Chemoresistant Cells Research Articles

MiR-185-5p May Modulate the Chemosensitivity of LUSC to Cisplatin via Targeting PCDHA11: Multi-omics Analysis and Experimental Validation.

Drug resistance is the major difficulty in treatment of lung squamous cell carcinoma (LUSC). This study aims to explore drug response-related miRNAs (DRmiRNAs) based on multi-omics research. We identified DRmiRNAs of LUSC with a multi-omics integrated system that combines expression data of microRNA, lncRNA, mRNA, methylation levels, somatic mutations. After identifying DRmiRNAs, we screened and validated of the target mRNAs of DRmiRNAs through Targetscan and the miRDB database. Then, Real-time PCR and Western blot assays were used to estimate the expression of DRmiRNAs and target protein, and the dual-luciferase assays were used to confirm the interaction of DRmiRNAs and target mRNA. Furthermore, CCK-8 (Cell Counting Kit-8) assays were used to evaluate cell proliferation and drug sensitivity. After integrated analysis, hsa-miR-185-5p was identified as DRmiRNA based on multi-omics data. Through Targetscan and miRDB database, the possible target mRNAs were obtained and PCDHA11 was validated as a target mRNA of miR-185-5p by real-time PCR, Western blot assays and dual-luciferase assays. CCK-8 assays and clone formation assays showed that the proliferation of miR-185-5p mimics was significantly slower than that of miR-185-5p inhibitors, which means overexpression of miR-185-5p enhanced the anticancer effects of cisplatin, whereas the downregulation of miR-185-5p reduced the effects. Furthermore, the proliferation of silencing PCDHA11 was significantly slower than that of overexpression of PCDHA11, which means PCDHA11 overexpression weakened the anticancer effects of cisplatin, and silencing PCDHA11 enhanced the effects. This study demonstrated that miR-185-5p was involved in chemoresistance of LUSC cells to cisplatin partly via down-regulating PCDHA11, which may promote understanding the underlying molecular mechanisms of drug response.

A novel approach toward the multigram synthesis of a novel Bcl2-specific inhibitor, and evaluation of its biological activity

The antiapoptotic protein BCL2 is overexpressed in several cancers. It contributes to prolonged cell survival and chemoresistance, making it an excellent target for targeted cancer therapy. Over the years, several BCL2 inhibitors have been investigated extensively for their anticancer potential; however, most of them were abolished before clinical use due to their pan activity. There is only one FDA-approved BCL2-specific inhibitor, Venetoclax, currently used in clinics. Previously, we reported the characterization and development of a novel BCL2 inhibitor, Disarib, which is selective against BCL2 and predominantly binds to the BH1 domain of BCL2. Importantly, the efficacy of Disarib was equally good or better than Venetoclax in various in vitro and in vivo assays. In the present study, we report the development of a scalable and practical method for the large-scale synthesis of Disarib to support the ongoing pre-clinical studies. The parameters of each step were optimized through prior knowledge, helping in improving the yield and purity to 98.8 %. Further, we compared the efficiency of a large-scale synthesized Disarib in a GLP-certified laboratory with that of an in-house synthesized Disarib. The results showed that the biological activity, including Disarib-induced cytotoxicity and cell cycle progression, were comparable. Besides, the tumor regression efficacy and pharmacokinetics analysis of Disarib have shown comparable results when Disarib synthesized in both routes were tested. Thus, a robust and scalable synthetic pathway of Disarib has developed to address small-scale synthesis's limitations in supporting their expanded applications and use.

MSLN induced EMT, cancer stem cell traits and chemotherapy resistance of pancreatic cancer cells

Chemoresistance is one of the main reasons for poor prognosis of pancreatic cancer. The effects of mesothelin (MSLN) on chemoresistance in pancreatic cancer are still unclear. We aim to investigate potential roles of MSLN in chemoresistance and its relationship with proliferation, epithelial-mesenchymal transition (EMT) and cancer stemness of pancreatic cancer cells. Human pancreatic cancer cell lines ASPC-1 and Mia PaCa-2 with high and low expression of MSLN, respectively, were selected. The ASPC-1 with MSLN knockout (KO) and Mia PaCa-2 of MSLN overexpression (OE) were generated. The effects of MSLN on cell phenotypes, expression of EMT-related markers, clone formation, tumor sphere formation, and pathologic role of MSLN in tumorigenesis were detected. Sensitivity of tumor cells to gemcitabine was evaluated. The results showed that adhesion, proliferation, migration and invasion were decreased significantly in ASPC-1 with MSLN KO, whereas increased significantly in Mia PaCa-2 with MSLN OE. The size and the number of clones and tumor spheres were decreased in ASPC-1 with MSLN KO, and increased in Mia PaCa-2 with MSLN OE. In xenograft model, tumor volume was decreased (tumor grew slower) in MSLN KO group compared to control group, while increased in MSLN OE group. Mia PaCa-2 with MSLN OE had a higher IC50 of gemcitabine, while ASPC-1 with MSLN KO had a lower IC50. We concluded that MSLN could induce chemoresistance by enhancing migration, invasion, EMT and cancer stem cell traits of pancreatic cancer cells. Targeting MSLN could represent a promising therapeutic strategy for reversing EMT and chemoresistance in pancreatic cancer cells.

Targeting eIF5A2 reduces invasion and reverses chemoresistance in SCC-9 cells in vitro.

Eukaryotic translation initiation factor 5A2 (EIF5A2) has been reported to be involved in metastasis and chemotherapy resistance in many human cancers. However, the effect and mechanism of EIF5A2 in oral cancer cells are unknown. Here, we investigated the effects of targeting EIF5A2 on chemotherapy resistance in oral cancer cells in vitro. By using a lentiviral system, we investigated the effects of targeting EIF5A2 on the invasion, migration, growth, and chemosensitivity of SCC-9 cells to CDDP in vitro. Through the method of gene intervention, we explore the role of pro-apoptotic Bim and epithelial and mesenchymal marker E-cadherin protein in this process and the regulation of EIF5A2 on Bim and E-cadherin. Targeting EIF5A2 reduces invasion and migration in SCC-9 cells partly through upregulation of E-cadherin expression; Targeting EIF5A2 promotes cell apoptosis and inhibits cell survival as well as increasing chemosensitivity in SCC-9 cells through upregulation of Bim expression. EIF5A2 may be a novel potential therapeutic target for oral cancer by upregulation of Bim and E-cadherin.

Novel combinatorial autophagy inhibition therapy for triple negative breast cancers

BackgroundTriple negative breast cancer (TNBC) has the worst prognosis among breast cancer subtypes. It is characterized by lack of estrogen, progesterone and human epidermal growth factor 2 receptors, and thus, have limited therapeutic options. Autophagy has been found to be correlated with poor prognosis and aggressive behaviour in TNBC. This study aimed to target autophagy in TNBC via a novel approach to inhibit TNBC progression. MethodsImmunoblotting and confocal microscopy were carried out to examine the effect of tumor microenvironmental stressors on autophagy. Cellular proliferation and migration assays were used to test the effect of different autophagy inhibitors and standard chemotherapy alone or in combination. In vivo xenograft mouse model was utilized to assess the effect of autophagy inhibitors alone or in combination with Paclitaxel. High resolution mass spectrometry based proteomic analysis was performed to explore the mechanisms behind chemoresistance in TNBC. Lastly, immunohistochemistry was done to assess the correlation between autophagic related proteins and clinical characteristics in TNBC tissue specimens. ResultsMetabolic stressors were found to induce autophagy in TNBC cell lines. Autophagy initiation inhibitors, SAR405 and MRT68921, showed marked synergy in their anti-proliferative activity in both chemosensitive and chemoresistant TNBC cell models. Paradoxically, positive expression of autophagosome marker LC3 was shown to be associated with better overall survival of TNBC patients. ConclusionIn this study, a novel combination between different autophagy inhibitors was identified which inhibited tumor cell proliferation in both chemosensitive and chemoresistant TNBC cells and could result in development of novel treatment modality against TNBC.

Natural compounds targeting YAP/TAZ axis in cancer: Current state of art and challenges

Cancer has become a burgeoning global healthcare concern marked by its exponential growth and significant economic ramifications. Though advancements in the treatment modalities have increased the overall survival and quality of life, there are no definite treatments for the advanced stages of this malady. Hence, understanding the diseases etiologies and the underlying molecular complexities, will usher in the development of innovative therapeutics. Recently, YAP/TAZ transcriptional regulation has been of immense interest due to their role in development, tissue homeostasis and oncogenic transformations. YAP/TAZ axis functions as coactivators within the Hippo signaling cascade, exerting pivotal influence on processes such as proliferation, regeneration, development, and tissue renewal. In cancer, YAP is overexpressed in multiple tumor types and is associated with cancer stem cell attributes, chemoresistance, and metastasis. Activation of YAP/TAZ mirrors the cellular "social" behavior, encompassing factors such as cell adhesion and the mechanical signals transmitted to the cell from tissue structure and the surrounding extracellular matrix. Therefore, it presents a significant vulnerability in the clogs of tumors that could provide a wide window of therapeutic effectiveness. Natural compounds have been utilized extensively as successful interventions in the management of diverse chronic illnesses, including cancer. Owing to their capacity to influence multiple genes and pathways, natural compounds exhibit significant potential either as adjuvant therapy or in combination with conventional treatment options. In this review, we delineate the signaling nexus of YAP/TAZ axis, and present natural compounds as an alternate strategy to target cancer.

Role of the Atypical MAPK ERK3 in Cancer Growth and Progression.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) whose structural and regulatory features are distinct from those of conventional MAPKs, such as ERK1/2. Since its identification in 1991, the regulation, substrates and functions of ERK3 have remained largely unknown. However, recent years have witnessed a wealth of new findings about ERK3 signaling. Several important biological functions for ERK3 have been revealed, including its role in neuronal morphogenesis, inflammation, metabolism, endothelial cell tube formation and epithelial architecture. In addition, ERK3 has been recently shown to play important roles in cancer cell proliferation, migration, invasion and chemoresistance in multiple types of cancers. Furthermore, accumulating studies have uncovered various molecular mechanisms by which the expression level, protein stability and activity of ERK3 are regulated. In particular, several post-translational modifications (PTMs), including ubiquitination, hydroxylation and phosphorylation, have been shown to regulate the stability and activity of ERK3 protein. In this review, we discuss recent findings regarding biochemical and cellular functions of ERK3, with a main focus on its roles in cancers, as well as the molecular mechanisms of regulating its expression and activity.

Interplay between paclitaxel, gap junctions, and kinases: unraveling mechanisms of action and resistance in cancer therapy.

This comprehensive review elucidates the multifaceted roles of paclitaxel, a key chemotherapeutic agent, in cancer therapy, with a focus on its interactions with gap junctions and related kinases. Paclitaxel, with its complex diterpene structure, mediates its anticancer effects predominantly through specific interactions with β-tubulin, instigating cell cycle arrest and triggering various cell death pathways, including apoptosis, pyroptosis, ferroptosis, and necroptosis. The paper systematically delineates the chemical attributes and action mechanisms of paclitaxel and its analogs, underscoring their capacity to disrupt microtubule dynamics, thereby leading to mitotic arrest and subsequent cell death induction. It also scrutinizes the pivotal role of gap junctions, composed of connexin proteins, in the modulation of cancer cell behavior and chemoresistance, especially in the milieu of paclitaxel administration. The review articulates how gap junctions can either suppress tumors or contribute to cancer progression, thereby influencing chemotherapy outcomes. Furthermore, the paper provides an in-depth analysis of how paclitaxel modulates gap junction-associated kinases via phosphorylation, influencing the drug's therapeutic efficacy and resistance profiles. By integrating insights from numerous key studies, the review offers a comprehensive understanding of the interplay between paclitaxel, gap junctions, and kinases, shedding light on potential approaches to augment paclitaxel's anti-tumor effectiveness and counteract chemoresistance in cancer treatment.

ELAVL2 loss promotes aggressive mesenchymal transition in glioblastoma

Glioblastoma (GBM), the most lethal primary brain cancer, exhibits intratumoral heterogeneity and molecular plasticity, posing challenges for effective treatment. Despite this, the regulatory mechanisms underlying such plasticity, particularly mesenchymal (MES) transition, remain poorly understood. In this study, we elucidate the role of the RNA-binding protein ELAVL2 in regulating aggressive MES transformation in GBM. We found that ELAVL2 is most frequently deleted in GBM compared to other cancers and associated with distinct clinical and molecular features. Transcriptomic analysis revealed that ELAVL2-mediated alterations correspond to specific GBM subtype signatures. Notably, ELAVL2 expression negatively correlated with epithelial-to-mesenchymal transition (EMT)-related genes, and its loss promoted MES process and chemo-resistance in GBM cells, whereas ELAVL2 overexpression exerted the opposite effect. Further investigation via tissue microarray analysis demonstrated that high ELAVL2 protein expression confers a favorable survival outcome in GBM patients. Mechanistically, ELAVL2 was shown to directly bind to the transcripts of EMT-inhibitory molecules, SH3GL3 and DNM3, modulating their mRNA stability, potentially through an m6A-dependent mechanism. In summary, our findings identify ELAVL2 as a critical tumor suppressor and mRNA stabilizer that regulates MES transition in GBM, underscoring its role in transcriptomic plasticity and glioma progression.

Low-Density Lipoproteins Increase Proliferation, Invasion, and Chemoresistance via an Exosome Autocrine Mechanism in MDA-MB-231 Chemoresistant Cells

Dyslipidemias involving high concentrations of low-density lipoproteins (LDLs) increase the risk of developing triple-negative breast cancer (TNBC), wherein cholesterol metabolism and protein translation initiation mechanisms have been linked with chemoresistance. Doxorubicin (Dox) treatment, a member of the anthracycline family, represents a typical therapeutic strategy; however, chemoresistance remains a significant challenge. Exosomes (Exs) secreted by tumoral cells have been implicated in cell communication pathways and chemoresistance mechanisms; the content of exosomes is an outcome of cellular cholesterol metabolism. We previously induced Dox resistance in TNBC cell models, characterizing a variant denominated as variant B cells. Our results suggest that LDL internalization in parental and chemoresistant variant B cells is associated with increased cell proliferation, migration, invasion, and spheroid growth. We identified the role of eIF4F translation initiation factor and the down-regulation of tumor suppressor gene PDCD4, an inhibitor of eIF4A, in chemoresistant variant B cells. In addition, the exomes secreted by variant B cells were characterized by the protein content, electronic microscopy, and cell internalization assays. Critically, exosomes purified from LDL-treated variant B cell promoted cell proliferation, migration, and an increment in lactate concentration. Our results suggest that an autocrine phenomenon induced by exosomes in chemoresistant cells may induce modifications on signaling mechanisms of the p53/Mdm2 axis and activation of p70 ribosomal protein kinase S6. Moreover, the specific down-regulated profile of chaperones Hsp90 and Hsp70 secretion inside the exosomes of the chemoresistant variant could be associated with this phenomenon. Therefore, autocrine activation mediated by exosomes and the effect of LDL internalization may influence changes in exosome chaperone content and modulate proliferative signaling pathways, increasing the aggressiveness of MDA-MB-231 chemoresistant cells.

Fusobacterium nucleatum induces chemoresistance in colorectal cancer by inhibiting pyroptosis via the Hippo pathway

ABSTRACT Chemotherapy resistance is one of the main reasons for the poor prognosis of colorectal cancer (CRC). Moreover, dysbiosis of gut bacteria was found to be a specific environmental risk factor. In this study, enrichment of F. nucleatum was elucidated to be significantly associated with CRC recurrence after chemotherapy. Functional experiments showed that F. nucleatum could inhibit pyroptosis induced by chemotherapy drugs, thereby inducing chemoresistance. Furthermore, mechanistic investigation demonstrated that F. nucleatum could regulate the Hippo pathway and promote the expression of BCL2, thereby inhibiting the Caspase-3/GSDME pyroptosis-related pathway induced by chemotherapy drugs and mediating CRC cell chemoresistance. Taken together, these results validated the significant roles of F. nucleatum in CRC chemoresistance, which provided an innovative theoretical basis for the clinical diagnosis and therapy of CRC.

Abstract 4639: Unraveling the oncogenic network of USP37 and USP14 in gynecological cancers: Decrypting the molecular landscape of chemoresistance

Abstract Background: Gynecological cancers (GC) involve female reproductive system malignancies, and acquired chemoresistance is a challenge in the treatment of GC. Protein deubiquitination, mediated by deubiquitinating enzymes (DUBs) like ubiquitin-specific proteases (USPs), holds therapeutic promise in cancer treatment. This study delves into the mechanistic understanding of DUBs, particularly in acquired chemoresistance in GC. Methodology: We initially examined TCGA database to assess the impact of Ubiquitin Specific Proteases (USPs), specifically USP37 and USP14, on prognosis in Gynaecological Cancers (GCs). Subsequently, we evaluated their expression in various GC cell lines at both protein and RNA levels. Cell proliferation and metastasis assays were performed to analyze the effect of depleting these USPs on survival and metastasis in GC cells under replication stress. Proteomics analysis was conducted on cells with altered expression of USP37 and USP14 to identify interacting proteins regulating proliferation and metastasis. To investigate DUBs role in chemoresistance, we generated chemoresistant (CR) cell lines with cisplatin exposure, characterized their viability and proliferation, conducted cell cycle and apoptosis analyses, and performed proteomic profiling to explore the USP37 and USP14 specific interactome in chemoresistant cell lines. Results: Examination of TCGA database revealed an association between elevated USP37 and USP14 expression and poor Overall Survival (OS) and Disease-free Survival (DFS) in Gynaecological Cancers (GC) cohort. Increased expression of USP37 and USP14 at RNA and protein levels was observed in GC cell lines. Inhibiting USP37 and USP14 reduced GC cell proliferation under replication stress and hindered metastasis. Proteomics analysis identified proteins linked to replication (PCNA), DNA damage (Chk-1), cell cycle, and metastasis. Cisplatin-resistant GC cell lines were successfully established, demonstrating prolonged survival and increased proliferative capacity after cisplatin treatment compared to chemosensitive (CS) cell lines. FACS analysis indicated altered cell cycle distribution and increased proliferation in CR cells. Comparative interactome analysis revealed altered protein sets associated with USP37 and USP14 in CR and CS GC cells. Conclusion: This study provides evidence that supports the participation of USP37 and USP14 in the onset of gynecological cancers (GC). We present evidence of alterations in the oncogenic network linked to USP37 and USP14 in GC. Additionally, we elucidate the roles of these deubiquitinating enzymes (DUBs) in chemoresistant cell lines. Citation Format: Ravi Chauhan, Ashna Gupta, Gunjan Dagar, Lakshay Malhotra, Ekta Rai, Muzafar A Macha, Ajaz A Bhat, Ethayathulla A Samath, Mayank Singh. Unraveling the oncogenic network of USP37 and USP14 in gynecological cancers: Decrypting the molecular landscape of chemoresistance [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 4639.

Abstract 543: Identifying drivers of chemoresistance in SQLC using forward and reverse genetic approaches

Abstract Non-small cell lung cancer, mainly lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (SQLC), accounts for about 85 % of all lung cancers. While LUAD treatment options have greatly improved, SQLC remains challenging, as targetable mutations are rare. Immunotherapy provides benefits, nevertheless, chemotherapy remains a backbone of systemic treatment strategies for SQLC patients. Accordingly, a major challenge for SQLC treatment is the development of chemoresistant tumors, oftentimes leading to cross-resistance to multiple drugs and limiting patient survival. Currently, the causal pathways enabling tumor cells to survive therapeutic stress and drivers of chemotherapy resistance are insufficiently known, which impedes the development of novel therapeutic strategies. We therefore leverage forward and reverse genetic approaches including large-scale chemo-genetic perturbations to systematically map the landscape of molecular drivers involved in chemotherapy resistance in SQLC. By integrating the resulting data we derive specific knowledge of the underlying processes, in order to identify druggable targets in chemoresistant tumors and contribute to improved patient care. To assess genes causally mediating chemotherapy resistance upon overexpression, we performed genome-wide CRISPR activation screens utilizing clinically used cisplatin and taxol. Additionally, we performed CRISPRa screens against four further drugs currently in clinical trials in order to distinguish genes causing cross-resistance to multiple drugs versus drug-specific mechanisms. In parallel, we generated chemoresistant clones by prolonged low-dose treatment, to analyze transcriptomic adaptions to chemoresistance and validate the CRISPRa screening hits. Initial findings show a large selection of unique and shared gene sets that were significantly enriched or reduced after treatment in the CRISPRa screen, indicating their involvement in the development of chemoresistance. Of note, transcriptomic analysis of chemoresistant cells shows an overlap with the CRISPRa screen specifically for the unfolded protein response (UPR) that is transcriptionally enriched in chemoresistant cells and a positive hit in the CRISPRa screen. Chemoresistant clones show increased resistance to tunicamycine induced ER-stress, indicating an increased ability to overcome the accumulation of misfolded protein under cytotoxic treatment through the UPR. Specific inhibition of the UPR during cytotoxic treatment shows promising results in chemoresistant cells. By combining our CRISPR activation screen and systematic analysis of chemoresistant cells we were able to identify the UPR as a driver and a druggable synthetically lethal dependency of chemoresistance in SQLC. This opens the possibility of studying the UPR as a potential target in the treatment of patients with chemoresistant tumors, improving patient outcome. Citation Format: Mareike Haarmann, Dennis Said Gadalla, Johannes Brägelmann. Identifying drivers of chemoresistance in SQLC using forward and reverse genetic approaches [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 543.

Abstract 2019: Primary cancer associated fibroblasts increase the chemoresistance of triple negative breast cancer cells by mitochondria metabolic upregulation

Abstract Breast cancers (BCs) are the most prevalent cancers occurring in women. Tumor reoccurrence attributed to chemotherapy resistance remains a main challenge in fighting disease relapse. Cancer associated fibroblasts (CAFs) are the most prominent component of the tumor microenvironment (TME) and have already been implicated in therapy resistances. Although the exact molecular mechanisms behind this resistance remain unclear. In this regard, we recently identified 4 CAF populations (S1-S4) in ovarian and BCs, and showed that only CAF-S1 promote immunotherapy resistance as well as metastasis. Based on these observations, we are now investigating the impact of CAF-S1 on the chemotherapy response of BCs. To study the interactions between CAF-S1 and BC cells, we developed a tumor on chip (ToC) device, mimicking the TME in 3D. Using ToC live imaging, we show that primary CAF-S1 mediate a chemoprotective effect over luminal and triple negative BC (TNBC) cells by significantly decreasing their apoptosis rate as early as 10h after Doxorubicin and 40h after Paclitaxel treatment. Interestingly, CAF-S1 display a highly heterogenous response to Doxorubicin, allowing the classification into chemo-sensitive and -resistant CAF-S1. Regardless of premature CAF-S1 cell death, the chemoprotective effect over BC cells is maintained for at least 3 days. This indicates a crucial CAF-S1 - cancer cell cross talk at early timepoints. Additionally, by applying machine learning algorithms on live imaging videos, we demonstrate that physical proximity of CAF-S1 to BC cells immediately after treatment initiation correlates with BC cell survival, even after several days of treatment. This again implies cancer cell priming by CAF-S1 as a possible mechanism of chemotherapy resistance. To investigate this molecular interplay further, we performed scRNA-seq of ToC extracted and treatment naïve CAF-S1 and TNBC cells as well as bulk RNA-seq of patient derived TNBC tumors before/after chemotherapy treatment. We identify mitochondrial metabolic pathways to be upregulated in TNBC cells after CAF-S1 co-culture inside ToC devices and in TNBC cells of chemo-resistant patients enriched in CAF-S1 at baseline. We are now validating the impact of target gene knock downs on the survival of TNBC cells in CAF-S1 co-cultures after chemotherapy treatment on chip. Overall, these results provide insights into molecular mechanisms by which CAF-S1 promote chemotherapy resistance of breast cancers. Citation Format: Isabella Hofer, Arianna Mencattini, Yann Kieffer, Eugenio Martinelli, Maria Carla Parrini, Fatima Mechta-Grigoriou. Primary cancer associated fibroblasts increase the chemoresistance of triple negative breast cancer cells by mitochondria metabolic upregulation [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 2019.

Abstract 5196: Stepwise molecular mechanisms involved in chemoresistance in bladder cancer cells

Abstract Bladder cancer (BC) is one of the most prevalent genitourinary tumors worldwide, with approximately 573,000 new cases reported. Approximately 80% of BC patients are diagnosed with non-muscle-invasive bladder cancer (NMIBC) with a high five-year survival rate, while the remaining 20% are diagnosed with muscle-invasive bladder cancer (MIBC). Although the surgical operation has been utilized to BC, patients with NMIBC frequently experience disease recurrence, of which about 20% progress to MIBC. However, the response rate of MIBC patients to gemcitabine treatment has been shown to be limited to less than 40% efficacy, and only a small number of patients have potential benefits.Chemotherapy resistance is an obstacle to cancer therapy and is considered a major cause of recurrence. Thus, understanding the mechanisms of chemoresistance is critical to improving the prognosis of patients. Here, we have established a stepwise gemcitabine-resistant T24 bladder cancer cell line to understand the molecular mechanisms of chemoresistance within cancer cells. The characteristics of the stepwise chemoresistance cell line were divided into 4 phases (parental, early, intermediate, and late phases). These four phase cells showed increasingly aggressive phenotypes in vitro and in vivo experiments with increasing phases and revealed the molecular properties of the biological process from parent cells to phased gemcitabine-resistant cell line (GRC). Taken together, through the analysis of gene expression profile data, we have characterized gene set of each phase indicating the response to anticancer drug treatment. Specifically, we identified a multigene signature (23-genes including GATA3, APOBEC3G, NT5E, MYC, STC1, FOXD1, SMAD9) and developed a chemoresistance score consisting of that could predict eventual responsiveness to gemcitabine treatment. We suggest that our findings improve prognosis by predicting to acquiring of chemotherapy resistance in bladder cancer patients. Citation Format: Jae-Jun Kim, Gi-Eun Yang, Tae Hwan Shin, Sun-Hee Leem. Stepwise molecular mechanisms involved in chemoresistance in bladder cancer cells [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 5196.

Abstract 5859: PLK1 predicts aggressive behavior in CRC patients and its inhibition reverse chemoresistance in CRC cells

Abstract Polo-like kinase 1 (PLK1) is a serine/threonine-protein kinase, that is involved in multiple stages of cell cycle progression in mammals. Dysregulation of PLK1 has been found in a variety of human cancers and was associated with poor prognoses in various cancers. However, its role in the pathogenesis of CRC from Middle Eastern ethnicity has not yet been explored. Therefore, we examined the expression of PLK1 protein in a large cohort of over 1000 Middle Eastern CRC cases by immunohistochemistry. Correlation with clinico-pathological parameters were performed. PLK1 overexpression was noted in 60.3% (693/1149) CRC cases and was significantly associated with larger tumor size (T3/T4 tumors), lymphnode metastasis, Stage III tumors, MSI-H status and p-ERK1/2 over-expression. Interestingly, on multivariate logistic regression analysis, we found that PLK1 was an independent predictor of lymphnode metastasis. However, no association was noted between PLK1 expression and clinical outcomes (DFS, RFS, OS, CSS). Our in vitro experimentation revealed that CRC cells expressing high levels of PLK1 exhibited resistance to 5-Fu treatment, whereas cells with low PLK1 expression demonstrated sensitivity to the treatment. To explore the role of PLK1 on chemoresistance, we inhibited PLK1 using specific inhibitor, volasertib and we showed that volasertib effectively reversed 5-Fu resistance in these cells. Furthermore, knockdown of PLK1 reduced the progression of epithelial-to-mesenchymal transition (EMT) and the stem cell-like properties in 5-Fu resistant cells, showing the role of PLK1 on inducing EMT and stemness in CRC. Interestingly, the forced expression of PLK1 triggered the activation of the CRAF-MEK-ERK-ZEB1 signaling cascade, whereas its inhibition led to the suppression of this cascade. In addition, we showed that silencing of ERK1/2 reversed the chemoresistance, EMT and stemness of PLK1 expressing CRC cell lines. Our findings underscore the significant role of PLK1 in conferring chemoresistance, and targeting PLK1 could represent a viable therapeutic approach for the treatment of patients with an aggressive subtype of CRC. Citation Format: Divya Padmaja, Maha Al-Rasheed, Pratheesh Kumar Poyil, Abdul K. Siraj, Sandeep Kumar Parvathareddy, Wael Haqawi, Fouad Al-Dayel, Khawla S. Al-Kuraya. PLK1 predicts aggressive behavior in CRC patients and its inhibition reverse chemoresistance in CRC cells [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 5859.

Abstract 1085: Exploiting bioenergetic vulnerabilities in chemotherapy resistant osteosarcoma

Abstract BACKGROUND: Osteosarcoma (OS) is the most common bone cancer in children and young adults. Standard treatment involves chemotherapy and surgical resection, yet nearly 4 in 10 patients experience disease relapse, and almost all of these patients are resistant to additional chemotherapy. Therefore, there is an urgent need to understand the biology of chemo-resistant OS and identify new therapeutic strategies to treat this therapy-resistant disease state. METHODS: Using a CRISPR-Cas9 based lineage tracing system, RNA sequencing, and Seahorse XF assays in chemo-naïve, -resistant, and -released human 143b and patient-derived 173x OS cell lines, we modeled the evolution of chemoresistance and release from the selective pressure of chemotherapy in osteosarcoma. The phenotype of each cell state was assessed through in vitro and ex vivo cell proliferation assays, a comparison of cell viability upon re-challenge to standard-of-care chemotherapy, and comparison of cell viability in response to novel therapies targeting alterations in cell respiration. RESULTS: Chemo-resistant OS cells demonstrate a decrease in expression of proliferation-associated gene pathways and shift their resources toward expression and maintenance of multi-drug resistance ABC transporters. Chemo-released cells maintain elevated expression of ABC transporters while re-activating proliferation. Across the spectrum of chemotherapy exposure, OS cells demonstrate changes in respiratory capacity, with unique bioenergetic phenotypes observed in chemo-naïve, -resistant, and -released cells. CONCLUSIONS: Chemo-resistant and chemo-released OS cells demonstrate unique bioenergetic patterns that correlate with gene expression signatures and growth phenotypes. Evolution imposed by the selective pressure of chemotherapy may render OS cells vulnerable to therapies that disrupt cellular energetics and exploit multi-drug resistant ABC transporters. Citation Format: Valerie U. Nguyen, Laurie Graves, Veronica Colmenares, Abol Macabangon, Casey Syal, Kelsey Fisher-Wellman, William C. Eward, So Young Kim, Jason A. Somarelli. Exploiting bioenergetic vulnerabilities in chemotherapy resistant osteosarcoma [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 1085.

Abstract 5805: A novel platform of diversified payloads to drive ADC innovation

Abstract First generation of antibody drug conjugates (ADCs) for anticancer therapy has been described more than two decades ago with gemtuzumab-ozogamicin receiving the first approval in 2000. Over the last 4 years, 8 new ADCs got FDA approval essentially due to the innovation on the linker improving the disposition in patients and the manufacturability together with the technical advantages in the engineering of biologics and the introduction of new cytotoxic payloads such as deruxtecan. In the future, next generation ADCs will be developed exploring payloads beyond the currently used cytotoxins to improve safety, efficacy and durability of responses and therefore the short- and long-term therapeutic benefit to patients. Nerviano Medical Sciences (NMS) generated a portfolio of novel and diversified payload linkers leveraging on its proprietary chemical collection that includes cytotoxic and targeted payloads. A rigorous screening funnel has been applied considering diversification in terms of MoAs, pharmacokinetic properties, mechanism of resistance and combinability with other therapies to identify the optimal match with specific tumor types and targets, generating a new class of ADCs with improved selectivity and efficacy for hard-to-treat cancers even in chemoresistant settings. NMS ADC platform incorporates 1) our patent protected payload linker NMS-P945, a duocarmycin like DNA minor groove binder and alkylating agent inducing DNA damage, acting in chemoresistant cells with bystander effect and immunogenic cell death properties and generating ADCs highly efficacious in mouse xenograft models and safe when administered in NHP, 2) novel anthracyclines (PNU-682 derivatives) with an improved stability and safety profile and 3) targeted molecules with different mechanism of action including payloads active in synthetic lethality contexts. Payload linkers with different functionalization generated from each payload have been evaluated for chemical stability, sensitivity to lysosomal protease cleavage, and conjugability to monoclonal antibodies. The ADCs, generated with proof-of-concept antibodies (e.g. Trastuzumab), were characterized in terms of Drug to Antibody Ratio (DAR), aggregation level, plasma stability, antigen binding and differential antiproliferative activity in target positive vs target negative tumor cells. The best products resulting from the screening funnel have been subjected to a multidimensional analysis taking into consideration payload properties, tumor features and antigen expression in selected populations with the aim of identifying tumor types particularly sensitive to the payload. Our multidimensional approach coupling new payload linker with innovative MoA to careful selection of patients will allow to reach unprecedented efficacy and safety profile. Citation Format: Paolo Orsini, Matteo Salsa, Simona Rizzi, Ulisse Cucchi, Daniela Faiardi, Alessia Burocchi, Antonella Ciavolella, Rosita Lupi, Fabio Gasparri, Barbara Valsasina. A novel platform of diversified payloads to drive ADC innovation [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 5805.

Abstract 1794: PAF1 reprograms metabolism in pancreatic cancer by interacting with HIF1α

Abstract Background: Reprogrammed cellular metabolism has been refocused on in the past decade, as many cancer cells rewire various metabolic pathways to facilitate their survival, unlimited cell growth, and division. RNA Polymerase II-Associated Factor 1 (PAF1)/Pancreatic Differentiation 2 (PD2) is a core subunit of the human PAF1 Complex (PAF1C) that regulates the RNA polymerase II function during transcriptional elongation in normal cells; however, its overexpression has been implicated in promoting pancreatic tumorigenesis and metastasis through epigenetic regulation, chemoresistance, radioresistance and maintenance of cancer stem cells. While most of these studies have provided evidence of the multifunctional nature of PAF1/PD2 in PDAC progression, no study has investigated the role of PAF1/PD2 during the metabolic rewiring of cancer. Methods: In this study, we sought to examine the role of PAF1/PD2 in the metabolic rewiring of cancer cells in pancreatic ductal adenocarcinoma (PDAC) and decipher how PAF1/PD2 mediates this metabolic reprogramming in PDAC. Pancreatic cancer cell lines were transfected with shRNAs to knock down PAF1/PD2. Metabolic genes regulated by PAF1/PD2 were identified by qPCR and western blot upon PAF1/PD2 depletion. Metabolic assays were performed to investigate the role of PAF1/PD2. Immunoprecipitations identified proteins that interact with PAF1/PD2. Confocal microscopy confirmed the co-localization of PAF1/PD2 with its protein partners. We performed chromatin immunoprecipitation (ChIP) - Polymerase chain reaction to confirm the binding of the PAF1 sub-complex to its target gene. Results: Our results showed that pancreatic cancer cells depleted PAF1/PD2 downregulate genes involved in aerobic glycolysis compared to control cells. Also, the lactate release assay indicated that more lactate was produced in control cells than in cells with PAF1/PD2 knockdown. Interestingly, we identified that HIF1α interacts with PAF1, specifically in pancreatic cancer cells. PAF1 and HIF1α co-localization was observed in pancreatic cancer cell lines. We also observed that the PAF1/PD2- HIF1α complex bound to the promoter region of LDHA to regulate the expression of LDHA in pancreatic cancer cells, reprogramming the metabolism to utilize the aerobic glycolysis pathway preferentially. Conclusions: In conclusion, our study shows that PAF1/PD2 rewires the metabolism of PDAC by interacting with HIF1 α to regulate the expression of LDHA, which is the rate-limiting step of aerobic glycolysis. Citation Format: Ayoola O. Ogunleye, Neelanjana Gayen, Saravanakumar Marimuthu, Rama Krishna Nimmakayala, Sanchita Rauth, Zahraa Alsafwani, Jesse L. Cox, Surinder K. Batra, Moorthy P. Ponnusamy. PAF1 reprograms metabolism in pancreatic cancer by interacting with HIF1α [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 1794.

Abstract 5214: HYAL4 splice variant induces treatment-resistance in advanced bladder cancer

Abstract Introduction and Objective: Precision-based treatment approaches could improve the prognosis of patients with bladder cancer (BC). We recently discovered a first-in-class human Chondroitinase (Chase) that degrades chondroitin sulfate. This Chase is a splice variant of HYAL4 (V1) and promotes a malignant phenotype and chemoresistance in BC cells. We evaluated V1/Chase expression and functions in BC and inhibitors to overcome V1/Chase-mediated Gemcitabine (Gem) resistance. Methods: Cohort 1: 653 voided urine specimens (BC, 160; non-BC, 493); cohort 2: 40 cystectomy specimens from MIBC patients who received adjuvant Gemcitabine plus cisplatin (G+C) treatment. Gene expression was measured by q-PCR and urine was assayed by the Chase test. V1 was expressed in BC cell lines or silenced and the transfectants were analyzed for sensitivity to Gem and Cisplatin. The mechanism of Gem resistance was evaluated in preclinical in vitro and in vivo models. Results: In cohort 1, Chase levels were 7-10-fold elevated in patients with BC compared to patients with benign genitourinary conditions, history of BC, or other cancers. Chase test detected BC with 94.4% and 87.4% specificity. Chase test had 84% accuracy in correctly diagnosing the cases with atypical cytology inference. Stem cell marker CD44 was the major substrate of Chase in BC cells. V1’s Chase activity induced CD44 cleavage and shedding. Intracellularly, CD44 cleavage induced JAK2/Stat3 pathway and subsequently upregulation of cytidine deaminase. CDA induces Gem metabolism and efflux of dFdU, an inactive Gem metabolite. HPLC analysis showed, V1 transfectants have increased levels of dFdU and higher efflux. STAT3 and CDA inhibitors synergistically re-sensitized V1-expressing cells to Gem. In cohort 2, V1, p-JAK2 and CDA expression predicted failure to G+C treatment (P<0.0001). JAK2, STAT3 and CDA inhibitors synergistically re-sensitized V1-expressing cells to Gem. V1-induced muscle invasive tumors that were metastatic. While V1-expressing tumors were resistant, combination of Gem with a CDA or JAK-2 inhibitor abrogated V1 tumor growth with minimal toxicity. Conclusion: V1/Chase is a potential diagnostic and prognostic biomarker for bladder cancer and drives muscle-invasion, metastasis, and Gem resistance in BC. Inhibitors of the Chase signaling pathway overcome V1-induced Gem resistance, suggesting a precision-based treatment approach to improve treatment response. Support: 1R01CA227277-05A1; 1R01CA283699-01 Citation Format: Karina Aguilar, Sunita Panda, Joshua Van Der Eerden, Luis Lopez, Martha Terris, Pablo Santamaria, Bal Lokeshwar, Vinata B. Lokeshwar. HYAL4 splice variant induces treatment-resistance in advanced bladder cancer [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 5214.