Cancer Cell Line Models Research Articles

Synthesis and biological evaluation of novel demethylzeylasteral derivatives as potential anticancer agents

Demethylzeylasteral (DEM), a class of terpenoids isolated from natural plants, frequently exhibits moderate or limited inhibitory effect on tumor growth across multiple cancer types. Thus, here we attempted to elevate the anti-tumor efficacy of DEM by altering active groups in its chemical structure. Initially, we synthesized a series of novel DEM derivatives 1–21 through performing a series of modifications of its phenolic hydroxyl groups at C-2/3, C-4 and C-29 positions. The anti-proliferative activities of these new compounds were subsequently assessed using three human cancer cell line models (A549, HCT116 and HeLa) and CCK-8 assay. Our data showed that compared to original DEM compound, derivative 7 exhibited remarkable inhibition effect on A549 (16.73 ± 1.07 μM), HCT116 (16.26 ± 1.94 μM) and HeLa (17.07 ± 1.09 μM), almost reaching to the same level of DOX. Moreover, the structure-activity relationships (SARs) of the synthesized DEM derivatives were discussed in detail. We found that treatment with derivative 7 only led to moderate cell cycle arrest at S-phase in a concentration-dependent manner. Meanwhile, derivative 7 treatment markedly induced apoptosis in tumor cells. Consistent with this observation, our subsequent docking analysis showed that derivative 7 is capable of activating caspase-3 through interaction with the His 121 and Gly 122 residues of the enzyme. Overall, we have developed a new series of DEM derivatives with elevated anti-tumor efficacy relative to its parent form. The results suggested that derivative 7 has great potential to be employed as an anticancer agent candidate for natural product-based cancer chemotherapy.

Abstract 3411: Proof of concept study of next generation drug screening platform for glioma patients

Abstract Precision medicine endeavors to match patients to therapies mostly on the basis of the genomic alterations in their tumors. However, it has been shown that ‘genomics only’ approach is often insufficient for best drug selection. Functional precision medicine has been recently emerging as it provides directly translatable information on which drug to choose among available drugs. Traditionally, cancer drugs are tested in cancer cell line models, but cell lines cannot represent individual clinical patients and are too biologically pronounced to be useful for drug screening purposes. Our patient-derived cell (PDC) drug screening system overcomes these points and can generally also correlate with genome changes. AVATAMED and CBmed, together with AimedBio, are collaborating in a Precision Cancer Project, set out next generation drug screening platform to screen Austrian glioma patients for proof-of-concept study. Fresh native tissue from both low- and high-grade glioma are collected. Tumor tissue is used for PDC isolation using mechanical and enzymatic tissue dissociation. To preserve the original tumor similarity, tissue is short term cultured under two weeks, and PDCs are seeded and treated with a panel of clinical- and preclinical drugs followed by viability assessment. We have cultured 65 PDCs from both low- and high-grade glioma. Tissue culture success rate and drug screening success rate was >85% suggesting that specimen logistics and tissue processing are well-established and conducted. PDCs were treated with 79 drugs that are commonly used in clinical setting. In order to evaluate the quality of the results, the screening was conducted at two different sites, AimedBio and CBmed, and the data were compared. Overall, high accuracy and reproducibility were confirmed by low Coefficient of Variation (CV) between replicates and high Z-factor between negative and positive controls. In addition, data generated from both sites showed high correlations. Collectively, our next-generation drug screening platform shows the potential to give the best therapeutic options to glioma patients. To this end, we are moving forward use this platform in clinical study. Citation Format: Nam-Gu Her, Amin El-Heliebi, Gi Ju Lee, San Ha Park, Barbara Prietl, Stephanie S. Ahn, Hong Boon Toh, Thomas R. Pieber, Do-Hyun Nam. Proof of concept study of next generation drug screening platform for glioma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3411.

Abstract 3878: Targeting Hippo-YAP, BRD4 and RAS-MAPK interplay in lung cancer to forestall drug resistance

Abstract Introduction: Identifying parallel survival pathways or compensatory mechanisms that limit the response to targeted therapy is critical to discover effective combinatorial strategies for the treatment of lung cancer and to prevent emergence of drug resistance Results: Using combination drug screening in a panel of cell line models of non-small cell lung cancer (NSCLC), we identified the tumor suppressor STK11 (aka LKB1) as a molecular biomarker of sensitivity to BET and RAS-MAPK pathway inhibitors combination in NSCLC. We found a small molecule inhibitor of BET (bromodomain and extra terminal) family proteins (BRD4, 3, 2), PLX51107, suppressed BRAF and KRAS mutant NSCLC cell proliferation and in combination with RAS (KRAS G12C), RAF or MEK inhibitor led to pronounced loss in cell viability with potent MYC downregulation and synergistic apoptosis induction in vitro and potent tumor regression or stasis in vivo in several KRAS mutant CDX and PDX mouse models of NSCLC. However, BRAF and KRAS mutant NSCLC cells with co-occurring mutations in STK11 were resistant to BET and RAS, RAF or MEK co-inhibition. To unravel the underlying mechanism of resistance to BET and RAS-MAPK inhibitors upon STK11 loss we modeled STK11 deficiency in BRAF and KRAS mutant NSCLC cells and tumors. We found that STK11 loss creates an unprecedented dependence on YAP/TEAD TFs. STK11 silencing in BRAF and KRAS mutant NSCLC cells resulted in nuclear transfer/activation of the HIPPO pathway effector, YAP, upregulation of the YAP target BIRC5 (aka Survivin) and rescued the synthetic lethality due to BET and RAS, RAF or MEK co-inhibition. Phenocopying STK11 loss, constitutively active YAP (YAP5SA), but not transcriptionally inactive YAP (YAPS94A), mitigated the deleterious effects of BET and RAS, RAF or MEK co-suppression in BRAF and KRAS mutant NSCLC cells with potent upregulation of the pro-survival factor BIRC5. Conversely, we found that co-silencing YAP or TEADs with BETs re-sensitized BRAF and KRAS mutant NSCLC cells with co-occurring mutations in STK11 to BET and RAS-MAPK inhibitors combination and caused synthetic lethality. Conclusion: This study uncovered a functional interaction between BET proteins, STK11, HIPPO-YAP and RAS-MAPK signaling pathways in lung cancer. These results will help identify combinatorial strategies for treating aberrant RAS-MAPK pathway driven lung cancer more effectively and forestall drug resistance. Citation Format: Nilanjana Chatterjee, Victor Olivas, Wei Wu, Ben Powell, Trever Bivona. Targeting Hippo-YAP, BRD4 and RAS-MAPK interplay in lung cancer to forestall drug resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3878.

Abstract 1413: Chromosome arm aneuploidies as genetic vulnerabilities of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. It has no approved targeted therapies due to the lack of the expression of key biomarkers, namely Estrogen and Progesterone receptors, and HER2 amplification. As a result, TNBC has the worst prognosis compared to other breast cancer subtypes. We previously showed that chromosome 4p (chr4p) loss is recurrent, correlates with poor prognosis, is an early event in tumor evolution, and confers a proliferative advantage. Here, we propose to leverage chr4p loss as a genetic vulnerability of TNBC by identifying its genetic interactions (GIs) with genes whose inactivation leads to synthetic lethality i.e. loss of viability of chr4p copy loss cells, but not of the chr4p copy neutral cells, or suppression i.e., positive GIs. We first classified breast cancer cell lines with TNBC enriched molecular subtype called basal-like, as chr4p copy loss (9) and chr4p copy neutral (7), using copy number data from CCLE. Associated with chr4p copy loss, the differential expression analysis carried out using CCLE data revealed transcriptomic changes that distinctly cluster the chr4p copy loss and chr4p copy neutral cell lines. We then used the publicly available CRISPR-based genome-wide gene inactivation data from the DepMap project and quantified chr4p loss-associated GIs using multiple scoring methods such as drugZ and MAGeCK. In total, we identified ~200 negative and ~70 positive statistically significant GIs involving genes annotated to biological processes such as translation, metabolism, and others. We have prioritized a subset of them for subsequent experimental validation using CRISPR-Cas9-based gene editing in TNBC cancer cell line models. Alongside, using the publicly available drug sensitivity data obtained from the PRISM project, we identified compounds that impair cell growth in a chr4p loss-specific manner, the strongest of which targeted redox balance. Collectively, the set of GIs, the compound sensitivities, and the computational methods we have generated are unique resources for developing precision oncology therapeutic strategies for TNBC, as well as for other cancers harboring chr4p loss. Citation Format: Rohan Dandage, Michael Schwartz, Lynn Karam, Alain Pacis, Traver Hart, Guillaume Bourque, Morag Park, Elena Kuzmin. Chromosome arm aneuploidies as genetic vulnerabilities of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1413.

Abstract 6143: Novel multiplexed assays of ferroptosis, pyroptosis and necroptosis biomarkers for translational studies

Abstract Background: The recent discovery that multiple chemo- and immuno-therapies activate non-apoptotic cell death pathways such as necroptosis, pyroptosis, and ferroptosis in cancer cells has necessitated development of pharmacodynamic biomarker assays to study their novel mechanisms of action. We describe the development, analytical validation, and proof-of-concept demonstration of assays for potential biomarkers of ferroptosis, pyroptosis and necroptosis pathways to support anti-cancer drug development. Methods: We developed multiplex sandwich immunoassays on the Luminex platform to quantify transferrin receptor (TfR), glutathione peroxidase 4 (GPx4), active caspase-3, cleaved and full-length gasdermins D and E (GSDMD, GSDME), MLKL oligomers, and MLKL heterodimers with RIPK1 and RIPK3. Novel antibodies to gasdermin cleavage neoepitopes were generated and their specificity validated with knockout (GSDMD) or non-expressing (GSDME) cell lines. We utilized well-established in vitro cancer cell line models (NCI-H522, THP-1, MV411, MB231, HT29) and inducers/inhibitors of pyroptosis, necroptosis and ferroptosis to test for expected directional changes of biomarker modulation. Results: Analytical validity of the measurements is demonstrated with acceptable reproducibility, precision, dilutional linearity, dilutional recovery, spike recovery, sample freeze-thaw and temperature stability. The fitness-for-purpose of the assays is demonstrated by increased cleavage of GSDME, loss of full-length GSDME, and activation of caspase-3 in NCI-H522 cells treated with doxorubicin; similarly, cleavage of GSDMD and loss of full-length GSDMD is shown in THP-1 cells primed with LPS and treated with nigericin, and in MV-4-11 cells treated with val-boroPro. HT29 cells treated with ZVAD + birinapant + TNFα were used to demonstrate increased MLKL oligomerization or heterodimer formation with RIPK1/RIPK3. Modulation of ferroptosis biomarkers TfR and GPx4 (SU-DHL-5, MDA-MB-231) in response to imidazole ketone erastin (IKE) or RSL3 was cell line-dependent. Studies are underway to validate modulation of select ferroptosis biomarkers using orthogonal LC-MS methods. Conclusions: We describe development and translational readiness of novel multiplex assays to interrogate drugs inducing ferroptosis, pyroptosis and necroptosis cell death pathways. Our data demonstrate utility of biomarkers of pyroptosis and necroptosis, whereas biomarkers of ferroptosis require further appraisal. In vivo pharmacodynamic studies of pyroptosis, ferroptosis and necroptosis inducers are ongoing to verify assay fitness-for-purpose with tumor biopsy specimens. This project was funded with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: William G. Herrick, Jeevan Govindharajulu, Ralph E. Parchment, James H. Doroshow, Apurva K. Srivastava. Novel multiplexed assays of ferroptosis, pyroptosis and necroptosis biomarkers for translational studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6143.

Abstract 4783: ADO is essential for redox homeostasis in liver cancer

Abstract 2-Aminoethanethiol dioxygenase (ADO) is a thiol dioxygenase that plays a direct role in both metabolism and protein stability. Oxidation of the metabolite cysteamine produces hypotaurine, while oxidation of N-terminal cysteines targets protein substrates for N-degron pathway-mediated degradation. Despite these known functions, the (patho)physiological roles of ADO remain obscure. By analyzing TCGA datasets we discovered that high ADO expression is associated with poor outcome for patients with hepatocellular carcinoma (HCC) (HR 1.2, p<0.001). HCC is linked to viral hepatitis, alcohol, metabolic syndrome, non-alcoholic fatty liver disease and chronic inflammation. It is the third leading cause of cancer-related deaths worldwide and is significantly more common in males than females. With poor outcomes, there is a need to better understand HCC biology and vulnerabilities. To assess the functional roles of ADO, we created ADO knockout mice as well as two liver cancer cell line models where ADO was depleted by siRNA, doxycycline-inducible shRNA, or CRISPR/Cas9. While male ADO knockout mice were viable, fertile and healthy, depletion of ADO in liver cancer models significantly reduced cancer cell proliferation and colony formation. ADO was also essential for supporting xenograft growth when implanted subcutaneously in immune-compromised mice. Taken together, this suggested that ADO depletion represents a cancer-specific vulnerability. Comprehensive metabolomic, transcriptomic and proteomic characterization of HCC cells isogenic for ADO demonstrated that loss of ADO resulted in dysregulation of glutathione, ascorbate, polyamine and proline metabolism. Consistent with this, ADO depleted cells had high levels of reactive oxygen species (ROS) measured by CellROX flowcytometry, and an increased ratio of oxidized to reduced glutathione, indicative of oxidative stress. Since mitochondria represent a substantial source of ROS, we assessed mitochondrial mass and function using MitotrackerTM flow cytometry and Seahorse stress tests, respectively. ADO depleted cancer cells had higher mitochondrial mass and higher maximal respiration rates compared to control cells. Finally, exogenously supplied antioxidants could rescue the survival of HCC cells with ADO depletion, demonstrating that the observed loss of viability is due to oxidative stress. This work shows that ADO is essential for redox homeostasis in liver cancer models and suggests that interfering with ADO function may represent a novel targeting strategy for HCC. Citation Format: Sandy C-E Lee, Andrea H. Pyo, Helia Mohammadi, Ji Zhang, Anna Dvorkin-Gheva, Lucie Malbeteau, Stephen Chung, Shahbaz Khan, Thomas Kislinger, Julie A. Reisz, Courtney Jones, Marianne Koritzinsky. ADO is essential for redox homeostasis in liver cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4783.

Transcription factor NKX2–1 drives serine and glycine synthesis addiction in cancer

BackgroundOne-third of cancers activate endogenous synthesis of serine/glycine, and can become addicted to this pathway to sustain proliferation and survival. Mechanisms driving this metabolic rewiring remain largely unknown.MethodsNKX2–1 overexpressing and NKX2–1 knockdown/knockout T-cell leukaemia and lung cancer cell line models were established to study metabolic rewiring using ChIP-qPCR, immunoblotting, mass spectrometry, and proliferation and invasion assays. Findings and therapeutic relevance were validated in mouse models and confirmed in patient datasets.ResultsExploring T-cell leukaemia, lung cancer and neuroendocrine prostate cancer patient datasets highlighted the transcription factor NKX2–1 as putative driver of serine/glycine metabolism. We demonstrate that transcription factor NKX2–1 binds and transcriptionally upregulates serine/glycine synthesis enzyme genes, enabling NKX2–1 expressing cells to proliferate and invade in serine/glycine-depleted conditions. NKX2–1 driven serine/glycine synthesis generates nucleotides and redox molecules, and is associated with an altered cellular lipidome and methylome. Accordingly, NKX2–1 tumour-bearing mice display enhanced tumour aggressiveness associated with systemic metabolic rewiring. Therapeutically, NKX2–1-expressing cancer cells are more sensitive to serine/glycine conversion inhibition by repurposed anti-depressant sertraline, and to etoposide chemotherapy.ConclusionCollectively, we identify NKX2–1 as a novel transcriptional regulator of serine/glycine synthesis addiction across cancers, revealing a therapeutic vulnerability of NKX2–1-driven cancers.Transcription factor NKX2–1 fuels cancer cell proliferation and survival by hyperactivating serine/glycine synthesis, highlighting this pathway as a novel therapeutic target in NKX2–1-positive cancers.

Identification of IRAK1BP1 as a candidate prognostic factor in lung adenocarcinoma.

Lung cancer is one of the major causes of cancer-related mortality worldwide. High-throughput RNA sequencing (RNA-seq) of surgically removed tumors has been used to identify new biomarkers of lung cancer; however, contamination by non-tumor cells in the tumor microenvironment significantly interferes with the search for novel biomarkers. Tumor organoids, as a pre-clinical cancer model, exhibit similar molecular characteristics with tumor samples while minimizing the interference from other cells. Here we analyzed six RNA-seq datasets collected from different organoid models, in which cells with oncogenic mutations were reprogrammed to mimic lung adenocarcinoma (LUAD) tumorigenesis. We uncovered 9 LUAD-specific biomarker genes by integrating transcriptomic data from multiple sources, and identified IRAK1BP1 as a novel predictor of LUAD disease outcome. Validation with RNA-seq and microarray data collected from multiple patient cohorts, as well as patient-derived xenograft (PDX) and lung cancer cell line models confirmed that IRAK1BP1 expression was significantly lower in tumor cells, and had no correlation with known markers oflung cancer prognosis. In addition, loss of IRAK1BP1 correlated with the group of LUAD patients with worse survival; and gene-set enrichment analysis using tumor and cell line data implicated that high IRAK1BP1 expression was associated with suppression of oncogenic pathways. In conclusion, we demonstrate that IRAK1BP1 is a promising biomarker of LUAD prognosis.

Abstract P3-07-16: Pre-clinical study of amcenestrant and HER2-targeted therapies in HER2+/ER+ breast cancer cell line models

Abstract Background: Up to 70% of HER2 over-expressing (HER2+) breast cancers also express estrogen receptor (ER). Dual targeting of HER2 and ER has shown pre-clinical and clinical benefit in HER2+/ER+ breast cancer. Rationale for these combinations is based on circumvention of resistance mediated by crosstalk between HER2 and ER signalling pathways. Fulvestrant is the only selective estrogen receptor degrader (SERD) approved by the FDA for the treatment of ER+ breast cancer. Development of alternative orally available SERDs with improved bioavailability, such as amcenestrant, is underway. The HER2-targeted tyrosine kinase inhibitors (TKIs) lapatinib, neratinib and tucatinib and the antibody drug conjugate T-DM1 are approved for the treatment of HER2+ breast cancer. This pre-clinical study aims to investigate the potential of combining the oral SERD amcenestrant with HER2-targeted therapies for the treatment of HER2+/ER+ breast cancer through identification of synergistic combinations. Methods: ER and HER2 expression levels were assessed in the HER2+/ER+ BT-474 and MDA-MB-361 cell lines by western blot. 5-day acid phosphatase-based assays were used to assess cell viability following treatment with neratinib, lapatinib, tucatinib, and T-DM1; alone and in combination with amcenestrant. All TKIs were commercially sourced, T-DM1 was obtained from Saint Vincent’s University Hospital. Amcenestrant was supplied by Sanofi. IC50 values were determined using Calcusyn, while matrix combination assays of the HER2-targeted therapies combined with amcenestrant were analysed using Combenefit software employing the Loewe model (LM) to determine synergy (Score > 0) or antagonism (Score < 0). All data are the result of a minimum of three independent experiments. Results: HER2 and ER expression was confirmed in the MDA-MB-361 and BT-474 cell lines. MDA-MB-361 cells express higher levels of ER and lower levels of HER2 and phosphorylated HER2 than BT474 cells. MDA-MB-361 exhibited a higher sensitivity to 1 µM amcenestrant alone (% cell viability = 53.3 ± 2.3 %) than the BT-474 (% viability = 85.3 ± 5%). A comparison of IC50 values for each TKI alone or in combination with 1 µM amcenestrant showed amcenestrant significantly lowered the IC50 values for neratinib and tucatinib in BT-474, and all three TKIs in MDA-MB-361 (Table 1). In matrix combination assays, all TKIs exhibited synergistic combinations, with the combination of neratinib and amcenestrant demonstrating the highest synergy values in MDA-MB-361 (LM max value = 25 ± 7) and in BT-474 (LM max value = 19 ± 7). The combination of amcenestrant and T-DM1 displayed additivity (LM value = 0) rather than synergy in the BT-474 cell line, likely due to the high potency of T-DM1 as a single agent. Conclusions: The combination of amcenestrant and HER2-targeting therapies such as neratinib shows potential as a dual-target therapeutic strategy for the treatment of HER2+/ER+ breast cancer and warrants further investigation. Table 1. IC50 values for TKIs alone and with the addition of 1 µM amcenestrant. Significance of chance in IC50 point calculated by Student’s T test, ns = p > 0.05, * = p < 0.05, ** = p < 0.01, SD = standard deviation Citation Format: Amira F. Mahdi, Niall Ashfield, Neil T. Conlon, John Crown, Denis M. Collins. Pre-clinical study of amcenestrant and HER2-targeted therapies in HER2+/ER+ breast cancer cell line models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-16.

Abstract P2-24-03: Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation

Abstract Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation Sayantani Sarkar Bhattacharya1, Taylor L. Witter1, Anh Q. T. Cong1, Elizabeth Bruinsma2, Swaathi Jayaraman2, Matthew P. Goetz2, John R. Hawse1, and Matthew J. Schellenberg1 1Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN USA 55905 2Department of Oncology, Mayo Clinic, Rochester, MN USA 55905 Background: Z-Endoxifen (ENDX), the active metabolite of tamoxifen (TAM) and a selective estrogen receptor modulator (SERM), exhibited high antitumor activity in endocrine-resistant hormone receptor-positive breast and other gynecologic cancer. ENDX has also been shown to be a protein kinase C (PKC) inhibitor. PKCs participate in diverse cellular functions and their activity is often elevated in breast tumors. Guided by mechanistic insights from our recently determined crystal structure of PKCβI, we sought to determine the effects of ENDX on PKCβI in a breast cancer cell line model. Methods: To determine how ENDX regulates PKC activity, we used a Z’LYTE kinase activity assay. This Fluorescence Resonance Energy Transfer (FRET) based biochemical method can detect differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. We probed changes in activity for conventional and novel PKCs, as well as the purified catalytic domain of conventional PKCs in vitro. Alongside, as kinase activity of PKC relies on its spatial assembly, therefore we studied its intracellular localization using live cell confocal imaging. MCF7 cells expressing YFP-tagged PKCβI were grown in a glass bottom chamber and treated with ENDX and other modulators for relevant time and doses for this study. Images were taken using Zeiss LSM 780 confocal laser scanning microscope and analyzed in Zeiss-ZEN microscope software and GraphPad Prism 9. Results: Our data from an in vitro kinase assay indicates that ENDX inhibits the kinase activity of conventional (PKCβI) and novel (PKC𝛿) PKC isoforms with a similar IC50, however PKCβI catalytic domain is less sensitive to ENDX. We also identified a multi-domain mechanism of PKC inhibition through an allosteric inhibitory mechanism. Our live cell imaging study demonstrated that ENDX promotes the recruitment of PKCβI to the cell membrane in both a dose and time-dependent manner. Moreover, this translocation can also be mitigated by co-treatment with inhibitors of the PKC-regulator PHLPP phosphatases. Conclusion: Taken together, these results suggest the allosteric effects of ENDX trigger PKCβI recruitment to the cell membrane, yet since ENDX also inhibits kinase activity it suggests that ENDX triggers a non-productive interaction with the enzyme and "breaks" the well-known mechanism of PKC activation upon binding the cell membrane. Furthermore, ENDX is likely to trigger dephosphorylation and ultimately degradation, suggesting that ENDX represents a new mechanistic basis for targeting and downregulating PKC in cancer cells. Hence, the current study provides an integrated pattern of highly specific treatments regimen that can exploit PKCβI as a repurposing clinical target in breast cancer. Citation Format: Sayantani Sarkar Bhattacharya, Taylor L. Witter, Anh T. Cong, Elizabeth S. Bruinsma, Swaathi Jayaraman, Matthew P. Goetz, John Hawse, Matthew Schellenberg. Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-03.

Abstract P6-10-13: An update to a Phase I trial of CFI-402257, an oral TTK inhibitor, in patients with advanced solid tumors with HER2-negative breast cancer expansion cohorts

Abstract Background: TTK (also known as MPS1), a dual-specificity serine-threonine kinase, is critical for the spindle assembly checkpoint, chromosome alignment, and error correction in mitosis. Inhibition of TTK causes premature mitotic exit with unattached chromosomes, to result in chromosomal missegregation, aneuploidy, and cell death. CFI-402257 is a potent and highly selective inhibitor of TTK. Robust suppression of tumor growth was achieved upon oral dosing of single agent CFI-402257 in ER+/HER2- cell line and patient derived xenograft models. CFI-402257 demonstrated enhanced cytotoxicity in CDK4/6 inhibitor resistant ER+ breast cancer cell line models compared to parental cell lines, including those with RB1 loss. CFI-402257 has previously exhibited monotherapy and combination efficacy with a tolerable safety profile in ER+/Her2- Breast cancer patients in an ongoing clinical study which is updated here. Methods: This is an ongoing phase I, multi-center, dose escalation study (3+3 design) to determine the safety, tolerability, and maximum tolerated dose of CFI-402257 and to evaluate anti-tumor activity at the recommended phase 2 dose (RP2D). CFI-402257 was dosed once daily on a continuous schedule in 28-day cycles at a starting dose of 5 mg. Dose escalation included patients (pts) with advanced solid tumors. Dose expansion at the RP2D included pts with advanced solid tumors (Cohort A), advanced Her2-negative (ER+ or TNBC) with 1-4 prior lines of chemotherapy for metastatic disease (Cohort B), and ER+/Her2- breast cancer in combination with Fulvestrant (500 mg IM Day 1, 15 and 29 and then every 28 days) who have had prior treatment with an aromatase inhibitor in combination with a CDK4/6 inhibitor (≥ 3 months) and ≤1 prior chemotherapy for metastatic disease (Cohort C). Results: At data cutoff of May 2, 2022, 87 pts were enrolled. 86 pts (66 pts receiving monotherapy and 20 patients receiving combination therapy received ≥1 dose of study therapy and were analyzed for safety. One pt was not dosed due to elevated liver enzymes prior to first dose. The median age for mono was 61 years (range, 35-81) and for combo was 54 (range, 31-70). The median number of prior regimens mono and combo was 5 (range, 0-17; and 1-9, respectively). Tumor types in mono were breast cancer (27 pts), ovarian cancer (7 pts), GI cancer (3 pts), pancreatic cancer (3 pts), and other (30 pts). 20 breast cancer pts were enrolled in the combo. To date, 11 dose levels have been studied (range: 5 to 294 mg) in mono. There were 4 dose limiting toxicities (neutropenia >7days at 168mg/day, febrile neutropenia at 210mg/day, and neutropenia and colitis at 294mg/day, all grade 3). The RP2D of 168 mg was established. 3 pts in mono (biliary obstruction, febrile neutropenia, and pancytopenia) and 0 pts in combo discontinued study due to adverse events (AEs).Treatment emergent adverse events (TEAE’s) occurring in ≥30% of pts were fatigue (31 pts; 47%), nausea (30 pts; 46%), decreased appetite (22 pts; 33%), and diarrhea (21 pts; 32%) in mono; and nausea (11 pts; 55%) and diarrhea (7 pts; 35%) in combo. 35% of mono and 39% of combo AEs were considered related to CFI-402257 by the investigators. Grade ≥3 AEs and serious AEs were reported in 25 pts (38%) and 17 pts (26%), respectively in mono; and 5 pts (25%) and 3 pts (15%) in combo. The investigator assessed best overall response rate (partial response [PR] or better within the efficacy population) of 6% (PR; hepatocellular carcinoma n=1, breast cancer n=2 from n=47) in mono and 18% (PR; n=2 breast cancer) in combo (n=11), with additional patients still to be assessed. Conclusion: CFI-402257 is well tolerated as mono and combination with fulvestrant. Efficacy signals are emerging with pts in the combo cohort demonstrating anti-tumor activity. Additional efficacy will be updated at the time of the presentation. Citation Format: John Hilton, Daniel Renouf, David W. Cescon, Aaron Hansen, Alibiruni Abdul Razak, Lee-Anne Stayner, Trisha A. Denny, Emily Roberts-Thomson, Dih-Yih Chen, Mark Bray, Philippe Bedard. An update to a Phase I trial of CFI-402257, an oral TTK inhibitor, in patients with advanced solid tumors with HER2-negative breast cancer expansion cohorts [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-13.

Abstract P3-10-02: Cell cycle dysregulation in breast cancer: why the details matter

Abstract Cell cycle dysregulation is a prerequisite for cancer formation. However, whether the type of cell cycle dysregulation event a cell incurs during transformation to malignancy influences the type of cancer that evolves or clinical outcome is unknown. In a comprehensive analysis of cell cycle dysregulation in breast cancer patient tumors, we associate mutations in each of four cell cycle checkpoint kinase genes, ATM, CHEK2, ATR and CHEK1, with known tumor characteristics and clinical outcome, and test these associations experimentally using transgenic mice, patient-derived xenografts and breast cancer cell line model systems. Results of this work demonstrate that dysregulation of specific cell cycle checkpoint kinases differently impacts the type of breast cancer that evolves in patients and in experimental model systems, and influences treatment responsiveness and disease progression. For instance, CHEK2 mutations associate preferentially with the incidence of metastatic, premenopausal estrogen receptor (ER)+/HER2- breast cancer in patient data (p=0.001) that is resistant to standard frontline therapy (HR=6.15, p=0.01). These associations appear causal when tested in an immune-competent genetically-engineered mouse model of Chk2 loss, in patient-derived xenograft, and in cell line experiments. On the other hand, ATR mutation by itself is not frequent in ER+/HER2- breast cancer, but co-incident mutation of ATR and TP53 is 2-fold enriched (p=0.002) and associates with metastatic progression (HR=2.01, p=0.007). Concordantly, ATR dysregulation induces metastatic phenotypes in ER+/HER- TP53 mutant, but not in TP53 wildtype, cell lines. Together, these results systematize the impact of individual cell cycle checkpoint kinases on the evolution of cancer subtypes, and on disease progression. Statement of Significance These findings reframe the paradigm of breast cancer classification through the lens of early cell cycle dysregulation events by demonstrating that cell cycle decisions during malignant transformation can direct the type of breast cancer that evolves, how it will respond to treatment, and whether it will metastasize. This work provides rationale for streamlined testing of checkpoint kinase dysregulation to improve precision diagnostics for cancer patients. Citation Format: Sinem Seker, Elena Oropeza, Sabrina Carrel, Aloran Mazumder, Nindo Punturi, Jonathan Lei, Meenakshi Anurag, Bora Lim, Matthew Bainbridge, Svasti Haricharan. Cell cycle dysregulation in breast cancer: why the details matter [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-10-02.

Evaluation of KRASG12C inhibitor responses in novel murine KRASG12C lung cancer cell line models.

The KRAS(G12C) mutation is the most common genetic mutation in North American lung adenocarcinoma patients. Recently, direct inhibitors of the KRASG12C protein have been developed and demonstrate clinical response rates of 37-43%. Importantly, these agents fail to generate durable therapeutic responses with median progression-free survival of ~6.5 months. To provide models for further preclinical improvement of these inhibitors, we generated three novel murine KRASG12C-driven lung cancer cell lines. The co-occurring NRASQ61L mutation in KRASG12C-positive LLC cells was deleted and the KRASG12V allele in CMT167 cells was edited to KRASG12C with CRISPR/Cas9 methods. Also, a novel murine KRASG12C line, mKRC.1, was established from a tumor generated in a genetically-engineered mouse model. The three lines exhibit similar in vitro sensitivities to KRASG12C inhibitors (MRTX-1257, MRTX-849, AMG-510), but distinct in vivo responses to MRTX-849 ranging from progressive growth with orthotopic LLC-NRAS KO tumors to modest shrinkage with mKRC.1 tumors. All three cell lines exhibited synergistic in vitro growth inhibition with combinations of MRTX-1257 and the SHP2/PTPN11 inhibitor, RMC-4550. Moreover, treatment with a MRTX-849/RMC-4550 combination yielded transient tumor shrinkage in orthotopic LLC-NRAS KO tumors propagated in syngeneic mice and durable shrinkage of mKRC.1 tumors. Notably, single-agent MRTX-849 activity in mKRC.1 tumors and the combination response in LLC-NRAS KO tumors was lost when the experiments were performed in athymic nu/nu mice, supporting a growing literature demonstrating a role for adaptive immunity in the response to this class of drugs. These new models of murine KRASG12C mutant lung cancer should prove valuable for identifying improved therapeutic combination strategies with KRASG12C inhibitors.

The paracaspase MALT1 is a downstream target of Smad3 and potentiates the crosstalk between TGF-β and NF-kB signaling pathways in cancer cells

TGF-β signaling mediates its biological effects by engaging canonical Smad proteins and crosstalking extensively with other signaling networks, including the NF-kB pathway. The paracaspase MALT1 is an intracellular signaling molecule essential for NF-kB activation downstream of several key cell surface receptors. Despite intensive research on TGF-β and NF-kB interactions, the significance of MALT1 in this context remains undecoded. Here we provide experimental evidence supporting that MALT1 functions to converge these pathways. Using A549 and Huh7 cancer cell line models, we report that TGF-β stimulation enhances MALT1 protein and transcript levels in a time- and dose-dependent manner. Systematic and selective perturbation of TGF-β signaling components identifies MALT1 as a downstream target of Smad3. Rescue experiments in SMAD3 knockout cells confirm that C-terminal phosphorylation of Smad3 is central to MALT1 induction. Corroborating these data, we document that the expression of SMAD3 and MALT1 genes are positively correlated in TCGA cohorts, and we trace the molecular basis of MALT1 elevation to promoter activation. Functional studies in parental as well as NF-kB p65 signaling reporter engineered cells conclusively reveal that MALT1 is paramount for TGF-β-stimulated nuclear translocation and transcriptional activation of NF-kB p65. Furthermore, we find that BCL10 is also implicated in TGF-β activation of NF-kB target genes, potentially coupling the TGF-β-MALT1-NF-kB signaling axis to the CARMA-BCL10-MALT1 (CBM) signalosome. The novel findings of this study indicate that MALT1 is a downstream target of the canonical TGF-β/Smad3 pathway and plays a critical role in modulating TGF-β and NF-kB crosstalk in cancer.

Comparison of Radiation Response between 2D and 3D Cell Culture Models of Different Human Cancer Cell Lines

Radiation therapy is one of the most effective tools in cancer therapy. However, success varies individually, necessitating improved understanding of radiobiology. Three-dimensional (3D) tumor spheroids are increasingly gaining attention, being a superior in vitro cancer model compared to 2D cell cultures. This in vitro study aimed at comparing radiation responses in 2D and 3D cell culture models of different human cancer cell lines (PC-3, LNCaP and T-47D) irradiated with varying doses (1, 2, 4, 6, 8 or 20 Gy) of X-ray beams. Radiation response was analyzed by growth analysis, various cell viability assays (e.g., clonogenic assay, resazurin assay) and amount of DNA damage (γH2AX Western Blot). Results showed decreasing cell proliferation with the increase of radiation doses for all cell lines in monolayers and spheroids of LNCaP and T-47D. However, significantly lower radiosensitivity was detected in spheroids, most pronounced in PC-3, evincing radiation resistance of PC-3 spheroids up to 8 Gy and significant growth inhibition only by a dose escalation of 20 Gy. Cell line comparison showed highest radiosensitivity in LNCaP, followed by T-47D and PC-3 in 2D, whereas, in 3D, T-47D showed highest sensitivity. The results substantiate the significant differences in radiobiological response to X-rays between 2D and 3D cell culture models.

Molecular Alterations and Putative Therapeutic Targeting of Planar Cell Polarity Proteins in Breast Cancer.

Treatment and outcomes of breast cancer, one of the most prevalent female cancers, have improved in recent decades. However, metastatic breast cancer remains incurable in most cases, and new therapies are needed to ameliorate prognosis. Planar cell polarity (PCP) is a characteristic of epithelial cells that form layers and is integral to the communication of these cells with neighboring cells. Dysfunction of PCP is observed in cancers and may confer a targetable vulnerability. The breast cancer cohorts from The Cancer Genome Atlas (TCGA) and the METABRIC study were interrogated for molecular alterations in genes of the PCP pathway. The groups with the most prevalent alterations were characterized, and survival was compared with counterparts not possessing PCP alterations. Breast cancer cell lines with PCP alterations from the Cancer Cell Line Encyclopedia (CCLE) were interrogated for sensitivity to drugs affecting PCP. Among genes of the PCP pathway, VANGL2, NOS1AP and SCRIB display amplifications in a sizable minority of breast cancers. Concomitant up-regulation at the mRNA level can be observed mostly in basal cancers, but it does not correlate well with the amplification status of the genes, as it can also be observed in non-amplified cases. In an exploration of cell line models, two of the four breast cancer cell line models with amplifications in VANGL2, NOS1AP and SCRIB display sensitivity to drugs inhibiting acyl-transferase porcupine interfering with the WNT pathway. This sensitivity suggests a possible therapeutic role of these inhibitors in cancers bearing the amplifications. Molecular alterations in PCP genes can be observed in breast cancers with a predilection for the basal sub-type. An imperfect correlation of copy number alterations with mRNA expression suggests that post-translational modifications are important in PCP regulation. Inhibitors of acyl-transferase porcupine may be rational candidates for combination therapy development in PCP-altered breast cancers.

TRIM9 Interacts with ZEB1 to Suppress Esophageal Cancer by Promoting ZEB1 Protein Degradation via the UPP Pathway.

Esophageal cancer remains one of the most lethal malignant diseases globally. Previous studies indicated that TRIM9 (Tripartite Motif Containing 9) is a potential marker in breast cancer patients. Therefore, in the current research, we intended to clarify the regulatory network of TRIM9 and its relative role in esophageal cancer patients. We aimed to elucidate the regulatory role of TRIM9 in esophageal cancer. Clinical tumor tissue samples combined with cancer cell line models were utilized to explore the TRIM9 expression pattern. Functional experiments including transwell assay, cell viability assay, and ubiquitination blocking experiments were performed to evaluate the role of the TRIM9/ZEB1 (zinc finger E-box binding homeobox 1) axis and UPP pathway in esophageal cancer progression and exacerbation. Both esophageal cancer samples and cell line models showed significantly suppressed levels of TRIM9. Functional experiments confirmed that TRIM9 overexpression inhibited the cell viability, invasiveness, and stem-like phenotype of cancer cells. Subsequent investigations suggested that TRIM9-ZEB1 interaction accelerated ZEB1 protein degradation through the modulation of the UPP pathway, which confirmed the protective role of TRIM9 in esophageal cancer progression and metastasis. This study concluded that TRIM9 was a tumor suppressor that interacted with ZEB1 and accelerated ZEB1 protein degradation via the ubiquitin-proteasome pathway (UPP). Our research emphasized TRIM9-ZEB1 interaction as a valuable target for esophageal cancer treatment in future development. More detailed studies are needed to further consolidate our findings.

Glucose Deprivation Promotes Cancer Cell Invasion by Varied Expression of EMT Structural Proteins and Regulatory Molecules in MDA-MB-231 Triple-Negative Breast Cancer Cells.

Cancer is one of the biggest leading causes of death worldwide. With increased accumulation of environmental factors like radiation and mutagens, causative mutation in the gene pool is increasing day by day in the human population. Metastasis and its resistance to a wide variety of treatments are the reasons which make cancer difficult to treat. It has already been reported that cancer cells introduce smart modifications in their metabolism and signaling pathways which sense changes in cancer microenvironment and help them survive under harsh conditions. The pattern of expression of these signaling molecules and their structural counterparts involved in cancer metastasis need to be investigated. We used different approaches to study the effect of several levels of glucose deprivation on cancer metastasis in the highly resistant breast cancer model cell line MDA-MB-231 grown in high- (25-mM) and low- (5-mM) glucose medium. Microscopic observations have shown that these cancer cells attach to the surface faster in the presence of high-compared to low-glucose concentrations. These observations were supported by varied expression of genes involved in this morphological transition. Under low-glucose concentration, the expression of epithelial-to-mesenchymal transition (EMT) structural proteins and regulatory molecules such as SLUG, ZEB, HIFα1A, STAT3, and VIM was downregulated, whereas expression of G9a (a histone-modifying enzyme, histone methyl transferase inhibitor), Snail, FBPase, MMP13, and PKM2 was upregulated. This resulted in the turning on of resistance mechanisms in MDA-MB-231 cancer cells enabling them to cope with the stressed tumor microenvironments leading to increased cancer invasion and migration. The increased invasion as shown by trans-well study and loose spheroids in 3D spheroid study showed how metastasis is triggered in MDA-MB-231 under glucose starvation. Our data suggests the devastating outcome of nutrient deprivation on cancer progression, which was previously thought to be supportive in cancer control.

A Rapid, Convergent Approach to the Identification of Exosome Inhibitors in Breast Cancer Models.

Targeting cancer cell exosome release and biogenesis represents a potentially efficient means to treat tumors and prevent cancer recurrence/metastasis; however, the complexity and time-consuming nature of currently employed methods to purify and characterize exosomes represent obstacles to progression. Herein, we describe a rapid, convergent, and cost-efficient strategy to analyze candidate U.S. Food and Drug Administration (FDA)-approved drugs that inhibit exosome release and/or biogenesis using breast cancer cell line models in the hope of repurposing them for the clinical treatment of metastatic tumors. We combined the ExoScreen assay based on AlphaScreenTM technology with the antibody-mediated detection of an atypical lipid (lysobisphosphatidic acid - LBPA) present in the intra-luminal vesicle/exosomal fraction to achieve both extracellular and intracellular information on exosome modulation after treatment. As proof of concept for this strategy, we identified docetaxel, biscurcumin, primaquine, and doxorubicin as potential exosome release inhibitors in the Her-2 positive MDA-MB-453 and luminal A MCF7 cell lines. Dinaciclib also functioned as an exosome release inhibitor in MCF7 cells. Further, we explored the expression of proteins involved in exosome biogenesis (TSG101, CD9 tetraspanin, Alix, SMase2) and release (Rab11, Rab27) to decipher and validate the possible molecular mechanisms of action of the identified exosome inhibitors. We anticipate that our approach could help to create robust high-throughput screening methodologies to accelerate drug repurposing when using FDA-approved compound libraries and to develop rationally-designed single/combination therapies (including nanomedicines) that can target metastasis progression by modulating exosome biogenesis or release in various tumor types.

Variation of extrachromosomal circular DNA in cancer cell lines

The presence of oncogene carrying eccDNAs is strongly associated with carcinogenesis and poor patient survival. Tumour biopsies and in vitro cancer cell lines are frequently utilized as models to investigate the role of eccDNA in cancer. However, eccDNAs are often lost during the in vitro growth of cancer cell lines, questioning the reproducibility of studies utilizing cancer cell line models. Here, we conducted a comprehensive analysis of eccDNA variability in seven cancer cell lines (MCA3D, PDV, HaCa4, CarC, MIA-PaCa-2, AsPC-1, and PC-3). We compared the content of unique eccDNAs between triplicates of each cell line and found that the number of unique eccDNA is specific to each cell line, while the eccDNA sequence content varied greatly among triplicates (∼ 0–1% eccDNA coordinate commonality). In the PC-3 cell line, we found that the large eccDNA (ecDNA) with MYC is present in high-copy number in an NCI cell line isolate but not present in ATCC isolates. Together, these results reveal that the sequence content of eccDNA is highly variable in cancer cell lines. This highlights the importance of testing cancer cell lines before use, and to enrich for subclones in cell lines with the desired eccDNA to get relatively pure population for studying the role of eccDNA in cancer.