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1.
NPJ Breast Cancer ; 10(1): 37, 2024 May 27.
Article in English | MEDLINE | ID: mdl-38802426

ABSTRACT

Triple negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases in the United States. Systemic neoadjuvant chemotherapy (NACT), with or without immunotherapy, is the current standard of care for patients with early-stage TNBC. However, up to 70% of TNBC patients have significant residual disease once NACT is completed, which is associated with a high risk of developing recurrence within two to three years of surgical resection. To identify targetable vulnerabilities in chemoresistant TNBC, we generated longitudinal patient-derived xenograft (PDX) models from TNBC tumors before and after patients received NACT. We then compiled transcriptomes and drug response profiles for all models. Transcriptomic analysis identified the enrichment of aberrant protein homeostasis pathways in models from post-NACT tumors relative to pre-NACT tumors. This observation correlated with increased sensitivity in vitro to inhibitors targeting the proteasome, heat shock proteins, and neddylation pathways. Pevonedistat, a drug annotated as a NEDD8-activating enzyme (NAE) inhibitor, was prioritized for validation in vivo and demonstrated efficacy as a single agent in multiple PDX models of TNBC. Pharmacotranscriptomic analysis identified a pathway-level correlation between pevonedistat activity and post-translational modification (PTM) machinery, particularly involving neddylation and sumoylation targets. Elevated levels of both NEDD8 and SUMO1 were observed in models exhibiting a favorable response to pevonedistat compared to those with a less favorable response in vivo. Moreover, a correlation emerged between the expression of neddylation-regulated pathways and tumor response to pevonedistat, indicating that targeting these PTM pathways may prove effective in combating chemoresistant TNBC.

2.
Int J Mol Sci ; 25(2)2024 Jan 20.
Article in English | MEDLINE | ID: mdl-38279277

ABSTRACT

Endometrial cancer is the most frequent malignant tumor of the female reproductive tract but lacks effective therapy. EphA2, a receptor tyrosine kinase, is overexpressed by various cancers including endometrial cancer and is associated with poor clinical outcomes. In preclinical models, EphA2-targeted drugs had modest efficacy. To discover potential synergistic partners for EphA2-targeted drugs, we performed a high-throughput drug screen and identified panobinostat, a histone deacetylase inhibitor, as a candidate. We hypothesized that combination therapy with an EphA2 inhibitor and panobinostat leads to synergistic cell death. Indeed, we found that the combination enhanced DNA damage, increased apoptosis, and decreased clonogenic survival in Ishikawa and Hec1A endometrial cancer cells and significantly reduced tumor burden in mouse models of endometrial carcinoma. Upon RNA sequencing, the combination was associated with downregulation of cell survival pathways, including senescence, cyclins, and cell cycle regulators. The Axl-PI3K-Akt-mTOR pathway was also decreased by combination therapy. Together, our results highlight EphA2 and histone deacetylase as promising therapeutic targets for endometrial cancer.


Subject(s)
Endometrial Neoplasms , Histone Deacetylase Inhibitors , Receptor, EphA2 , Animals , Female , Humans , Mice , Apoptosis , Cell Line, Tumor , Endometrial Neoplasms/drug therapy , Endometrial Neoplasms/genetics , Endometrial Neoplasms/pathology , Histone Deacetylase Inhibitors/therapeutic use , Panobinostat/pharmacology , Panobinostat/therapeutic use , Phosphatidylinositol 3-Kinases , Molecular Targeted Therapy , Receptor, EphA2/antagonists & inhibitors
3.
Blood ; 142(12): 1056-1070, 2023 09 21.
Article in English | MEDLINE | ID: mdl-37339579

ABSTRACT

TP 53-mutant acute myeloid leukemia (AML) remains the ultimate therapeutic challenge. Epichaperomes, formed in malignant cells, consist of heat shock protein 90 (HSP90) and associated proteins that support the maturation, activity, and stability of oncogenic kinases and transcription factors including mutant p53. High-throughput drug screening identified HSP90 inhibitors as top hits in isogenic TP53-wild-type (WT) and -mutant AML cells. We detected epichaperomes in AML cells and stem/progenitor cells with TP53 mutations but not in healthy bone marrow (BM) cells. Hence, we investigated the therapeutic potential of specifically targeting epichaperomes with PU-H71 in TP53-mutant AML based on its preferred binding to HSP90 within epichaperomes. PU-H71 effectively suppressed cell intrinsic stress responses and killed AML cells, primarily by inducing apoptosis; targeted TP53-mutant stem/progenitor cells; and prolonged survival of TP53-mutant AML xenograft and patient-derived xenograft models, but it had minimal effects on healthy human BM CD34+ cells or on murine hematopoiesis. PU-H71 decreased MCL-1 and multiple signal proteins, increased proapoptotic Bcl-2-like protein 11 levels, and synergized with BCL-2 inhibitor venetoclax in TP53-mutant AML. Notably, PU-H71 effectively killed TP53-WT and -mutant cells in isogenic TP53-WT/TP53-R248W Molm13 cell mixtures, whereas MDM2 or BCL-2 inhibition only reduced TP53-WT but favored the outgrowth of TP53-mutant cells. Venetoclax enhanced the killing of both TP53-WT and -mutant cells by PU-H71 in a xenograft model. Our data suggest that epichaperome function is essential for TP53-mutant AML growth and survival and that its inhibition targets mutant AML and stem/progenitor cells, enhances venetoclax activity, and prevents the outgrowth of venetoclax-resistant TP53-mutant AML clones. These concepts warrant clinical evaluation.


Subject(s)
Antineoplastic Agents , Leukemia, Myeloid, Acute , Humans , Animals , Mice , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Proto-Oncogene Proteins c-bcl-2 , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Apoptosis , Stem Cells/metabolism , Cell Line, Tumor
4.
Mol Cancer Ther ; 22(8): 962-975, 2023 08 01.
Article in English | MEDLINE | ID: mdl-37310170

ABSTRACT

Mutations in KRAS are found in more than 50% of tumors from patients with metastatic colorectal cancer (mCRC). However, direct targeting of most KRAS mutations is difficult; even the recently developed KRASG12C inhibitors failed to show significant benefit in patients with mCRC. Single agents targeting mitogen-activated protein kinase kinase (MEK), a downstream mediator of RAS, have also been ineffective in colorectal cancer. To identify drugs that can enhance the efficacy of MEK inhibitors, we performed unbiased high-throughput screening using colorectal cancer spheroids. We used trametinib as the anchor drug and examined combinations of trametinib with the NCI-approved Oncology Library version 5. The initial screen, and following focused validation screens, identified vincristine as being strongly synergistic with trametinib. In vitro, the combination strongly inhibited cell growth, reduced clonogenic survival, and enhanced apoptosis compared with monotherapies in multiple KRAS-mutant colorectal cancer cell lines. Furthermore, this combination significantly inhibited tumor growth, reduced cell proliferation, and increased apoptosis in multiple KRAS-mutant patient-derived xenograft mouse models. In vivo studies using drug doses that reflect clinically achievable doses demonstrated that the combination was well tolerated by mice. We further determined that the mechanism underlying the synergistic effect of the combination was due to enhanced intracellular accumulation of vincristine associated with MEK inhibition. The combination also significantly decreased p-mTOR levels in vitro, indicating that it inhibits both RAS-RAF-MEK and PI3K-AKT-mTOR survival pathways. Our data thus provide strong evidence that the combination of trametinib and vincristine represents a novel therapeutic option to be studied in clinical trials for patients with KRAS-mutant mCRC. SIGNIFICANCE: Our unbiased preclinical studies have identified vincristine as an effective combination partner for the MEK inhibitor trametinib and provide a novel therapeutic option to be studied in patients with KRAS-mutant colorectal cancer.


Subject(s)
Colonic Neoplasms , Colorectal Neoplasms , Mitogen-Activated Protein Kinase Kinases , Vincristine , Animals , Humans , Mice , Cell Line, Tumor , Colonic Neoplasms/drug therapy , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mutation , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , TOR Serine-Threonine Kinases/metabolism , Vincristine/pharmacology , Vincristine/therapeutic use
5.
Br J Cancer ; 128(11): 2013-2024, 2023 06.
Article in English | MEDLINE | ID: mdl-37012319

ABSTRACT

BACKGROUND: Cisplatin (CDDP) is a mainstay treatment for advanced head and neck squamous cell carcinomas (HNSCC) despite a high frequency of innate and acquired resistance. We hypothesised that tumours acquire CDDP resistance through an enhanced reductive state dependent on metabolic rewiring. METHODS: To validate this model and understand how an adaptive metabolic programme might be imprinted, we performed an integrated analysis of CDDP-resistant HNSCC clones from multiple genomic backgrounds by whole-exome sequencing, RNA-seq, mass spectrometry, steady state and flux metabolomics. RESULTS: Inactivating KEAP1 mutations or reductions in KEAP1 RNA correlated with Nrf2 activation in CDDP-resistant cells, which functionally contributed to resistance. Proteomics identified elevation of downstream Nrf2 targets and the enrichment of enzymes involved in generation of biomass and reducing equivalents, metabolism of glucose, glutathione, NAD(P), and oxoacids. This was accompanied by biochemical and metabolic evidence of an enhanced reductive state dependent on coordinated glucose and glutamine catabolism, associated with reduced energy production and proliferation, despite normal mitochondrial structure and function. CONCLUSIONS: Our analysis identified coordinated metabolic changes associated with CDDP resistance that may provide new therapeutic avenues through targeting of these convergent pathways.


Subject(s)
Antineoplastic Agents , Head and Neck Neoplasms , Humans , Cisplatin/metabolism , Squamous Cell Carcinoma of Head and Neck , Kelch-Like ECH-Associated Protein 1/genetics , NF-E2-Related Factor 2/genetics , Drug Resistance, Neoplasm/genetics , Cell Line, Tumor , Glucose , Antineoplastic Agents/pharmacology
6.
PLoS One ; 18(3): e0281063, 2023.
Article in English | MEDLINE | ID: mdl-36952536

ABSTRACT

Metastatic colorectal cancer (mCRC) is the second leading cause of cancer deaths in the United States. More than 50% of patients with mCRC harbor mutations of the oncogenic driver RAS (KRAS or NRAS). Because directly targeting most mutations of RAS is technically challenging, researchers have concentrated on targeting MEK, a downstream mediator of RAS. However, targeting MEK as single-agent therapy is ineffective in patients with mCRC. We hypothesize that combining a MEK inhibitor with other agents can enhance the efficacy of MEK targeting in mCRC. Unbiased high-throughput screening (HTS) was performed to identify drugs that enhance the efficacy of MEK inhibitors. HTS was performed with KRAS-mutated CRC cells using the MEK inhibitor trametinib as a "backbone" and two "clinically ready" compound libraries approved by the U.S. Food and Drug Administration or in clinical trials. HTS demonstrated that the combination of the SRC inhibitor dasatinib and trametinib was synergistic in CRC cells in vitro (MTT and colony formation assays). Analysis of markers for cell proliferation and apoptosis using fluorescence-activated cell sorting, reverse-phase protein array, or Western blotting demonstrated decreased cell proliferation and increased cell death when targeting both SRC and MEK as compared to single agents in multiple CRC cell lines. However, combining dasatinib and trametinib in vivo at doses in mice equivalent to doses used in humans failed to significantly enhance the antitumor activity of trametinib when compared to that of trametinib alone. These results underscore the importance of performing careful preclinical in vivo validation studies using clinically relevant doses as a prerequisite for translating in vitro findings to the clinic.


Subject(s)
Colorectal Neoplasms , Proto-Oncogene Proteins p21(ras) , Animals , Humans , Mice , Cell Line, Tumor , Cell Proliferation , Colorectal Neoplasms/pathology , Dasatinib/pharmacology , Dasatinib/therapeutic use , Mitogen-Activated Protein Kinase Kinases , Protein Kinase Inhibitors/therapeutic use , Pyridones/pharmacology , Pyridones/therapeutic use , Xenograft Model Antitumor Assays , Genes, src
7.
Int J Mol Sci ; 24(4)2023 Feb 15.
Article in English | MEDLINE | ID: mdl-36835335

ABSTRACT

EphA2 tyrosine kinase is upregulated in many cancers and correlated with poor survival of patients, including those with endometrial cancer. EphA2-targeted drugs have shown modest clinical benefit. To improve the therapeutic response to such drugs, we performed a high-throughput chemical screen to discover novel synergistic partners for EphA2-targeted therapeutics. Our screen identified the Wee1 kinase inhibitor, MK1775, as a synergistic partner to EphA2, and this finding was confirmed using both in vitro and in vivo experiments. We hypothesized that Wee1 inhibition would sensitize cells to EphA2-targeted therapy. Combination treatment decreased cell viability, induced apoptosis, and reduced clonogenic potential in endometrial cancer cell lines. In vivo Hec1A and Ishikawa-Luc orthotopic mouse models of endometrial cancer showed greater anti-tumor responses to combination treatment than to either monotherapy. RNASeq analysis highlighted reduced cell proliferation and defective DNA damage response pathways as potential mediators of the combination's effects. In conclusion, our preclinical findings indicate that Wee1 inhibition can enhance the response to EphA2-targeted therapeutics in endometrial cancer; this strategy thus warrants further development.


Subject(s)
Antineoplastic Agents , Endometrial Neoplasms , Molecular Targeted Therapy , Protein Kinase Inhibitors , Protein-Tyrosine Kinases , Receptor, EphA2 , Animals , Female , Humans , Mice , Antineoplastic Agents/therapeutic use , Apoptosis , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation , Endometrial Neoplasms/drug therapy , Protein Kinase Inhibitors/therapeutic use , Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor, EphA2/antagonists & inhibitors
8.
Front Pharmacol ; 13: 1049640, 2022.
Article in English | MEDLINE | ID: mdl-36561339

ABSTRACT

Drug repurposing can overcome both substantial costs and the lengthy process of new drug discovery and development in cancer treatment. Some Food and Drug Administration (FDA)-approved drugs have been found to have the potential to be repurposed as anti-cancer drugs. However, the progress is slow due to only a handful of strategies employed to identify drugs with repurposing potential. In this study, we evaluated GPCR-targeting drugs by high throughput screening (HTS) for their repurposing potential in triple-negative breast cancer (TNBC) and drug-resistant human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC), due to the dire need to discover novel targets and drugs in these subtypes. We assessed the efficacy and potency of drugs/compounds targeting different GPCRs for the growth rate inhibition in the following models: two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and two HER2+ BC cell lines (BT474 and SKBR3), sensitive or resistant to lapatinib + trastuzumab, an effective combination of HER2-targeting therapies. We identified six drugs/compounds as potential hits, of which 4 were FDA-approved drugs. We focused on ß-adrenergic receptor-targeting nebivolol as a candidate, primarily because of the potential role of these receptors in BC and its excellent long-term safety profile. The effects of nebivolol were validated in an independent assay in all the cell line models. The effects of nebivolol were independent of its activation of ß3 receptors and nitric oxide production. Nebivolol reduced invasion and migration potentials which also suggests its inhibitory role in metastasis. Analysis of the Surveillance, Epidemiology and End Results (SEER)-Medicare dataset found numerically but not statistically significant reduced risk of all-cause mortality in the nebivolol group. In-depth future analyses, including detailed in vivo studies and real-world data analysis with more patients, are needed to further investigate the potential of nebivolol as a repurposed therapy for BC.

9.
Sci Rep ; 12(1): 16109, 2022 09 27.
Article in English | MEDLINE | ID: mdl-36168036

ABSTRACT

Computational models have been successful in predicting drug sensitivity in cancer cell line data, creating an opportunity to guide precision medicine. However, translating these models to tumors remains challenging. We propose a new transfer learning workflow that transfers drug sensitivity predicting models from large-scale cancer cell lines to both tumors and patient derived xenografts based on molecular pathways derived from genomic features. We further compute feature importance to identify pathways most important to drug response prediction. We obtained good performance on tumors (AUROC = 0.77) and patient derived xenografts from triple negative breast cancers (RMSE = 0.11). Using feature importance, we highlight the association between ER-Golgi trafficking pathway in everolimus sensitivity within breast cancer patients and the role of class II histone deacetylases and interlukine-12 in response to drugs for triple-negative breast cancer. Pathway information support transfer of drug response prediction models from cell lines to tumors and can provide biological interpretation underlying the predictions, serving as a steppingstone towards usage in clinical setting.


Subject(s)
Everolimus , Triple Negative Breast Neoplasms , Cell Line , Cell Line, Tumor , Heterografts , Histone Deacetylases , Humans , Machine Learning , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics
10.
Nucleic Acids Res ; 50(16): 9548-9567, 2022 09 09.
Article in English | MEDLINE | ID: mdl-36039764

ABSTRACT

The AP1 transcription factor ΔFOSB, a splice variant of FOSB, accumulates in the brain in response to chronic insults such as exposure to drugs of abuse, depression, Alzheimer's disease and tardive dyskinesias, and mediates subsequent long-term neuroadaptations. ΔFOSB forms heterodimers with other AP1 transcription factors, e.g. JUND, that bind DNA under control of a putative cysteine-based redox switch. Here, we reveal the structural basis of the redox switch by determining a key missing crystal structure in a trio, the ΔFOSB/JUND bZIP domains in the reduced, DNA-free form. Screening a cysteine-focused library containing 3200 thiol-reactive compounds, we identify specific compounds that target the redox switch, validate their activity biochemically and in cell-based assays, and show that they are well tolerated in different cell lines despite their general potential to bind to cysteines covalently. A crystal structure of the ΔFOSB/JUND bZIP domains in complex with a redox-switch-targeting compound reveals a deep compound-binding pocket near the DNA-binding site. We demonstrate that ΔFOSB, and potentially other, related AP1 transcription factors, can be targeted specifically and discriminately by exploiting unique structural features such as the redox switch and the binding partner to modulate biological function despite these proteins previously being thought to be undruggable.


Subject(s)
Cysteine , Proto-Oncogene Proteins c-fos , Proto-Oncogene Proteins c-fos/metabolism , Cysteine/genetics , Cysteine/metabolism , Gene Expression Regulation , DNA/genetics , DNA/metabolism , Oxidation-Reduction , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism
11.
Clin Cancer Res ; 28(20): 4479-4493, 2022 10 14.
Article in English | MEDLINE | ID: mdl-35972731

ABSTRACT

PURPOSE: Human papillomavirus (HPV) causes >5% of cancers, but no therapies uniquely target HPV-driven cancers. EXPERIMENTAL DESIGN: We tested the cytotoxic effect of 864 drugs in 16 HPV-positive and 17 HPV-negative human squamous cancer cell lines. We confirmed apoptosis in vitro and in vivo using patient-derived xenografts. Mitotic pathway components were manipulated with drugs, knockdown, and overexpression. RESULTS: Aurora kinase inhibitors were more effective in vitro and in vivo in HPV-positive than in HPV-negative models. We hypothesized that the mechanism of sensitivity involves retinoblastoma (Rb) expression because the viral oncoprotein E7 leads to Rb protein degradation, and basal Rb protein expression correlates with Aurora inhibition-induced apoptosis. Manipulating Rb directly, or by inducing E7 expression, altered cells' sensitivity to Aurora kinase inhibitors. Rb affects expression of the mitotic checkpoint genes MAD2L1 and BUB1B, which we found to be highly expressed in HPV-positive patient tumors. Knockdown of MAD2L1 or BUB1B reduced Aurora kinase inhibition-induced apoptosis, whereas depletion of the MAD2L1 regulator TRIP13 enhanced it. TRIP13 is a potentially druggable AAA-ATPase. Combining Aurora kinase inhibition with TRIP13 depletion led to extensive apoptosis in HPV-positive cancer cells but not in HPV-negative cancer cells. CONCLUSIONS: Our data support a model in which HPV-positive cancer cells maintain a balance of MAD2L1 and TRIP13 to allow mitotic exit and survival in the absence of Rb. Because it does not affect cells with intact Rb function, this novel combination may have a wide therapeutic window, enabling the effective treatment of Rb-deficient cancers.


Subject(s)
Alphapapillomavirus , Oncogene Proteins, Viral , Papillomavirus Infections , Uterine Cervical Neoplasms , ATPases Associated with Diverse Cellular Activities/metabolism , ATPases Associated with Diverse Cellular Activities/pharmacology , ATPases Associated with Diverse Cellular Activities/therapeutic use , Adenosine Triphosphatases , Apoptosis , Aurora Kinases/metabolism , Aurora Kinases/therapeutic use , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Female , Humans , Oncogene Proteins, Viral/genetics , Papillomaviridae/genetics , Papillomavirus E7 Proteins/genetics , Papillomavirus Infections/complications , Papillomavirus Infections/drug therapy , Papillomavirus Infections/genetics , Retinoblastoma Protein/genetics , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology
12.
SLAS Discov ; 27(3): 175-184, 2022 04.
Article in English | MEDLINE | ID: mdl-35314378

ABSTRACT

High-throughput viability screens are commonly used in the identification and development of chemotherapeutic drugs. These systems rely on the fidelity of the cellular model systems to recapitulate the drug response that occurs in vivo. In recent years, there has been an expansion in the utilization of patient-derived materials as well as advanced cell culture techniques, such as multi-cellular tumor organoids, to further enhance the translational relevance of cellular model systems. Simple quantitative analysis remains a challenge, primarily due to the difficulties of robust image segmentation in heterogenous 3D cultures. However, explicit segmentation is not required with the advancement of deep learning, and it can be used for both continuous (regression) or categorical classification problems. Deep learning approaches are additionally benefited by being fully data-driven and highly automatable, thus they can be established and run with minimal to no user-defined parameters. In this article, we describe the development and implementation of a regressive deep learning model trained on brightfield images of patient-derived organoids and use the terminal viability readout (CellTiter-Glo) as training labels. Ultimately, this has led to the generation of a non-invasive and label-free tool to evaluate changes in organoid viability.


Subject(s)
Cell Culture Techniques , Organoids , Cell Survival , Humans
13.
Cancer Metab ; 10(1): 6, 2022 Feb 22.
Article in English | MEDLINE | ID: mdl-35193687

ABSTRACT

BACKGROUND: Primary and posttreatment resistance to BRAFV600 mutation-targeting inhibitors leads to disease relapse in a majority of melanoma patients. In many instances, this resistance is promoted by upregulation of mitochondrial oxidative phosphorylation (OxPhos) in melanoma cells. We recently showed that a novel electron transport chain (ETC) complex I inhibitor, IACS-010759 (IACS), abolished OxPhos and significantly inhibited tumor growth of high-OxPhos, BRAF inhibitor (BRAFi)-resistant human melanomas. However, the inhibition was not uniform across different high OxPhos melanomas, and combination with BRAFi did not improve efficacy. METHODS: We performed a high-throughput unbiased combinatorial drug screen of clinically relevant small molecules to identify the most potent combination agent with IACS for inhibiting the growth of high-OxPhos, BRAFi-resistant melanomas. We performed bioenergetics and carbon-13 metabolite tracing to delineate the metabolic basis of sensitization of melanomas to the combination treatment. We performed xenograft tumor growth studies and Reverse-Phase Protein Array (RPPA)-based functional proteomics analysis of tumors from mice fed with regular or high-fat diet to evaluate in vivo molecular basis of sensitization to the combination treatment. RESULTS: A combinatorial drug screen and subsequent validation studies identified Atorvastatin (STN), a hydroxymethylglutaryl-coenzyme A reductase inhibitor (HMGCRi), as the most potent treatment combination with IACS to inhibit in vitro cell growth and induce tumor regression or stasis of some BRAFi-resistant melanomas. Bioenergetics analysis revealed a dependence on fatty acid metabolism in melanomas that responded to the combination treatment. RPPA analysis and carbon-13 tracing analysis in these melanoma cells showed that IACS treatment decreased metabolic fuel utilization for fatty acid metabolism, but increased substrate availability for activation of the mevalonate pathway by HMGCR, creating a dependence on this pathway. Functional proteomic analysis showed that IACS treatment inhibited MAPK but activated AKT pathway. Combination treatment with STN counteracted AKT activation. CONCLUSIONS: STN and other clinically approved HMGCRi could be promising combinatorial agents for improving the efficacy of ETC inhibitors like IACS in BRAFi-resistant melanomas.

14.
iScience ; 24(11): 103227, 2021 Nov 19.
Article in English | MEDLINE | ID: mdl-34712924

ABSTRACT

Transcription is a highly regulated sequence of stochastic processes utilizing many regulators, including nuclear receptors (NR) that respond to stimuli. Endocrine disrupting chemicals (EDCs) in the environment can compete with natural ligands for nuclear receptors to alter transcription. As nuclear dynamics can be tightly linked to transcription, it is important to determine how EDCs affect NR mobility. We use an EPA-assembled set of 45 estrogen receptor-α (ERα) ligands and EDCs in our engineered PRL-Array model to characterize their effect upon transcription using fluorescence in situ hybridization and fluorescence recovery after photobleaching (FRAP). We identified 36 compounds that target ERα-GFP to a transcriptionally active, visible locus. Using a novel method for multi-region FRAP analysis we find a strong negative correlation between ERα mobility and inverse agonists. Our findings indicate that ERα mobility is not solely tied to transcription but affected highly by the chemical class binding the receptor.

15.
Mol Cancer Ther ; 20(12): 2352-2361, 2021 12.
Article in English | MEDLINE | ID: mdl-34583979

ABSTRACT

CRM1 inhibitors have demonstrated antitumor effects in ovarian and other cancers; however, rational combinations are largely unexplored. We performed a high-throughput drug library screen to identify drugs that might combine well with selinexor in ovarian cancer. Next, we tested the combination of selinexor with the top hit from the drug screen in vitro and in vivo Finally, we assessed for mechanisms underlying the identified synergy using reverse phase protein arrays (RPPA). The drug library screen assessing 688 drugs identified olaparib (a PARP inhibitor) as the most synergistic combination with selinexor. Synergy was further demonstrated by MTT assays. In the A2780luc ip1 mouse model, the combination of selinexor and olaparib yielded significantly lower tumor weight and fewer tumor nodules compared with the control group (P < 0.04 and P < 0.03). In the OVCAR5 mouse model, the combination yielded significantly fewer nodules (P = 0.006) and markedly lower tumor weight compared with the control group (P = 0.059). RPPA analysis indicated decreased expression of DNA damage repair proteins and increased expression of tumor suppressor proteins in the combination treatment group. Collectively, our preclinical findings indicate that combination with selinexor to expand the utility and efficacy of PARP inhibitors in ovarian cancer warrants further exploration.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , High-Throughput Screening Assays/methods , Hydrazines/therapeutic use , Ovarian Neoplasms/drug therapy , Phthalazines/therapeutic use , Piperazines/therapeutic use , Triazoles/therapeutic use , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cell Line, Tumor , Disease Models, Animal , Female , Humans , Hydrazines/pharmacology , Mice , Mice, Nude , Ovarian Neoplasms/pathology , Phthalazines/pharmacology , Piperazines/pharmacology , Triazoles/pharmacology
16.
Nat Commun ; 12(1): 5389, 2021 09 10.
Article in English | MEDLINE | ID: mdl-34508101

ABSTRACT

Conditional overexpression of histone reader Tripartite motif containing protein 24 (TRIM24) in mouse mammary epithelia (Trim24COE) drives spontaneous development of mammary carcinosarcoma tumors, lacking ER, PR and HER2. Human carcinosarcomas or metaplastic breast cancers (MpBC) are a rare, chemorefractory subclass of triple-negative breast cancers (TNBC). Comparison of Trim24COE metaplastic carcinosarcoma morphology, TRIM24 protein levels and a derived Trim24COE gene signature reveals strong correlation with human MpBC tumors and MpBC patient-derived xenograft (PDX) models. Global and single-cell tumor profiling reveal Met as a direct oncogenic target of TRIM24, leading to aberrant PI3K/mTOR activation. Here, we find that pharmacological inhibition of these pathways in primary Trim24COE tumor cells and TRIM24-PROTAC treatment of MpBC TNBC PDX tumorspheres decreased cellular viability, suggesting potential in therapeutically targeting TRIM24 and its regulated pathways in TRIM24-expressing TNBC.


Subject(s)
Carcinosarcoma/genetics , Carrier Proteins/genetics , Mammary Neoplasms, Experimental/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , Triple Negative Breast Neoplasms/genetics , Animals , Breast/pathology , Carcinosarcoma/pathology , Carrier Proteins/metabolism , Clinical Trials as Topic , Female , Gene Expression Regulation, Neoplastic , Humans , Mammary Glands, Animal/pathology , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Transgenic , Nuclear Proteins/metabolism , Primary Cell Culture , Proto-Oncogene Proteins c-met/genetics , RNA-Seq , Single-Cell Analysis , Transcription Factors/metabolism , Triple Negative Breast Neoplasms/pathology , Whole Genome Sequencing , Xenograft Model Antitumor Assays
18.
Nat Commun ; 12(1): 4262, 2021 07 12.
Article in English | MEDLINE | ID: mdl-34253738

ABSTRACT

The epithelial-mesenchymal transition (EMT) has been implicated in conferring stem cell properties and therapeutic resistance to cancer cells. Therefore, identification of drugs that can reprogram EMT may provide new therapeutic strategies. Here, we report that cells derived from claudin-low mammary tumors, a mesenchymal subtype of triple-negative breast cancer, exhibit a distinctive organoid structure with extended "spikes" in 3D matrices. Upon a miR-200 induced mesenchymal-epithelial transition (MET), the organoids switch to a smoother round morphology. Based on these observations, we developed a morphological screening method with accompanying analytical pipelines that leverage deep neural networks and nearest neighborhood classification to screen for EMT-reversing drugs. Through screening of a targeted epigenetic drug library, we identified multiple class I HDAC inhibitors and Bromodomain inhibitors that reverse EMT. These data support the use of morphological screening of mesenchymal mammary tumor organoids as a platform to identify drugs that reverse EMT.


Subject(s)
Antineoplastic Agents/pharmacology , Epithelial-Mesenchymal Transition/drug effects , Mammary Neoplasms, Animal/pathology , Mesoderm/pathology , Organoids/pathology , Animals , Azacitidine/pharmacology , Benzamides/pharmacology , Drug Screening Assays, Antitumor , Epigenesis, Genetic , Female , Gene Expression Regulation, Neoplastic , Image Processing, Computer-Assisted , Mammary Neoplasms, Animal/genetics , Mice, Inbred BALB C , MicroRNAs/genetics , MicroRNAs/metabolism , Neoplasm Proteins/metabolism , Organoids/drug effects , Pyrimidines/pharmacology , Reproducibility of Results , Small Molecule Libraries/pharmacology
19.
J Clin Endocrinol Metab ; 106(10): 2962-2978, 2021 09 27.
Article in English | MEDLINE | ID: mdl-34120183

ABSTRACT

BACKGROUND: Despite the use of aggressive multimodality treatment, most anaplastic thyroid carcinoma (ATC) patients die within a year of diagnosis. Although the combination of BRAF and MEK inhibitors has recently been approved for use in BRAF-mutated ATC, they remain effective in a minority of patients who are likely to develop drug resistance. There remains a critical clinical need for effective systemic agents for ATC with a reasonable toxicity profile to allow for rapid translational development. MATERIAL AND METHODS: Twelve human thyroid cancer cell lines with comprehensive genomic characterization were used in a high-throughput screening (HTS) of 257 compounds to select agents with maximal growth inhibition. Cell proliferation, colony formation, orthotopic thyroid models, and patient-derived xenograft (PDX) models were used to validate the selected agents. RESULTS: Seventeen compounds were effective, and docetaxel, LBH-589, and pralatrexate were selected for additional in vitro and in vivo analysis as they have been previously approved by the US Food and Drug Administration for other cancers. Significant tumor growth inhibition (TGI) was detected in all tested models treated with LBH-589; pralatrexate demonstrated significant TGI in the orthotopic papillary thyroid carcinoma model and 2 PDX models; and docetaxel demonstrated significant TGI only in the context of mutant TP53. CONCLUSIONS: HTS identified classes of systemic agents that demonstrate preferential effectiveness against aggressive thyroid cancers, particularly those with mutant TP53. Preclinical validation in both orthotopic and PDX models, which are accurate in vivo models mimicking tumor microenvironment, may support initiation of early-phase clinical trials in non-BRAF mutated or refractory to BRAF/MEK inhibition ATC.


Subject(s)
Antineoplastic Agents/pharmacology , High-Throughput Screening Assays , Protein Kinase Inhibitors/pharmacology , Thyroid Carcinoma, Anaplastic/drug therapy , Thyroid Neoplasms/drug therapy , Animals , Carcinogenicity Tests , Cell Line, Tumor , Disease Models, Animal , Humans , Proto-Oncogene Proteins B-raf/drug effects , Tumor Microenvironment/drug effects
20.
Sci Rep ; 11(1): 10461, 2021 05 17.
Article in English | MEDLINE | ID: mdl-34002003

ABSTRACT

Loss of primary cilia in cells deficient for the tumor suppressor von Hippel Lindau (VHL) arise from elevated Aurora Kinase A (AURKA) levels. VHL in its role as an E3 ubiquitin ligase targets AURKA for degradation and in the absence of VHL, high levels of AURKA result in destabilization of the primary cilium. We identified NVP-BEZ235, a dual PI3K/AKT and mTOR inhibitor, in an image-based high throughput screen, as a small molecule that restored primary cilia in VHL-deficient cells. We identified the ability of AKT to modulate AURKA expression at the transcript and protein level. Independent modulation of AKT and mTOR signaling decreased AURKA expression in cells confirming AURKA as a new signaling node downstream of the PI3K cascade. Corroborating these data, a genetic knockdown of AKT in cells deficient for VHL rescued the ability of these cells to ciliate. Finally, inhibition of AKT/mTOR using NVP-BEZ235 was efficacious in reducing tumor burden in a 786-0 xenograft model of renal cell carcinoma. These data highlight a previously unappreciated signaling node downstream of the AKT/mTOR pathway via AURKA that can be targeted in VHL-null cells to restore ciliogenesis.


Subject(s)
Aurora Kinase A/metabolism , Carcinoma, Renal Cell/drug therapy , Cilia/drug effects , Imidazoles/pharmacology , Kidney Neoplasms/drug therapy , Quinolines/pharmacology , von Hippel-Lindau Disease/drug therapy , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Cell Line, Tumor , Cilia/pathology , Gene Knockdown Techniques , Humans , Imidazoles/therapeutic use , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Quinolines/therapeutic use , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , Tumor Burden/drug effects , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Xenograft Model Antitumor Assays , von Hippel-Lindau Disease/complications , von Hippel-Lindau Disease/genetics , von Hippel-Lindau Disease/pathology
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