Platinum-resistant Models Research Articles

A Stem-like Patient-Derived Ovarian Cancer Model of Platinum Resistance Reveals Dissociation of Stemness and Resistance.

To understand chemoresistance in the context of cancer stem cells (CSC), a cisplatin resistance model was developed using a high-grade serous ovarian cancer patient-derived, cisplatin-sensitive sample, PDX4. As a molecular subtype-specific stem-like cell line, PDX4 was selected for its representative features, including its histopathological and BRCA2 mutation status, and exposed to cisplatin in vitro. In the cisplatin-resistant cells, transcriptomics were carried out, and cell morphology, protein expression, and functional status were characterized. Additionally, potential signaling pathways involved in cisplatin resistance were explored. Our findings reveal the presence of distinct molecular signatures and phenotypic changes in cisplatin-resistant PDX4 compared to their sensitive counterparts. Surprisingly, we observed that chemoresistance was not inherently linked with increased stemness. In fact, although resistant cells expressed a combination of EMT and stemness markers, functional assays revealed that they were less proliferative, migratory, and clonogenic-features indicative of an underlying complex mechanism for cell survival. Furthermore, DNA damage tolerance and cellular stress management pathways were enriched. This novel, syngeneic model provides a valuable platform for investigating the underlying mechanisms of cisplatin resistance in a clinically relevant context, contributing to the development of targeted therapies tailored to combat resistance in stem-like ovarian cancer.

Abstract 945: In vivo anti-tumor activity of onvansertib, a PLK1 inhibitor, combined with gemcitabine or carboplatin in platinum- resistant ovarian carcinoma patient-derived xenograft models

Abstract Background: The standard treatment for high grade ovarian carcinoma (HGOC) is cytoreductive surgery followed by a platinum based therapy. Despite an initial high rate of complete remission, most of the patients relapse with a platinum resistant disease. Patients with resistant tumors have few options, including monotherapy with gemcitabine, and new effective therapeutic alternatives are needed. The Polo-like kinase 1 (PLK1) is a master regulator of mitosis and recent evidences suggest its role in interfering with several DNA repair mechanisms. We investigated the effect of onvansertib (ONVA), a highly selective ATP-competitor PLK1 inhibitor, in combination with carboplatin (CARBO) and gemcitabine (GEM) in platinum-resistant HGOC patient-derived xenografts (PDXs). Methods: The combination of ONVA with either CARBO or GEM was investigated in two platinum-resistant HGOC PDXs, one intrinsically resistant to platinum (MNHOC#315), the other made resistant after repeated in vivo treatments with cisplatin (MNHOC#266R). Tumors were subcutaneously (s.c.; MNHOC#315) or orthotopically (i.p.; MNHOC#266R) transplanted in nude mice, mice were treated for 4 weeks with vehicle, single agents or drug combinations and followed for tumor growth (s.c. model) and survival. Results: CARBO- and GEM-ONVA combinations were well tolerated, with a maximum body weight loss of 6.6%. Both combinations showed strong anti-tumor activities in the two tested models. In MNHOC#315 PDX, CARBO- and GEM-ONVA combinations caused a tumor stabilization that extended beyond the dosing period and resulted in a higher tumor growth inhibition as compared to vehicle and single agents treated mice. In i.p. MNHOC#266R PDX, single agents were inactive, while a statistically significant increase in survival was observed in CARBO/ONVA and in GEM/ONVA treated mice as compared to vehicle treated mice, with a 6.6- and 7-fold increase in mean survival time, respectively. Pharmacodynamic studies are ongoing to decipher the mechanisms underlying the synergic effects of both combinations. Conclusions: The combinations of ONVA with either CARBO or GEM showed potent anti-tumor activities in two platinum-resistant HGOC PDXs. Additional studies in other HGOC platinum-resistant models, both in vivo and in vitro, as well pharmacodynamic studies are ongoing to validate these results and to get insights in the mechanisms underlying their efficacy. Citation Format: Michela Chiappa, Federica Guffanti, Ilaria Mengoli, Maya Ridinger, Giovanna Damia. In vivo anti-tumor activity of onvansertib, a PLK1 inhibitor, combined with gemcitabine or carboplatin in platinum- resistant ovarian carcinoma patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 945.

Abstract 3264: Ex vivo treatment in high grade serous ovarian cancer demonstrates the benefit of EZH2 inhibition in combination with standard therapy

Abstract While high-grade serous ovarian cancers (HGSOC) are initially sensitive to platinum-based therapy, resistance to chemotherapy remains a major challenge. Recently, the role of EZH2 in establishing a chemo-resistant state has been described in small cell lung cancer with a potential mechanism thought to involve the silencing of SLFN111. Preclinical studies showed that EZH2 inhibition results in re-expression of SLFN11 protein and restores sensitivity to DNA damaging agents. A study in ovarian cancer2 emphasized the need to investigate the effect further. Here we describe the response of platinum-resistant HGSOC patient-derived xenograft (PDX) tumors to CPI-0209, an investigational new drug that is currently being explored for its potential as a reversible second generation EZH2 inhibitor as monotherapy and in combination with carboplatin or PARP inhibition. Four luciferized PDXs (DF101, DF149, DF181, and DF216) were selected based on known platinum sensitivity and BRCA mutation status. PDX tumor ascites were harvested from mice at the IVIS signal intensity of 1011. Xenograft-derived organotypic tumor spheroids (xDOTS3) were loaded into 3D devices (AIM Biotech), treated with either CPI-0209, carboplatin, or olaparib alone, or with CPI-0209 for 7 days prior to adding carboplatin or olaparib for an additional 5-11 days. H3K27me3 was assessed by immunofluorescence. Expression of SLFN11 following treatment was assessed by western blot. Activity was measured at endpoint by dual-labeling fluorescence microscopy. EZH2-dependent target engagement via demethylation of H3K27me3 was seen in all models. While DF181 (BRIP1m) showed resistance to all treatments, DF216 (BRCAwt) showed a consistent trend in reduction in live area in the combination of carboplatin or olaparib with CPI-0209 vs single agent, and significant reduction (>20%; P<0.05) vs control. DF101 (BRCAm) and DF149 (BRCAwt), which showed no evidence of sensitivity to carboplatin and CPI-0209 monotherapy, showed sensitivity to olaparib which was not enhanced by the addition of CPI-0209. Further assessment of DF216 via western blot analysis showed elevated expression of SLFN11 after 7 days of EZH2i treatment. Since PDX in vivo studies are time-consuming and costly, it is impractical to use them for large drug screens. Here we show that our short-term and efficient xDOTS platform allows for rapid evaluation of multiple targeted therapies or therapeutic combinations. The results suggested that CPI-0209 directed combination therapy could enhance activity over standard chemotherapy alone in a platinum-resistant model of ovarian cancer ex vivo. Increased SLFN11 protein level supported the importance of EZH2-SLFN11 axis. Further exploration of CPI-0209 combination in ovarian cancer in known genomic contexts is warranted. Citation Format: Ha V. Vo, Qing Zeng, David A. Barbie, Prafulla C. Gokhale, Elizabeth Adams, Cloud P. Paweletz, Elena Ivanova. Ex vivo treatment in high grade serous ovarian cancer demonstrates the benefit of EZH2 inhibition in combination with standard therapy [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 3264.

Targeting CX3CR1 Suppresses the Fanconi Anemia DNA Repair Pathway and Synergizes with Platinum.

Simple SummaryChemotherapeutics exerting their antiproliferative actions by introducing DNA crosslinks, such as platinum drugs, are used to treat numerous cancers. Unfortunately, their therapeutic potential is limited due to adverse side effects and acquired resistance, the latter often associated with enhanced DNA repair capacity. Thus, targeting DNA repair is a promising strategy to lower effective doses and associated side effects, and to restore sensitivity to treatment. The C-X3-C motif chemokine receptor 1 (CX3CR1) is an emerging anticancer target which expression correlates with worse overall survival in cancer patients undergoing DNA damaging treatments. Here we show for the first time that the clinical-phase small molecule inhibitor KAND567 targeting CX3CR1 augments the efficacy of DNA crosslinking chemotherapeutics in cancer cell lines, including platinum resistant models, by interference of the Fanconi anemia DNA repair pathway. Hence, the interplay between CX3CR1 and FA repair provides novel potential therapeutic opportunities in cancers treated with DNA crosslinking agents.The C-X3-C motif chemokine receptor 1 (CX3CR1, fractalkine receptor) is associated with neoplastic transformation, inflammation, neurodegenerative diseases and aging, and the small molecule inhibitor KAND567 targeting CX3CR1 (CX3CR1i) is evaluated in clinical trials for acute systemic inflammation upon SARS-CoV-2 infections. Here we identify a hitherto unknown role of CX3CR1 in Fanconi anemia (FA) pathway mediated repair of DNA interstrand crosslinks (ICLs) in replicating cells. FA pathway activation triggers CX3CR1 nuclear localization which facilitates assembly of the key FA protein FANCD2 into foci. Interfering with CX3CR1 function upon ICL-induction results in inability of replicating cells to progress from S phase, replication fork stalling and impaired chromatin recruitment of key FA pathway factors. Consistent with defective FA repair, CX3CR1i results in increased levels of residual cisplatin-DNA adducts and decreased cell survival. Importantly, CX3CR1i synergizes with platinum agents in a nonreversible manner in proliferation assays including platinum resistant models. Taken together, our results reveal an unanticipated interplay between CX3CR1 and the FA pathway and show for the first time that a clinical-phase small molecule inhibitor targeting CX3CR1 might show benefit in improving responses to DNA crosslinking chemotherapeutics.

Targeting Dormant Ovarian Cancer Cells In Vitro and in an In Vivo Mouse Model of Platinum Resistance.

Spheroids exhibit drug resistance and slow proliferation, suggesting involvement in cancer recurrence. The protein kinase C inhibitor UCN-01 (7-hydroxystaurosporine) has shown higher efficacy against slow proliferating and/or quiescent ovarian cancer cells. In this study, tumorigenic potential was assessed using anchorage-independent growth assays and spheroid-forming capacity, which was determined with ovarian cancer cell lines as well as primary ovarian cancers. Of 12 cell lines with increased anchorage-independent growth, 8 formed spheroids under serum-free culture conditions. Spheroids showed reduced proliferation (P < 0.0001) and Ki-67 immunostaining (8% vs. 87%) relative to monolayer cells. Spheroid formation was associated with increased expression of mitochondrial pathway genes (P ≤ 0.001) from Affymetrix HT U133A gene expression data. UCN-01, a kinase inhibitor/mitochondrial uncoupler that has been shown to lead to Puma-induced mitochondrial apoptosis as well as ATP synthase inhibitor oligomycin, demonstrated effectiveness against spheroids, whereas spheroids were refractory to cisplatin and paclitaxel. By live in vivo imaging, ovarian cancer xenograft tumors were reduced after primary treatment with carboplatin. Continued treatment with carboplatin was accompanied by an increase in tumor signal, whereas there was little or no increase in tumor signal observed with subsequent treatment with UCN-01 or oltipraz. Taken together, our findings suggest that genes involved in mitochondrial function in spheroids may be an important therapeutic target in preventing disease recurrence.

Combining PARP with ATR inhibition overcomes PARP inhibitor and platinum resistance in ovarian cancer models

Ovarian cancer (OVCA) inevitably acquires resistance to platinum chemotherapy and PARP inhibitors (PARPi). We show that acquisition of PARPi-resistance is accompanied by increased ATR-CHK1 activity and sensitivity to ATR inhibition (ATRi). However, PARPi-resistant cells are remarkably more sensitive to ATRi when combined with PARPi (PARPi-ATRi). Sensitivity to PARPi-ATRi in diverse PARPi and platinum-resistant models, including BRCA1/2 reversion and CCNE1-amplified models, correlate with synergistic increases in replication fork stalling, double-strand breaks, and apoptosis. Surprisingly, BRCA reversion mutations and an ability to form RAD51 foci are frequently not observed in models of acquired PARPi-resistance, suggesting the existence of alternative resistance mechanisms. However, regardless of the mechanisms of resistance, complete and durable therapeutic responses to PARPi-ATRi that significantly increase survival are observed in clinically relevant platinum and acquired PARPi-resistant patient-derived xenografts (PDXs) models. These findings indicate that PARPi-ATRi is a highly promising strategy for OVCAs that acquire resistance to PARPi and platinum.

Oxaliplatin Pt(IV) prodrugs conjugated to gadolinium-texaphyrin as potential antitumor agents

Described here is the development of gadolinium(III) texaphyrin-platinum(IV) conjugates capable of overcoming platinum resistance by 1) localizing to solid tumors, 2) promoting enhanced cancer cell uptake, and 3) reactivating p53 in platinum-resistant models. Side by side comparative studies of these Pt(IV) conjugates to clinically approved platinum(II) agents and previously reported platinum(II)-texaphyrin conjugates demonstrate that the present Pt(IV) conjugates are more stable against hydrolysis and nucleophilic attack. Moreover, they display high potent antiproliferative activity in vitro against human and mouse cell cancer lines. Relative to the current platinum clinical standard of care (SOC), a lead Gd(III) texaphyrin-Pt(IV) prodrug conjugate emerging from this development effort was found to be more efficacious in subcutaneous (s.c.) mouse models involving both cell-derived xenografts and platinum-resistant patient-derived xenografts. Comparative pathology studies in mice treated with equimolar doses of the lead Gd texaphyrin-Pt(IV) conjugate or the US Food and Drug Administration (FDA)-approved agent oxaliplatin revealed that the conjugate was better tolerated. Specifically, the lead could be dosed at more than three times (i.e., 70 mg/kg per dose) the tolerable dose of oxaliplatin (i.e., 4 to 6 mg/kg per dose depending on the animal model) with little to no observable adverse effects. A combination of tumor localization, redox cycling, and reversible protein binding is invoked to explain the relatively increased tolerability and enhanced anticancer activity seen in vivo. On the basis of the present studies, we conclude that metallotexaphyrin-Pt conjugates may have substantial clinical potential as antitumor agents.

Synthesis of Peptoid-Based Class I-Selective Histone Deacetylase Inhibitors with Chemosensitizing Properties.

There is increasing evidence that histone deacetylase (HDAC) inhibitors can (re)sensitize cancer cells for chemotherapeutics via "epigenetic priming". In this work, we describe the synthesis of a series of class I-selective HDAC inhibitors with 2-aminoanilides as zinc-binding groups. Several of the synthesized compounds revealed potent inhibition of the class I HDAC isoforms HDAC1, HDAC2, and/or HDAC3 and promising antiproliferative effects in the human ovarian cancer cell line A2780 and the human squamous carcinoma cell line Cal27. Selected compounds were investigated in a cellular model of platinum resistance. In particular, compound 2a revealed potent chemosensitizing properties and full reversal of cisplatin resistance in Cal27CisR cells. This effect is related to a synergistic increase in caspase 3/7 activation and induction of apoptosis. Thus, this work demonstrates that pan-HDAC inhibition or dual class I/class IIb inhibition is not required for full reversal of cisplatin resistance.

Preclinical Studies Comparing Efficacy and Toxicity of DNA Repair Inhibitors, Olaparib, and AsiDNA, in the Treatment of Carboplatin-Resistant Tumors.

Purpose: Carboplatin is used to treat many cancers, but occurrence of drug resistance and its high toxicity remain a clinical hurdle limiting its efficacy. We compared the efficacy and toxicity of DNA repair inhibitors olaparib or AsiDNA administered alone or in combination with carboplatin. Olaparib acts by inhibiting PARP-dependent repair pathways whereas AsiDNA inhibits double-strand break repair by preventing recruitment of enzymes involved in homologous recombination and non-homologous end joining.Experimental Design: Mice with MDA-MB-231 tumors were treated with carboplatin or/and olaparib or AsiDNA for three treatment cycles. Survival and tumor growth were monitored. Toxicities of treatments were assayed in C57BL/6 immunocompetent mice. Circulating blood hematocrits, bone marrow cells, and organs were analyzed 10 and 21 days after end of treatment using flow cytometry and microscopy analysis. Resistance occurrence was monitored after cycles of treatments with combination of AsiDNA and carboplatin in independent BC227 cell cultures.Results: Olaparib or AsiDNA monotherapies decreased tumor growth and increased mean survival of grafted animals. The combination with carboplatin further increased survival. Carboplatin toxicity resulted in a decrease of most blood cells, platelets, thymus, and spleen lymphocytes. Olaparib or AsiDNA monotherapies had no toxicity, and their combination with carboplatin did not increase toxicity in the bone marrow or thrombocytopenia. All animals receiving carboplatin combined with olaparib developed high liver toxicity with acute hepatitis at 21 days. In vitro, carboplatin resistance occurs after three cycles of treatment in all six tested cultures, whereas only one became resistant (1/5) after five cycles when carboplatin was associated to low doses of AsiDNA. All selected carboplatin-resistant clones retain sensitivity to AsiDNA.Conclusion: DNA repair inhibitor treatments are efficient in the platinum resistant model, MDA-MB-231. The combination with carboplatin improves survival. The association of carboplatin with olaparib is associated with high liver toxicity, which is not observed with AsiDNA. AsiDNA could delay resistance to carboplatin without increasing its toxicity.

Cisplatin-induced immune modulation in ovarian cancer mouse models with distinct inflammation profiles.

The backbone of ovarian cancer treatment is platinum-based chemotherapy and aggressive surgical debulking. New therapeutic approaches using immunotherapy via immune checkpoint blockade, which have demonstrated clinical efficacy in other tumor types, have been less promising in ovarian cancer. To increase their clinical efficacy, checkpoint inhibitors are now being tested in clinical trials in combination with chemotherapy.Here, we evaluated the impact of cisplatin on tumor immunogenicity and its in vivo roles when used alone or in combination with anti-PD-L1, in two novel murine ovarian cancer cell models. The 2F8 and its platinum-resistant derivative 2F8cis model, display distinct inflammatory profiles and chemotherapy sensitivities, and mirror the primary and recurrent human disease, respectively. Acute and chronic exposure to cisplatin enhances tumor immunogenicity by increasing calreticulin, MHC class I, antigen presentation and T cell infiltration. Cisplatin also upregulates PD-L1 expression in vitro and in vivo, demonstrating a dual, paradoxical immune modulatory effect and supporting the rationale for combination with immune checkpoint blockade. One of the pathways activated by cisplatin treatment is the cGAS/STING pathway. Chronic cisplatin treatment led to upregulation of cGAS and STING proteins in 2F8cis compared to parental 2F8 cells, while acute exposure to cisplatin further increases cGAS and STING levels in both 2F8 and 2F8cis cells. Overexpression of cGAS/STING modifies tumor immunogenicity by upregulating PD-L1, MHC I and calreticulin in tumor cells. Anti-PD-L1 alone in a platinum-sensitive model or with cisplatin in a platinum-resistant model increases survival. These studies have high translational potential in ovarian cancer.

Mathematical Modeling Predicts Response to Chemotherapy and Drug Combinations in Ovarian Cancer.

Platinum-based chemotherapy constitutes the backbone of clinical care in advanced solid cancers such as high-grade serous ovarian cancer (HGSOC) and has prolonged survival of millions of patients with cancer. Most of these patients, however, become resistant to chemotherapy, which generally leads to a fatal refractory disease. We present a comprehensive stochastic mathematical model and simulator approach to describe platinum resistance and standard-of-care (SOC) therapy in HGSOC. We used pre- and posttreatment clinical data, including 18F-FDG-PET/CT images, to reliably estimate the model parameters and simulate "virtual patients with HGSOC." Treatment responses of the virtual patients generated by our mathematical model were indistinguishable from real-life patients with HGSOC. We demonstrated the utility of our approach by evaluating the survival benefit of combination therapies that contain up to six drugs targeting platinum resistance mechanisms. Several resistance mechanisms were already active at diagnosis, but combining SOC with a drug that targets the most dominant resistance subpopulation resulted in a significant survival benefit. This work provides a theoretical basis for a cancer treatment paradigm in which maximizing platinum's killing effect on cancer cells requires overcoming resistance mechanisms with targeted drugs. This freely available mathematical model and simulation framework enable rapid and rigorous evaluation of the benefit of a targeted drug or combination therapy in virtual patients before clinical trials, which facilitates translating preclinical findings into clinical practice.Significance: These findings present a comprehensive mathematical model for platinum resistance and standard-of-care therapy in a solid cancer, allowing virtual evaluation of novel therapy regimens. Cancer Res; 78(14); 4036-44. ©2018 AACR.

Abstract 990: Single-cell profiling of small cell lung cancer circulating tumor cell-derived xenograft models reveals intratumoral heterogeneity among mediators of chemoresistance

Abstract Small cell lung cancer (SCLC) accounts for 14% of lung cancer diagnoses in the United States and is characterized by rapid onset of chemoresistance and poor clinical outcomes. Once considered a homogeneous disease, recent analyses of SCLC have identified intratumoral heterogeneity (ITH) with respect to NOTCH signaling, ASCL1/NEUROD1 balance and MYC amplification - all of which are potential mechanisms underlying SCLC's aggressive and refractory biology. Unfortunately, patient-derived models of SCLC with which to better characterize the molecular profiles of refractory SCLC are scarce. To address this, we generated circulating tumor cell-derived xenograft (CDX) models from liquid biopsies of patients with treatment-naïve or relapsed SCLC. Each CDX model underwent pathological review to confirm tumors were consistent with SCLC based on histology and standard immunohistochemical markers (e.g., TTF1, chromogranin A, synaptophysin, NCAM). Sequencing of these models revealed mutations typical of SCLC (e.g. TP53, RB1), which were maintained in vivo over multiple passages. Importantly, each model's in vivo response to cisplatin matched the patient's platinum response at the time of CDX generation. At the proteomic level, platinum-resistant models exhibited mTOR activation, increased SOX2 and ATM, and reduced E-cadherin, suggesting a shift toward EMT and cancer stem cell expansion may contribute to resistance. To investigate ITH, we analyzed single-cell gene expression profiles by RNAseq using a droplet-based Chromium Single Cell system that analyzed a filtered subsample of 2000 cells per tumor. Consistent with SCLC, all CDX models contained large numbers of cells expressing neuroendocrine-specific genes (SYP, CHGA). However, Principle Component Analysis revealed that cells from chemosensitive CDX models had distinct expression profiles from resistant models. Using our published EMT gene signature, we found that resistant models had higher proportions of mesenchymal (vs. epithelial) cells. Several other distinctions between sensitive and resistant models were detected at the single-cell level but not in bulk RNA and protein analyses, suggesting that single-cell resolution can identify occult platinum-resistant subpopulations. For example, higher proportions of ASCL1- and DLL3-expressing cells were associated with platinum sensitivity, whereas a shift toward predominant NEUROD1-expression was observed with resistance. Cells expressing each of these three genes were identified across all tumors, suggesting platinum-sensitive and resistant subpopulations are ubiquitous but that even subtle shifts in the fractional distribution of these subsets can exert significant impact on response. These data support further use of single-cell analysis to explore the role of ITH as a driver of drug resistance in SCLC. Citation Format: C. Allison Stewart, Carl M. Gay, Yuanxin Xi, Siva V., Junya Fujimoto, Pan Tong, Lixia Diao, Lerong Li, Mohan Bolisetty, Neda Kalhor, Patrice Lawson, Mayra Vasquez, Hai Tran, Ignacio I. Wistuba, Bonnie Glisson, Jianjun Zhang, Stephen G. Swisher, Jack A. Roth, John V. Heymach, Paul Robson, Jing Wang, Lauren A. Byers. Single-cell profiling of small cell lung cancer circulating tumor cell-derived xenograft models reveals intratumoral heterogeneity among mediators of chemoresistance [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 990.

CHK1 Inhibition in Small-Cell Lung Cancer Produces Single-Agent Activity in Biomarker-Defined Disease Subsets and Combination Activity with Cisplatin or Olaparib

Effective targeted therapies for small-cell lung cancer (SCLC), the most aggressive form of lung cancer, remain urgently needed. Here we report evidence of preclinical efficacy evoked by targeting the overexpressed cell-cycle checkpoint kinase CHK1 in SCLC. Our studies employed RNAi-mediated attenuation or pharmacologic blockade with the novel second-generation CHK1 inhibitor prexasertib (LY2606368), currently in clinical trials. In SCLC models in vitro and in vivo, LY2606368 exhibited strong single-agent efficacy, augmented the effects of cisplatin or the PARP inhibitor olaparib, and improved the response of platinum-resistant models. Proteomic analysis identified CHK1 and MYC as top predictive biomarkers of LY2606368 sensitivity, suggesting that CHK1 inhibition may be especially effective in SCLC with MYC amplification or MYC protein overexpression. Our findings provide a preclinical proof of concept supporting the initiation of a clinical efficacy trial in patients with platinum-sensitive or platinum-resistant relapsed SCLC. Cancer Res; 77(14); 3870-84. ©2017 AACR.

Abstract 4437: DNA methylation analysis in cisplatin resistant ovarian cancer cells and recurrent ovarian cancer patient samples

Abstract Acquisition of platinum resistance following first line platinum therapy is commonly observed in ovarian cancer patients and is a major obstacle to clinical effectiveness. Currently there are no options available to prevent platinum resistance; however, studies show that demethylating agents may resensitize patients to platinum therapy thereby demonstrating DNA methylation as a critical contributor to the development of platinum resistance. Using a cellular model of platinum resistance we have previously shown that resistant cells have increased DNA methyltransferase (DNMT) activity as well as increased global DNA methylation. These observations were dependent on cisplatin induced activation of the Epidermal Growth Factor Receptor (EGFR) which we previously identified as a novel a regulator of DNMT activity and DNA methylation. Inhibition of the EGFR conferred during a platinum resistance paradigm decreased DNMT activity, decreased global methylation and attenuated resistance in our platinum resistant model. We hypothesized that further genome wide analysis of DNA methylation in resistant, parental and EGFR inhibited cell lines at specific CG sites would reveal targets of methylation important for the development of platinum resistance. Using the Illumina 450K methylation array, we found few significant alterations in DNA methylation for given genes between control and cisplatin resistant (CPR) cells. However, additional DNA methylation analysis with the 450K methylation array of patient tumor samples at initial diagnosis as well as after relapse, while not significant, showed similar trends in the data for certain genes namely CSRNP3, LOC400904, LYPD6, MTMR9L, NIPAL4 and RBP7. We are still evaluating the effects of EGFR inhibition in vitro on DNA methylation patterns. We suggest that while the in vitro model of platinum resistance corresponds to patient samples in some aspects, ovarian cancer cells display a great deal of heterogeneity in DNA methylation as described by others and a much larger sample size is necessary to draw concrete conclusions. Current studies are evaluating gene expression in control and resistant cells to validate whether alterations in DNA methylation coincide with changes in expression. Citation Format: Michaela L. Granados, Laurie G. Hudson, Li Luo, Sabrina L. Samudio-Ruiz. DNA methylation analysis in cisplatin resistant ovarian cancer cells and recurrent ovarian cancer patient samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4437.

Contributions of the Epidermal Growth Factor Receptor to Acquisition of Platinum Resistance in Ovarian Cancer Cells.

Acquisition of platinum resistance following first line platinum/taxane therapy is commonly observed in ovarian cancer patients and prevents clinical effectiveness. There are few options to prevent platinum resistance; however, demethylating agents have been shown to resensitize patients to platinum therapy thereby demonstrating that DNA methylation is a critical contributor to the development of platinum resistance. We previously reported the Epidermal Growth Factor Receptor (EGFR) is a novel regulator of DNA methyltransferase (DNMT) activity and DNA methylation. Others have shown that EGFR activation is linked to cisplatin treatment and platinum resistance. We hypothesized that cisplatin induced activation of the EGFR mediates changes in DNA methylation associated with the development of platinum resistance. To investigate this, we evaluated EGFR signaling and DNMT activity after acute cisplatin exposure. We also developed an in vitro model of platinum resistance to examine the effects of EGFR inhibition on acquisition of cisplatin resistance. Acute cisplatin treatment activates the EGFR and downstream signaling pathways, and induces an EGFR mediated increase in DNMT activity. Cisplatin resistant cells also showed increased DNMT activity and global methylation. EGFR inhibition during repeated cisplatin treatments generated cells that were more sensitive to cisplatin and did not develop increases in DNA methylation or DNMT activity compared to controls. These findings suggest that activation of EGFR during platinum treatment contributes to the development of platinum resistance. Furthermore, EGFR inhibition may be an effective strategy at attenuating the development of platinum resistance thereby enhancing the effectiveness of chemotherapeutic treatment in ovarian cancer.

Abstract POSTER-THER-1427: Epidermal growth factor receptor mediated alterations in DNA methylation associated with the development of platinum resistance in ovarian cancer cells

Abstract Acquisition of platinum resistance following first line platinum-taxane therapy occurs in a large portion of ovarian cancer patients and continues to be a major challenge in clinical effectiveness. To date, there are limited interventions available to prevent or reverse platinum resistance; however, there has been some advances in the use of demethylating agents in the resensitization of patients to platinum based therapy. This highlights epigenetic alterations, in particular DNA methylation, as critical contributors to the acquisition of drug resistance in ovarian cancer. The Epidermal Growth Factor Receptor (EGFR) has also been linked to chemoresistance and is known to be activated by cisplatin treatment. We previously published that activation of the EGFR can increase DNA methyltransferase (DMNT) activity and, over extended activation, can significantly increase global DNA methylation in ovarian cancer cells. We hypothesized that cisplatin induced activation of the EGFR could mediate changes in DNA methylation associated with the development of platinum resistance. To investigate this, we used an in vitro model of platinum resistance and evaluated EGFR signaling and DNMT activity after acute cisplatin and in platinum resistant cells. We found that cisplatin treatment activates the EGFR and downstream signaling pathways JAK and AKT. Cisplatin also increased DNMT activity and this increase was dependent on EGFR activation. Over repeated sequential treatments of cisplatin, we successfully developed platinum resistant cells that possessed significant increases in DNMT activity and had moderate, but significant, increases in global DNA methylation. Preliminary data using the EGFR inhibitor Erlotinib during the development of platinum resistant cells suggest that the EGFR plays an integral role in the acquisition of platinum resistance. This work implicates EGFR driven alterations to DNMT activity and DNA methylation as a novel mechanism for development of platinum resistance. Further studies are currently being done to evaluate potential alterations in activation of downstream signaling pathways, levels of DNA methyltransferases and other changes in expression associated with platinum resistance. Citation Format: Sabrina L. Samudio-Ruiz, Michaela L. Granados, Laurie G. Hudson. Epidermal growth factor receptor mediated alterations in DNA methylation associated with the development of platinum resistance in ovarian cancer cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1427.

Critical role of Wnt/β-catenin signaling in driving epithelial ovarian cancer platinum resistance.

Resistance to platinum-based chemotherapy is the major barrier to treating epithelial ovarian cancer. To improve patient outcomes, it is critical to identify the underlying mechanisms that promote platinum resistance. Emerging evidence supports the concept that platinum-based therapies are able to eliminate the bulk of differentiated cancer cells, but are unable to eliminate cancer initiating cells (CIC). To date, the relevant pathways that regulate ovarian CICs remain elusive. Several correlative studies have shown that Wnt/β-catenin pathway activation is associated with poor outcomes in patients with high-grade serous ovarian cancer (HGSOC). However, the functional relevance of these findings remain to be delineated. We have uncovered that Wnt/β-catenin pathway activation is a critical driver of HGSOC chemotherapy resistance, and targeted inhibition of this pathway, which eliminates CICs, represents a novel and effective treatment for chemoresistant HGSOC. Here we show that Wnt/β-catenin signaling is activated in ovarian CICs, and targeted inhibition of β-catenin potently sensitized cells to cisplatin and decreased CIC tumor sphere formation. Furthermore, the Wnt/β-catenin specific inhibitor iCG-001 potently sensitized cells to cisplatin and decreased stem-cell frequency in platinum resistant cells. Taken together, our data is the first report providing evidence that the Wnt/β-catenin signaling pathway maintains stem-like properties and drug resistance of primary HGSOC PDX derived platinum resistant models, and therapeutic targeting of this pathway with iCG-001/PRI-724, which has been shown to be well tolerated in Phase I trials, may be an effective treatment option.

Abstract B46: Epigenetic alterations associated with platinum resistance in an ovarian cancer cell model

Abstract Platinum resistance in ovarian cancer continues to be an impediment to clinical effectiveness. While multiple alterations associated with platinum resistance have been proposed, mechanisms responsible for those alterations, particularly epigenetic alterations, are still under investigation. Epigenetic alterations, specifically epigenetic silencing induced by DNA methylation, to genes involved in drug response is a common occurrence in platinum resistance. We have previously published that activation of the Epidermal Growth Factor Receptor (EGFR) in ovarian cancer cells increases activity of DNA methyltransferases (DNMTs) and extended EGFR activation can also increase global DNA methylation. In a concurrent abstract, we show that acute cisplatin treatment activates the EGFR and increases DNMT activity. Our overarching hypothesis for this work is that the EGFR is a key player in the development of platinum resistance by its ability to regulate DNMT activity and DNA methylation and thereby could be a therapeutic target for preventing acquisition of platinum resistance during chemotherapeutic treatment. Here, we show data obtained using a cellular model of platinum resistance that we have generated in the lab. We show that OVCA 433 cells treated repeatedly with increasing doses of cisplatin acquire resistance and demonstrate resistance in a 3D model which is more indicative of the ovarian cancer cell microenvironment. These cisplatin resistant cells (CPR) also display significantly increased DNMT activity compared to passage control (non-resistant) cells, with modest increases in global DNA methylation. While hyperactivation of the EGFR is not observed in this chronic model of cisplatin treatment, we do see a significant decrease in Copper transporter 1 (CTR1) levels in CPR cells. CTR1 is particularly important in cisplatin drug uptake, thus we believe that platinum resistance in CPR cells is due to downregulation of CTR1 potentially by DNA methylation. Current studies are evaluating DNA methylation of CTR1 and other genes that may be contributing to the development of platinum resistance. Future studies inhibiting the EGFR during cisplatin treatment will be imperative in linking cisplatin induced EGFR activation to the changes in DNMT activity and methylation that we are observing in our model of platinum resistance. This work implicates EGFR driven alterations to DNMT activity and DNA methylation as a novel mechanism for development of platinum resistance. Citation Format: Sabrina L. Samudio-Ruiz, Michaela L. Granados, Laurie G. Hudson. Epigenetic alterations associated with platinum resistance in an ovarian cancer cell model. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B46.