Anticancer Therapeutic Target Research Articles

In vivo CRISPR screens identify Mga as an immunotherapy target in triple-negative breast cancer

Immune evasion is not only critical for tumor initiation and progression, but also determines the efficacy of immunotherapies. Through iterative in vivo CRISPR screens with seven syngeneic tumor models, we identified core and context-dependent immune evasion pathways across cancer types. This valuable high-confidence dataset is available for the further understanding of tumor intrinsic immunomodulators, which may lead to the discovery of effective anticancer therapeutic targets. With a focus on triple-negative breast cancer (TNBC), we found that Mga knock-out significantly enhances antitumor immunity and inhibits tumor growth. Transcriptomics and single-cell RNA sequencing analyses revealed that Mga influences various immune-related pathways in the tumor microenvironment. Our findings suggest that Mga may play a role in modulating the tumor immune landscape, though the precise mechanisms require further investigation. Interestingly, we observed that low MGA expression in breast cancer patients correlates with a favorable prognosis, particularly in those with active interferon-γ signaling. These observations provide insights into tumor immune escape mechanisms and suggest that further exploration of MGA's function could potentially lead to effective therapeutic strategies in TNBC.

Plectalibertellenone A suppresses colorectal cancer cell motility and glucose metabolism by targeting TGF-β/Smad and Wnt pathways.

Colorectal cancer (CRC) is the second most common cause of cancer-related death and represents a serious worldwide health problem. CRC metastasis decreases the survival rate of cancer patients, underscoring the need to identify novel anticancer agents and therapeutic targets. Here, we introduce Plectalibertellenone A (B) as a promising agent for the inhibition of CRC cell motility and glucose metabolism and explore its mechanism of action in CRC cells. Plectalibertellenone A suppressed TGF-β gene expression and the activation of the TGF-β/Smad signaling pathway, leading to reverse epithelial to mesenchymal transition (EMT) by modulating the expressions of EMT markers and transcriptional factors such as E-cadherin, N-cadherin, vimentin, Slug, Snail, Twist, and ZEB1/2. Furthermore, disruption of Wnt signaling inhibited CRC motility and glucose metabolism including glycolysis and oxidative phosphorylation, primarily affecting glycolytic enzymes, GLUT1, HK2, PKM2, LDHA, and HIF-1α under hypoxic condition. Therefore, Plectalibertellenone A is a potential drug candidate that can be developed into a promising anticancer treatment to prevent CRC metastasis and inhibit glucose metabolism.

Unraveling the Mystery of Energy-Sensing Enzymes and Signaling Pathways in Tumorigenesis and Their Potential as Therapeutic Targets for Cancer.

Cancer research has advanced tremendously with the identification of causative genes, proteins, and signaling pathways. Numerous antitumor drugs have been designed and screened for cancer therapeutics; however, designing target-specific drugs for malignant cells with minimal side effects is challenging. Recently, energy-sensing- and homeostasis-associated molecules and signaling pathways playing a role in proliferation, apoptosis, autophagy, and angiogenesis have received increasing attention. Energy-metabolism-based studies have shown the contribution of energetics to cancer development, where tumor cells show increased glycolytic activity and decreased oxidative phosphorylation (the Warburg effect) in order to obtain the required additional energy for rapid division. The role of energy homeostasis in the survival of normal as well as malignant cells is critical; therefore, fuel intake and expenditure must be balanced within acceptable limits. Thus, energy-sensing enzymes detecting the disruption of glycolysis, AMP, ATP, or GTP levels are promising anticancer therapeutic targets. Here, we review the common energy mediators and energy sensors and their metabolic properties, mechanisms, and associated signaling pathways involved in carcinogenesis, and explore the possibility of identifying drugs for inhibiting the energy metabolism of tumor cells. Furthermore, to corroborate our hypothesis, we performed meta-analysis based on transcriptomic profiling to search for energy-associated biomarkers and canonical pathways.

Human NQO1 as a Selective Target for Anticancer Therapeutics and Tumor Imaging.

Human NAD(P)H-quinone oxidoreductase1 (HNQO1) is a two-electron reductase antioxidant enzyme whose expression is driven by the NRF2 transcription factor highly active in the prooxidant milieu found in human malignancies. The resulting abundance of NQO1 expression (up to 200-fold) in cancers and a barely detectable expression in body tissues makes it a selective marker of neoplasms. NQO1 can catalyze the repeated futile redox cycling of certain natural and synthetic quinones to their hydroxyquinones, consuming NADPH and generating rapid bursts of cytotoxic reactive oxygen species (ROS) and H2O2. A greater level of this quinone bioactivation due to elevated NQO1 content has been recognized as a tumor-specific therapeutic strategy, which, however, has not been clinically exploited. We review here the natural and new quinones activated by NQO1, the catalytic inhibitors, and the ensuing cell death mechanisms. Further, the cancer-selective expression of NQO1 has opened excellent opportunities for distinguishing cancer cells/tissues from their normal counterparts. Given this diagnostic, prognostic, and therapeutic importance, we and others have engineered a large number of specific NQO1 turn-on small molecule probes that remain latent but release intense fluorescence groups at near-infrared and other wavelengths, following enzymatic cleavage in cancer cells and tumor masses. This sensitive visualization/quantitation and powerful imaging technology based on NQO1 expression offers promise for guided cancer surgery, and the reagents suggest a theranostic potential for NQO1-targeted chemotherapy.

Research Progress of Long Non-coding RNA-ZFAS1 in Malignant Tumors.

Long non-coding RNAs (lncRNAs), although incapable of encoding proteins, play crucial roles in multiple layers of gene expression regulation, epigenetic modifications, and post-transcriptional regulation. Zinc finger antisense 1 (ZFAS1), a lncRNA located in the 20q13 region of the human genome, exhibits dual functions as an oncogene or tumor suppressor in various human malignancies. ZFAS1 plays a crucial role in cancer progression, metastasis, invasion, apoptosis, cell cycle regulation, and drug resistance through complex molecular mechanisms. Additionally, ZFAS1 has a long half-life of over 16 h, demonstrating exceptional stability, and making it a potential biomarker. This review integrates recent studies on the role and molecular mechanisms of ZFAS1 in malignancies and summarizes its clinical significance. By summarizing the role of ZFAS1 in cancer, we aim to highlight its potential as an anti-cancer biomarker and therapeutic target.

Generation and preclinical evaluation of a human heavy-chain-only antibody recognizing the membrane-bound tumor-associated antigen mesothelin

ObjectiveMesothelin (MSLN) is an attractive target for anticancer therapeutics and bioimaging reagents that utilize antibodies. This study was aimed at developing a novel human anti-MSLN single-domain antibody that exclusively binds to the membrane-attached MSLN using transgenic mice generating human heavy-chain-only antibodies (HCAbs) and exploring the resulting HCAbs as imaging tools.MethodsWe introduced a doxycycline-inducible human MSLN gene in genetically modified mice expressing human HCAbs. This new method of non-invasive immunization by antigen induction results in MSLN antigen production in its native conformation on the cell surface. Screening of 2,000 HCAbs from the resulting immune library yielded numerous binders, from which we chose 19G6 as the lead antibody. This antibody was 111Indium radiolabeled and tested in a xenotransplantation tumor model with OVCAR-3 cells.ResultsThe 19G6 antibody shows nanomolar affinity toward membrane-bound MSLN and does not recognize soluble MSLN. The human MSLN-positive tumors were visualized in an in vivo mouse model. The non-labeled antibody prevented binding when provided in excess, showing tumor specificity.Conclusion19G6 with a human Fc is a promising tumor-cell tracer in vivo. This HCAb can also be engineered into a smaller and shorter-lived tracer (only the VH domain) or combined with other target-binding domains to form multispecific modalities for tumor immunotherapy.

Insights into the DNA and RNA Interactions of Human Topoisomerase III Beta Using Molecular Dynamics Simulations.

Human topoisomerase III beta (hTOP3B) is the only topoisomerase in the human cell that can act on both DNA and RNA substrates. Recent findings have emphasized the physiological importance of hTOP3B and consolidated it as a valuable drug target for antiviral and anticancer therapeutics. Although type IA topoisomerases of different organisms have been studied over the years, the step-by-step interaction of hTOP3B and nucleic acid substrates is still not well understood. Due to the lack of hTOP3B-RNA structures as well as DNA/RNA covalent complexes, computational investigations have been limited. In our study, we utilized molecular dynamics (MD) simulations to study the interactions between hTOP3B and nucleic acids to get a closer look into the residues that play a role in binding DNA or RNA and facilitate catalysis, along with the differences and similarities when hTOP3B interacts with DNA compared to RNA. For this, we generated multiple models of hTOP3B complexed with DNA and RNA sequences using the hTOP3B crystal structure and 8-mer single-stranded DNA and RNA sequences. These models include both covalent and noncovalent complexes, which are then subjected to MD simulations and analyzed. Our findings highlight the complexes' stability, sequence preference, and interactions of the binding pocket residues with different nucleotides. Our work demonstrates that hTOP3B forms stable complexes with both DNA and RNA and provides a better understanding of the enzyme's interaction with different nucleic acid substrate sequences.

A Comprehensive Pan-Cancer Analysis of Cytochrome C Oxidase Assembly Factor 1 (COA1) Reveals Instrumental Role of Mitochondrial Protein Assembly in Cancer that Modulates Disease Progression and Prognostic Outcome.

Cytochrome c oxidase assembly factor 1 (COA1), a mitochondrial respiratory chain complex assembly factor protein of inner mitochondrial membrane (IMM), is involved in translating many mitochondrial components and assembling nuclear-encoded components within mitochondria. Given the lack of extensive research on COA1 in cancer, this study undertakes a comprehensive pan-cancer analysis of COA1, which is overexpressed across various cancer types, shedding light on its multifaceted role in tumorigenesis, prognosis, and tumor microenvironment (TME) modulation. Leveraging bioinformatics tools and public databases, we elucidated its potential as a diagnostic cancer biomarker as well as a target for novel anti-cancer therapeutics. Gene expression analysis using "TIMER2.0", "UALCAN" and "GEPIA2" platforms, supported by protein expression data, revealed a significant correlation between COA1 upregulation and poor prognosis in Kaplan-Meir analysis, underscoring its clinical relevance. Additionally, genetic mutation analysis of COA1 with the help of "cBioPortal" warrants further exploration into its functional significance. Moreover, our investigation of the tumor microenvironment unveiled the interplay of COA1 with fibroblast and T cell infiltration implicating the role of COA1 in the tumor immune microenvironment. Furthermore, COA1-related gene enrichment study in "GeneMANIA" and pathway cross-talk analysis with Gene Ontology (GO) gene sets established comprehensive clarifications about the molecular pathways and protein networks associated with COA1 deregulation. Overall, this study lays a sturdy foundation to support future research endeavors targeting COA1, unraveling the molecular mechanisms underlying COA1 deregulation, and exploring its therapeutic potential in cancer.

ECRG2/SPINK7 Tumor Suppressor as Modulator of DNA Damage Response.

Esophageal Cancer-Related Gene 2 (ECRG2), also known as Serine Peptidase Inhibitor Kazal type 7 (SPINK7), is a novel tumor suppressor gene from the SPINK family of genes that exhibits anticancer potential. ECRG2 was originally identified during efforts to discover genes involved in esophageal tumorigenesis. ECRG2 was one of those genes whose expression was absent or reduced in primary human esophageal cancers. Additionally, absent or reduced ECRG2 expression was also noted in several other types of human malignancies. ECRG2 missense mutations were identified in various primary human cancers. It was reported that a cancer-derived ECRG2 mutant (valine to glutamic acid at position 30) failed to induce cell death and caspase activation triggered by DNA-damaging anticancer drugs. Furthermore, ECRG2 suppressed cancer cell proliferation in cultured cells and grafted tumors in animals and inhibited cancer cell migration/invasion and metastasis. ECRG2 also was identified as a negative regulator of Hu-antigen R (HuR), an oncogenic RNA-binding protein that is known to regulate mRNA stability and the expression of transcripts corresponding to many cancer-related genes. ECRG2 function is important also for the regulation of inflammatory responses and the maintenance of epithelial barrier integrity in the esophagus. More recently, ECRG2 was discovered as one of the newest members of the pro-apoptotic transcriptional targets of p53. Two p53-binding sites (BS-1 and BS-2) were found within the proximal region of the ECRG2 gene promoter; the treatment of DNA-damaging agents in cancer cells significantly increased p53 binding to the ECRG2 promoter and triggered a strong ECRG2 promoter induction following DNA damage. Further, the genetic depletion of ECRG2 expression significantly impeded apoptotic cell death induced by DNA damage and wild-type p53 in cancer cells. These findings suggest that the loss of ECRG2 expression, commonly observed in human cancers, could play important roles in conferring anticancer drug resistance in human cancers. Thus, ECRG2 is a novel regulator in DNA damage-induced cell death that may also be a potential target for anticancer therapeutics.

Discovery of a Novel and Potent Cyclin-Dependent Kinase 8/19 (CDK8/19) Inhibitor for the Treatment of Cancer.

The mediator kinases CDK8 and CDK19 control the dynamic transcription of selected genes in response to various signals and have been shown to be hijacked to sustain hyperproliferation by various solid and liquid tumors. CDK8/19 is emerging as a promising anticancer therapeutic target. Here, we report the discovery of compound 12, a novel small molecule CDK8/19 inhibitor. This molecule demonstrated not only decent enzymatic and cellular activities but also remarkable selectivity in CDK and kinome panels. Besides, compound 12 also displayed favorable ADME profiles including low CYP1A2 inhibition, acceptable clearance, and high oral bioavailability in multiple preclinical species. Robust in vivo PD and efficacy studies in mice models further demonstrated its potential use as mono- and combination therapy for the treatment of cancers.

Jolkinolide B synergistically potentiates the antitumor activity of GPX4 inhibitors via inhibiting TrxR1 in cisplatin-resistant bladder cancer cells

Glutathione peroxidase 4 (GPX4) is a promising anticancer therapeutic target; however, the application of GPX4 inhibitors (GPX4i) is limited owing to intrinsic or acquired drug resistance. Hence, understanding the mechanisms underlying drug resistance and discovering molecules that can overcome drug resistance are crucial. Herein, we demonstrated that GPX4i killed bladder cancer cells by inducing lipid reactive oxygen species-mediated ferroptosis and apoptosis, and cisplatin-resistant bladder cancer cells were also resistant to GPX4i, representing a higher half-maximal inhibitory concentration value than that of parent bladder cancer cells. In addition, thioredoxin reductase 1 (TrxR1) overexpression was responsible for GPX4i resistance in cisplatin-resistant bladder cancer cells, and inhibiting TrxR1 restored the sensitivity of these cells to GPX4i. In vitro and in vivo studies revealed that Jolkinolide B (JB), a natural diterpenoid and previously identified as a TrxR1 inhibitor, potentiated the antiproliferative efficacy of GPX4i (RSL3 and ML162) against cisplatin-resistant bladder cancer cells. Furthermore, GPX4 knockdown and inhibition could augment JB-induced paraptosis and apoptosis. Our results suggest that inhibiting TrxR1 can effectively improve GPX4 inhibition-based anticancer therapy. A combination of JB and GPX4i, which is well-tolerated and has several anticancer mechanisms, may serve as a promising therapy for treating bladder cancer.

Abstract 1800: Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis in human cells

Abstract The genes that are required for the growth of mammalian cells can depend on an interplay of cell-intrinsic factors and environmental context. However, there is often little investigation into how nutrient availability impacts gene essentiality. Moreover, efforts such as DepMap have catalogued genes that contribute to cancer cell fitness using in vitro CRISPR screens in hundreds of cancer cell lines cultured in traditional media that poorly recapitulate the nutrient availability of human blood. Previously, we developed Human Plasma-Like Medium (HPLM), a physiologic medium designed to better model the nutrient availability of human blood. We recently tested the hypothesis that nutrient availability influences gene essentiality. By performing paired CRISPR-based screens in human blood cancer cells, we identified sets of genes that are differentially required to support cell growth in HPLM versus traditional media such as RPMI. Among the strongest scoring HPLM-essential genes was NAD kinase (NADK), which encodes an enzyme critical for the generation of cytosolic NADPH. NADK has been previously suggested as an anti-cancer therapeutic target, given the central importance of NADPH for enabling proliferative metabolism through macromolecule synthesis and ROS management. Interestingly, NADK has been annotated as an essential gene in only 1% of the cell lines included in DepMap, suggesting that the essentiality of NADK may have been largely masked by the nutrient conditions used to generate the DepMap. To investigate the conditional CRISPR phenotype for NADK, we engineered NADK-knockout cells and found that they showed a 50% stronger growth defect versus control cells in HPLM relative to RPMI. Next, we used localization and activity studies to confirm that cytosolic NAD+ kinase activity is required for the conditionally essential role of NADK. We then employed systematic approaches in media engineering and metabolomics to identify the medium component(s) that contribute to the NADK gene-nutrient interaction and to gain insights into the conditionally essential role of NADK. Through these studies, we traced the cause of NADK essentiality to an inhibition of folate-dependent nucleotide synthesis linked to differential folic acid availability. Finally, we examined the pre-translational potential of NADK as an anti-cancer therapeutic target by demonstrating that the reported NADK inhibitor thionicotinamide largely phenocopies NADK-knockout in a folic-acid dependent manner in the K562 cell line. Collectively, our work improves understanding of how NADK contributes to human cell growth and reveals how the relative importance of cytosolic NAD+ kinase activity can depend on nutrient availability. Citation Format: Kyle M. Flickinger, Kimberly S. Huggler, Jason R. Cantor. Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis in human cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1800.

Abstract 4119: CXCR4/CB2 heterodimer antagonizes CXCR4-mediated metastasis and tumor growth in vivo

Abstract The G-protein-coupled chemokine receptor (GPCR), CXCR4, is ubiquitously expressed in malignant tumor tissues, and controls migration and other tumor survival mechanisms upon interaction with its chemokine agonist, stromal cell-derived 1α (SDF1α). The CXCR4/SDF1α axis is relevant to shaping the tumor microenvironment mainly the metastatic spread of primary tumors to distal organs and immune response; thus, expression of CXCR4 in tumors is vital to tumor aggressiveness, survival probability, and metastasis-associated mortality. CXCR4 signals through a homodimer, but also forms heterodimers with other chemokine and non-chemokine GPCRs. We’ve demonstrated that CXCR4 forms a heterodimer with the GPCR, cannabinoid receptor 2 (CB2), in human breast and prostate cell lines in vitro. The cannabinoid system has earned interest as anticancer therapeutic targets by modulating growth, migration, angiogenesis, etc. In our model, CXCR4-mediated, pro-tumor signaling was reduced by the physical heterodimer, and the dimer was induced by simultaneous treatment with agonists (SDF1α and AM1241) for respective receptors. The resultant heterodimer negatively interfered with migration, cytoskeletal rearrangement, motility associated proteins, and signaling that coordinates movement. Given that cannabinoids have been shown to reduce metastasis and profession of clinical tumors, we hypothesize that a heterodimer of CXCR4 and CB2 disrupts CXCR4-mediated metastasis, and ultimately, decrease tumor progression in vivo.Herein, we tested our hypothesis in an in vivo model of spontaneous metastasis NSG male mice. Luciferase-containing human prostate cancer cells (DU145 RFP/Luc) were implanted subcutaneously. Once tumors reached 40mm3, mice were randomized by volume, weight and age, and divided into 4 treatments: vehicle, SDF1α, AM124, and SDF1α/AM124(combo) for daily intraperitoneal injections. Tumors reaching 400mm3 were excised, and treatments continued for 3 weeks. Spontaneous metastasis was monitored via IVIS imaging at 2- and 3-weeks post-tumor removal. We observed reduced tumor growth, and decreased incidence rates of metastasis to the lung in the combination treatment group, which, based on our in vitro data, is representative of a CXCR4/CB2 heterodimer. This novel definition of heterodimerization could reveal and exploit new mechanisms for CXCR4 regulation in tumor metastasis. The CXCR4/CB2 heterodimer may attenuate responses triggered individually by CXCR4 without toxicity and side effects experienced with current CXCR4 antagonists. Citation Format: Nakea Michelle Pennant, Alifiani B. Hartono, Shiqin Liu, Christopher Massey, Chung Lee, Tanya Stoyanova, Cimona V. Hinton. CXCR4/CB2 heterodimer antagonizes CXCR4-mediated metastasis and tumor growth in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4119.

Abstract 5136: Development of a highly specific NAPRT monoclonal antibody for the detection of tumors sensitive to pharmacological inhibition of NAMPT

Abstract Introduction: A key component of targeting synthetic lethality in cancer is identifying specific biomarkers, such as sources of NAD+ synthesis, that differentiate between healthy and diseased tissue. In mammals, NAD+ can be synthesized from nicotinamide via nicotinamide phosphoribosyltransferase (NAMPT) or from nicotinic acid (niacin, NA) via nicotinic acid phosphoribosyltransferase (NAPRT). NAPRT loss has been identified across tumor indications as a synthetic lethal sensitizer to small molecule inhibitors of NAMPT. Given the reliance of cancer cells on NAD+ synthesis, blocking NAD+ production is a compelling target for anti-cancer therapeutics. However, identifying tumors with NAPRT loss has been a challenge as NAPRT can be expressed by as many as 14 transcripts, only some of which code for the active enzyme. This complicates transcript-based analyses and necessitates proteomic assessment. Here, we have developed a mouse monoclonal antibody (4A5D7) to specifically detect active NAPRT and to identify indications enriched for NAPRT loss in patients through an IHC-based assay. We further confirm that loss of NAPRT makes cells susceptible to NAMPT inhibition, and ultimately show that our antibody can identify patient-derived xenograft (PDX) models that respond to NAMPT inhibition. Methods We utilized a hybridoma system to produce antibodies against epitopes in the NAPRT active site; clones were subsequently screened for their ability to selectively recognize active NAPRT. After identifying clone 4A5D7, we validated our proprietary antibody against FFPE specimens across multiple cancer indications (TNBC, NSCLC, SCLC, CRC, gastric, ovarian, and prostate). Stained samples were scored by a pathologist and assessed for percent of positive tumor cells, nuclear and/or cytoplasmic staining, and staining intensity. CCLE and TCGA datasets were mined for indications enriched for suspected NAPRT-loss and sensitivity to NAMPT inhibitors. We then selected cell lines and pan-tumor PDX tissue microarrays (TMAs) from indications of interest and characterized samples by western blot and IHC to determine NAPRT status. Cell lines and PDX tumor tissue were exposed to increasing ranges of NAMPTi in the presence or absence of NAPRT-substrate NA to validate our approach. Results: We validated that our proprietary NAPRT monoclonal antibody clone 4A5D7 only detects functional NAPRT and demonstrated improved performance over previous antibodies. We used our antibody to characterize cell lines and PDX tumor samples, and subsequently show that NAPRT-null cell lines and tumor samples are susceptible to NAMPT inhibition, while samples with functional NAPRT can be rescued by NA. Conclusion: Our proprietary antibody can identify NAPRT null tumors and identify samples that will respond to NAMPT inhibition, a critical advancement for patient selection in clinical trials. Citation Format: Josh Spurrier, Emmanuel S. Burgos, Mark R. Lundquist, Keiichiro Tanaka, Samuel Brook, Matt Gozzi, Jae-Sung Yi, Joseph Edmonds, Mitch Raponi. Development of a highly specific NAPRT monoclonal antibody for the detection of tumors sensitive to pharmacological inhibition of NAMPT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5136.

Abstract 2091: Pharmacological inhibition of chaperone-mediated autophagy via NCoR1/RARα targeting is effective against non-small cell lung cancer

Abstract Dysregulation of autophagy, the lysosomal degradation of intracellular components, has been universally described in cancer. While activity of macroautophagy changes in a context-dependent manner in cancers, the upregulation of chaperone-mediated autophagy (CMA) is well-described in all solid tumors studied to date. Upregulated CMA directly contributes to both tumor growth and protection against anti-oncogenic interventions in cancers. Selective degradation of anti-oncogenic and pro-apoptotic proteins, such as mutant p53 and PUMA, misfolded proteins, and glycolytic enzymes by CMA allows for increased survival, reduced cell death, increased stress resistance, and enhancement of the Warburg effect in cancer cells, respectively. As such, inhibition of CMA is an attractive target for anti-cancer therapeutics, but the field currently lacks protein targets specific to CMA and selective CMA inhibitors. We have previously demonstrated the regulatory effect of retinoic acid receptor alpha (RARα) and its coregulator, nuclear receptor corepressor 1 (NCoR1), on CMA through transcriptional upregulation of the CMA effector proteins (Bourdenx et al. Cell 2021, Gomez-Sintes et al, Nat. Commun. 2022). Here, we identified a high NCoR1/RARα expression ratio in human non-small cell lung cancer (NSCLC) tumors compared to healthy lung tissue. We further illustrated a dependency on NCoR1 expression and its interaction with RARα to maintain high CMA activity in NSCLC cell lines. Provided the reliance on NCoR1 repression of RARα for high CMA activity in NSCLC, we hypothesized that small molecule targeting of the druggable NCoR1/RARα interaction to disrupt the interaction may result in suppression of CMA effector proteins and CMA inhibition. Through a series of in-silico screens for predicted NCoR1/RARα targeting, and functional screens for CMA inhibition, we identified CIM7, a first-in-class potent and selective CMA inhibitor. We validated CIM7 function through binding to RARα and subsequent disruption of NCoR1 binding, resulting in alterations to CMA-related gene expression. CIM7 inhibits CMA in vivo with no observed toxicity in normal tissues. We additionally demonstrated the therapeutic potential of CIM7 through its ability to reduce tumor growth in vivo in mouse NSCLC xenografts. Our findings support the NCoR1/RARα interaction as a major regulator of CMA in NSCLC and demonstrate the potential of pharmacologic CMA inhibition as an anti-cancer therapeutic strategy. Citation Format: Mericka McCabe, Rabia R. Khawaja, Rajanya Bhattacharyya, Antonio Diaz, Thomas P. Garner, Kristen Lindenau, Rebecca Sereda, Olaya Santiago-Fernández, Ana Maria Cuervo, Evripidis Gavathiotis. Pharmacological inhibition of chaperone-mediated autophagy via NCoR1/RARα targeting is effective against non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2091.

AURKB promotes bladder cancer progression by deregulating the p53 DNA damage response pathway via MAD2L2

BackgroundBladder cancer (BC) is the most common urinary tract malignancy. Aurora kinase B (AURKB), a component of the chromosomal passenger protein complex, affects chromosomal segregation during cell division. Mitotic arrest-deficient 2-like protein 2 (MAD2L2) interacts with various proteins and contributes to genomic integrity. Both AURKB and MAD2L2 are overexpressed in various human cancers and have synergistic oncogenic effects; therefore, they are regarded as emerging therapeutic targets for cancer. However, the relationship between these factors and the mechanisms underlying their oncogenic activity in BC remains largely unknown. The present study aimed to explore the interactions between AURKB and MAD2L2 and how they affect BC progression via the DNA damage response (DDR) pathway.MethodsBioinformatics was used to analyze the expression, prognostic value, and pro-tumoral function of AURKB in patients with BC. CCK-8 assay, colony-forming assay, flow cytometry, SA-β-gal staining, wound healing assay, and transwell chamber experiments were performed to test the viability, cell cycle progression, senescence, and migration and invasion abilities of BC cells in vitro. A nude mouse xenograft assay was performed to test the tumorigenesis ability of BC cells in vivo. The expression and interaction of proteins and the occurrence of the senescence-associated secretory phenotype were detected using western blot analysis, co-immunoprecipitation assay, and RT-qPCR.ResultsAURKB was highly expressed and associated with prognosis in patients with BC. AURKB expression was positively correlated with MAD2L2 expression. We confirmed that AURKB interacts with, and modulates the expression of, MAD2L2 in BC cells. AURKB knockdown suppressed the proliferation, migration, and invasion abilities of, and cell cycle progression in, BC cells, inducing senescence in these cells. The effects of AURKB knockdown were rescued by MAD2L2 overexpression in vitro and in vivo. The effects of MAD2L2 knockdown were similar to those of AURKB knockdown. Furthermore, p53 ablation rescued the MAD2L2 knockdown-induced suppression of BC cell proliferation and cell cycle arrest and senescence in BC cells.ConclusionsAURKB activates MAD2L2 expression to downregulate the p53 DDR pathway, thereby promoting BC progression. Thus, AURKB may serve as a potential molecular marker and a novel anticancer therapeutic target for BC.

Natural product-inspired molecules for covalent inhibition of SHP2 tyrosine phosphatase

Natural products have been playing indispensable roles in the development of lifesaving drug molecules. They are also valuable sources for covalent protein modifiers. However, they often are scarce in nature and have complex chemical structures, which are limiting their further biomedical development. Thus, natural product-inspired small molecules which still contain the essence of the parent natural products but are readily available and amenable for structural modification, are important and desirable in searching for lead compounds for various disease treatment. Inspired by the complex and diverse ent-kaurene diterpenoids with significant biological activities, we have created a synthetically accessible and focused covalent library by incorporating the bicyclo[3.2.1]octane α-methylene ketone, which is considered as the pharmacophore of ent-kaurene diterpenoids, as half of the structure, and replacing the other half with much less complex but more druglike scaffolds. From this library, we have identified and characterized selective covalent inhibitors of protein tyrosine phosphatase SHP2, an important anti-cancer therapeutic target. The success of this study demonstrated the importance of creating and evaluating natural product-inspired library as well as their application in targeting challenging disease targets.

Biological Significance of EphB4 Expression in Cancer.

Eph receptors and their Eph receptor-interacting (ephrin) ligands comprise a vital cell communication system with several functions. In cancer cells, there was evidence of bilateral Eph receptor signaling with both tumor-suppressing and tumor-promoting actions. As a member of the Eph receptor family, EphB4 has been linked to tumor angiogenesis, growth, and metastasis, which makes it a viable and desirable target for drug development in therapeutic applications. Many investigations have been conducted over the last decade to elucidate the structure and function of EphB4 in association with its ligand ephrinB2 for its involvement in tumorigenesis. Although several EphB4-targeting drugs have been investigated, and some selective inhibitors have been evaluated in clinical trials. This article addresses the structure and function of the EphB4 receptor, analyses its possibility as an anticancer therapeutic target, and summarises knowledge of EphB4 kinase inhibitors. To summarise, EphB4 is a difficult but potential treatment option for cancers.

Homeostatic status of thyroid hormones and brain water movement as determinant factors in biology of cerebral gliomas: a pilot study using a bioinformatics approach.

The expression and localization of the water channel transporters, aquaporins (AQPs), in the brain are substantially modified in gliomas during tumorigenesis, cell migration, edema formation, and resolution. We hypothesized that the molecular changes associated with AQP1 and AQP4 in the brain may potentially be anticancer therapeutic targets. To test this hypothesis, a bioinformatics analysis of publicly available data from international consortia was performed. We used RNA-seq as an experimental strategy and identified the number of differential AQP1 and AQP4 transcript expressions in glioma tissue compared to normal brain tissue. AQPs genes are overexpressed in patients with glioma. Among the glioma subtypes, AQP1 and AQP4 were overexpressed in astrocytoma (low-grade glioma) and classical (high-grade glioma). Overall survival analysis demonstrated that both AQP genes can be used as prognostic factors for patients with low-grade glioma. Additionally, we observed a correlation between the expression of genes involved in the tyrosine and thyroid hormone pathways and AQPs, namely: PNMT, ALDH1A3, AOC2, HGDATP1B1, ADCY5, PLCB4, ITPR1, ATP1A3, LRP2, HDAC1, MED24, MTOR, and ACTB1 (Spearman's coefficient = geq 0.20 and p-value = ≤ 0.05). Our findings indicate that the thyroid hormone pathways and AQPs 1 and 4 are potential targets for new anti-tumor drugs and therapeutic biomarkers for malignant gliomas.

Mechanical activation and expression of HSP27 in epithelial ovarian cancer

Understanding the complex biomechanical tumor microenvironment (TME) is of critical importance in developing the next generation of anti-cancer treatment strategies. This is especially true in epithelial ovarian cancer (EOC), the deadliest of the gynecologic cancers due to recurrent disease or chemoresistance. However, current models of EOC progression provide little control or ability to monitor how changes in biomechanical parameters alter EOC cell behaviors. In this study, we present a microfluidic device designed to permit biomechanical investigations of the ovarian TME. Using this microtissue system, we describe how biomechanical stimulation in the form of tensile strains upregulate phosphorylation of HSP27, a heat shock protein implicated in ovarian cancer chemoresistance. Furthermore, EOC cells treated with strain demonstrate decreased response to paclitaxel in the in vitro vascularized TME model. The results provide a direct link to biomechanical regulation of HSP27 as a mediator of EOC chemoresistance, possibly explaining the failure of such therapies in some patients. The work presented here lays a foundation to elucidating mechanobiological regulation of EOC progression, including chemoresistance and could provide novel targets for anti-cancer therapeutics.