Mechanisms Of Resistance In Cancer Research Articles

Discovering novel mechanisms of taxane resistance in human breast cancer by whole-exome sequencing.

Taxanes are important drugs used in the treatment of breast cancer; however, some cancer types are taxane-resistant. The aim of the present study was to investigate the underlying mechanisms of taxane resistance using whole-exome sequencing (WES). Six patients with breast cancer whose tumors responded well to anthracycline treatment but grew rapidly during neoadjuvant taxane-based chemotherapy, were included in the present study. WES of samples from these patients was carried out to identify somatic mutations of candidate genes thought to affect taxane resistance, and the candidate proteins were structurally modeled. The mRNA and protein expression levels of these candidate genes in other breast cancers treated with taxanes were also examined. Nine variants common to all six patients were identified and two of these [R552P in V-type proton ATPase catalytic subunit A (ATP6V1A) and T114P in apolipoprotein B MRNA editing enzyme catalytic subunit 3F (APOBEC3F)] were selected. The results also showed that, protein-structure visualization suggested that these mutations may cause structural changes. The Kaplan-Meier analyses revealed that higher APT6V1A and APOBEC3F expression levels were significantly associated with poorer disease-free survival (DFS) and overall survival. Moreover, multivariate analysis identified high ATP6V1A mRNA expression as an independent risk factor for poor DFS. Two specific mutations that might affect taxane resistance were identified. Thus, these results suggest that breast cancer patients receiving taxanes who have high ATP6V1A or APOBEC3F expression levels may have shorter survival.

Identification of key genes involved in tamoxifen-resistant breast cancer using bioinformatics analysis.

BackgroundThe purpose of the present study was to investigate the molecular mechanisms of tamoxifen resistance in breast cancer and to identify potential targets for antitamoxifen resistance.MethodsDifferentially expressed genes (DEGs) in tamoxifen-resistant and tamoxifen-sensitive breast cancer cells were assessed using the GSE67916 dataset acquired from the Gene Expression Omnibus database. Gene ontology (GO) and pathway enrichment analyses were applied to investigate the functions and pathways of the DEGs. Subsequently, the protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING), and subnetworks were further analyzed by Molecular Complex Detection (MCODE). The PPI network and subnetworks were visualized using Cytoscape software.ResultsIn total, 438 DEGs were identified, of which 300 were upregulated and 138 were downregulated. The DEGs were significantly enriched in the protein binding, cellular response to estradiol stimulus, and immune response GO terms while the most significant pathways included the mitogen-activated protein kinase (MAPK) signaling pathway in cancer. The PPI network of DEGs was constructed with 288 nodes and 629 edges, and 2 subnetworks were screened out from the entire network.ConclusionsA number of significant hub DEGs were identified based on their degree of connectivity in the PPI network, , included MAPK1 (node degree 36), ESR1 (node degree 27), SMARCA4 (node degree 27), RANBP2 (node degree 25), and PRKCA (node degree 21). These critical hub genes were found to be related to tamoxifen resistance in breast cancer. The results of this study further the understanding of tamoxifen resistance at the molecular level and identify potential therapeutic targets for tamoxifen-resistant breast cancer.

Abstract P026: NUC-7738 alters oxidative phosphorylation and causes terminal differentiation in acute myeloid leukemia cells

Abstract Background NUC-7738, a phosphoramidate transformation of 3’deoxyadenosine (3’dA), is specifically designed to generate the active anti-cancer metabolite 3’-deoxyadenosine monophosphate (3’-dAMP) directly in cells, bypassing key cancer resistance mechanisms of transport, activation and breakdown. NUC-7738 is currently in a Phase I clinical study to assess safety and determine the recommended dose in patients with advanced solid tumors and lymphoma. Acute myeloid leukemia (AML) cells exhibit impaired differentiation, uncontrolled proliferation and are highly dependent on mitochondrial metabolic processes for survival and chemoresistance. The aim of this study was to determine the effect of NUC-7738 on oxidative phosphorylation (OXPHOS) proteins and resulting changes in mitochondria morphology and activation of the intrinsic apoptotic pathway. Methods AML cell lines OCI-AML3 and HL-60 were treated with NUC-7738 for 48 hours. Cell differentiation was determined by morphologic modification with Giemsa stain and assessing the expression of surface markers CD11b and CD163 by flow cytometry. Immunoblotting was performed for Mitochondrial Electron Transport Chain Complex I subunit NDUFB8, Complex II subunit SDHB, Complex III subunit UQCRC2, Complex IV subunit COX II and ATP synthase subunit ATP5A. Mitochondria morphology was assessed by confocal microscopy with MitoTracker-Deep Red CMXRos dye. Annexin-V, DAPI, Bcl-2, cytochrome c, and cleaved-caspase 7 were assessed by flow cytometry to determine intrinsic apoptosis. Results NUC-7738 increased the proportion of CD11b+ and CD163+ cells in a dose-dependent manner from 6.1% to 28.7% and 9.6% to 26.9%, respectively. CD11b positivity was also increased in HL-60 cells from 14.5% to 31.1%. NUC-7738 induced morphological differentiation as indicated by decreased nucleo-cytoplasmic ratio, appearance of multi-lobed nuclei and vacuolization of the cytoplasm in OCI-AML3 cells. Decreased nucleo-cytoplasmic ratio was also evident in HL-60 cells. NUC-7738 decreased the protein expression of all 5 complexes of oxidative phosphorylation in OCI-AML3 cells at 48 hours and this metabolic disruption was also evident from distinct changes in mitochondria morphology. NUC-7738 activated the intrinsic apoptotic pathway, causing sequestration of BCL-2 and release of cytochrome c, resulting in a decrease in the percentage of live OCI-AML3 cells from 76% to 37% at 48 hours. Conclusion Through the ability of NUC-7738 to inhibit OXPHOS complexes with consequent terminal differentiation and activation of mitochondrial-mediated apoptosis, it exploits the mitochondrial characteristics of certain cancer cells, including their greater dependence on OXPHOS for survival than normal cells. NUC-7738 offers a potential treatment option for patients with a variety of malignancies. Citation Format: Akbar M. Shahid, In Hwa Um, Oliver J. Read, David J. Harrison. NUC-7738 alters oxidative phosphorylation and causes terminal differentiation in acute myeloid leukemia cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P026.

Individualized Proteogenomics Reveals the Mutational Landscape of Melanoma Patients in Response to Immunotherapy.

Simple SummaryMelanoma is the most aggressive form of skin cancer, with a rapidly increasing incidence rate. Due to ineffective treatment options in the late stage melanoma, patients have an overall poor prognosis. Over the last decades, the role of the immune system in the control of tumor progression has been established and immune checkpoint inhibitors (ICi) have shown remarkable clinical activity. While current trials suggest durable responses in patient under ICi therapy, there is increasing evidence pointing towards existence of innate and acquired resistance to ICi therapy; and it is now clear that personalized medicine will be critical for effective patient therapy. Proteogenomics is a powerful tool to study the mode of action of disease-associated mutations at the genome, transcriptome, proteome and PTM level. Here, we applied a proteogenomic workflow to study melanoma samples from human tumors. Such workflow may be applicable to other patient-derived samples and different cancer types.Immune checkpoint inhibitors are used to restore or augment antitumor immune responses and show great promise in the treatment of melanoma and other types of cancers. However, only a small percentage of patients are fully responsive to immune checkpoint inhibition, mostly due to tumor heterogeneity and primary resistance to therapy. Both of these features are largely driven by the accumulation of patient-specific mutations, pointing to the need for personalized approaches in diagnostics and immunotherapy. Proteogenomics integrates patient-specific genomic and proteomic data to study cancer development, tumor heterogeneity and resistance mechanisms. Using this approach, we characterized the mutational landscape of four clinical melanoma patients. This enabled the quantification of hundreds of sample-specific amino acid variants, among them many that were previously not reported in melanoma. Changes in abundance at the protein and phosphorylation site levels revealed patient-specific over-represented pathways, notably linked to melanoma development (MAPK1 activation) or immunotherapy (NLRP1 inflammasome). Personalized data integration resulted in the prediction of protein drug targets, such as the drugs vandetanib and bosutinib, which were experimentally validated and led to a reduction in the viability of tumor cells. Our study emphasizes the potential of proteogenomic approaches to study personalized mutational landscapes, signaling networks and therapy options.

EGFR-mediated Rad51 expression potentiates intrinsic resistance in prostate cancer via EMT and DNA repair pathways

AimTo study the role of EGFR signaling in regulation of intrinsic resistance in prostate cancer. Materials and methodsRadioresistant prostate carcinoma DU145 and PC-3 cells were used to study the effect of shRNA-mediated knockdown of EGFR on intrinsic radioresistance mechanisms. Semi-quantitative PCR, western blotting, growth kinetics, colony formation, transwell migration, invasion and trypan blue assays along with inhibitors erlotinib, NU7441, B02, PD98059 and LY294002 were used. Key findingsEGFR knock-down induced morphological alterations along with reduction in clonogenic potential and cell proliferation in DU145 cells. Migratory potential of prostate cancer cells were reduced concomitant with upregulation of epithelial marker, E-cadherin and decreased expression of mesenchymal markers, vimentin and snail. Further, EGFR knock-down decreased the expression of Rad51 and DNA-PK at mRNA as well as protein levels. Likewise, erlotinib, an EGFR inhibitor, and NU7441, a DNA-PK inhibitor increased the expression of E-cadherin and decreased the level of vimentin. Both these inhibitors also decreased the levels of DNA damage regulatory protein Rad51. Further, Rad51 inhibitor, B02, inhibited the clonogenic potential, cell migration and reduced the expression of vimentin, Ku70 and Ku80, and also, B02 radiosensitized DU145 cells. EGFR-regulated expression of Rad51 was found to be mediated via PI3K/Akt and Erk1/2 pathways. SignificanceEGFR was found to regulate DNA damage repair, survival and EMT responses in prostate cancer cells through transcriptional regulation of Rad51. A novel role of EGFR-Erk1/2/Akt-Rad51 axis through modulation of EMT and DNA repair pathways in prostate cancer resistance mechanisms is suggested.

Mechanisms of drug resistance in ovarian cancer

Ovarian cancer is the fifth most common cancer affecting women and the most lethal cancer among the gynecological cancers. Because of the lack of specific symptoms and no special screening tools, it is recognised in an advanced stage. In addition, drug resistance in ovarian cancer is so frequent, that genes and cross-talks between some important pathways are still analysed. In this review, the major and recently identified molecular mechanisms of drug resistance, including platinum, taxane, bevacizumab and PARPi resistance mechanisms in ovarian cancer from relevant literature have been investigated.

VKNG-1 Antagonizes ABCG2-Mediated Multidrug Resistance via p-AKT and Bcl-2 Pathway in Colon Cancer: In Vitro and In Vivo Study.

Simple SummaryMultidrug resistance or chemoresistance is a phenomenon where cells exhibit resistance to drugs that are pharmacologically and structurally distinct. ABCG2, a member of ABC transporter superfamily has been widely reported to be a principal cause of MDR in various cancers via pumping out various antineoplastic drugs. VKNG-1, a phenyltetrazole analogue selectively inhibits the ABCG2 transporter and reverses resistance to standard anticancer drugs both in vitro and in vivo in ABCG2-overexpressing colon cancers. This study presents the importance of VKNG-1 as a modulator of the ABCG2 transporter. In cancer patients, a combination of VKNG-1 and ABCG2 substrate drugs could be a beneficial treatment option for cells with high ABCG2 expression.The emergence of multidrug resistance (MDR) to chemotherapeutic drugs is a major problem in the therapy of cancer. Knowledge of the mechanisms of drug resistance in cancer is necessary for developing efficacious therapies. ATP-binding cassette (ABC) transporters are transmembrane proteins that efflux chemotherapeutic drugs from cancer cells, thereby producing MDR. Our research efforts have led to the discovery of VKNG-1, a compound that selectively inhibits the ABCG2 transporter and reverses resistanctabe to standard anticancer drugs both in vitro and in vivo. VKNG-1, at 6 µM, selectively inhibited ABCG2 transporter and sensitized ABCG2-overexpressing drug-resistant cancer cells to the ABCG2 substrate anticancer drugs mitoxantrone, SN-38, and doxorubicin in ABCG2-overexpressing colon cancers. VKNG- 1 reverses ABCG2-mediated MDR by blocking ABCG2 efflux activity and downregulating ABCG2 expression at the mRNA and protein levels. Moreover, VKNG-1 inhibits the level of phosphorylated protein kinase B (PKB/p-AKT), and B-cell lymphoma-2 (Bcl-2) protein which may overcome resistance to anticancer drugs. However, the in vitro translocation of ABCG2 protein did not occur in the presence of 6 µM of VKNG-1. In addition, VKNG-1 enhanced the anticancer efficacy of irinotecan in ABCG2- overexpressing mouse tumor xenografts. Overall, our results suggest that VKNG-1 may, in combination with certain anticancer drugs, represent a treatment to overcome ABCG2-mediated MDR colon cancers.

The Novel Nucleoside Analogue ProTide NUC-7738 Overcomes Cancer Resistance Mechanisms In Vitro and in a First-In-Human Phase I Clinical Trial.

Nucleoside analogues form the backbone of many therapeutic regimens in oncology and require the presence of intracellular enzymes for their activation. A ProTide is comprised of a nucleoside fused to a protective phosphoramidate cap. ProTides are easily incorporated into cells whereupon the cap is cleaved and a preactivated nucleoside released. 3'-Deoxyadenosine (3'-dA) is a naturally occurring adenosine analogue with established anticancer activity in vitro but limited bioavailability due to its rapid in vivo deamination by the circulating enzyme adenosine deaminase, poor uptake into cells, and reliance on adenosine kinase for its activation. In order to overcome these limitations, 3'-dA was chemically modified to create the novel ProTide NUC-7738. We describe the synthesis of NUC-7738. We determine the IC50 of NUC-7738 using pharmacokinetics (PK) and conduct genome-wide analyses to identify its mechanism of action using different cancer model systems. We validate these findings in patients with cancer. We show that NUC-7738 overcomes the cancer resistance mechanisms that limit the activity of 3'-dA and that its activation is dependent on ProTide cleavage by the enzyme histidine triad nucleotide-binding protein 1. PK and tumor samples obtained from the ongoing first-in-human phase I clinical trial of NUC-7738 further validate our in vitro findings and show NUC-7738 is an effective proapoptotic agent in cancer cells with effects on the NF-κB pathway. Our study provides proof that NUC-7738 overcomes cellular resistance mechanisms and supports its further clinical evaluation as a novel cancer treatment within the growing pantheon of anticancer ProTides.

Long noncoding RNA ZMIZ1‐AS1 promotes gefitinib resistance via binding to hnRNPA1

AbstractTo gain an insight into the molecular mechanisms of gefitinib resistance in nonsmall cell lung cancer, we screened out the long noncoding RNA related to gefitinib resistance through microarray data in gefitinib‐sensitive and resistant cells. lncRNA ZMIZ1‐AS1 was significantly upregulated in HCC827/GR cells by screening the microarray data. Further real‐time‐qPCR results were consistent with the microarray data. Cell viability assay and flow cytometry showed that ZMIZ1‐AS1 influenced the sensitivity of HCC827/GR cells to gefitinib. RNA pull‐down assay demonstrated that hnRNPA1 was a specific binding protein for ZMIZ1‐AS1. Our results help to reveal the role and mechanism of lncRNA in the secondary resistance of gefitinib and provide a new therapeutic target for gefitinib therapy.

59TiP Phase III study of NUC-1031 + cisplatin vs gemcitabine + cisplatin for first-line treatment of patients with advanced biliary tract cancer (NuTide:121)

Biliary tract cancer (BTC) is an aggressive disease with a poor prognosis. Gemcitabine + cisplatin (GemCis) is the accepted global standard of care; however, key cancer resistance mechanisms associated with the transport, activation and breakdown of gemcitabine are known to limit its clinical activity. NUC-1031 is a phosphoramidate transformation of gemcitabine designed to overcome these key resistance mechanisms and generate much higher levels of the active anti-cancer metabolite, dFdCTP, in cells.

MEK inhibition overcomes resistance to EphA2-targeted therapy in uterine cancer

BackgroundOur pilot clinical study of EphA2 inhibitor (dasatinib) plus paclitaxel and carboplatin showed interesting clinical activity in endometrial cancer with manageable toxicity. However, the underlying mechanisms of dasatinib resistance in uterine cancer are unknown. Here, we investigated potential mechanisms underlying resistance to EphA2 inhibitors in uterine cancer and examined the anti-tumor activity of EphA2 inhibitors alone and in combination with a MEK inhibitor. MethodsWe evaluated the antitumor activity of EphA2 inhibitors plus a MEK inhibitor using in vitro and in vivo orthotopic models of uterine cancer. ResultsEphA2 inhibitor induced MAPK in dasatinib-resistant uterine cancer cells (HEC-1A and Ishikawa) and BRAF/CRAF heterodimerization in HEC-1A cells. EphA2 inhibitor and trametinib significantly increased apoptosis in cancer cells resistant to EphA2 inhibitors compared with controls (p < 0.01). An in vivo study with the orthotopic HEC-1A model showed significantly greater antitumor response to combination treatment compared with dasatinib alone (p < 0.01). Combination treatment increased EphrinA1 and BIM along with decreased pMAPK, Jagged 1, and c-MYC expression in dasatinib-resistant cells. In addition, Spearman analysis using the TCGA data revealed that upregulation of EphA2 was significantly correlated with JAG1, MYC, NOTCH1, NOTCH3 and HES1 expression (p < 0.001, r = 0.25–0.43). Specifically, MAP3K15 and the NOTCH family genes were significantly related to poor clinical outcome in patients with uterine cancer. ConclusionsThese findings indicate that the MAPK pathway is activated in dasatinib-resistant uterine cancer cells and that EphrinA1-mediated MEK inhibition overcomes dasatinib resistance. Dual targeting of both EphA2 and MEK, combined with chemotherapy, should be considered for future clinical development.

PO-1931 Identifying resistance mechanisms in breast cancer in patient-derived organoids and 3D cell culture

PO-1931 Identifying resistance mechanisms in breast cancer in patient-derived organoids and 3D cell culture

Breast cancer resistance mechanisms: challenges to immunotherapy.

The clinical implementation of immunotherapy has profoundly transformed cancer treatment. Targeting the immune system to mount anti-tumor responses can elicit a systemically durable response. Employing immune checkpoint blockade (ICB) has suppressed tumor growth and vastly improved patient overall and progression-free survival in several cancer types, most notably melanoma and non-small cell lung carcinoma. Despite widescale clinical success, ICB response is heterogeneously efficacious across tumor types. Many cancers, including breast cancer, are frequently refractory to ICB. In this review, we will discuss the challenges facing immunotherapy success and address the underlying mechanisms responsible for primary and acquired breast cancer resistance to immunotherapy. Even in initially ICB-responsive tumors, many acquire resistance due to tumor-specific alterations, loss of tumor-specific antigens, and extrinsic mechanisms that reshape the immune landscape within the tumor microenvironment (TME). The tumor immune interaction circumvents the benefits of immunotherapy; tumors rewire the tumor-suppressive functions of activated immune cells within their stroma to propagate tumor growth and progression. The breast cancer immune TME is complex and the mechanisms driving resistance to ICB are multifaceted. Continued study in both preclinical models and clinical trials should help elucidate these mechanisms so they can be targeted to benefit more breast cancer patients.

From Micro to Long: Non-Coding RNAs in Tamoxifen Resistance of Breast Cancer Cells.

Simple SummaryBreast cancer is a disease that affects thousands of women around the world. Adequate treatment depends on the characterization of breast cancer subtypes. Tumors that are positive for the estrogen receptor represent the most common subtypes and have the best prognosis. However, many patients relapse due to resistance to tamoxifen, one of the main drugs used for these subtypes. In this study, our goal is to discuss a class of molecules that have been directly linked to the tamoxifen resistance process but is still underexplored: the non-coding RNAs (ncRNAs). We have reviewed ncRNAs that have been associated with different processes of resistance to tamoxifen in order to stimulate a discussion about the importance of knowing and understanding the role of these molecules in breast cancer and their relevance to clinical applications.Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer mortality among women. Two thirds of patients are classified as hormone receptor positive, based on expression of estrogen receptor alpha (ERα), the main driver of breast cancer cell proliferation, and/or progesterone receptor, which is regulated by ERα. Despite presenting the best prognosis, these tumors can recur when patients acquire resistance to treatment by aromatase inhibitors or antiestrogen such as tamoxifen (Tam). The mechanisms that are involved in Tam resistance are complex and involve multiple signaling pathways. Recently, roles for microRNAs and lncRNAs in controlling ER expression and/or tamoxifen action have been described, but the underlying mechanisms are still little explored. In this review, we will discuss the current state of knowledge on the roles of microRNAs and lncRNAs in the main mechanisms of tamoxifen resistance in hormone receptor positive breast cancer. In the future, this knowledge can be used to identify patients at a greater risk of relapse due to the expression patterns of ncRNAs that impact response to Tam, in order to guide their treatment more efficiently and possibly to design therapeutic strategies to bypass mechanisms of resistance.

Drug Resistance Mechanism of Non-small Cell Lung Cancer after EGFR-TKI Treatment: Transformation of Small Cell Lung Cancer

Drug Resistance Mechanism of Non-small Cell Lung Cancer after EGFR-TKI Treatment: Transformation of Small Cell Lung Cancer

Reproductive tract neoplasia in adult female Asian elephants (Elephas maximus).

Recent reports have highlighted a lower-than-expected prevalence of neoplasia in elephants and suggested mechanisms for cancer resistance. But despite infrequent reports in the literature, uterine neoplasia is common in managed Asian elephants (Elephas maximus). This study is an archival review of reproductive tract neoplasia in 80 adult female Asian elephant mortalities in managed care facilities in the United States from 1988 to 2019. Neoplasms occurred in 64/80 (80%) of cases. Most were in the uterus (63/64; 98%) with only a single case of ovarian neoplasia. Myometrial leiomyomas were present in 57/63 (90%) cases with uterine neoplasia. Uterine adenocarcinoma was present in 8/63 (13%) cases. Remaining cases included endometrial adenoma (2), focal carcinoma in situ in endometrial polyps (1), anaplastic carcinoma (1), endometrial hemangioma (1), primitive neuroectodermal tumor (PNET; 1), and angiosarcoma (1). One case with uterine adenocarcinoma had a separate pelvic mass histologically characterized as an anaplastic sarcoma. Distant metastases were documented in 5/8 (63%) cases of uterine adenocarcinoma, and in the uterine anaplastic carcinoma, PNET, and angiosarcoma. Four uterine adenocarcinomas and one carcinoma in situ were examined immunohistochemically for pan-cytokeratin, vimentin, and estrogen receptor. In all, neoplastic cells were pan-cytokeratin positive and vimentin negative, and in 2 cases were immunoreactive for estrogen receptor. Results show that female reproductive tract neoplasia, particularly of the uterus, is common in Asian elephants and is not limited to leiomyomas. Importantly, uterine neoplasms have the potential to impact fecundity and may represent obstacles to conservation in managed care.

Abstract 1081: Genome-wide CRISPR screens reveal Hippo pathway activation as a resistance mechanism in BRAF mutant colon cancer

Abstract BRAF V600E mutations occur in a subset of colon cancers. These are typically resistant to chemotherapy and are associated with a poor outcome. Combination treatment with BRAF and EGFR inhibitors is superior to standard chemotherapy and has recently received FDA approval, however the early emergence of drug resistance is a significant clinical problem. Clinical studies of resistant patients have identified mutations and amplifications in the MAPK pathway as important resistance drivers. To identify novel non-MAPK dependent resistance mechanisms, genome-wide CRISPR/Cas9 knockout screens were performed to identify genes causing resistance to a combination BRAF/MEK/EGFR inhibitor regimen in the BRAF mutant HT29 and LS411N colon cancer cell lines. A number of strong resistance hits were identified but importantly, only 3 genes (CSK, ARID1A and STK11) were detected as significantly enriched in both cell lines screened. Two of these, CSK and ARID1A, have been shown to play a role in activation of the Hippo signaling pathway. The generation of CSK knockout colon cell lines confirmed resistance to BRAF/MEK/EGFR inhibition both in vitro and in vivo using xenograft models. Furthermore, since CSK is a negative regulator of SRC, re-sensitisation of resistant BRAF mutant CSK knockout cells was achieved by adding Src inhibitors (Dasatinib and Saracatinib) to the combination therapy. Nuclear localisation of the transcription factors YAP1 and WWTR1 and binding to TEAD family members are required for Hippo pathway activation and we confirmed significantly increased nuclear YAP1/WWTR1 in CSK knockout cells. Furthermore, YAP1/WWTR1 nuclear localisation in these cells was reversed by treatment with Src inhibitors. Novel pharmacological TEAD inhibitors have recently been developed and will also be used to confirm that resistance can be overcome by specifically targeting the Hippo pathway. Expression profiling of CSK knockout cells revealed significant enrichment of pathways associated with Hippo signaling. Genes involved in regulating Hippo pathway activation were also identified as CRISPR screen resistance hits in both lung and head and neck cancer cell lines, suggesting that this may be an important mechanism of resistance among other tumour types and not limited to the colon. Here we show that activation of the Hippo pathway is a potential MAPK-independent resistance mechanism in BRAF mutant colon cancer, readily reversible by rational pharmacological targeting. Given the development of specific Hippo pathway inhibitors and plans for their use in clinical trials, activation of Hippo signaling should be considered in resistant BRAF mutant colon cancer where alterations in the MAPK pathway are not detected. Citation Format: Jonathan S. Brammeld, Hannah Thorpe, Mar Arias Garcia, Stacey Price, Jamie Young, Matthias Pfeifer, Barbara Lupo, Kosuke Yusa, Livio Trusolino, Mathew Garnett, Andrea Bertotti, Chris Bakal, Ultan McDermott. Genome-wide CRISPR screens reveal Hippo pathway activation as a resistance mechanism in BRAF mutant colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1081.

Abstract 1421: Targeting cell-adaptive mechanisms of drug resistance in lethal prostate cancer

Abstract Precision oncology therapies are significantly hindered by the emergence of genetically or epigenetically-distinct cellular sub-populations, presenting either pre-existing or acquired drug resistance. For example, patients affected by advanced prostate cancer (PC), which is initially driven by aberrant activation of androgen receptor (AR) signals, often respond to Androgen Deprivation Therapy (ADT). Unfortunately, long-term treatment leads to the emergence of ADT-resistant tumors, termed Castration Resistant Prostate Cancer (CRPC), and poor outcome. Next-generation AR inhibitors extend CRPC patient survival, but frequently lead to the emergence of an even more aggressive Neuroendocrine PC (NEPC) phenotype, with exceedingly poor prognosis. Progression to NEPC is often determined by epigenetic-mediated reprogramming, rather than genomic alterations, resulting in cell-adaptive resistance, driven by aberrant activation of a handful of Master Regulator (MR) proteins. Consequently, better and more mechanistic understanding of cell-adaptive resistance is necessary to develop effective therapeutic strategies for CRPC. To address this challenge, we devised an RNA-based, high-throughput methodology, to systematically elucidate drug resistance mechanisms with single-cell resolution, and we applied it to study diverse, clonal populations that emerge following long-term treatment of PC LnCAP cells with next-generation AR inhibitors. Specifically, we identified Master Regulator (MR) proteins leading to the emergence of drug resistance, as well as clinically-available inhibitors able to reversing their activity, which were experimentally validated. We used PLATE-Seq, a low-cost, high-throughput RNA-Seq technology to profile more than 80 clonal, ADT-resistant populations, with single-cell resolution, and the VIPER algorithm to elucidate the MR proteins that mechanistically regulate their cell-adaptation programs. Our analysis revealed distinct cell-adaptive responses, associated with profound epigenetic reprogramming of prostate cancer cells. We used OncoTreat to prioritize a list of more than 400 FDA-approved and late-stage investigational drugs. Sensitivity to predicted drugs was confirmed in vitro and in vivo studies, in xenograft models derived from ADT- resistant clones are underway. We propose that this framework may represent a relevant advance to the study of cell-adaptive mechanism of drug resistance. Critically, this methodology is not limited to prostate cancer and can be applied to study arbitrary drug-resistance phenotypes. Citation Format: Prem Subramaniam, Alessandro Vasciaveo, Juan Arriaga, Jordan Kesner, Cory Abate-Shen, Andrea Califano. Targeting cell-adaptive mechanisms of drug resistance in lethal prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1421.

Abstract 2998: Targeting resistance mechanisms in muscle invasive bladder cancer using the chicken egg chorioallantoic membrane patient-derived xenograft model

Abstract Introduction: Non-metastatic muscle invasive urothelial bladder cancer (MIBC) has a poor prognosis and standard of care (SOC) consists of neoadjuvant cisplatin-based chemotherapy (NAC) combined with cystectomy. Patients receiving SOC have at best &amp;lt;10% improvement in five-year overall survival. Residual invasive cancer following NAC has no SOC and has high mortality risk. This major clinical problem underscores gaps in our understanding resistance mechanisms and a need for reliable pre-clinical models. The chicken embryo chorioallantoic membrane (CAM) represents a rapid, scalable, and cost-effective alternative in vivo patient-derived xenograft (PDX) platform. It leverages an easily accessible engraftment scaffold and vascular-rich, immunosuppressed environments for the engraftment of PDX tumors and subsequent functional studies. Our previous proteogenomic data on MIBCs with known chemo-resistant outcomes obtained prior to NAC (pre-NAC) suggests a benefit from treatment with kinase inhibitors (KI). Use of the CAM model in this study aims to address the MIBC pre-clinical model deficit and allow for the rapid screening of therapeutics against NAC-resistant PDXs using selected KI. Methods: We employed the CAM model to expand primary MIBC tumors and tested concordance between cisplatin-based chemotherapy response of patients to matching PDX tumors. We also tested selected KI response on chemotherapy-resistant bladder cancers. PDX growth was assessed by tumor surface area quantification following a week of growth with chemotherapy and KI treatment. Hematoxylin and eosin staining coupled with in situ hybridization, immunohistochemistry for Ki67, and cleaved PARP helped identify tumor cells on the CAM as well as their proliferative and apoptotic indexes. Results: Our initial results show pre-NAC primary MIBC tumors grown on the CAM phenocopy cisplatin-based chemotherapy resistance observed in parent tumor specimens. Histological analysis confirmed engraftment and growth of bladder tumors on the CAM. Patient tumor specimens acquired after chemotherapy treatment (post-NAC) and exhibiting NAC resistance were engrafted successfully on the CAM. Such CAM PDX tumors displayed decreased tumor growth size and proliferation in response to treatment with a dual EGFR and HER2 inhibitor, but had no significant response to either CDK4/6 or FGFR inhibition. Conclusion: Our data suggests concordance between Cisplatin-based chemotherapy resistance phenotypes in primary patient tumors and CAM PDX models. Further, proteogenomic informed KI use on MIBC CAM PDX models suggests a benefit from integration of rapid in vivo testing of novel therapeutics to inform more complex, pre-clinical mouse PDX experiments for more effective clinical trial design aimed at achieving optimal precision medicine for patients with limited treatment options. Citation Format: Hugo Villanueva, Patricia Castro, Andrew G. Sikora, Seth P. Lerner. Targeting resistance mechanisms in muscle invasive bladder cancer using the chicken egg chorioallantoic membrane patient-derived xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2998.

Prospective Evaluation of Clinical Outcomes Using a Multiplex Liquid Biopsy Targeting Diverse Resistance Mechanisms in Metastatic Prostate Cancer.

PURPOSENearly all men with prostate cancer treated with androgen receptor (AR) signaling inhibitors (ARSIs) develop resistance via diverse mechanisms including constitutive activation of the AR pathway, driven by AR genomic structural alterations, expression of AR splice variants (AR-Vs), or loss of AR dependence and lineage plasticity termed neuroendocrine prostate cancer. Understanding these de novo acquired ARSI resistance mechanisms is critical for optimizing therapy.MATERIALS AND METHODSA novel liquid biopsy technology was used to collect mRNA from circulating tumor cells (CTCs) to measure expression of AR-Vs, AR targets, and neuroendocrine prostate cancer markers. An institutional review board–approved prospective cohort (N = 99) was used to identify patterns of gene expression. Two prospective multicenter phase II clinical trials of ARSIs for men with castration-resistant prostate cancer (ClinicalTrials.gov: NCT01942837 [enzalutamide, N = 21] and NCT02025010 [abiraterone, N = 27]) were used to further validate these findings.RESULTSHierarchical clustering of CTC transcripts identified two distinct clusters. Cluster 2 (C2) exhibited increased expression of AR-regulated genes and was associated with worse overall survival (median 8.6 v 22.4 months; P < .01; hazard ratio [HR] = 3.45 [1.9 to 6.14]). In multivariable analysis, C2 was prognostic independent of other clinicopathologic variables. AR-V status was not significant when accounting for C2. Upon further validation in pooled multicenter phase II trials, C2 was associated with worse overall survival (15.2 months v not reached; P < .01; HR = 8.43 [2.74 to 25.92]), prostate-specific antigen progression-free survival (3.6 v 12 months; P < .01; HR = 4.64 [1.53 to 14.11]), and radiographic progression-free survival (2.7 v 40.6 months; P < .01; HR = 4.64 [1.82 to 17.41]).CONCLUSIONWe demonstrate that a transcriptional profile detectable in CTCs obtained from liquid biopsies can serve as an independent prognostic marker beyond AR-V7 in patients with metastatic prostate cancer and can be used to identify the emergence of multiple ARSI resistance mechanisms. This is currently being investigated in additional prospective trials.