 had benign PI3K mutation and 4 were not evaluable for response. Of the remaining 25 patients with PI3K and/or KRAS pathway, 11 (44%) had either objective response or SD ≥4 months. Eighteen of them had molecular alteration only in the PI3K pathway. Seven of 18 (39%) had objective response or SD ≥4 months. Importantly, all 3 patients who had molecular alterations in both the PI3K and RAS pathway achieved SD ≥4 months, while 3 of the 4 patients with exclusive resistant KRAS mutation had progressive disease. Collectively, of the 13 patients with either objective response or SD ≥4 months, 11 (85%) patients had molecular alteration in the PI3K and/or RAS pathway. Conclusion: Temsirolimus in combination with metformin was well tolerated. Anti-tumor activity was seen in patients with advanced/refractory endometrial cancer, particularly in patients with molecular alterations in the PI3K and/or RAS pathway that warrants further study. Citation Format: Aung Naing, Daniel Karp, Sarina A. Piha-Paul, Shubham Pant, Vivek Subbiah, Diane C. Bodurka, Siqing Fu, Amir A. Jazaeri, Shumei Kato, Kathleen Schmeler, Alpa Nick, Yali Yang, Fechukwu O. Akhmedzhanov, Senait Fessahaye, Jing Gong, Bettzy Stephen, Amber M. Johnson, Pamela T. Soliman, Anil K. Sood, Funda Meric-Bernstam, Karen H. Lu. Temsirolimus in combinaton with metformin in patients with advanced or refractory endometrial cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT163.

The role of immune checkpoint inhibitors (ICPIs) in ovarian cancer continues to be refined. The presence of intratumoral T cells in ovarian tumors correlates with improved survival and progression, suggesting that immune modulation, such as by ICPIs, may be beneficial. However, a better understanding of the immune effects following ICPI use, particularly in those with a long progression free survival, is needed. Thus, we used imaging mass cytometry (IMC) to compare the tumor immune landscape of patients with ovarian cancer before and after a CTLA4 immune checkpoint inhibitor treatment to determine the association between alterations in immune landscapes and progression free survival. Fine needle biopsies were obtained from 8 patients with recurrent platinum-resistant ovarian carcinoma enrolled in a phase 2 randomized trial evaluating the efficacy of Tremelimumab and Durvalumab (CTLA4 and PDL1 checkpoint inhibitors, respectively) in recurrent ovarian cancer treatment. Each patient had a pre-treatment biopsy and on-treatment biopsy after 3 cycles of CTLA4 inhibitor therapy. Progression free time (PFS) was recorded with 3 patients with a long PFS (>180 days) and 5 with short PFS ( Formalin fixed paraffin embedded (FFPE) tissue sections were stained for IMC analysis via Fluidigm protocol with 34 metal-tagged antibodies to detect various cell and immune related markers. The IMC data was obtained using the Fluidigm Helios CyTOF instrument and Hyperion Imaging System laser ablation module. A novel image informatics pipeline through Matlab was used to assess the image intensity of each marker and cell location. Using the developed image informatics pipeline, cell types and locations were analyzed for 16 samples from 8 patients. In all patients, mean CD8+ T cell densities had an increased trend in on-treatment vs pre-treatment samples (205.6 v 129.9 cells/mm2, p=0.086). An analysis of cell location found that in all patients on-treatment, the mean number of CD8+ T cells adjacent to M2 macrophages increased significantly (0.09 v. 0.17 cells, p = 0.0460) as well as the mean number of CD8+ T cells adjacent to B7H4+ tumor cells (0.035 v. 0.01 cells, p = 0.046). Furthermore, patients with a long PFS had a significantly higher number of CD8+ cells neighboring B cells on-treatment than short PFS (0.12 v. 0.27 cells, p=0.028). Using IMC and novel image informatics pipeline, we found that patients with recurrent ovarian cancer treated with Tremelimumab with a long PFS mounted a significant immune response compared to patients with a short PFS, with a higher number of CD8+ T cells neighboring B cells after treatment. For all patients, the immune landscape altered after treatment. To our knowledge, this is the first known use of IMC to demonstrate immune response in ovarian cancer following ICPI therapy. Overall, IMC and image informatics pipeline are robust tools that simultaneously analyze multiple biomarkers and spatial location of cells to better assess the tumor immune microenvironment and cellular interactions. Citation Format: Han Cun, Emily M. Hinchcliff, Ying Zhu, Sammy Ferri-Borgogno, Rita Cheng, Jared K. Burks, Stephen T. Wong, Amir A. Jazaeri, Samuel C. Mok. Identification of a novel biomarker response in a prospective clinical trial of immune checkpoint blockade in recurrent ovarian carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3267.

High-grade serous ovarian carcinoma (HGSOC) is the most common and lethal form of epithelial ovarian cancer. While HGSOCs respond well to platinum and taxane chemotherapy, the majority of patients eventually relapse with treatment-resistant tumors. This new and ominous resistance property of recurrent tumors is generally thought to be acquired in a process aided by the mutagenic activities of the chemotherapy itself. However, using advanced and robust technology that enables the direct cloning of cancer stem cells (CSCs) from HGSOCs, our study challenges the notion that chemotherapy resistance is acquired. In particular, patient-specific “libraries” of CSCs generated from therapy-naive tumors show that, while the majority of the CSCs are rapidly killed by platinum-taxane treatment, a discrete subset is highly resistant to these drugs to which they had no prior exposure. More telling is that these pre-existing, platinum-taxane resistant CSCs also display a profound “polyresistance” to a wide range of unrelated chemotherapeutic drugs. Importantly, gene expression profiles of polyresistant CSCs are highly uniform within a particular patient and clearly distinct from those of sensitive clones. This finding suggests that resistant clones within a patient carry the same gene signature, suggesting that resistance is a singular and knowable event in each patient. Comparing the resistance gene expression signatures across patients is revealing a very limited set of common pathways involving nuclear receptors that likely hold the key to the polyresistance phenomena. Taken together, our study supports the concept that these poly-resistant variants exist and can be cloned from most if not all HGOC patients. Further, the cloning of these polyresistant cells sets in motion the possibility that such variants can be exploited to identify both key pathways of polyresistance and drugs that specifically and pre-emptively eliminate these clones to prevent onset of recurrent disease. Citation Format: Jingzhong Xie, Yusuke Yamamoto, Audrey-Ann Liew, Bailiang Wang, Rajasekaran Mahalingam, Marcin Duleba, Wei Rao, Suchan Niroula, Kristina Goller, Yanting Zhang, Shan Wang, Amir Jazaeri, Peter Davies, Suzy Torti, Giulio Draetta, Matthew Anderson, Molly Brewer, Christopher Crum, Frank McKeon, Wa Xian. Pre-existing, poly-resistant cancer stem cells in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A78.

INTRODUCTION: High-grade ovarian cancer (HGOC) shows excellent responses to standard-of-care surgery and paclitaxel/carboplatin therapy only to relapse 6-24 months later with typically resistant disease. While the origin of this recurrent, resistant disease is unclear, most believe it is acquired by the action of chemotherapeutics. Using novel stem cell technology that enables the cloning of cancer stem cells (CSCs) from epithelial cancers, we have generated large libraries of CSCs from multiple cases of HGOC. And while the vast majority of these CSC clones are killed by standard-of-care chemotherapeutic drugs, a minor fraction shows profound resistance not only to paclitaxel/carboplatin but to a wide range of structurally unrelated chemotherapeutic drugs to which these cells had no prior exposure. We describe screens for drugs that selectively target this resistant CSC population. METHODS: Libraries of 10- to 100,000 CSC clones were generated from individual, therapy naïve, HGOC resections using technology we developed for cloning so-called “adult” stem cells from normal columnar epithelia (Wang et al., 2015, Nature, 522, 173). RESULTS: Paclitaxel/carboplatin resistant CSCs were identified in CSC libraries derived from therapy naïve tumors at ratios of 1:50 to 1:300. By copy number variation, these resistant variant clones proved distinct from the bulk of CSCs, and by gene expression analysis varied from sensitive clones by more than 700 differentially expressed genes. Independent resistant clones from the same library clustered with other resistant clones by both copy number variation and gene expression profiles, suggesting the possibility that resistance within a single tumor is dominated by a single type of resistant CSCs. Clones resistant to paclitaxel/carboplatin were screened in a 384-well format against a wide range of experimental drug-like molecules. These pre-existing resistant clones also proved to be profoundly resistant to a large number of structurally unrelated chemotherapeutic drugs. This same screening program identified drugs that act alone or in combination with paclitaxel to eliminate these resistant clones, suggesting a route to personalized medicine for addressing the problem of recurrent disease in HGOC. CONCLUSIONS: Tumors from patients with HGOC possess clonogenic CSCs including variants that are resistant to a broad spectrum of chemotherapeutics to which they have not been exposed. It is likely that such CSCs would survive standard-of-care chemotherapy and contribute to the recurrent disease seen in HGOC. We have identified known and experimental drugs that specifically eliminate these resistant variants and the overall platform represents a potential strategy to addressing the problem of recurrent disease in these patients. Citation Format: Jingzhong Xie, Yusuke Yamamoto, Marcin Duleba, Bailiang Wang, Rajasekaran Mahalingam, Shan Wang, Wei Rao, Suchan Niroula, Clifford Stephan, Peter Davies, Amir Jazaeri, Giulio Draetta, Molly Brewer, Matt L. Anderson, Christopher P. Crum, Frank McKeon, and Wa Xian. PRE-EXISTENCE OF POLY-RESISTANT CANCER STEM CELLS IN HIGH-GRADE OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-115.

Introduction: The objective was to identify molecular mechanisms and protein networks involved in synthetic lethality as well as mechanisms of adaptive changes to the combination of olaparib (O) combined with inhibitors of the PI3K-AKT axis. Methods: 110 patients with recurrent ovarian, endometrial, and triple negative breast cancer were treated with a combination of O and vistusertib (V) or capivasertib (C) (mTORC1/2 and AKT inhibitors, respectively). We have previously reported durable clinical efficacy of these combinations, regardless of BRCA status. Tumor samples were collected pre-treatment and 30 days post-treatment and reverse-phase protein array (RPPA) was performed. Baseline expression was assessed and change in expression was calculated. Bioinformatics and statistical methods were developed to assess activity of signaling pathways. Pathway scores were determined based on the expression of sets of proteins known to be involved in a specific pathway, including PI3K, RAS/RAF, and DNA damage repair. Association between protein expression and treatment efficacy was determined. Results: We analyzed a total of 55 paired samples from patients treated with O/V (n=25) and O/C (n=30). These included 20 endometrial (37% PR, 31.5% SD, 31.5% PD), 21 ovarian (10% PR, 55% SD, 35% PD) and 14 triple negative breast (15% PR, 23% SD, 62% PD) tumors. Response to O/V was associated with low pre-treatment mTOR pathway activity and led to altered immune activity based on decreased CD4 and PD1 expression. Resistance to O/V was associated with high pre-treatment cell proliferation (high CCNB1, CDK1 and PLK1 protein expression). In contrast, response to O/C was associated with adaptive responses indicated by decreased mTOR activity and induction of the DNA damage checkpoint (high phospho-CHK1, -WEE1 and -CDC2). Resistance was associated with high pre-treatment RTK activity levels (high phospho-HER3, -EGFR, -IGFR, -MET) and AKT-independent activation of mTOR in the on-treatment samples. Conclusion: Analysis of pre- and on-treatment samples from responders and non-responders to PARP and PI3K pathway combination therapy identified biomarkers of patients likely to benefit from therapy and helps to understand molecular mechanisms involved in response and resistance to these drug combinations. Citation Format: Marilyne Labrie, Zhenlin Ju, Jennifer K. Litton, Tae-Beom Kim, Sanghoon Lee, Ken Chen, Pamela T. Soliman, Michael Frumovitz, Larissa A. Meyer, Stacy Moulder, Amir A. Jazaeri, Karen H. Lu, Anil K. Sood, Robert L. Coleman, Gordon B. Mills, Shannon N. Westin. Exploration of markers of synergistic lethality of PARP and PI3K-Akt-mTOR inhibitors in women's cancers [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 2070.

The nearly ubiquitous development of chemoresistant disease remains a major obstacle against improving outcomes for patients with ovarian cancer. In this investigation, we evaluated the preclinical activity of MLN4924, an investigational inhibitor of the NEDD8-activating enzyme, in ovarian cancer cells. Efficacy of MLN4924 both alone and in combination with platinum was assessed. Overall, single-agent MLN4924 exhibited moderate activity in ovarian cancer cell lines. However, the combination of MLN4924 with cisplatin or carboplatin produced synergistic effects in SKOV3 and ES2 cells, as well as in primary ovarian cancer cell lines established from high-grade serous, clear cell, and serous borderline ovarian tumors. The efficacy of cisplatin plus MLN4924 was also evident in several in vitro models of platinum-resistant ovarian cancer. Mechanistically, the combination of cisplatin and MLN4924 was not associated with DNA re-replication, altered platinum-DNA adduct formation, abrogation of FANCD2 monoubiquitination, or CHK1 phosphorylation. An siRNA screen was used to investigate the contribution of each member of the cullin RING ligase (CRL) family of E3 ubiquitin ligases, the best-characterized downstream mediators of MLN4924's biologic effects. Cisplatin-induced cytotoxicity was augmented by depletion of CUL3, and antagonized by siCUL1 in both ES2 and SKOV3 ovarian cancer cells. This investigation identifies inhibition of neddylation as a novel mechanism for overcoming platinum resistance in vitro, and provides a strong rationale for clinical investigations of platinum and MLN4924 combinations in ovarian cancer. Mol Cancer Ther; 12(10); 1958–67. ©2013 AACR.

High-grade ovarian cancer is extremely sensitive to chemotherapy and yet usually lethal due to recurrent disease. To address the underlying mechanism of chemotherapy resistance in this disease, we employed novel technology developed to clone normal adult epithelial stem cells to generate libraries of patient-specific, clonogenic tumor cells. These clones possess hallmarks of “cancer stem cells” including long-term self-renewal and recapitulation of tumors of appropriate histology in immunodeficient mice. Significantly, these clonogenic tumor cells possess the overall pattern of genomic alterations of the whole tumor and yet display clone-specific patterns of variation reflecting intratumor heterogeneity that is stable despite months of continuous propagation. Our analyses at the clonal level have revealed a subset that display an intrinsic, pretherapy resistance to paclitaxel and hypervariable genomics in contrast to the bulk of clones sampled from the library. These intrinsically resistant clones share genomic and gene expression profiles with those surviving paclitaxel treatment of the whole library, suggesting a role for intrinsically resistant cells in recurrent disease. Remarkably, known drugs that interfere with signaling pathways enriched in both intrinsically resistant and paclitaxel survivor clones are synthetically lethal with standard-of-care chemotherapy. The targeting of intrinsically resistant clones from patient-specific libraries of cancer stem cells offers new strategies for preempting recurrent disease. Citation Format: Guoqiang Chang, Jinxiu Li, Bailiang Wang, Yusuke Yamamoto, Rajasekaran Mahalingam, Marcin Duleba, Wei Rao, Jingzhong Xie, Shan Wang, Amir Jazaeri, Peter Davies, Suzy Torti, Giulio Draetta, Matthew Anderson, Molly Brewer, Christopher Crum, Frank McKeon, Wa Xian. Cloning and vulnerability of intrinsically resistant subset of ovarian cancer stem cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B05.

Introduction: High-grade ovarian cancer (HGOC) shows excellent responses to standard-of-care surgery and paclitaxel/carboplatin therapy only to relapse 6-24 months later with typically resistant disease. While the origin of this recurrent, resistant disease is unclear, most believe it is acquired by the action of chemotherapeutics. Using novel stem cell technology that enables the cloning of cancer stem cells (CSCs) from epithelial cancers, we have generated large libraries of CSCs from multiple cases of HGOC. And while the vast majority of these CSC clones are killed by standard-of-care chemotherapeutic drugs, a minor fraction shows profound resistance not only to paclitaxel/carboplatin but to a wide range of structurally unrelated chemotherapeutic drugs to which these cells had no prior exposure. We describe screens for drugs that selectively target this resistant CSC population. Methods: Libraries of 10- to 100,000 CSC clones were generated from individual, therapy naïve, HGOC resections using technology we developed for cloning so-called “adult” stem cells from normal columnar epithelia (Wang et al., 2015). Results: Paclitaxel/carboplatin resistant CSCs were identified in CSC libraries derived from therapy naïve tumors at ratios of 1:50 to 1:300. By copy number variation, these resistant variant clones proved distinct from the bulk of CSCs, and by gene expression analysis varied from sensitive clones by more than 700 differentially expressed genes. Independent resistant clones from the same library clustered with other resistant clones by both copy number variation and gene expression profiles, suggesting the possibility that resistance within a single tumor is dominated by a single type of resistant CSCs. Clones resistant to paclitaxel/carboplatin were screened in a 384-well format against a wide range of experimental drug-like molecules. These pre-existing resistant clones also proved to be profoundly resistant to a large number of structurally unrelated chemotherapeutic drugs. This same screening program identified drugs that act alone or in combination with paclitaxel to eliminate these resistant clones, suggesting a route to personalized medicine for addressing the problem of recurrent disease in HGOC. Conclusions: Tumors from patients with HGOC possess clonogenic CSCs including variants that are resistant to a broad spectrum of chemotherapeutics to which they have not been exposed. It is likely that such CSCs would survive standard-of-care chemotherapy and contribute to the recurrent disease seen in HGOC. We have identified known and experimental drugs that specifically eliminate these resistant variants and the overall platform represents a potential strategy to addressing the problem of recurrent disease in these patients. Citation Format: Wa Xian, Frank McKeon, Christopher Crum, Jingzhong Xie, Bailiang Wang, Yusuke Yamamoto, Suzy Torti, Molly Brewer, Matthew Anderson, Amir Jazaeri, Giulio Draetta, Shan Wang, Rajasekaran Mahalingam, Hong Chen, Marcin Duleba, Wei Rao. Pre-existence of poly-resistant cancer stem cells in high-grade ovarian cancer [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 1994.

Adoptive cell therapy (ACT) may be effective in treating immunogenic tumors with high mutational load, such as melanoma, and virally-associated tumors, like cervical cancer, with several patients in studies performed at various institutions achieving durable, complete responses for years. HPV infection increases mutational load, thus providing additional neoantigen targets ideal for the polyclonal nature of ACT. As outcomes for patients with recurrent, metastatic or persistent cervical cancer remain extremely poor, there is an enormous need for novel immunotherapeutic approaches with curative potential such as ACT. Clinical trial C-145-04 (NCT03108495) is a prospective, phase II multicenter, open-label study evaluating the efficacy of a single autologous tumor infiltrating lymphocyte infusion (LN-145) followed by IL-2 after a non-myeloablative lymphodepletion (NMA-LD) regimen in patients with recurrent, metastatic, or persistent cervical cancer, who have failed at least one prior systemic therapy. The clinical trial requires resection of an adequate size tumor lesion, which is then shipped to a central GMP manufacturing facility for tumor infiltrating lymphocyte (TIL) extraction, expansion, and preparation of the final infusion product (LN-145). One week prior to LN-145 shipment and infusion, patients undergo NMA-LD consisting of cyclophosphamide (60 mg/kg) daily x 2 days followed by fludarabine (25 mg/m2) daily x 5 days. LN-145 is infused 24 hours after the last dose of fludarabine followed by up to 6 doses of IL-2 (600,000 IU/kg) every 8-12 hours. The primary endpoint is the ORR per RECIST v1.1. Secondary endpoints include complete response, duration of response, disease control rate, progression free- and overall survival and safety. Patients must, in addition to the tumor targeted for excision for TIL manufacture, have an additional measurable lesion for assessment of response. Other major eligibility criteria include: adequate bone marrow, liver, pulmonary, cardiac and renal function; ECOG performance status of 0 or 1. Systemic steroids greater than 10 mg/day prednisone equivalents are prohibited as are a history of serious immunotherapy-related adverse events. Citation Format: Amir Jazaeri, Robert Edwards, Robert Wenham, Koji Matsuo, Gini F. Fleming, David M. O'Malley, Brian Slomovitz, Bradley Monk, Robert J. Brown, Sam Suzuki, Igor Gorbatchevsky, Maria Fardis, Emese Zsiros. A phase 2 multicenter study to evaluate the efficacy and safety using autologous tumor infiltrating lymphocytes (LN-145) in patients with recurrent metastatic or persistent cervical carcinoma [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 CT172.

Purpose: The goal of this study was to determine whether distinct gene expression profiles are associated with intrinsic and/or acquired chemoresistance in epithelial ovarian carcinoma. Experimental Design: Gene expression profiles were generated from 21 primary chemosensitive tumors and 24 primary chemoresistant tumors using cDNA-based microarrays. Gene expression profiles of both groups of primary tumors were then compared with those of 15 ovarian carcinomas obtained following platinum-based chemotherapy (“postchemotherapy” tumors). A theme discovery tool was used to identify functional categories of genes involved in drug resistance. Results: Comparison of primary chemosensitive and chemoresistant tumors revealed differential expression of 85 genes (P < 0.001). Comparison of gene expression profiles of primary chemosensitive tumors and postchemotherapy tumors revealed more robust differences with 760 genes differentiating the two groups (P < 0.001). In contrast, only 230 genes were differentially expressed between primary chemoresistant and postchemotherapy groups (P < 0.001). Common to both gene lists were 178 genes representing transcripts differentially expressed between postchemotherapy tumors and all primary tumors irrespective of intrinsic chemosensitivity. The gene expression profile of postchemotherapy tumors compared with that of primary tumors revealed statistically significant overrepresentation of genes encoding extracellular matrix–related proteins. Conclusions: These data show that gene expression profiling can discriminate primary chemoresistant from primary chemosensitive ovarian cancers. Gene expression profiles were also identified that correlate with states of intrinsic and acquired chemoresistance and that represent targets for future investigation and potential therapeutic interventions.

ARN-3261 is an orally bioavailable small molecule inhibitor of the Salt Inducible Kinase 2 (SIK2, 11 nM) and SIK3 (19 nM). Three isoforms of SIK (SIKs) proteins have been reported: SIK1 (SNF1LK), SIK2 (QIK), and SIK3 (QSK). They are the Ser/Thr centrosome kinase family members required for bipolar mitotic spindle formation. The overexpression of SIK2 kinase in 30% of ovarian cancer specimens allows a novel, clinically important new method of treating ovarian cancer by blocking SIK2 kinase activity. In addition to a role in ovarian cancer, SIK2 and SIK3 are prevalent in several other tumor types, including breast, prostate diffuse large B-cell lymphoma, and melanoma cancers. Inhibition of SIK2 has been reported to cause centrosome splitting in interphase, while SIK2 depletion blocked centrosome separation in mitosis and sensitized ovarian cancers to paclitaxel in culture and in vivo Xenograft models. Depletion of SIK2 also delayed G1/S transition and reduced AKT phosphorylation. Higher levels of expression of SIK2 have been shown to be highly correlated with poor survival in patients with high-grade serous ovarian cancers. Using the homology structure of SIK2, fragment-based lead optimization strategies, and screening and structure-activity relationship efforts, we have discovered ARN-3261, a first-in-class novel, selective inhibitor of SIK2 that could prove useful in treating ovarian, endometrial, primary peritoneal, fallopian tube, and triple negative breast cancers. ARN-3261 specifically inhibited SIK2-expressed SKOv3 cells with an IC50 of 92 nM. ARN3261 was effective against ovarian, breast cancer cell lines alone and in combination with Paclitaxel and Cisplatin. ARN-3261 also inhibited ovarian tumor growth significantly at 70% in SKOv3 human ovarian cancer Xenografts in Ncr nu/nu mice in a dose dependent manner at 20, 40, 60, and 100 mg/kg orally. Moreover, ARN-3261 has exhibited excellent in vivo pharmacokinetic, pharmacodynamics, and correlative PK/PD and ADME characteristics. Preliminary in vitro and in vivo tumor up-take studies suggest that ARN-3261 blocks centrosome separation by inhibiting SIK2, thereby enhancing the sensitivity of Paclitaxel. Encouraged by these results, we initiated the IND enabling GLP-Toxicology and safety studies to bring ARN-3261 to the clinic for First-In-Human (FIH) Phase 1 trials. The non-clinical pharmacology data along with Phase 1 POC clinical trial plans will be presented. Citation Format: Hariprasad Vankayalapati, Venkatakrishnareddy Yerramreddy, Jinhua Zhou, Jeffrey A. Handler, Rajendra P. Appalaneni, Ramamohan R. Kancherla, Roy J. Wu, Hiroshi Takemori, Angelique Whitehurst, Amir Anthony Jazaeri, Robert L. Coleman, Zhen Lu, Robert C. Bast. Discovery of ARN-3261 as a potent, selective, orally available SIK2 inhibitor for treating ovarian, endometrial, primary peritoneal, fallopian tube, and triple negative breast cancers [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 LB-296. doi:10.1158/1538-7445.AM2017-LB-296

Resistance to anchorage independent cell death (anoikis) is one of the features of metastasis and can be regulated by a diverse array of growth factors. The protein encoded by GDF2 is BMP9 and is a member of the Bone Morphogenetic protein family and the TGFβ superfamily with defined roles in angiogenesis and emerging yet controversial roles in carcinogenesis. We used normal and oncogenic epithelial systems to examine the role of GDF2 in ovarian and breast epithelia. We find that both normal and transformed epithelial cells are competent to activate the Smad1/5 signaling cascade in response to GDF2. This activation occurs via specific complex formation between the Type I receptor Serine threonine kinases Alk3 and Alk6 and the Type II receptor serine threonine kinase BMPRII. We find that in breast and ovarian epithelial cells GDF2 specifically regulates anchorage independent growth by increasing anoikis sensitivity that is dependent on GDF2's ability to sustain SMAD1/5 activation via Alk3 and Alk6. Analysis of cell lines and patient data indicates epigenetic regulation of GDF2 expression in ovarian and breast cancers. These findings reveal an antimetastatic role for GDF2 in ovarian and breast cancer models of disease with significant implications for the use of epigenetic drugs currently in clinical trials. Citation Format: Archana Varadaraj, Pratik Patel, Anne Serrao, Tirthankar Bandyopadhyay, Nam Y. Lee, Amir Jazaeri, Susan Murphy, Karthikeyan Mythreye. GDF2 promotes anoikis susceptibility in ovarian and breast epithelia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3912. doi:10.1158/1538-7445.AM2015-3912

The inability to successfully treat women with ovarian cancer is due in large part to the advanced stage of disease at diagnosis and the development of platinum resistance. T-type calcium channels have recently garnered interest as potential targets for the treatment of many cancers. We investigated the hypothesis that inhibiting T-type calcium channels would sensitize ovarian cancer cells to platinum based chemotherapy using cell culture and mouse models. Sequential treatment of cultured ovarian cancer cell lines or primary cells obtained from patients with mibefradil followed by carboplatin showed a dose-dependent decrease in cell number that was greater than observed with either drug alone. Importantly, treatment of platinum-resistant tumor cells with mibefradil followed by carboplatin also decreased cell number. Moreover, either simultaneous administration of both drugs or treatment with carboplatin followed by mibefradil did not alter cellular response to carboplatin. Based on these findings, we tested the effect of mibefradil and carboplatin in a mouse model of peritoneal ovarian cancer metastasis with platinum-resistant ovarian cancer cells. Tumor-bearing mice were subjected to 3 cycles of Interlaced TherapyTM, which consists of 5 days of mibefradil treatment followed by one dose of carboplatin and 2 days off. We found that while neither drug alone significantly affected tumor growth compared to control, the mice that received the dual treatment had a dramatic reduction in tumor burden. Together, the data provide pre-clinical evidence supporting the use of T-type calcium channel blockers with platinum-based chemotherapy to treat ovarian cancer. Citation Format: Eli V. Casarez, Lloyd Gray, Amir A. Jazaeri, Jill K. Slack-Davis. Inhibition of T-type calcium channels sensitizes ovarian cancer cell growth and metastasis to platinum-based chemotherapy. [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 945. doi:10.1158/1538-7445.AM2013-945

Purpose: The objective of this investigation was to evaluate the preclinical activity of MLN4924, an investigational inhibitor of the NEDD8-activating enzyme, against chemoresistant and chemosensitive ovarian cancer cells. ExperimentalDesign: Efficacy of MLN4924 both alone and in combination with cisplatin was assessed using short-term tetrazolium based and longer term focus formation assays. Mechanistic studies included cell cycle analysis, evaluation of DNA-platinum adduct formation (using an ELISA), and evaluation of DNA damage repair pathways. In addition, we employed an siRNA screen to assess the contribution of each member of the Cullin RING-Ligase (CRL) family of E3 ubiquitin ligases, the best characterized downstream mediators of MLN4924’s biological effects. Results: Single agent MLN4924 exhibited moderate activity in ovarian cancer cell lines. The combination of MLN4924 with cisplatin or carboplatin resulted in significant platinum sensitization in SKOV3 and ES2 cells, as well as, in several primary ovarian cancer cell lines established from high grade serous, clear cell, and serous borderline ovarian tumors. Furthermore, the platinum sensitizing effect of MLN4924 was also observed in several in vitro models of platinum resistant ovarian cancer (using both established and primary chemoresistant ovarian cancer cells). Mechanistically, we show that depletion of CUL3 and CUL5 augment, while knockdown of CUL1 and CUL4A antagonize cisplatin-induced cytotoxicity in ovarian cancer cells. The combination of cisplatin and MLN4924 was not associated with DNA re-replication, altered platinum-DNA adduct formation, or abrogation of FANCD2 monoubiquitination. Conclusions: Our investigation reveals a novel mechanism for overcoming platinum resistance in vitro, and provides a strong rationale for clinical investigations of platinum and MLN4924 combinations in ovarian cancer. This work also highlights that different CRL ubiquitination pathways can have distinct and opposing effects on platinum sensitivity and resistance in ovarian cancer cells. Citation Format: Amir A. Jazaeri, Etsuko Shibata, Jonghoon Park, Jennifer Bryant, Mark R. Conaway, Susan C. Modesitt, Peter G. Smith, Michael Milhollen, Allison J. Berger, Anindya Dutta. Overcoming platinum resistance in ovarian cancer using the novel compound MLN4924. [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 3380. doi:10.1158/1538-7445.AM2013-3380

Platinum drugs are the most active agents for treatment of ovarian cancer. While most patients initially respond to this treatment, the majority eventually become resistant and ultimately succumb to the disease. Despite much investigation, in vitro elucidated mechanisms of platinum resistance have not correlated with those involved in clinical chemoresistance. Traditionally, in vitro models have consisted of established ovarian cancer cell lines that have been propagated in culture for decades. Chemoresistance is then modeled by growing cells in the presence of increasing concentrations of platinum or other cytotoxic drugs. As an alternative approach, we developed primary cell lines from normal human fallopian tube epithelial cells (n=5) and serous ovarian cancers (n=15). The latter group included tumors isolated from chemo-naïve (n=9) and chemo-exposed (n=6) patients. Each primary cell line was treated with vehicle and cisplatin (50uM) for 24hrs and expression profiling was performed using Affymetrix arrays. Differentially expressed genes were identified using BRB ArrayTools (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 801. doi:1538-7445.AM2012-801

Objective: Ubiquitination is a key posttranslational modification involved in the tight regulation of biological processes including cell-cycle regulation and DNA replication and repair. Our objective was to investigate the expression and function of CDT2, a substrate recognition factor for the CUL4 E3 ubiquitin ligase family (CRL4-CDT2) in ovarian cancer. Methods: CDT2 expression was evaluated using realtime RT-PCR in 26 serous ovarian cancers 11 normal ovaries, and 14 fallopian tube fimbria specimens and confirmed by Western blotting on a subset of the above. Immunohistochemistry was performed using tissue microarrays consisting of over 130 independent ovarian cancer samples of various histologies, as well as, normal fallopian tube samples using antibodies to CDT2 and p21. Each samples was represented at least in triplicate and scored semi-quantitatively. CDT2 knockdown was accomplished using siRNA and overexpression via retroviral transduction. Cell growth was evaluated using MTT assays. Statistical analysis were performed using either parametric (t test) as well as non-parametric (Mann-Whitney and Kruskal Wallis) methods as indicated. Comparison of p21 and CDT2 expression was performed using Spearman's correlation. Results: CDT2 mRNA expression was approximately 9 fold higher in serous ovarian cancers compared to normal ovary and fallopian tube fimbria samples (P Conclusions: The results of this ongoing investigation are the first to suggest an oncogenic role for the CRL4-CDT2 ubiquitin ligase in ovarian cancer. The mechanism of action remains unknown but does not appear to involve p21 degradation. 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 608. doi:10.1158/1538-7445.AM2011-608

Objective: The MDS1 and EVI1 complex locus (MECOM) located at the 3q26 locus is amplified in many cancers and gives rise to a number of alternatively spliced transcripts and proteins including EVI1 and MDS1-EVI1 (ME). EVI1 functions as an oncogene in human and murine myeloproliferative disorders and myelogenous leukemias. Amplification and over expression of EVI1 and ME have been demonstrated in ovarian cancers, and these proteins have been proposed as potential therapeutic targets that may also play a role in chemoresistance. Our objective was to evaluate the expression and biological function of EVI1 and one of its splice isoform known as EVI1s or Δ324 in ovarian cancer. Methods: Expression of EVI1 isoforms was evaluated in serous ovarian cancer samples, normal ovaries, benign ovarian neoplasms, and fallopian tube fimbria (emerging as the likely site of origin of many serous ovarian cancers) using realtime RT-PCR, and immunoblotting. Alterations in EVI1 and EVI1s expression were induced by retroviral transduction and isoform selective siRNA. Proliferation was measured using MTT and BrdU assays. Dose response to paclitaxel and cisplatin was measured following isoform selective knockdown of EVI1 and EVI1s using a histone release apoptosis ELISA. Results: EVI1 and EVI1s transcripts were present in normal ovaries, fimbria, benign ovarian neoplasms, and papillary serous ovarian cancers, with highest expression of both isoforms noted in the ovarian cancer samples. There was no change in relative expression of EVI1s among the different tissue types examined. In contrast, EVI1 protein isoforms were essentially undetectable in normal ovarian tissues. While highest average expression of ME, EVI1, and EVI1s protein isoforms was observed in serous ovarian cancer samples, considerable variability was noted between different serous ovarian cancer samples. In addition, variable expression of the same EVI1 protein isoforms was also observed in normal fallopian tube fimbria and several benign ovarian neoplasms. Forced expression of EVI1 and EVI1s did not result in any increased proliferation in OVCAR8 ovarian cancers (that lack endogenous EVI1 expression) nor in HEK293T cells. Furthermore, no effects on proliferation were observed following knockdown of EVI1, EVI1s, or all EVI1 transcripts. In dose response assays there was no significant effect on cisplatin and paclitaxel induced apoptosis following EVI1 knockdown. Daunorubicin induced DNA damage as determined by phospho-H2AX expression was also unaltered following isoform selective EVI1 siRNA. Conclusion: There is a lack of correlation between EVI1 transcript and protein isoforms in gynecologic tissues. Our results suggest that EVI1 protein expression in some serous ovarian cancers may be related to their derivation from fallopian tube origin, and that alteration of EVI1 and EVI1s protein levels doesn't influence ovarian cancer cell growth, apoptosis, or response to chemotherapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5480.

Cancer growth and metastasis involve an active interaction between primary tumor cells and the tumor's microenvironment, which consists of fibroblasts, vascular cells and inflammatory cells. Such interactions between the cancer and the cells within the microenvironment induce differential expression of genes that act to foster the growth, invasion and spread of tumor cells. This study utilizes pair-wise primary cultures of fibroblasts from normal and cancerous human endometrial tissues of the same patients to investigate the potential factors in the tumor microenvironment and their role in carcinogenesis. Both microRNA and mRNA profiling were performed on paired normal and cancer fibroblasts. SatB2 emerged as a promising candidate when its expression was shown to be significantly increased in fibroblasts surrounding the tumor tissues in all but one patients tested (n=8). Further investigation documented that SatB2 is a target for miR31, via 2 binding sites located within the SATB2 3’ UTR region. In concordance, miR31 is down-regulated in tumor fibroblasts, which further implicates the potential role of SatB2 in promoting a favorable environment for tumors. Ectopic expression of SATB2 in normal fibroblasts does not affect the growth rate of an endometrial cancer cell line (EC-1) in co-culture experiments; however, it did increase the motility of EC-1 cells towards these fibroblasts by multiple folds in both in vitro migration and invasion assays. A reduction of SATB2 expression in cancer activated fibroblasts (CAFs) by shRNA attenuated the migratory ability of EC-1 cells, further supporting the role of SatB2 in tumor aggression. Our observations also indicated that the conditioned media from CAFs were sufficient to induce the motility changes in the EC-1 cells. We believe the elucidation of SatB2 pathway(s) will assist in uncovering the intricate connections between tumors and their microenvironments, leading to new targets for treating tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1423.

The dynamic interactions between tumor cells and their corresponding microenvironment play an important role in carcinogenesis. Fibroblasts are a dominant feature within a tumor stroma and have undergone a “reprogramming” to become cancer-activated fibroblasts (CAFs). In our study we established primary cell cultures of fibroblasts from matched pairs of normal human endometrium and endometrial cancer tissues and analyzed the differential expressions of mRNA and microRNA. We found that the WNT pathway was activated in CAFs and confirmed these results via Topflash luciferase reporter assays. Wingless-type (WNT) proteins are a highly conserved family of secreted glycoproteins that have been implicated in the carcinogenesis of many cancers. We demonstrated that one of the reasons for this WNT pathway activation was the methylation-dependent silencing of the secreted frizzled-related protein 4 (SFRP4), an antagonist to the WNT signaling pathway. An additional reason for WNT pathway activation was found in the microRNA analysis. These experiments showed downregulation of miR-148a, which is a predicted regulator of the founder member of the WNT family, WNT1. We observed that CAFs have increased WNT1 protein expression in three of the five endometrial cancer CAFs. We confirmed that miR-148a directly targets WNT1 by cloning the WNT1 3'UTR into a luciferase reporter construct and showed that co-transfection with miR-148a results in a decrease of luciferase activity. When mutations to the miR-148a binding sites were introduced into the same luciferase reporter construct, the downregulation of luciferase activity by miR-148a was prevented. We conclude that the observed activation of the WNT pathway in endometrial cancer CAFs may be regulated by the miR-148a and contribute to the progression of the endometrial cancer tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1430.