Platinum-resistant Cells Research Articles

Upregulation of miRNA-450b-5p targets ACTB to affect drug resistance and prognosis of ovarian cancer via the PI3K/Akt signaling pathway.

Ovarian cancer (OC) is the most malignant gynecologic cancer, and chemoresistance is a major cause of treatment failure in patients with OC. The understanding of microRNA (miRNA) in cancer is limited, and the role of miRNA (miR)-450b-5p in cancer drug resistance is unknown. In this study, we aim to evaluate the role of miR-450b-5p in drug-resistant OC and its underlying mechanisms. MiR-450b-5p expression was assessed in drug-sensitive and resistant OC cells via quantitative real-time polymerase chain reaction. Cell viability was evaluated using the Cell Counting Kit-8 assay. Progression-free survival (PFS) and overall survival (OS) curves were generated using the Kaplan-Meier method and the log-rank test. Target genes of miR-450b-5p were identified from the Cancer MIRNome database. Co-expressed genes were obtained from The Cancer Genome Atlas and Cancer Genome cBioportal for pathway enrichment and functional clustering analysis. The miRNA-450b-5p expression was significantly increased in A2780 and SKOV3 OC-resistant cells and significantly increased by 17-fold in the A2780-CBP-Lv-miR-450b-5p cells compared to A2780-CBP and A2780-CBP-Lv-NC cells. The up-regulated expression of miR-450b-5p increased the cell viability and half maximal inhibitory concentration (IC50) of A2780 platinum-resistant cells and was associated with poor OS. We obtained 33 potential target genes of miR-450b-5p and beta-actin (ACTB) might be a potential target of miR-450b-5p. Low expression of ACTB predicted poor OS and PFS. We obtained 362 common genes co-expressed with ACTB, which involved 4 critical pathways. PI3K acted as an upstream pathway of the other three pathways, which ultimately responded to drug resistance regulation in OC. The genes enriched in four pathways were cross-analyzed and 13 overlapping genes were obtained. These 13 genes were also significantly and positively co-expressed with ACTB at both protein and mRNA levels. High expression of miRNA-450b-5p might affect drug resistance and prognosis in OC by targeting 13 co-expressed genes of ACTB directly through the PI3K/Akt signaling pathway. Thus, miR-450b-5p might provide a new therapeutic target for drug resistance in OC.

Circ_0002722-induced regulation of YAP promotes platinum resistance in oral squamous cell carcinoma: Implications for verteporfin therapy

Oral squamous cell carcinoma (OSCC) poses a significant public health burden due to its high prevalence and poor prognosis. Platinum resistance is one of the major challenges in OSCC treatment. Yes-associated protein (YAP) has been identified as a pivotal player in OSCC tumorigenesis and progression. Circular RNA (circRNA) has been implicated in chemoresistance in various cancers by regulation the function of microRNA. Nevertheless, the specific mechanisms linking circRNA to YAP expression in OSCC remain poorly understood. In this study, we detected the YAP and circRNA hsa_circ_0002722 (circ_0002722) expression by western blot (WB) and quantitative polymerase chain reaction (qPCR). We found that YAP and circ_0002722 were up-regulated in platinum resistance in OSCC tissues. Furthermore, transfection of circ_0002722 siRNA into platinum-resistant cells revealed that circ_0002722 acted as a regulator of miR-1305, which influenced YAP expression and thereby affected platinum sensitivity. In vivo experiments corroborated the synergistic effects of cisplatin and verteporfin (a YAP inhibitor) in combating platinum resistance. Targeting YAP emerges as a promising therapeutic strategy for addressing platinum resistance in OSCC, with circ_0002722 serving as a potential therapy target and valuable diagnostic marker. These findings shed light on the underlying mechanisms of platinum resistance, paving the way for the development of effective treatment approaches.

Cell-intrinsic platinum response and associated genetic and gene expression signatures in ovarian cancer cell lines and isogenic models.

Ovarian cancers are still largely treated with platinum-based chemotherapy as the standard of care, yet few biomarkers of clinical response have had an impact on clinical decision making as of yet. Two particular challenges faced in mechanistically deciphering platinum responsiveness in ovarian cancer have been the suitability of cell line models for ovarian cancer subtypes and the availability of information on comparatively how sensitive ovarian cancer cell lines are to platinum. We performed one of the most comprehensive profiles to date on 36 ovarian cancer cell lines across over seven subtypes and integrated drug response and multiomic data to improve on our understanding of the best cell line models for platinum responsiveness in ovarian cancer. RNA-seq analysis of the 36 cell lines in a single batch experiment largely conforms with the currently accepted subtyping of ovarian cancers, further supporting other studies that have reclassified cell lines and demonstrate that commonly used cell lines are poor models of high-grade serous ovarian carcinoma. We performed drug dose response assays in the 32 of these cell lines for cisplatin and carboplatin, providing a quantitative database of IC 50 s for these drugs. Our results demonstrate that cell lines largely fall either well above or below the equivalent dose of the clinical maximally achievable dose (C max ) of each compound, allowing designation of cell lines as sensitive or resistant. We performed differential expression analysis for high-grade serous ovarian carcinoma cell lines to identify gene expression correlating with platinum-response. Further, we generated two platinum-resistant derivatives each for OVCAR3 and OVCAR4, as well as leveraged clinically-resistant PEO1/PEO4/PEO6 and PEA1/PEA2 isogenic models to perform differential expression analysis for seven total isogenic pairs of platinum resistant cell lines. While gene expression changes overall were heterogeneous and vast, common themes were innate immunity/STAT activation, epithelial to mesenchymal transition and stemness, and platinum influx/efflux regulators. In addition to gene expression analyses, we performed copy number signature analysis and orthogonal measures of homologous recombination deficiency (HRD) scar scores and copy number burden, which is the first report to our knowledge applying field-standard copy number signatures to ovarian cancer cell lines. We also examined markers and functional readouts of stemness that revealed that cell lines are poor models for examination of stemness contributions to platinum resistance, likely pointing to the fact that this is a transient state. Overall this study serves as a resource to determine the best cell lines to utilize for ovarian cancer research on certain subtypes and platinum response studies, as well as sparks new hypotheses for future study in ovarian cancer.

FANCD2 expression affects platinum response and further characteristics of high grade serous ovarian cancer in cells with different genetic backgrounds

High-grade serous ovarian cancer (HGSOC) is the most prevalent subtype of ovarian cancer and demonstrates 5-year survival of just 40%. One of the major causes of mortality is the development of tumour resistance to platinum-based chemotherapy, which can be modulated by dysregulation of DNA damage repair pathways. We therefore investigated the contribution of the DNA interstrand crosslink repair protein FANCD2 to chemosensitivity in HGSOC. Increased FANCD2 protein expression was observed in some cell line models of platinum resistant HGSOC compared with paired platinum sensitive models. Knockdown of FANCD2 in some cell lines, including the platinum resistant PEO4, led to increased carboplatin sensitivity. Investigation into mechanisms of FANCD2 regulation showed that increased FANCD2 expression in platinum resistant cells coincides with increased expression of mTOR. Treatment with mTOR inhibitors resulted in FANCD2 depletion, suggesting that mTOR can mediate platinum sensitivity via regulation of FANCD2. Tumours from a cohort of HGSOC patients showed varied nuclear and cytoplasmic FANCD2 expression, however this was not significantly associated with clinical characteristics. Knockout of FANCD2 was associated with increased cell migration, which may represent a non-canonical function of cytoplasmic FANCD2. We conclude that upregulation of FANCD2, possibly mediated by mTOR, is a potential mechanism of chemoresistance in HGSOC and modulation of FANCD2 expression can influence platinum sensitivity and other tumour cell characteristics.

TCEB3 initiates ovarian cancer apoptosis by mediating ubiquitination and degradation of MCL-1.

Platinum resistance remains a major contributor to the poor prognosis of ovarian cancer. Anti-apoptotic protein myeloid cell leukemia-1 (MCL-1) has emerged as a promising target for overcoming drug resistance, but different cancer cells utilize distinct protein degradation pathways to alter MCL-1 level. We systematically investigated E3 ligases to identify novel candidates that mediate platinum resistance in ovarian cancer. Transcription Elongation Factor B (TCEB3) has been identified as a novel E3 ligase recognition subunit that targets MCL-1 in the cytoplasm during platinum treatment other than its traditional function of targeting the Pol II in the nuclear compartment. TCEB3 expression is downregulated in platinum-resistant cell lines and this low expression is associated with poor prognosis. The ubiquitination of MCL-1 induced by TCEB3 leads to cell death in ovarian cancer. Moreover, platinum treatment increased the cytoplasm proportion of TCEB3, and the cytoplasm localization of TCEB3 is important for its targeting of MCL-1. This study emphasizes the dual function of TCEB3 in homeostasis maintenance and in cell fate determination under different conditions, and provides a new insight into drug resistance in ovarian cancer.

Novel Titanocene Y derivative with albumin affinity exhibits improved anticancer activity against platinum resistant cells

The antitumor activity of Ti(IV)-based compounds put them in the spotlight for cancer treatment in the past, but their lack of stability in vivo due to a high rate of hydrolysis has hindered their development as antitumor drugs. As a possible solution for this problem, we have reported a synthesis strategy through which we combined a titanocene fragment, a tridentate ligand, and a long aliphatic chain. This strategy allowed us to generate a titanium compound (Myr-Ti) capable of interacting with albumin, highly stable in water and with cytotoxic activity in tumor cells[1]. Following a similar strategy, now we report the synthesis of a new compound (Myr-TiY) derived from titanocene Y that shows antitumoral activity in a cisplatin resistant model with a 50% inhibitory concentration (IC50) of 41–76 μM. This new compound shows high stability and a strong interaction with human serum albumin. Myr-TiY has a significant antiproliferative and proapoptotic effect on the tested cancer cells and shows potential tumor selectivity when assayed in non-tumor human epithelial cells being more selective (1.3–3.8 times) for tumor cells than cisplatin. These results lead us to think that the described synthesis strategy could be useful to generate compounds for the treatment of both cisplatin-sensitive and cisplatin-resistant cancers.

Abstract B095: Tenascin C promotes ovarian cancer stem cells interactions with the niche

Abstract Background: Effective treatment of ovarian cancer (OC) is greatly impacted by advanced, late-stage diagnosis which usually presents with intra-peritoneal metastases, chemoresistance and high recurrence. The development of intra-peritoneal metastases is correlated with the formation of multicellular spheroids disseminated in the peritoneal fluid. Spheroid formation is promoted by a change of cell adhesion properties, enhanced secretion of extracellular matrix (ECM) components that stiffen the tumor stroma and promote the aberrant activation of oncogenic processes, including survival, invasion, and stemness. Tenascin C (TNC) is one of the molecules secreted in the ECM and correlates with migration and metastatic progression in a variety of carcinomas. However, the functional role of TNC in spheroid formation and in the maintenance of the platinum resistant ovarian cancer stem phenotype has not been investigated yet. We tested the hypothesis that the increased expression of TNC and its secretion in the ECM compartment promotes survival of OCSCs, supports OC spheroid formation and tumor progression. Methods: We compared TNC expression in OC cells grown as spheroids and monolayers and in chemoresistant vs sensitive OC cells by realtime-PCR and Western Blot (WB). TNC blockade by siRNA knockdown (KD) was evaluated in combination with carboplatin on spheroid assay, colony formation, and by qPCR of stemness related gene expression. Chromatin immunoprecipitation (ChIP) assay detected the interaction between the β-catenin and the TNC promoter. Results: OC cells grown as spheroids showed a significant increase in TNC expression compared to monolayers. Our results also demonstrated an increase in TNC expression levels in platinum resistant OC cell lines when compared to the platinum sensitive counterpart. A comparison of normal adjacent ovarian epithelium and primary OC tumors on a multitissue array revealed that TNC expression coincided with a pro-tumorigenic phenotype (n=88, P<0.001), correlating with poor outcome. TNC-KD in combination with carboplatin led to a significant decrease in the expression levels of stemness-related genes, spheroid and colony formation. Mechanistically, b-catenin-KD decreased TNC expression levels and β-catenin coimmunoprecipitated with the TNC promoter, suggesting that TNC is a direct β-catenin target. Additionally, OC spheroids treated with the Wnt ligand WNT-3A showed increased TNC expression compared to control untreated cells, as demonstrated by WB. Importantly, the enhanced expression of TNC was followed by its secretion in the ECM compartment, as observed via immunofluorescence imaging, implying a direct role of TNC in the ECM-rearrangement which is responsible for CSC maintenance and spheroid formation. Conclusions: TNC is strongly linked to chemoresistance and is regulated by the oncogenic β-catenin pathway. Our data suggest that strategies aimed at interfering with TNC expression and/or secretion along with carboplatin treatment could be a potential combination therapy in OC. Citation Format: Amber Rogers, Maya Shebeshi, Morganna Gordon, Rula Atwani, Virginie Lazar, Salvatore Condello. Tenascin C promotes ovarian cancer stem cells interactions with the niche [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B095.

Abstract A066: TUSC2 immunogene therapy enhances checkpoint blockade through increased cytotoxic immune activation in chemo-resistant small cell lung cancer (SCLC) in humanized mice

Abstract Immunotherapy has been effective but has had limited effect in clinical trials for SCLC. We identified TUSC2, a potent tumor suppressor gene, as a novel immunogene, which mediates both apoptosis and induces a strong antitumor immune response. TUSC2 expression is reduced in almost all SCLC tumors and absent in 41%. We investigated the antitumor immune response to TUSC2 gene therapy in combination with immune checkpoint blockade (ICB) against a chemo-resistant inflamed SCLC tumors in humanized mice. Colony formation was markedly reduced in platinum resistant inflamed H841 cells transfected with TUSC2. Humanized mice were generated by transplanting fresh human cord blood derived CD34 stem cells into sub-lethally irradiated NSG mice(hu-NSG). The level of engraftment of human CD45, CD3 T, CD19 B, NK cells was verified before tumor implantation at > 25%. Subcutaneous tumors were developed in hu-NSG mice and treated with intravenous injections of TUSC2 loaded cationic lipid nanoparticles (quaratusugene ozeplasmid abbreviated quar oze) with or without pembrolizumab (pembro; anti-PD1). A dramatic antitumor effect was mediated by quar oze, whereas pembro remained ineffective. A synergistic antitumor effect was found when quar oze was combined with pembro. The antitumor effect of the quar oze + pembro was associated with increased infiltration of human TILs, CD3 T, cytotoxic T, and NK cells, and a decreased number of human regulatory T (Treg) cells, and PD1 expressing exhausted CD8 T cells in the tumor microenvironment. Quar oze was also combined with atezolizumab (atezo; anti-PD-L1) and the antitumor effect on H841 lung metastases in hu-NSG mice was tested. Metastases were treated with quar oze and atezo alone or in combination. Although quar oze alone and atezo alone showed a strong antitumor effect in controlling metastases, a greater antitumor effect was found when quar oze was combined with atezo. Infiltration of human CD45, CD3 T, CD8 T and NK cells increased in the quar oze and quar oze +atezo treated groups compared with others. A significantly higher number of CD8 T effector memory and lower number of naïve T cells were found in the quar oze +atezo which were associated with increased activated CD8 T cells (CD69+, PD1+ CD8 T cells). HLA-DR+ dendritic cells were increased, whereas myeloid derived suppressor cells were strongly inhibited by the combination. Quar oze was also combined with carboplatin+atezo which showed enhanced antitumor efficacy compared with carbo+atezo or quar oze alone. When quar oze was combined with carboplatin, a greater effect was found in inhibiting H841 cell growth, colony formation, and induction of apoptosis. Quar oze was combined with NPRL2, another tumor suppressor gene, in H841 tumors in humanized mice. Synergistic antitumor efficacy of the dual gene therapy occurred with the quar oze +NPRL2 treatment. Taken together, these data suggest that TUSC2 gene therapy in combination with ICB induces strong antitumor activity on a chemo-resistant inflamed SCLC through cytotoxic CD8 T and NK cell activation. Citation Format: Ismail M Meraz, Mourad Majidi, Renduo Song, Feng Meng, Lihui Gao, Qi Wang, Jing Wang, Elizabeth Shpall, Mark Berger, Hemant Kumar, Jack A Roth. TUSC2 immunogene therapy enhances checkpoint blockade through increased cytotoxic immune activation in chemo-resistant small cell lung cancer (SCLC) in humanized mice [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A066.

Targeting RAS-ERK pathway alterations with MEK inhibitors to improve chemosensitivity in high grade serous ovarian cancers

ObjectiveAssess if MEK inhibitor blockade of RAS-ERK pathway adaptive response in high grade serous ovarian cancers (HGSOC) improves platinum sensitivity. MethodsThree HGSOC cell lines and three patient derived organoid (PDOs) samples from ascites of platinum resistant HGSOC patients were collected. Cell lines and PDOs were exposed to carboplatin and MEK inhibitors cobimetinib or trametinib. Cytotoxic effects of MEK inhibitors alone or combined with carboplatin were established. Western blots demonstrated RAS-ERK pathway blockage after MEK inhibitor treatment. RNA sequencing assessed gene expression after MEK inhibitor treatment. Cell line NF1 gene knockdown was performed with corresponding chemosensitivity levels. ResultsHigh carboplatin IC50 levels indicated platinum resistance in cell lines and PDOs. Cobimetinib induced cytotoxicity in cell lines and PDOs, while trametinib was less effective. Western blot confirmed MEK-ERK pathway blockage at minimal concentrations of MEK inhibitors in cell lines and PDOs. Phosphorylated-ERK levels of untreated cells indicated higher levels of RAS-ERK pathway activation in OVSAHO and OVCAR7 compared to OVCAR3. OVSAHO harbors a NF1 mutation and had highest levels of RAS-ERK activation. Cotreatment with carboplatin and MEK inhibitors showed varying synergistic cytotoxic effects at different combinations. Synergistic effect was most prominent in the OVSAHO carboplatin and cobimetinib combination. RNA sequencing identified downregulation of c-MYC and FOXM1 gene expression after MEK inhibitor treatment. NF1 gene knockdown showed an acquired increased IC50 compared to parental cells. ConclusionMEK inhibitors block RAS-ERK pathways in platinum resistant HGSOC cells and PDOs. MEK inhibitors with carboplatin have select synergistic effects which may indicate a strategy to improve platinum sensitivity.

MYC drives platinum resistant SCLC that is overcome by the dual PI3K-HDAC inhibitor fimepinostat

BackgroundSmall cell lung cancer (SCLC) is an aggressive neuroendocrine cancer with an appalling overall survival of less than 5% (Zimmerman et al. J Thor Oncol 14:768-83, 2019). Patients typically respond to front line platinum-based doublet chemotherapy, but almost universally relapse with drug resistant disease. Elevated MYC expression is common in SCLC and has been associated with platinum resistance. This study evaluates the capacity of MYC to drive platinum resistance and through screening identifies a drug capable of reducing MYC expression and overcoming resistance.MethodsElevated MYC expression following the acquisition of platinum resistance in vitro and in vivo was assessed. Moreover, the capacity of enforced MYC expression to drive platinum resistance was defined in SCLC cell lines and in a genetically engineered mouse model that expresses MYC specifically in lung tumors. High throughput drug screening was used to identify drugs able to kill MYC-expressing, platinum resistant cell lines. The capacity of this drug to treat SCLC was defined in vivo in both transplant models using cell lines and patient derived xenografts and in combination with platinum and etoposide chemotherapy in an autochthonous mouse model of platinum resistant SCLC.ResultsMYC expression is elevated following the acquisition of platinum resistance and constitutively high MYC expression drives platinum resistance in vitro and in vivo. We show that fimepinostat decreases MYC expression and that it is an effective single agent treatment for SCLC in vitro and in vivo. Indeed, fimepinostat is as effective as platinum-etoposide treatment in vivo. Importantly, when combined with platinum and etoposide, fimepinostat achieves a significant increase in survival.ConclusionsMYC is a potent driver of platinum resistance in SCLC that is effectively treated with fimepinostat.

Single-cell RNA-sequencing analysis reveals divergent transcriptome events between platinum-sensitive and platinum-resistant high-grade serous ovarian carcinoma.

Tumor resistance is one of the main reasons leading to the failure of ovarian cancer treatment. Overcoming platinum resistance remains the greatest challenge in the management of high-grade serous ovarian carcinoma (HGSC). Small conditional RNA-sequencing is a powerful method for exploring the complexity of the cellular components and their interactions in the tumor microenvironment. We profiled the transcriptomes of 35,042 cells from two platinum-sensitive and three platinum resistance HGSC clinical cases downloaded from Gene Expression Omnibus (GSE154600) and annotated tumor cells as platinum-resistant or sensitive based on the clinical trait. The study systematically investigated the inter-tumoral (using differential expression analysis, CellChat, and SCENIC) and intra-tumoral heterogeneity (using enrichment analysis such as gene set enrichment analysis, as well as gene set variation analysis, weighted gene correlation network analysis, and Pseudo-time analysis) of HGSC. A cellular map of HGSC generated by profiling 30,780 cells was revisualized using Uniform Manifold Approximation and Projection. The inter-tumoral heterogeneity was demonstrated with intercellular ligand-receptor interactions of major cell types and regulons networks. FN1, SPP1, and COLLAGEN play important roles in the cross-talk between tumor cells and the tumor microenvironment. HOXA7, HOXA9_extended, TBL1XR1_extended, KLF5, SOX17, and CTCFL regulons consistent with the distribution of platinum-resistant HGSC cells were the high activity regions. The intra-tumoral heterogeneity of HGSC was presented with corresponding functional pathway characteristics, tumor stemness features, and the cellular lineage transition from platinum-sensitive to resistant condition. Epithelial-mesenchymal transition played an important role in platinum resistance, whereas oxidative phosphorylation was the opposite. There was a small subset of cells in platinum-sensitive samples that had transcriptomic characteristics similar to platinum-resistant cells, suggesting that the progression of platinum resistance in ovarian cancer is inevitable. The present study describes a view of HGSC at single-cell resolution that reveals the characteristics of the HGSC heterogeneity and provides a useful framework for future investigation of platinum-resistant.

Abstract 1679: Unraveling the function of TRAF2 and NCK interacting kinase (TNIK) in high-grade serous ovarian cancer

Abstract The large majority of high grade serous ovarian cancer (HGSOC) patients will eventually become resistant to platinum-based chemotherapy, highlighting the urgent need to develop novel therapeutics targeting recurrent, platinum-resistant disease. To study this, our lab generated two patient derived platinum-resistant tumor spheres from platinum-sensitive primary patient cells to better understand the biology behind recurrent disease and identify novel therapeutic targets. Utilizing an artificial intelligence (AI) powered model, we screened a library that consisted of compounds predicted to target known or postulated resistance mechanisms and involved a novel therapeutic target. From this screen, we identified a TNIK kinase inhibitor as the only compound to effectively kill HGSOC spheres while sparing non-malignant fallopian tube cells. TNIK plays a key role in Wnt pathway activation, which our lab has previously shown to be critical in driving cancer stemness and epithelial ovarian cancer platinum resistance. Additionally, patient data shows that TNIK is amplified in ovarian cancer more than any other cancer type and is highly activated in ovarian cancer patients. We found that this compound was able to decrease overall Wnt activity in our platinum-resistant cells and could resensitize our platinum-resistant cells to cisplatin. This work demonstrates the abilities of using AI to expedite the drug discovery process as well as uncover novel therapeutic targets which may lead to future therapeutic strategies for HGSOC patients who no longer respond to standard of care platinum chemotherapy. Citation Format: Noah Puleo, Michele Cusato, Dylan Carvette, Chantelle Husdon, Analisa DiFeo. Unraveling the function of TRAF2 and NCK interacting kinase (TNIK) in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1679.

Abstract 5886: Targeting IDO1 and TDO2 is crucial for the modulation of immune effector in platinum resistant non-small cell lung cancer

Abstract The impact of non-small cell lung cancer (NSCLC) tumor metabolism on the immune microenvironment is not well understood in the context of platinum resistance. We have identified and characterized crucial metabolic differences between cisplatin-resistant (CR) and cisplatin-sensitive (CS) NSCLC cells in which CR cells possessed higher indoleamine 2,3-dioxygenase-1 (IDO1) activity as determined by an increase in extracellular kynurenine concentration in vitro and in vivo. Utilizing in vitro co-culture models (n=6), we showed that IDO-mediated kynurenine production from CR cells suppressed natural killer group 2 member D (NKG2D) on natural killer (NK) and CD8+ T cells, but enhanced immunosuppressive regulatory T cell (Treg) and myeloid-derived suppressor cells (MDSC) populations. Utilizing syngeneic (n=7) and humanized (n=5) mouse models, mice with CR tumors exhibited a more highly immunosuppressive environment that blocked the antitumor immune response. Inhibition of IDO1 by the selective IDO1 inhibitor epacadostat (200 mg/kg P.O. once a day for 15 days) attenuated tumor growth in these CR tumor-bearing mice. Interestingly, we found an induction of tryptophan 2,3-dioxygenase-2 (TDO2) enzyme in epacadostat-treated mice. To overcome this compensatory effect induced by IDO1 inhibition, mice were treated with a novel IDO1/TDO2 dual inhibitor (AT-0174; Antido Therapeutics Pty Ltd, Melbourne, Australia) at 170 mg/kg P.O. once a day for 15 days. Dual inhibitors suppressed tumor growth to a greater degree than IDO1 inhibitors in CR conditions, and significantly enhanced immune effector populations as well as suppressed immunosuppressive Tregs and MDSC population in CR tumors. In addition, increased expression of programmed death-ligand 1 (PD-L1) was observed in CR cells, we assessed the effects of combination treatment of AT-0174 + anti-PD1 antibody. This combined therapy produced a profound anti-tumor effect in CR tumors and extended survival in mice. Our data support an increase in the therapeutic efficacy of blocking both IDO1/TDO2 in treating CR lung cancer by enhancing tumor immune surveillance. Thus, inhibiting the kynurenine pathway may be a more suitable therapeutic approach in a subgroup of lung cancer patients who failed platinum therapy and lead to improved outcomes in the treatment and management of NSCLC. Support by Dept. of Veterans. Citation Format: Sydney Spector, Chunjing Wu, George Theodore, Jonathan Dan Nguyen, Emily Kim, Ashley Garcia, Niramol Savaraj, Diane Lim, Lynn Feun, Medhi Wangpaichitr. Targeting IDO1 and TDO2 is crucial for the modulation of immune effector in platinum resistant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5886.

Abstract 5808: Integrin-linked kinase regulates Wnt transcriptional activity in platinum resistant ovarian cancer stem cells

Abstract Ovarian cancer (OC) is the most lethal gynecological cancer, characterized by chemo-resistance and fatal tumor recurrence after primary treatment. The metastatic progression is mediated by exfoliation of tumor cells into the peritoneal cavity, the formation of compact OC spheroids, and their implantation onto the overlying peritoneal surface. Spheroid formation is promoted by a change of cell adhesion properties, enhanced secretion of extracellular matrix (ECM) components, such as fibronectin (FN), that stiffen the tumor stroma, promote the aberrant activation of integrins, and recruit the adaptor protein integrin-linked kinase (ILK). ILK drives cytoskeletal assembly and modulates key processes, including survival, invasion, and stemness. However, the functional role of ILK in chemoresistance of ovarian cancer stem cells (OCSCs) remains incompletely understood. We tested the hypothesis that the formation of FN-integrin complexes at the cell membrane promotes survival of OCSCs and supports OC spheroid formation by activating ILK and downstream oncogenic signaling. In OCSCs compared with non-CSC, ILK, FN, and integrin β1 mRNA expression levels were increased (P<0.001) and further enriched in OC spheroids compared with monolayers (P<0.01). ILK blockade with the specific inhibitor cpd-22 suppressed spheroid proliferation and tumor initiating capacity in xenograft mouse model. Furthermore, key oncogenic signaling pathways, in particular decreased β-catenin signaling essential to sustaining cancer cell stemness, were altered in OC spheroids treated with cpd-22. Of the genes examined, expression of Wnt receptor Frizzled 7 (Fzd7) was the mostly highly downregulated in OC spheroids treated with cpd-22, indicating a direct correlation between ILK activation and Wnt signaling. By using co-immunoprecipitation and proximity ligation assay, we demonstrated that ILK co-localizes with Fzd7 in OC spheroids. Mechanistically, treatment of OC spheroids with the combination of carboplatin and ILK-Fzd7 blockade decreased β-catenin signaling, inhibited phospho-Akt at Ser473 and increased levels of cleaved-caspase-3 compared to single agent alone, resulting in apoptosis. By regulating β-catenin signaling, Fzd7 expression and ILK activation are essential for OCSCs survival. Targeting Fzd7-ILK clusters may represent a new therapeutic approach to eradicate OCSC and ultimately improve patient outcomes. Citation Format: Rula Atwani, Virginie Lazar, George Earl Sandusky, Salvatore Condello. Integrin-linked kinase regulates Wnt transcriptional activity in platinum resistant ovarian cancer stem cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5808.

Proteomic analysis reveals CAAP1 negatively correlates with platinum resistance in ovarian cancer

The present study sought to investigate the correlation between CAAP1 and platinum resistance in ovarian cancer and to preliminarily explore the potential biological function of CAAP1. Proteomic analysis was used to analyze differentially expressed proteins in platinum-sensitive and -resistant tissue samples of ovarian cancer. The Kaplan-Meier plotter was used for prognostic analysis. Immunohistochemistry assay and chi-square test were employed to explore the relationship between CAAP1 and platinum resistance in tissue samples. Lentivirus transfection, immunoprecipitation-mass spectrometry, and bioinformatics analysis were used to determine the potential biological function of CAAP1. Based on results, the expression level of CAAP1 was significantly higher in platinum-sensitive tissues compared to that in resistant tissues. Chi-square test demonstrated that there is a negative correlation between high expression of CAAP1 and platinum resistance. Overexpression of CAAP1 increased cis‑platinum sensitivity of the A2780/DDP cell line likely via the mRNA splicing pathway by interacting with the splicing factor AKAP17A. In summary, there is a negative correlation between high expression of CAAP1 and platinum resistance. CAAP1 might be a potential biomarker for platinum resistance in ovarian cancer. SignificancePlatinum resistance is a key factor affecting the survival of ovarian cancer patients. Understanding the mechanisms of platinum resistance is highly important for ovarian cancer management. Here, we performed the DIA- and DDA-based proteomics to analyze differentially expressed proteins in tissue and cell samples of ovarian cancer. We found that the protein identified as CAAP1, which was first reported to be involved in the regulation of apoptosis, may be negatively correlates with platinum resistance in ovarian cancer. In addition, we also found that CAAP1 enhanced the sensitivity of platinum-resistant cells to cis‑platinum via the mRNA splicing pathway by interacting with the splicing factor AKAP17A. Our data would be useful to reveal novel molecular mechanisms of platinum resistance in ovarian cancer.

Half sandwich-type osmium, ruthenium, iridium and rhodium complexes with bidentate glycosyl heterocyclic ligands induce cytostasis in platinum-resistant ovarian cancer cells and bacteriostasis in Gram-positive multiresistant bacteria.

The toxicity of and resistance to platinum complexes as cisplatin, oxaliplatin or carboplatin calls for the replacement of these therapeutic agents in clinical settings. We have previously identified a set of half sandwich-type osmium, ruthenium and iridium complexes with bidentate glycosyl heterocyclic ligands exerting specific cytostatic activity on cancer cells but not on non-transformed primary cells. The apolar nature of the complexes, conferred by large, apolar benzoyl protective groups on the hydroxyl groups of the carbohydrate moiety, was the main molecular feature to induce cytostasis. We exchanged the benzoyl protective groups to straight chain alkanoyl groups with varying length (3 to 7 carbon units) that increased the IC50 value as compared to the benzoyl-protected complexes and rendered the complexes toxic. These results suggest a need for aromatic groups in the molecule. The pyridine moiety of the bidentate ligand was exchanged for a quinoline group to enlarge the apolar surface of the molecule. This modification decreased the IC50 value of the complexes. The complexes containing [(η6-p-cymene)Ru(II)], [(η6-p-cymene)Os(II)] or [(η5-Cp*)Ir(III)] were biologically active unlike the complex containing [(η5-Cp*)Rh(III)]. The complexes with cytostatic activity were active on ovarian cancer (A2780, ID8), pancreatic adenocarcinoma (Capan2), sarcoma (Saos) and lymphoma cell lines (L428), but not on primary dermal fibroblasts and their activity was dependent on reactive oxygen species production. Importantly, these complexes were cytostatic on cisplatin-resistant A2780 ovarian cancer cells with similar IC50 values as on cisplatin-sensitive A2780 cells. In addition, the quinoline-containing Ru and Os complexes and the short chain alkanoyl-modified complexes (C3 and C4) proved to be bacteriostatic in multiresistant Gram-positive Enterococcus and Staphylococcus aureus isolates. Hereby, we identified a set of complexes with submicromolar to low micromolar inhibitory constants against a wide range of cancer cells, including platinum resistant cells and against multiresistant Gram-positive bacteria.

Platinum-Resistant Ovarian Cancer Is Vulnerable to the cJUN-XRCC4 Pathway Inhibition.

DNA double-strand breaks (DSBs) caused by platinum drugs are dangerous lesions that kill cancer cells in chemotherapy. Repair of DSB by homologous recombination (HR) and nonhomologous end joining (NHEJ) is frequently associated with platinum resistance in ovarian cancer. While the role of the HR pathway and HR-targeting strategy in platinum resistance is well studied, dissecting and targeting NHEJ machinery to overcome platinum resistance in ovarian cancer remain largely unexplored. Here, through an NHEJ pathway-focused gene RNAi screen, we found that the knockdown of XRCC4 significantly sensitized cisplatin treatment in the platinum-resistant ovarian cancer cell lines. Moreover, upregulation of XRCC4 is observed in a panel of platinum-resistant cell lines relative to the parental cell lines, as well as in ovarian cancer patients with poor progression-free survival. Mechanistically, the increased sensitivity to cisplatin upon XRCC4 knockdown was caused by accumulated DNA damage. In cisplatin-resistant ovarian cancer, the JNK-cJUN complex, activated by cisplatin, translocated into the nucleus and promoted the transcription of XRCC4 to confer cisplatin resistance. Knockdown of XRCC4 or treatment of the JNK inhibitor led to the attenuation of cisplatin-resistant tumor growth in the xenograft mouse models. These data suggest targeting XRCC4 is a potential strategy for ovarian cisplatin resistance in ovarian cancer.

Platinum-resistance in epithelial ovarian cancer: an interplay of epithelial–mesenchymal transition interlinked with reprogrammed metabolism

BackgroundEpithelial ovarian cancer is the most lethal gynaecological cancer worldwide. Chemotherapy resistance represents a significant clinical challenge and is the main reason for poor ovarian cancer prognosis. We identified novel expression of markers related to epithelial mesenchymal transitions (EMT) in a carboplatin resistant ovarian cancer cell line by proteomics. This was validated in the platinum resistant versus sensitive parental cell lines, as well as platinum resistant versus sensitive human ovarian cancer patient samples. The prognostic significance of the different proteomics-identified marker proteins in prognosis prediction on survival as well as their correlative association and influence on immune cell infiltration was determined by public domain data bases.MethodsWe explored the proteomic differences between carboplatin-sensitive OVCAR5 cells (parental) and their carboplatin-resistant counterpart, OVCAR5 CBPR cells. qPCR and western blots were performed to validate differentially expressed proteins at the mRNA and protein levels, respectively. Association of the identified proteins with epithelial–mesenchymal transition (EMT) prompted the investigation of cell motility. Cellular bioenergetics and proliferation were studied to delineate any biological adaptations that facilitate cancer progression. Expression of differentially expressed proteins was assessed in ovarian tumors obtained from platinum-sensitive (n = 15) versus platinum-resistant patients (n = 10), as well as matching tumors from patients at initial diagnosis and following relapse (n = 4). Kaplan–Meier plotter and Tumor Immune Estimation Resource (TIMER) databases were used to determine the prognostic significance and influence of the different proteomics-identified proteins on immune cell infiltration in the tumor microenvironment (TME).ResultsOur proteomics study identified 2422 proteins in both cell lines. Of these, 18 proteins were upregulated and 14 were downregulated by ≥ twofold (p < 0.05) in OVCAR5 CBPR cells. Gene ontology enrichment analysis amongst upregulated proteins revealed an overrepresentation of biological processes consistent with EMT in the resistant cell line. Enhanced mRNA and/or protein expression of the identified EMT modulators including ITGA2, TGFBI, AKR1B1, ITGAV, ITGA1, GFPT2, FLNA and G6PD were confirmed in OVCAR5 CBPR cells compared to parental OVCAR5 cell line. Consistent with the altered EMT profile, the OVCAR5 CBPR cells demonstrated enhanced migration and reduced proliferation, glycolysis, and oxidative phosphorylation. The upregulation of G6PD, AKR1B1, ITGAV, and TGFβ1 in OVCAR5 CBPR cells was also identified in the tumors of platinum-resistant compared to platinum-sensitive high grade serous ovarian cancer (HGSOC) patients. Matching tumors of relapsed versus newly diagnosed HGSOC patients also showed enhanced expression of AKR1B1, ITGAV, TGFβ1 and G6PD protein in relapsed tumors. Among the identified proteins, significant enhanced expression of GFPT2, FLNA, TGFBI (CDGG1), ITGA2 predicted unfavorable prognosis in ovarian cancer patients. Further analysis suggested that the expression of TGFBI to correlate positively with the expression of identified and validated proteins such as GFPT2, FLNA, G6PD, ITGAV, ITGA1 and ITGA2; and with the infiltration of CD8+ T cells, macrophages, neutrophils, and dendritic cells in the TME.ConclusionsOur research demonstrates proteomic-based discovery of novel EMT-related markers with an altered metabolic profile in platinum-resistant versus sensitive ovarian cancer cell lines. The study also confirms the expression of selected identified markers in the tumors of platinum-resistant versus sensitive, and in matching relapsed versus newly diagnosed HGSOC patients. The study provides insights into the metabolic adaptation of EMT-induced carboplatin resistant cells that confers on them reduced proliferation to provide effective migratory advantage; and the role of some of these identified proteins in ovarian cancer prognosis. These observations warrant further investigation of these novel target proteins in platinum-resistant patients.

Abstract IA003: SC144 and the next generation IL6/GP130/STAT3 inhibitors for cancer prevention

Abstract IL6/GP130/STAT3 signaling plays a crucial role in multiple diseases, and all components of this signaling cascade are directly or indirectly implicated in tumorigenesis. Therefore, blockade of this signaling axis is expected to provide novel therapeutic opportunities to treat various diseases including cancer. IL6 binds to IL6R and GP130 to activate downstream signaling pathways to promote proliferation, survival and metastasis of cancer cells, and to stimulate angiogenesis on endothelial cells. Although GP130 is positioned at the junction of this oncogenic signaling network and is essential for activation of the network, there are no small-molecule inhibitors of GP130 under clinical development. Previously, we identified SC144 as a first-in-class, efficacious, safe, and orally active inhibitor of GP130. SC144 selectively inhibits the activation of downstream signaling pathways induced by GP130 ligands (IL6, LIF), with no significant effects on the activation by non-GP130 ligands, such as IFN-γ, SDF-1α, and PDGF. SC144 exhibits cytotoxicity in a panel of platinum-sensitive and platinum-resistant cells, with no significant cytotoxicity to human normal epithelial cells. In mouse xenograft models, SC144 significantly inhibited tumor growth through GP130 inhibition and induction of necrosis in the tumor. No toxicity was evident in normal tissues. Furthermore, SC144 showed significant in vivo efficacy in immune competent syngeneic mouse models. Unfortunately, clinical development of SC144 was delayed due to its poor solubility and metabolic instability. During the past two years, we have identified metabolic liabilities of SC144 and have undertaken an extensive medicinal chemistry lead optimization campaign to produce analogs with increased solubility and metabolic stability. The new analogs show favorable properties in single- and repeat-dosing pharmacokinetic (PK) studies. As a result, we have in hand a series of 2nd-generation analogs that are orally active, water-soluble, and display desirable PK properties suitable for advanced preclinical studies to support IND filing. Citation Format: Nouri Neamati. SC144 and the next generation IL6/GP130/STAT3 inhibitors for cancer prevention [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA003.

Dinaciclib as an effective pan-cyclin dependent kinase inhibitor in platinum resistant ovarian cancer.

Ovarian cancer (OC) is amongst the most lethal of common cancers in women. Lacking in specific symptoms in the early stages, OC is predominantly diagnosed late when the disease has undergone metastatic spread and chemotherapy is relied on to prolong life. Platinum-based therapies are preferred and although many tumors respond initially, the emergence of platinum-resistance occurs in the majority of cases after which prognosis is very poor. Upregulation of DNA damage pathways is a common feature of platinum resistance in OC with cyclin dependent kinases (CDKs) serving as key regulators of this process and suggesting that CDK inhibitors (CDKis) could be effective tools in the treatment of platinum resistant and refractory OC. The aim of this study was to evaluate the efficacy of CDKis in platinum resistant OC models and serve as a predictor of potential clinical utility. The efficacy of CDKi, dinaciclib, was determined in wildtype and platinum resistant cell line pairs representing different OC subtypes. In addition, dinaciclib was evaluated in primary cells isolated from platinum-sensitive and platinum-refractory tumors to increase the clinical relevance of the study. Dinaciclib proved highly efficacious in OC cell lines and primary cells, which were over a thousand-fold more sensitive to the CDKi than to cisplatin. Furthermore, cisplatin resistance in these cells did not influence sensitivity to dinaciclib and the two drugs combined additively in both platinum-sensitive and platinum-resistant OC cells suggesting a potential role for pan-CDKis (CDKis targeting multiple CDKs), such as dinaciclib, in the treatment of advanced and platinum-resistant OC.