Low-passage Cell Research Articles

Direct-Current Electrical Field Stimulation of Patient-Derived Colorectal Cancer Cells.

Several cues for a directional migration of colorectal cancer cells were identified as being crucial in tumor progression. However, galvanotaxis, the directional migration in direct-current electrical fields, has not been investigated so far. Therefore, we asked whether direct-current electrical fields could be used to mobilize colorectal cancer cells along field vectors. For this purpose, five patient-derived low-passage cell lines were exposed to field strengths of 150-250 V/m in vitro, and migration along the field vectors was investigated. To further study the role of voltage-gated calcium channels on galvanotaxis and intracellular signaling pathways that are associated with migration of colorectal cancer cells, the cultures were exposed to selective inhibitors. In three out of five colorectal cancer cell lines, we found a preferred cathodal migration. The cellular integrity of the cells was not impaired by exposure of the cells to the selected field strengths. Galvanotaxis was sensitive to inhibition of voltage-gated calcium channels. Furthermore, signaling pathways such as AKT and MEK, but not STAT3, were also found to contribute to galvanotaxis in our in vitro model system. Overall, we identify electrical fields as an important contributor to the directional migration of colorectal cancer cells.

Passage number of cancer cell lines: Importance, intricacies, and way-forward.

Cancer cell lines play a crucial role as invaluable models in cancer research, facilitating the examination of cancer progression as well as the advancement of diagnostics and treatments. While they may not perfectly replicate the original tumor, they generally exhibit similar characteristics. Low-passage cancer cell lines are generally preferred due to their closer resemblance to the original tumor, as long-term culturing can alter the genetic and molecular profiles of a cell line thereby highlighting the importance of monitoring the passage number (PN). Variations in proliferation, migration, gene expression, and drug sensitivity can be linked to PN differences. PN can also influence DNA methylation levels, metabolic profiles, and the expression of genes/or proteins in cancer cell lines. When conducting research on cancer cell lines, it is crucial for researchers to carefully select the appropriate PN to maintain consistency and reliability of results. Moreover, to ensure dependability and replicability, scientists ought to actively track the growth, migration, and gene/or protein profiles of cancer cell lines at specific PNs. This approach enables the identification of the most suitable range of PNs for experiments, guaranteeing consistent and precise results. Additionally, such efforts serve to minimize disparities and uphold the integrity of research. In this review, we have laid out recommendations for laboratories to overcome these PN discrepancies when working with cancer cell lines.

Epigenetic modifiers either individually or in specific combinations impair viability of patient-derived glioblastoma cell line while exhibit moderate effect on normal stem cells growth.

Glioblastomas (GBM), also known as glioblastoma multiforme, are the most aggressive type of brain cancer. Currently, there is no effective treatment for GBM, highlighting the pressing need for new therapeutic strategies. In a recent study, we demonstrated that specific combinations of epigenetic modifiers significantly affect the metabolism and proliferation rate of the two most aggressive GBM cell lines, D54 and U-87. Importantly, these combinations exhibited minimal effects on the growth of normal stem cells. In this study, we extended our investigation to include a patient-derived GBM stem cell line. Our results showed that the combinations of modulators of histone and DNA covalent modifying enzymes that synergistically suppress D54 and U87 cell line growth also impair the viability of the patient-derived GBM stem cell line. These findings suggest that epigenetic modifiers alone or in specific combinations exhibit a cytotoxic effect on established and low-passage patient-derived GBM cell lines, and thus could be a promising therapeutic approach for this type of brain cancer.

Establishment, characterization, and drug screening of low-passage patient individual non-small cell lung cancer in vitro models including the rare pleomorphic subentity.

For pre-clinical drug development and precision oncology research, robust cancer cell models are essential. Patient-derived models in low passages retain more genetic and phenotypic characteristics of their original tumors than conventional cancer cell lines. Subentity, individual genetics, and heterogeneity greatly influence drug sensitivity and clinical outcome. Here, we report on the establishment and characterization of three patient-derived cell lines (PDCs) of different subentities of non-small cell lung cancer (NSCLC): adeno-, squamous cell, and pleomorphic carcinoma. The in-depth characterization of our PDCs included phenotype, proliferation, surface protein expression, invasion, and migration behavior as well as whole-exome and RNA sequencing. Additionally, in vitro drug sensitivity towards standard-of-care chemotherapeutic regimens was evaluated. The pathological and molecular properties of the patients' tumors were preserved in the PDC models HROLu22, HROLu55, and HROBML01. All cell lines expressed HLA I, while none were positive for HLA II. The epithelial cell marker CD326 and the lung tumor markers CCDC59, LYPD3, and DSG3 were also detected. The most frequently mutated genes included TP53, MXRA5, MUC16, and MUC19. Among the most overexpressed genes in tumor cells compared to normal tissue were the transcription factors HOXB9, SIM2, ZIC5, SP8, TFAP2A, FOXE1, HOXB13, and SALL4; the cancer testis antigen CT83; and the cytokine IL23A. The most downregulated genes on the RNA level encode the long non-coding RNA LANCL1-AS1, LINC00670, BANCR, and LOC100652999; the regulator of angiogenesis ANGPT4; the signaling molecules PLA2G1B and RS1; and the immune modulator SFTPD. Furthermore, neither pre-existing therapy resistances nor drug antagonistic effects could be observed. In summary, we successfully established three novel NSCLC PDC models from an adeno-, a squamous cell, and a pleomorphic carcinoma. Of note, NSCLC cell models of the pleomorphic subentity are very rare. The detailed characterization including molecular, morphological, and drug-sensitivity profiling makes these models valuable pre-clinical tools for drug development applications and research on precision cancer therapy. The pleomorphic model additionally enables research on a functional and cell-based level of this rare NCSLC subentity.

Psymberin, a marine-derived natural product, induces cancer cell growth arrest and protein translation inhibition.

Colorectal cancer (CRC) is the third most prevalent form of cancer in the United States and results in over 50,000 deaths per year. Treatments for metastatic CRC are limited, and therefore there is an unmet clinical need for more effective therapies. In our prior work, we coupled high-throughput chemical screens with patient-derived models of cancer to identify new potential therapeutic targets for CRC. However, this pipeline is limited by (1) the use of cell lines that do not appropriately recapitulate the tumor microenvironment, and (2) the use of patient-derived xenografts (PDXs), which are time-consuming and costly for validation of drug efficacy. To overcome these limitations, we have turned to patient-derived organoids. Organoids are increasingly being accepted as a "standard" preclinical model that recapitulates tumor microenvironment cross-talk in a rapid, cost-effective platform. In the present work, we employed a library of natural products, intermediates, and drug-like compounds for which full synthesis has been demonstrated. Using this compound library, we performed a high-throughput screen on multiple low-passage cancer cell lines to identify potential treatments. The top candidate, psymberin, was further validated, with a focus on CRC cell lines and organoids. Mechanistic and genomics analyses pinpointed protein translation inhibition as a mechanism of action of psymberin. These findings suggest the potential of psymberin as a novel therapy for the treatment of CRC.

The Chick Embryo Xenograft Model for Malignant Pleural Mesothelioma: A Cost and Time Efficient 3Rs Model for Drug Target Evaluation.

Malignant pleural mesothelioma (MPM) has limited treatment options and poor prognosis. Frequent inactivation of the tumour suppressors BAP1, NF2 and P16 may differentially sensitise tumours to treatments. We have established chick chorioallantoic membrane (CAM) xenograft models of low-passage MPM cell lines and protocols for evaluating drug responses. Ten cell lines, representing the spectrum of histological subtypes and tumour suppressor status, were dual labelled for fluorescence/bioluminescence imaging and implanted on the CAM at E7. Bioluminescence was used to assess viability of primary tumours, which were excised at E14 for immunohistological staining or real-time PCR. All MPM cell lines engrafted efficiently forming vascularised nodules, however their size, morphology and interaction with chick cells varied. MPM phenotypes including local invasion, fibroblast recruitment, tumour angiogenesis and vascular remodelling were evident. Bioluminescence imaging could be used to reliably estimate tumour burden pre- and post-treatment, correlating with tumour weight and Ki-67 staining. In conclusion, MPM-CAM models recapitulate important features of the disease and are suitable to assess drug targets using a broad range of MPM cell lines that allow histological or genetic stratification. They are amenable to multi-modal imaging, potentially offering a time and cost-efficient, 3Rs-compliant alternative to rodent xenograft models to prioritise candidate compounds from in vitro studies.

DDDR-27. A PATIENT-DERIVED DRUG SCREENING PLATFORM PREDICTS RESPONSE TO TEMOZOLOMIDE AND IDENTIFIES POTENTIAL NEW TREATMENTS FOR GLIOBLASTOMA

Abstract BACKGROUND Major obstacles that have impeded the development of effective new therapies for GBM include inter- and intratumoral heterogeneity, the blood-brain-barrier and use of sub-optimal cell line-based preclinical models. Taking these hurdles into account, we have set up a patient-derived GBM drug-screening platform. We optimized protocols to improve cell culture success rate and retrospectively assessed the predictive power of our assay for patient response to TMZ. A large panel of GBM cells was screened for sensitivity to available oncological agents. Drugs of interest were selected based on favorable physicochemical properties for BBB crossing and potent activity in (a subset of) GBM cultures. Finally, we determined the success rate of performing a small-scale screen with 20 selected agents within 4 weeks of receiving tumor tissue. RESULTS By combining both CUSA and tissue piece-derived dissociation protocols, culture success increased to 95%, ensuring representation of the near-complete spectrum of GBM subtypes. Single-cell sequencing studies confirmed heterogeneity in our low-passage cell cultures. In vitro screening of TMZ on a large cohort (n = 55) identified 3 response categories (responders/intermediates/non-responders) for which Cox regression analysis revealed significantly different overall survival curves of corresponding patients. Screening of 107 FDA-approved anticancer agents on 45 GBM cultures underscored the tremendous intertumoral heterogeneity in drug sensitivities. We identified 20 potent agents each effective at clinically-achievable concentrations in (a subset of) GBM cultures and having favorable BBB penetration properties (CNS-MPO score). Screening of these agents on a per patient basis within 4 weeks of receiving tissue was successful in 18 out of 24 (75%) tested tumors. In the remaining cases the tumor cells grew very slowly and longer culture times were required. CONCLUSION Our drug screening platform offers a tool to predict TMZ response and assess sensitivity to candidate treatments, either for GBM subsets or on a per patient basis.

Genomic and Transcriptomic Analyses of NF1-Mutant Melanoma Identify Potential Targeted Approach for Treatment

There is currently no targeted therapy to treat NF1-mutant melanomas. In this study, we compared the genomic and transcriptomic signatures of NF1-mutant and NF1 wild-type melanoma to reveal potential treatment targets for this subset of patients. Genomic alterations were verified using qPCR, and differentially expressed genes were independently validated using The Cancer Genome Atlas data and immunohistochemistry. Digital spatial profiling with multiplex immunohistochemistry and immunofluorescence were used to validate the signatures. The efficacy of combinational regimens driven by these signatures was tested through invitro assays using low-passage cell lines. Pathogenic NF1 mutations were identified in 27% of cases. NF1-mutant melanoma expressed higher proliferative markers MK167 and CDC20 than NF1 wild-type (P= 0.008), which was independently validated both in The Cancer Genome Atlas dataset (P= 0.01, P= 0.03) and with immunohistochemistry (P=0.013, P= 0.036), respectively. Digital spatial profiling analysis showed upregulation of LY6E within the tumor cells (false discovery rate < 0.01, log2 fold change > 1), confirmed with multiplex immunofluorescence showing colocalization of LY6E in melanoma cells. The combination of MAPK/extracellular signal‒regulated kinase kinase and CDC20 coinhibition induced both cytotoxic and cytostatic effects, decreasing CDC20 expression in multiple NF1-mutant cell lines. In conclusion, NF1-mutant melanoma is associated with a distinct genomic and transcriptomic profile. Our data support investigating CDC20 inhibition with MAPK pathway inhibitors as a targeted regimen in this melanoma subtype.

In-Vitro Use of Verteporfin for Photodynamic Therapy in Glioblastoma

BackgroundStummer et al. established fluorescence-guided surgery (FGS) for glioblastoma (GBM) using 5-aminolevulinic acid (5-ALA). Its metabolite, protoporphyrin IX (PPIX), is also a photosensitizer and can be used for photodynamic therapy (PDT) using a laser beam of 635 nm. The porphyrin derivate verteporfin (VP) was discovered to have properties to penetrate the brain, pharmacologically target glioma cells, and is approved for PDT of choroidal neovascularization in wet age-related macular degeneration at 689 nm. ObjectiveTo elucidate whether GBM cell lines are susceptible to PDT with second-generation photosensitizer VP. MethodsHuman glioma cell lines LN229, HSR-GBM1, and a low-passage patient-derived GBM cell line P1 were treated with variable concentrations of VP for 24 h, followed by PDT at 689 nm using a diode laser light. Cell viability was measured using the MTT assay and VP uptake was measured using a desktop cytometer. ResultsSignificantly higher cell death following PDT with VP compared to VP treatment alone or no treatment was detected in all cell models (LN229, HSR-GBM1, P1). Flowcytometric measurements revealed a concentration-dependent cellular uptake of VP after 24 h incubation up to 99% at 10 µM (HSR-GBM1). ConclusionThis study demonstrates that PDT with VP causes cell death in GBM cells at marginal concentrations. Additionally, red spectrum fluorescence was detected at therapeutic concentrations in all cell lines, validating the cellular uptake of VP in GBM cells. VP, therefore, is not only a potential drug for targeting GBM pharmacologically but can be used as an optical imaging dye in surgery and photosensitizer to make GBM susceptible to PDT.

Abstract 1179: Rational combinations with the dual RAF/MEK inhibitor VS-6766 for treatment of cutaneous melanoma harboring BRAF, NRAS, NF1 or CRAF mutations

Abstract VS-6766 is a unique dual RAF/MEK inhibitor which blocks MEK activity without the compensatory MEK activation that limits the efficacy of MEKi. VS-6766 produced clinical responses as a single agent in gynecological cancers and KRAS mutant non-small cell lung cancer (NSCLC) (Guo Lancet Oncology 2020). Clinical responses were also observed with VS-6766 in combination with the focal adhesion kinase (FAK) inhibitor defactinib in patients with low-grade serous ovarian cancer and KRAS mutant NSCLC. In patients with advanced cutaneous melanoma, mutations in the RAS/RAF/MEK/ERK (MAPK) pathway occur mainly in BRAF (41%), NRAS (27%), NF1 (25%) and CRAF (2.6%) (AACR Genie v10). Although several selective BRAFV600 inhibitors (BRAFi) are FDA-approved alone or in combination with MEK-only inhibitors (MEKi) for melanomas with BRAFV600E/K, there is still a need for agents to improve response rate, duration of response, and tolerability. There are no targeted therapy options for melanoma patients carrying NRAS or NF1 mutations following progression on immune checkpoint inhibitors. Using low passage cell lines derived from patients with metastatic melanoma and extensively profiled for genomic alterations together with commercially available immortalized human melanoma cell lines, all of which carried mutations in the MAPK pathway, we tested the activity of VS-6766 alone or in combination with other agents. In vitro proliferation assays showed that VS-6766 is as potent as BRAFi in BRAFV600E melanoma cell lines and is more potent than pan-RAF inhibitors in melanoma cell lines bearing NRAS, NF1 or CRAF mutations. We next tested rational combinations of VS-6766 with other agents in specific genetic backgrounds. In BRAFV600E melanoma cell lines, combination of VS-6766 with BRAFi (encorafenib, vemurafenib, dabrafenib) showed greater synergy than combination of MEKi (binimetinib, cobimetinib, trametinib) with BRAFi. Since ~65% of BRAF or NRAS mutant melanomas co-express mutations in the PI3K/AKT/mTOR pathway, we tested the combination of VS-6766 with the mTOR inhibitor everolimus. VS-6766 was synergistic with everolimus in reducing the viability of melanoma cells harboring BRAF or NRAS mutations. Because CDK4/6 pathway activation has been correlated with poor progression-free survival in melanoma patients treated with BRAFi combined with MEKi, we tested the combination of VS-6766 with the CDK4/6 inhibitor abemaciclib. We found that VS-6766 was synergistic with abemaciclib in reducing viability of melanoma cell lines. Additional combinations with VS-6766 are currently being tested and will be reported. These preclinical data support clinical testing of VS-6766 in rational combinations for treatment of cutaneous melanoma with BRAF, NRAS, NF1 or CRAF mutations. In clinical trials, a recommended phase 2 dose has been defined for the combination of VS-6766 with everolimus. Citation Format: Antonella Bacchiocchi, Silvia Coma, Sanjib Chowdhury, Mario Sznol, Ruth Halaban, Jonathan A. Pachter. Rational combinations with the dual RAF/MEK inhibitor VS-6766 for treatment of cutaneous melanoma harboring BRAF, NRAS, NF1 or CRAF mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1179.

Abstract 1466: MYC localizes to the nuclear pore to promote DNA repair during replication stress

Abstract Genomic instability is a hallmark of cancer which promotes oncogenic mutations in pancreatic ductal adenocarcinoma (PDAC), leading to more aggressive tumors with greater potential for drug resistance. Deregulation of the master transcription factor, MYC, is found in virtually all PDAC tumors and promotes genomic alterations by increasing replication stress, augmenting DNA repair pathways, and promoting cell survival. To resolve replication stress, stalled forks are trafficked to the nuclear pores where the necessary machinery accumulates. Activated MYC is spatially reorganized to the nuclear pores as well, a mechanism that is exacerbated in cancer. Whether MYC activity at the nuclear pores contributes to resolving replication stress in PDAC remains to be known. To investigate this mechanism, we are creating genomic and proteomic tools to determine MYC’s function at the nuclear pores during replication stress induced by olaparib treatment in PDAC cells. We found that olaparib significantly increases MYC accumulation at the nuclear pore in low-passage cell lines we established from PDAC patient tumors. Furthermore, we showed that MYC interacts with the nuclear pore resident SUMO protease, SENP1, which plays a key role in resolving stalled forks. Our preliminary data suggest a role for MYC resolving replication stress at nuclear pores, and could indicate MYC activity as a therapeutic target in olaparib-resistant PDAC. Citation Format: Gabriel Cohn, Colin J. Daniel, Daniel F. Liefwalker, Rosalie C. Sears. MYC localizes to the nuclear pore to promote DNA repair during replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1466.

The identification of the anthracycline aclarubicin as an effective cytotoxic agent for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer, mainly due to its delayed diagnosis and lack of effective therapeutic options. Therefore, it is imperative to find novel treatment options for PDAC. Here, we tested a series of conventional chemotherapeutics together with anthracycline compounds as single agents or in combination, determining their effectivity against established commercial and patient-derived, low-passage PDAC cell lines. Proliferation and colony formation assays were performed to determine the anticancer activity of anthracyclines; aclarubicin and doxorubicin, on commercial and patient-derived, low-passage PDAC cell lines. In addition, the effect of standard-of-care drugs gemcitabine and individual components of FOLFIRINOX were also investigated. To evaluate which mechanisms of cell death were involved in drug response, cleavage of poly(ADP-ribose)polymerase was evaluated by western blot. Aclarubicin showed superior antitumor activity compared to other anthracyclines and standard of care drugs (gemcitabine and individual components of FOLFIRINOX) in a patient-derived, low-passage PDAC cell line and in commercial cell lines. Importantly, the combination of gemcitabine and aclarubicin showed a synergistic effect at a dose range where the single agents by themselves were ineffective. In parallel, evaluation of the antitumor activity of aclarubicin demonstrated an apoptotic effect in all PDAC cell lines. Aclarubicin is cytotoxic for commercial and patient-derived low-passage PDAC cell lines, at doses lower than peak serum concentrations for patient treatment. Our findings support a (re)consideration of aclarubicin as a backbone of new combination regimens for pancreatic cancer patients.

Microsatellite Status and IκBα Expression Levels Predict Sensitivity to Pharmaceutical Curcumin in Colorectal Cancer Cells.

Simple SummaryThe global burden of colorectal cancer is high. Chemotherapy has been the backbone of colorectal cancer therapy for decades. Toxic side effects and frequently occurring drug resistances remain challenging problems. Therefore, exploring natural compounds with low or even no toxicity holds great potential. However, natural curcumin is poorly absorbed, limiting its clinical use. Therefore, our focus was to screen different molecular types of colorectal cancer to find the ones with the highest sensitivity to curcumin. We observed very individual responses to curcumin for various colorectal cancer cell lines. Most curcumin-sensitive cell lines were of the microsatellite-stable molecular type, and expressed high baseline levels of the IκBα protein. Contrarily, curcumin-resistant lines were mainly microsatellite instable, with low baseline IκBα levels. Considering all of the data obtained, we conclude that patients with microsatellite-stable tumors and high baseline IκBα protein expression would benefit from treatment with novel curcumin formulations and derivatives.Clinical utilization of curcumin in colorectal cancer (CRC) was revived as a result of the development of novel curcumin formulations with improved bioavailability. Additionally, identification of biomarkers for curcumin sensitivity would also promote successful clinical applications. Here, we wanted to identify such biomarkers in order to establish a predictive model for curcumin sensitivity. Thirty-two low-passage CRC cell lines with specified tumor characteristics were included. Curcumin suppressed cell proliferation, yet sensitivity levels were distinct. Most curcumin-sensitive CRC cell lines were microsatellite stable and expressed high levels of IκBα. The predictive capacity of this biomarker combination possessed a statistical significance of 72% probability to distinguish correctly between curcumin-sensitive and -resistant CRC cell lines. Detailed functional analyses were performed with three sensitive and three resistant CRC cell lines. As curcumin’s mode of action, inhibition of NF-κB p65 activation via IκBα was identified. In consequence, we hypothesize that novel curcumin formulations—either alone or, more likely, in combination with standard therapeutics—can be expected to prove clinically beneficial for CRC patients with high IκBα expression levels.

Abstract PO-081: Studying MYC's contribution to replication stress at the nuclear pore

Abstract Genomic instability is a hallmark of cancer which promotes oncogenic mutations in pancreatic ductal adenocarcinoma (PDAC), leading to more aggressive tumors with greater potential for drug resistance. Deregulation of the master transcription factor, MYC, is found in virtually all PDAC tumors and promotes genomic alterations by increasing replication stress, augmenting DNA repair pathways, and promoting cell survival. To resolve replication stress, stalled forks are trafficked to the nuclear pores where the necessary machinery accumulates. Activated MYC is spatially reorganized to the nuclear pores as well, a mechanism that is exacerbated in cancer. Whether MYC activity at the nuclear pores contributes to resolving replication stress in PDAC remains to be known. To investigate this mechanism, we are creating genomic and proteomic tools to determine MYC’s function at the nuclear pores during replication stress induced by olaparib treatment in PDAC cells. We found that olaparib significantly increases MYC accumulation at the nuclear pore in low-passage cell lines we established from PDAC patient tumors. Furthermore, we showed that MYC interacts with the nuclear pore resident SUMO protease, SENP1, which plays a key role in resolving stalled forks. Our preliminary data suggest a role for MYC resolving replication stress at nuclear pores, and could indicate MYC activity as a therapeutic target in olaparib-resistant PDAC. Citation Format: Gabriel M. Cohn, Colin J. Daniel, Daniel F. Liefwalker, Rosalie C. Sears. Studying MYC's contribution to replication stress at the nuclear pore [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-081.

Anti-invasive efficacy and survival benefit of the YAP-TEAD inhibitor verteporfin in preclinical glioblastoma models.

Glioblastoma (GBM) remains a largely incurable disease as current therapy fails to target the invasive nature of glioma growth in disease progression and recurrence. Here, we use the FDA-approved drug and small molecule Hippo inhibitor Verteporfin (VP) to target YAP-TEAD activity, known to mediate convergent aspects of tumor invasion/metastasis, and assess the drug's efficacy and survival benefit in GBM models. Up to 8 low-passage patient-derived GBM cell lines with distinct genomic drivers, including 3 primary/recurrent pairs, were treated with VP or vehicle (VEH) to assess in vitro effects on proliferation, migration, invasion, YAP-TEAD activity, and transcriptomics. Patient-derived orthotopic xenograft (PDX) models were used to assess VP's brain penetrance and effects on tumor burden and survival. VP treatment disturbed YAP/TAZ-TEAD activity; disrupted transcriptome signatures related to invasion, epithelial-to-mesenchymal, and proneural-to-mesenchymal transition, phenocopying TEAD1-knockout effects; and impaired tumor migration/invasion dynamics across primary and recurrent GBM lines. In an aggressive orthotopic PDX GBM model, short-term VP treatment consistently diminished core and infiltrative tumor burden, which was associated with decreased tumor expression of Ki67, nuclear YAP, TEAD1, and TEAD-associated targets EGFR, CDH2, and ITGB1. Finally, long-term VP treatment appeared nontoxic and conferred survival benefit compared to VEH in 2 PDX models: as monotherapy in primary (de novo) GBM and in combination with Temozolomide chemoradiation in recurrent GBM, where VP treatment associated with increased MGMT methylation. We demonstrate combined anti-invasive and anti-proliferative efficacy for VP with survival benefit in preclinical GBM models, indicating potential therapeutic value of this already FDA-approved drug if repurposed for GBM patients.

Abstract 3014: A preclinical platform of PDX-derived cell lines as a tool for pharmacological screening and functional studies

Abstract Despite considerable progress in understanding the biology and genetics of cancer progression, the development of effective therapies against cancer need physiological and predictive preclinical models. In this context, patient-derived xenograft (PDX) models has become a standard tool as they reproduce accurately the behavior of tumor of origin, in term of histological and molecular phenotype and response to chemotherapy. Although PDXs in vivo models are indispensable for preclinical studies, they suffer from some limitations due to study costs related to tumor maintenance on mice, variable engraftment rate, growth delay and limited throughput for large-scale drug screening. To address this problem and propose a time a cost effective preclinical screening tool, we developed a panel of PDX-derived low-passage 2D cell lines as a convenient in vitro pre-screening platform to profile compound activity. Different PDX models including breast, lung, colon, melanoma, glioblastoma and hepatoblastoma were tested for their capacity to generate cell lines maintaining the characteristics of the parental PDX tumor and usable for in vitro assays. Today, we succeeded with a series of 50 PDX models with 83% success rate. Tumor cells isolated from PDX tumor tissue were cultured under different media and matrix conditions, allowing at least 5 passages in culture. A Short Tandem Repeat (STR) comparison profile was done with the parental PDX before performing a master bank. We performed short term 2D cytotoxicity assays and long term colony assays to compare cell lines in vitro drug sensitivity with their parental PDX in vivo drug response and overall, the results show that this panel reproduced the drug response profile of the original PDXs with chemotherapies, PARP inhibitors, an ADC (T-DM1) and FGFR-targeting therapies. Moreover, cellular models engrafted back onto mice showed in vivo response to chemotherapies similar to that of the parental PDX confirming the identical behavior of cell line/PDX couples. As the use of cellular models is still considered as a standard for early preclinical test to evaluate drug response before moving to in vivo assays, our PDX-derived cell line platform appeared to be a robust and relevant tool. Furthermore, since the main concern when using in vitro models is the representativeness of the results obtained when transposed to in vivo models, the similarities between cell lines and parental PDX should maximize success of further in vivo preclinical drug development. Citation Format: Olivier Deas, Sophie Banis, Kathleen Flosseau, Lea Sinayen, Enora Le Ven, Jean-Gabriel Judde, Stefano Cairo. A preclinical platform of PDX-derived cell lines as a tool for pharmacological screening and functional studies [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 3014.

Compound NSC84167 selectively targets NRF2-activated pancreatic cancer by inhibiting asparagine synthesis pathway

Nuclear factor erythroid 2-related factor 2 (NRF2) is aberrantly activated in about 93% of pancreatic cancers. Activated NRF2 regulates multiple downstream molecules involved in cancer cell metabolic reprogramming, translational control, and treatment resistance; however, targeting NRF2 for pancreatic cancer therapy remains largely unexplored. In this study, we used the online computational tool CellMinerTM to explore the NCI-60 drug databases for compounds with anticancer activities correlating most closely with the mRNA expression of NQO1, a marker for NRF2 pathway activity. Among the >100,000 compounds analyzed, NSC84167, termed herein as NRF2 synthetic lethality compound-01 (NSLC01), was one of the top hits (r = 0.71, P < 0.001) and selected for functional characterization. NSLC01 selectively inhibited the viabilities of four out of seven conventional pancreatic cancer cell lines and induced dramatic apoptosis in the cells with high NRF2 activation. The selective anticancer activity of NSLC01 was further validated with a panel of nine low-passage pancreatic patient-derived cell lines, and a significant reverse correlation between log(IC50) of NSLC01 and NQO1 expression was confirmed (r = −0.5563, P = 0.024). Notably, screening of a panel of nine patient-derived xenografts (PDXs) revealed six PDXs with high NQO1/NRF2 activation, and NSLC01 dramatically inhibited the viabilities and induced apoptosis in ex vivo cultures of PDX tumors. Consistent with the ex vivo results, NSLC01 inhibited the tumor growth of two NRF2-activated PDX models in vivo (P < 0.01, n = 7–8) but had no effects on the NRF2-low counterpart. To characterize the mechanism of action, we employed a metabolomic isotope tracer assay that demonstrated that NSLC01-mediated inhibition of de novo synthesis of multiple amino acids, including asparagine and methionine. Importantly, we further found that NSLC01 suppresses the eEF2K/eEF2 translation elongation cascade and protein translation of asparagine synthetase. In summary, this study identified a novel compound that selectively targets protein translation and induces synthetic lethal effects in NRF2-activated pancreatic cancers.

Targeting CD30 Expression in Adult T-Cell Leukemia-Lymphoma (ATLL)

▪Introduction:Adult T-cell leukemia-lymphoma (ATLL) is caused by HTLV-1, and carries a dismal prognosis urging the development of new therapies. CD30 expression has been reported at variable frequencies in ATLL. The anti-CD30 monomethyl-auristatin-E conjugate brentuximab vedotin (BV), can be an effective treatment option for Hodgkin lymphoma, anaplastic T-cell lymphomas, and various peripheral T-cell lymphoma types. However, the impact role of BV in ATLL has not been established, which warrants an in-depth characterization of CD30 expression and clinical investigation of BV in this disease.Methods:ATLL specimens from 107 patients (pts) were classified according to Shimoyama criteria (A=acute, C=chronic, L=lymphomatous, and S=smoldering) and analyzed for CD30 expression. Unfavorable chronic (UC) had elevated LDH level < twice the normal value. Cases were confirmed or analyzed by immunohistochemistry at our institution using anti-human CD30 antibody (Dako) on formalin-fixed paraffin-embedded tissue or cellblock sections prepared from cytospun CD4+-enriched leukemic cells. The cutoff positive value was 20%. Outcomes were compared according to prior treatment. Soluble(s) CD30 levels were measured in sera of 21 pts by ELISA. Low-passage ATLL cell lines (ATLL-84c and ATLL-97c established from pts with A-type) were treated with BV, and apoptosis was measured by annexin V and FACS analysis. Five pts with relapsed CD30+ ATLL (L-type=3, A-type=2) were treated with BV.Results:ATLL specimens obtained from 95 (L=51, A=37, UC=6, and S=1) untreated pts, and 13 (L=7, and A=6) pre-treated patients were analyzed. The percent frequencies of CD30+ cases within untreated subtypes were L=41%, A=30%, UC=17%, and S=0%, as compared to L=14% and A=17% in the pre-treated group (Figure 1). There was a trend towards a higher frequency of CD30 expression in L-type vs. A-type (p=0.37, two-sided Fisher's test). Sera analyzed from 21 pts revealed positive CD30 levels regardless of CD30tumor status, suggesting ATLL cells may shed the receptor (Figure 2). The overall response (OR) and complete response (CR) rates in A/UC-type pts (n=31) treated with AZT (zidovudine) plus interferon-α (AI) were 59% (13/22) and 32% (7/22) in the CD30- group (n=22), compared to 33% (3/9) and 0% in CD30+ group (n=9) respectively (Figure 3). BV had no significant clinical significant impact in 4 out of 5 (80%) CD30+ treated pts (2 L and 2 A). A transient hematologic response was observed in one CD30+ A-type patient, followed by leukemic progression with CD30- cells (Figure 4). By contrast, BV induced a sustained CR lasting 2 years in an L-type patient with diffuse homogenously CD30+ expression (Figure 5). In vitro treatment of patient-derived ATLL cell lines confirmed BV induced more apoptosis in cells expressing higher CD30 (ATLL-84c) as compared to cells low in CD30 expression (ATLL97c).Conclusions:CD30 expression in ATLL is heterogeneous and occurs at variable frequencies, and tends to be higher in L-type, and lower in A/UC-types that respond to AZT-interferon therapy. Our data suggest CD30 may represent an elusive target in A-type ATLL, thus warranting further optimization of BV in leukemic subtypes. The analysis of additional cases is still ongoing and the final results will be presented at the meeting.This project has been accomplished with the support of “ASH HONORS award” achieved in 2016. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Histone Deacetylase Inhibitors Abolish HBZ Protein Expression and Induce Cell Death in HTLV-1 Related Adult T-Cell Leukemia-Lymphoma

Background:Adult T-cell leukemia-lymphoma (ATL) is a malignancy with dismal prognosis caused by HTLV-1. ATL is incurable by conventional drugs. Histone deacetylase (HDAC) inhibitors (HDIs) are broadly active anti-neoplastic agents that can be cautiously exploited in the treatment of viral related lymphomas. HTLV-1 usually establishes lifelong latency in human CD4+ T-cells, which in turn propagate and persist as result of the multifaceted functions of HTLV-1 basic zipper factor (HBZ) through various signaling pathways that promote growth and proliferation, and block apoptosis and cell senescence. All ATL cells harbor at least one copy of latent intact HTLV-1 provirus in most cases, and unlike Tax, all tumors express HBZ from the negative 3‘ proviral strand via SP-1 activity. The HTLV-1 promoter is negatively regulated by HDACs at the 5‘ long terminal repeat (LTR), and by HBZ, which disrupts Tax positive interaction with the transcriptional activator p300/CBP, thus blocking viral transcription. Therefore, treatment of HTLV-1 infected cells with HDIs can induce expression of Tax protein, which is highly immunogenic, and which could promote immune-mediated clearance of ATL cells in vivo.Methods:Low-passage IL-2 dependent ATL cell lines established from patients with acute type ATL and freshly isolated ATL cells were treated with several HDIs (VPA, sodium butyrate, vorinostat, belinostat, panabinostat, and mocetinostat) at various doses and time points. Apoptosis was measured by annexin V and FACS analysis. Expression of HBZ protein, Tax, acetyl and total histone 3 subunits (H3), BIM, caspase 3, caspase 9, Jun B, c-Jun, Jun D, and Sp-1 were measured by Western blots before and after HDI treatment. HBZ and Tax mRNA expression was measured by quantitative RT-PCR. SP-1 binding to HTLV-1 specific oligonucleotide consensus sequence was measured by gel shift assay using nuclear protein extracts before and after HDI treatment.Results:HDIs sequentially increased acetylated H3 levels, blocked HBZ protein, upregulated Tax expression, stabilized BIM, and induced cleavage of pro-caspases 9 and 3 (intrinsic apoptotic pathway), resulting in apoptosis of ATL cells. HDIs tested (VPA and belinostat) blocked expression of Jun-B, which is known to be upregulated by HBZ, while no significant effects were observed on c-Jun and Jun D protein levels. HDIs tested (VPA, belinostat, and panabinostat) did not significantly alter spliced-HBZ RNA levels or affect SP-1 binding to HBZ promoter-specific oligonucleotide sequence, suggesting a post-translational mechanism may contribute to abrogation of HBZ in ATL cells.Conclusions:Our data demonstrate that HDIs target ATL cells in part by abrogating HBZ protein, which could negatively impact the longevity of ATL and HTLV-1 cells in vivo. Long-term clinical use of HDIs might be beneficial for eliminating residual ATL cells after definitive treatment. A pilot clinical trial testing belinostat-based consolidation therapy for aggressive leukemic ATL types is currently ongoing at our institution (available at Clinicaltrials.gov , NCT02737046). DisclosuresNo relevant conflicts of interest to declare.

Heat Shock Protein 90 Inhibitor Effects on Pancreatic Cancer Cell Cultures.

Pancreatic ductal adenocarcinoma is one of the deadliest cancers for which few curative therapies are available to date. Heat shock protein 90 (Hsp90) inhibitors have shown activity against numerous cancers in vitro; therefore, we tested whether they could be used to target pancreatic ductal adenocarcinoma. Inhibitors of Hsp90 ATPase activity were applied on low-passage pancreatic cell line cultures (Panc10.05, Panc215, A6L) in a dose-response manner, and the inhibitor in vitro effect on cell growth was evaluated. Seven of novel Hsp90 inhibitors based on resorcinol fragment and 5 commercially available Hsp90 inhibitors (17-AAG, AT-13387, AUY-922, ganetespib, and rifabutin) as well as control compound triptolide were tested yielding IC50 values in 2- and 3-dimensional assays. The novel Hsp90 inhibitors exhibited strong effects on all 3 tested pancreatic cell line cultures (Panc10.05, Panc215, A6L) reaching the IC50 of 300 to 600 nM in 2- and 3-dimensional assays. Novel Hsp90 inhibitors can be developed as antipancreatic cancer agents. Their chemical structures are simpler, and they are likely to exhibit lower side effects than the much more complex inhibitors used as controls.