Active Mutant Research Articles

Abstract PO1-24-05: EZH2 histone methyltransferase activity promotes spindle metaplastic breast carcinoma metastasis and is induced by CCN6 knockout/b-catenin/TCF axis

Abstract Background: Metaplastic breast carcinomas (mBrCAs) are a highly aggressive subtype of triple negative breast cancer with histological evidence of deregulated differentiation towards non-glandular components. Previous studies have demonstrated that human mBrCA often exhibit activation of differentiation pathways, including canonical Wnt/b-catenin and EZH2-mediated transcriptional repression. Our lab has identified CCN6 as a tumor suppressor in mBrCA. MMTV-Cre;Ccn6fl/fl (CCN6KO) mice develop spindle mBrCAs and CCN6 is reduced/lost in 68% of human mBrCAs. Recently, we have demonstrated that the tumor suppressor function of CCN6 in spindle mBrCA requires activation of Wnt/b-catenin signaling pathway. Here, we tested the hypothesis that CCN6 KO leads to the upregulation of EZH2 histone methyltransferase promoting mBrCA. Furthermore, we investigated the requirement for the activation of the Wnt/b-catenin pathway in this mechanism. Methods: To test the effect of CCN6/b-catenin on EZH2 gene and protein expression we performed IHC, IF, qRT-PCR, ChIP-Seq, RNA-seq, invasion and adhesion assays, EZH2 reporter assay and immunoblots in mBrCA cell lines and MMTV-Cre;Ccn6fl/fl tumors. To investigate the role of CCN6KO-induced b-catenin activation on EZH2 activity and neoplastic functions we employed three independent approaches: i) Expression of a dominant-negative Tcf4 (dnTcf4) rescued with EZH2-WT, dSET and dNLS domain mutants versus vector in MMTV-Cre;Ccn6fl/fl tumor-derived cells; ii) Expression of a constitutively active mutant (S33Y) b-catenin in concert with treatment with recombinant human CCN6 (rhCCN6; 500 ug/ml) versus control; iii) Syngeneic orthotopic mammary tumor transplants of MMTV-Cre;Ccn6fl/fl were used for in vivo rescue experiments with rhCCN6 or BSA. To assess the therapeutic benefit of inhibiting EZH2 methyltransferase activity, MMTV-Cre;Ccn6fl/fl tumor cells (CCN6KO cells) were implanted orthotopically or intracardially in FVB mice, followed by treatment with EPZ-6438 (a selective EZH2 methyltransferase inhibitor) or vehicle. We tested CCN6, b-catenin, and EZH2 expression by IHC in a cohort of 27 human mBrCA tumor samples. Results: CCN6KO-induced b-catenin/TCF activation mediates EZH2 transcriptional upregulation and the deposition of repressive H3K27me3 in spindle mBrCAs. We found that the invasion program triggered by CCN6KO-induced Wnt/b-catenin requires EZH2 catalytic activity as WT-EZH2 (but not dSET-EZH2) rescued the reduced invasion of CCN6KO-dnTcf4 cells. RNA-seq and 3H3K27 ChIP-seq of CCN6KO-dnTcf4 cells transduced with EZH2-WT or dSET-EZH2 identify specific CCN6KO-b-catenin/Tcf targets that require EZH2 transcriptional repressor function. In vivo, administration of CCN6 protein to MMTV-Cre;Ccn6fl/fl tumor transplants reduces tumor growth and nuclear b-catenin, EZH2 and 3H3K27 in the tumors. Pharmacologic inhibition of EZH2 reduces the growth and metastasis of CCN6KO mBrCA tumors and improves survival. We identify a subset of human spindle mBrCA (54%) that display a CCN6Low/nuclear b-cat/EZH2High phenotype. Conclusion: We found a critical role for EZH2 activation in CCN6-deficient mBrCA tumor phenotypes via b-catenin/TCF Wnt canonical signaling. We demonstrate the effectiveness of pharmacological inhibition of EZH2 methyltransferase activity in reducing primary tumor growth and distant metastasis in mouse models of spindle mBrCA. In clinical samples, low CCN6 is significantly associated with activated b-catenin and high EZH2 in spindle mBrCAs compared to other subtypes. These data reveal a novel tumor suppressor mechanism of CCN6 and provide compelling evidence supporting the potential therapeutic value of CCN6 restoration, b-catenin or EZH2 inhibition as promising approaches for the treatment of spindle mBrCAs. Citation Format: Maria Gonzalez, Ahmad Eido, GIUSEPPINA AUGIMERI, Celina Kleer. EZH2 histone methyltransferase activity promotes spindle metaplastic breast carcinoma metastasis and is induced by CCN6 knockout/b-catenin/TCF axis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-05.

Abstract PO2-24-07: Investigation into potential Estrogen Receptor (ER)-mediated regulation of Phosphoserine Aminotransferase (PSAT1)

Abstract Breast cancer is one of the leading causes of death among women. Expression of different proteins, such as hormone receptors (HR), are used to classify different types of breast cancer (i.e., HR+BC or triple negative breast cancer (TNBC)). While activation of the estrogen receptor (ER) in HR+BC is known to regulate a vast number of downstream targets in promoting breast cancer, there may be additional ER-regulated processes that are still unknown. The serine synthetic pathway has been demonstrated to be crucial in the proliferation and survival of select subtypes of breast cancer and phosphoserine aminotransferase 1 (PSAT1) is a key regulatory step. Previous studies have demonstrated higher PSAT1 levels in cells lacking ER and we now show a negative correlation between ERα protein levels and PSAT1 transcripts in human patient samples. This indicates that ER may potentially regulate PSAT1 expression. To initially investigate this potential relationship, either WT or constitutively active mutant ERα was expressed in TNBC cells (MDA-MB-231), which have high endogenous PSAT1 expression and no ER. Immunoblot was used to verify ERα levels and to assess effects on PSAT1. We observed a qualitative trend in decreasing PSAT1 levels with re-expression of ERα in the TNBC cells. While the variabilities in these results preclude any conclusion of a ERα:PSAT1 regulatory axis, the lack of a quantifiable effect on PSAT1 levels is postulated to be due to the transient nature of ERα expression/suppression in these systems. A more profound effect would be expected under durable changes in ERα levels and is a focus on-going studies. Citation Format: Mary Sumlut, James Wittliff, Brian Clem. Investigation into potential Estrogen Receptor (ER)-mediated regulation of Phosphoserine Aminotransferase (PSAT1) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-07.

Identification of a 1-acyl-glycerol-3-phosphate acyltransferase from Mycobacterium tuberculosis, a key enzyme involved in triacylglycerol biosynthesis.

Latent tuberculosis, caused by dormant Mycobacterium tuberculosis (Mtb), poses a threat to global health through the incubation of undiagnosed infections within the community. Dormant Mtb, which is phenotypically tolerant to antibiotics, accumulates triacylglycerol (TAG) utilizing fatty acids obtained from macrophage lipid droplets. TAG is vital to mycobacteria, serving as a cell envelope component and energy reservoir during latency. TAG synthesis occurs by sequential acylation of glycerol-3-phosphate, wherein the second acylation step is catalyzed by acylglycerol-3-phosphate acyltransferase (AGPAT), resulting in the production of phosphatidic acid (PA), a precursor for the synthesis of TAG and various phospholipids. Here, we have characterized a putative acyltransferase of Mtb encoded by Rv3816c. We found that Rv3816c has all four characteristic motifs of AGPAT, exists as a membrane-bound enzyme, and functions as 1-acylglycerol-3-phosphate acyltransferase. The enzyme could transfer the acyl group to acylglycerol-3-phosphate (LPA) from monounsaturated fatty acyl-coenzyme A of chain length 16 or 18 to produce PA. Complementation of Escherichia coli PlsC mutant invivo by Rv3816c confirmed that it functions as AGPAT. Its active site mutants, H43A and D48A, were incapable of transferring the acyl group to LPA invitro and were not able to rescue the growth defect of E. coli PlsC mutant invivo. Identifying Rv3816c as AGPAT and comparing its properties with other AGPAT homologs is not only a step toward understanding the TAG biosynthesis in mycobacteria but has the potential to explore it as a drug target.

Expression and functional significance of phosphoenolpyruvate carboxykinase 1 in uveal melanoma

Uveal melanoma (UVM), an uncommon yet potentially life-threatening ocular cancer, arises from melanocytes in the uveal tract of the eye. The exploration of novel oncotargets for UVM is of paramount importance. In this study, we show that PCK1 (phosphoenolpyruvate carboxykinase 1) expression is upregulated in various UVM tissues as well as in primary UVM cells and immortalized lines. Furthermore, bioinformatics studies reveal that PCK1 overexpression in UVM correlates with advanced disease stages and poor patient survival. Genetic silencing (utilizing viral shRNA) or knockout (via CRISPR/Cas9) of PCK1 significantly curtailed cell viability, proliferation, cell cycle progression, and motility, while provoking apoptosis in primary and immortalized UVM cells. Conversely, ectopic overexpression of PCK1, achieved through a viral construct, bolstered UVM cell proliferation and migration. Gαi3 expression and Akt phosphorylation were reduced following PCK1 silencing or knockout, but increased after PCK1 overexpression in UVM cells. Restoring Akt phosphorylation through a constitutively active mutant Akt1 (S473D) ameliorated the growth inhibition, migration suppression, and apoptosis induced by PCK1 silencing in UVM cells. Additionally, ectopic expression of Gαi3 restored Akt activation and counteracted the anti-UVM cell effects by PCK1 silencing. In vivo, the growth of subcutaneous xenografts of primary human UVM cells was significantly inhibited following intratumoral injection of adeno-associated virus (aav) expressing PCK1 shRNA. PCK1 depletion, Gαi3 downregulation, Akt inhibition, proliferation arrest, and apoptosis were detected in PCK1-silenced UVM xenografts. Collectively, our findings demonstrate that PCK1 promotes UVM cell growth possibly by modulating the Gαi3-Akt signaling pathway.

A tryptophan-derived uremic metabolite-Ahr-Pdk4 axis governs skeletal muscle mitochondrial energetics in chronic kidney disease.

Chronic kidney disease (CKD) causes an accumulation of uremic metabolites that negatively impact skeletal muscle function. Tryptophan-derived uremic metabolites are agonists of the aryl hydrocarbon receptor (AHR) which has been shown to be activated in the blood of CKD patients. This study investigated the role of the AHR in skeletal muscle pathology of CKD. Compared to control participants with normal kidney function, AHR-dependent gene expression (CYP1A1 and CYP1B1) was significantly upregulated in skeletal muscle of patients with CKD (P=0.032) and the magnitude of AHR activation was inversely correlated with mitochondrial respiration (P<0.001). In mice with CKD, muscle mitochondrial oxidative phosphorylation (OXPHOS) was significantly impaired and strongly correlated with both the serum level of tryptophan-derived uremic metabolites and AHR activation. Muscle-specific deletion of the AHR significantly improved mitochondrial OXPHOS in male mice with the greatest uremic toxicity (CKD+probenecid) and abolished the relationship between uremic metabolites and OXPHOS. The uremic metabolite-AHR-mitochondrial axis in skeletal muscle was further confirmed using muscle-specific AHR knockdown in C57BL6J that harbour a high-affinity AHR allele, as well as ectopic viral expression of constitutively active mutant AHR in mice with normal renal function. Notably, OXPHOS changes in AHRmKO mice were only present when mitochondria were fueled by carbohydrates. Further analyses revealed that AHR activation in mice led to significant increases in Pdk4 expression (P<0.05) and phosphorylation of pyruvate dehydrogenase enzyme (P<0.05). These findings establish a uremic metabolite-AHR-Pdk4 axis in skeletal muscle that governs mitochondrial deficits in carbohydrate oxidation during CKD.

Inhibition of adenylyl cyclase by GTPase-deficient Gαi is mechanistically different from that mediated by receptor-activated Gαi

Signal transduction through G protein-coupled receptors (GPCRs) has been a major focus in cell biology for decades. Numerous disorders are associated with GPCRs that utilize Gi proteins to inhibit adenylyl cyclase (AC) as well as regulate other effectors. Several early studies have successfully defined the AC-interacting domains of several members of Gαi by measuring the loss of activity upon homologous replacements of putative regions of constitutive active Gαi mutants. However, whether such findings can indeed be translated into the context of a receptor-activated Gαi have not been rigorously verified. To address this issue, an array of known and new chimeric mutations was introduced into GTPase-deficient Q204L (QL) and R178C (RC) mutants of Gαi1, followed by examinations on their ability to inhibit AC. Surprisingly, most chimeras failed to abolish the constitutive activity brought on by the QL mutation, while some were able to eliminate the inhibitory activity of RC mutants. Receptor-mediated inhibition of AC was similarly observed in the same chimeric constructs harbouring the pertussis toxin (PTX)-resistant C351I mutation. Moreover, RC-bearing loss-of-function chimeras appeared to be hyper-deactivated by endogenous RGS protein. Molecular docking revealed a potential interaction between AC and the α3/β5 loop of Gαi1. Subsequent cAMP assays support a cooperative action of the α3/β5 loop, the α4 helix, and the α4/β6 loop in mediating AC inhibition by Gαi1-i3. Our results unveiled a notable functional divergence between constitutively active mutants and receptor-activated Gαi1 to inhibit AC, and identified a previously unknown AC-interacting domain of Gαi subunits. These results collectively provide valuable insights on the mechanism of AC inhibition in the cellular environment.

A 50-gene biomarker identifies estrogen receptor-modulating chemicals in a microarray compendium

High throughput transcriptomics (HTTr) profiling has the potential to rapidly and comprehensively identify molecular targets of environmental chemicals that can be linked to adverse outcomes. We describe here the construction and characterization of a 50-gene expression biomarker designed to identify estrogen receptor (ER) active chemicals in HTTr datasets. Using microarray comparisons, the genes in the biomarker were identified as those that exhibited consistent directional changes when ER was activated (4 ER agonists; 4 ESR1 gene constitutively active mutants) and opposite directional changes when ER was suppressed (4 antagonist treatments; 4 ESR1 knockdown experiments). The biomarker was evaluated as a predictive tool using the Running Fisher algorithm by comparison to annotated gene expression microarray datasets including those evaluating the transcriptional effects of hormones and chemicals in MCF-7 cells. Depending on the reference dataset used, the biomarker had a predictive accuracy for activation of up to 96%. To demonstrate applicability for HTTr data analysis, the biomarker was used to identify ER activators in a set of 15 chemicals that are considered potential bisphenol A (BPA) alternatives examined at up to 10 concentrations in MCF-7 cells and analyzed by full-genome TempO-Seq. Using benchmark dose (BMD) modeling, the biomarker genes stratified the ER potency of BPA alternatives consistent with previous studies. These results demonstrate that the ER biomarker can be used to accurately identify ER activators in transcript profile data derived from MCF-7 cells.

Newly discovered clouting interplay between matrix metalloproteinases structures and novel quaternary Ammonium K21: computational and in-vivo testing

Aims and objectivesTo analyze anti-MMP mode of action of Quaternary Ammonium Silane (QAS, codenamed as k21) by binding onto specific MMP site using computational molecular simulation and Anti-Sortase A (SrtA) mode of action by binding onto specific site using computational molecular simulation.Materials and methodsIn silico Molecular Dynamics (MD) was used to determine the interactions of K21 inside the pocket of the targeted protein (crystal structure of fibroblast collagenase-1 complexed to a diphenyl-ether sulphone based hydroxamic acid; PDB ID: 966C; Crystal structure of MMP-2 active site mutant in complex with APP-derived decapeptide inhibitor. MD simulations were accomplished with the Desmond package in Schrödinger Drug Discovery Suite. Blood samples (~ 0.5 mL) collected into K2EDTA were immediately transferred for further processing using the Litron MicroFlow® PLUS micronucleus analysis kit for mouse blood according to the manufacturer’s instructions. Bacterial Reverse Mutation Test of K21 Molecule was performed to evaluate K21 and any possible metabolites for their potential to induce point mutations in amino acid-requiring strains of Escherichia coli (E. coli) (WP2 uvrA (tryptophan-deficient)).ResultsMolecular Simulation depicted that K21 has a specific pocket binding on various MMPs and SrtA surfaces producing a classical clouting effect. K21 did not induce micronuclei, which are the result of chromosomal damage or damage to the mitotic apparatus, in the peripheral blood reticulocytes of male and female CD-1 mice when administered by oral gavage up to the maximum recommended dose of 2000 mg/kg. The test item, K21, was not mutagenic to Salmonella typhimurium (S. typhimurium) strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2 uvrA in the absence and presence of metabolic activation when tested up to the limit of cytotoxicity or solubility under the conditions of the test.ConclusionK21 could serve as a potent protease inhibitor maintaining the physical and biochemical properties of dental structures.

Abstract 1998: ABCB1 promotes uveal melanoma cell resistance to FR900359 and identifies a tumor cell subpopulation with a distinct gene expression signature

Abstract Uveal melanoma (UM) is the leading form of intraocular cancer in adults and the second most common form of melanoma. Although rare, UM has a median survival rate of ~1 year after metastasis occurs. Metastatic UM is largely refractory to treatment and there are no effective pharmacological therapies, resulting in poor overall survival. In &amp;gt;90% UM cases, the oncogenic initiator proteins are constitutively active mutant GNAQ or GNA11 proteins (GNAQ/11). FR900359 (FR) is a natural compound that inhibits GNAQ/11 and has anti-proliferative effects on UM cells. To identify key oncogenic mechanisms downstream of mutant GNAQ/11, we performed a Sleeping Beauty (SB) transposon forward genetic screen to identify drivers of FR resistance. The SB screen identified ATP-binding cassette sub-family B member 1 (ABCB1) as one of the top genes upregulated by SB in FR-resistant UM cell populations. We determined that although ABCB1 does not provide immediate resistance to FR, the overexpression of ABCB1 leads to emergence of resistant colonies at a much higher rate than spontaneous resistance, validating ABCB1 as contributors to FR resistance. Unexpectedly, immunofluorescence revealed that a relatively small fraction of UM cells strongly expresses the transgenes after stable transfection. Immunoblotting confirmed that ABCB1 expression was initially modest in ABCB1-transfected populations but increased in the cells that developed resistance to FR. To examine the kinetics of FR resistance, population doubling versus time (PDVT) assays were performed in UM cells expressing ABCB1 or empty vector control treated with FR or vehicle. FR potently suppressed the proliferation of vector control cells. Drug-naïve ABCB1-transfected cells developed resistance to FR over time but accomplished fewer population doublings than vehicle-treated cells in the same time interval. Once transgenic populations emerged as resistant populations in FR, they grew similarly in drug or vehicle. Ongoing RNA-sequencing analysis from PDVT samples has identified distinct gene expression signatures associated with FR treatment and ABCB1 expression. These findings are expected to illuminate tumor cell heterogeneity amongst UM cell populations that may be related to drug resistance and metastasis. Citation Format: Sarina Dion Murray, Jesse D. Riordan, Eric R. Anderson, Michael D. Onken, Kendall J. Blumer, Adam J. Dupuy, Christopher S. Stipp. ABCB1 promotes uveal melanoma cell resistance to FR900359 and identifies a tumor cell subpopulation with a distinct gene expression signature [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 1998.

Abstract 2828: Combining neratinib with trastuzumab deruxtecan to treat HER2;TP53 co-mutated breast cancers

Abstract In metastatic breast cancer, HER2 activating mutations frequently co-occur with mutations in the TP53 gene and patients with these two co-occuring mutations have a lower response rate to neratinib. In order to address this clinical problem, we bred HER2 activating mutant transgenic mice with two different alleles of TP53. We used mice with the TP53 R172H mutation, which is the murine homolog of the human hotspot TP53 R175H mutation, and mice with TP53flox/flox allele, which mimics TP53 truncating mutations seen in human breast cancers. All three H53 genotypes (H53null, H53172/WT, and H53172/null) developed breast cancers faster than H mice, with H53172/null mice developing breast tumors the fastest, at just 6 weeks after adenoviral-Cre injection into the mammary fat pad. We developed organoids from these murine breast cancers and found that all H53 genotype organoids were less sensitive to neratinib compared to H, however, neratinib was able to synergize with trastuzumab deruxtecan (T-DXd), likely through neratinib mediated increased internalization of T-DXd. Prior literature studies suggest TP53 mutant cancer cells may have therapeutic vulnerabilities to the topoisomerase 1 (Top1) inhibitors. Organoids from all three H53 genotypes were sensitive to the Top1 inhibitor, exatecan. H53172/WT behaved like H organoids with G1 phase cell cycle arrest and induction of p21 expression after exatecan treatment. In contrast, H53null and H53172/null failed to induce p21 and arrested at late-S/G2 phase. Additional animal experiments with these H53 genotypes are in progress and results will be presented at the AACR Annual Meeting. Abbreviation Genotype Median tumor formation (weeks) Neratinib IC50 (nM) Neratinib +T-DXd H HER2V777L 28 10 Not determined H53null HER2V777L;TP53flox/flox 10 240 Synergy H53172/WT HER2V777L;TP53R172H/WT 11 100 Synergy H53172/null HER2V777L;TP53R172H/flox 6 150 Synergy Citation Format: Xiaoqing (Cathy) Cheng, Maureen Highkin, Jacob Hsia, Julie L. Prior, Ian S. Hagemann, Ron Bose. Combining neratinib with trastuzumab deruxtecan to treat HER2;TP53 co-mutated breast cancers [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 2828.

Abstract 393: RAS and PI3K activation promote glioblastoma progression and ischemia-associated tumor cell death

Abstract Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor in adults. Despite the association of tumor necrosis and a poor prognosis in GBM, the mechanisms underlying tumor necrosis formation remain unclear, likely due to the lack of appropriate experimental models to examine the development of tumor necrosis. In our study, we developed mouse models with tumor necrosis by intracranially injecting human GBM cells expressing KRAS or PI3K active mutants into immunodeficient mice. Surprisingly, despite all tumors displayed similar sizes, the control tumors expressing an empty vector (EV) did not exhibit tumor necrosis. This intriguing observation led us to explore the potential role of KRAS and PI3K in promoting cell death within the tumor microenvironment. Ischemia, which is characterized by insufficient nutrients and hypoxia, is often found in GBM. Under an in vitro ischemia mimetic condition, we found that the KRAS or PI3K activated GBM cells showed more cell death compared to EV cells. Mechanistically, ischemia activates the p38 MAPK-MK2 pathway to induce cell death, a process further enhanced by KRAS or PI3K. Notably, we observed elevated expression of p-MK2 in the peri-necrotic areas of GBMs harboring KRAS or PI3K mutants compared to the cellular tumor zones. Moreover, we revealed that ischemia induces the unfolded protein response (UPR) pathway. Among UPR-related molecules, KRAS and PI3K upregulate ERN1, which encodes the transmembrane protein kinase IRE1, in ischemic conditions. Inhibition of ERN1 not only prevented ischemia-induced cell death but also inhibited the p38 MAPK-MK2 pathway. Furthermore, we observed an increase in the expression of pro-inflammatory cytokine IL-8 under ischemic conditions, a response that was prevented by inhibitors of p38 MAPK or MK2. Lastly, we found that ischemia induces ATP release and calreticulin exposure, indicating the cell death possessing certain immunogenic cell death features. In summary, our findings revealed a pivotal role of KRAS and PI3K in inducing cell death in the ischemic GBM tumor microenvironment through activating the IRE1-p38 MAPK-MK2 pathway. The cell death under this circumstance displays pro-inflammatory and immunogenic properties. Therefore, the IRE1-p38 MAPK-MK2 pathway may be exploited for the treatment of GBM in which RAS or PI3K is activated. Citation Format: Soo Yeon Kim, Miaolu Tang, Stephen Chih, Jessica Thorpe, Tong Lu, Hong-Gang Wang, Wei Li. RAS and PI3K activation promote glioblastoma progression and ischemia-associated tumor cell death [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 393.

Abstract 1568: Her2 promotes early dissemination of breast cancer by suppressing the p38 pathway through upregulation of Wip1

Abstract (a)Metastasis is the leading cause of cancer-related deaths. Increasing evidence shows that cancer cells can disseminate from early primary lesions that are impalpable, but the pathways are poorly understood. Her2 promotes breast cancer early dissemination by suppressing p38, but how Her2 downregulates p38 is unclear. In this study, we investigated the mechanism underlying suppression of p38 by Her2 in breast cancer early dissemination using cell models and xenograft models of early lesions of breast cancer. (b)We studied the role of Wip1, a p38 phosphatase, in disseminating phenotypes in cell line models of breast cancer early lesions, including cell motility measured in transwell assays, cell invasion measured by percentage of organoids with outward invading cells, and epithelial-to-mesenchymal transition (EMT) measured by levels of EMT markers in Western blotting analysis and immunofluorescence (IF) staining assays. (c)We demonstrate that high levels of Wip1 correlated with Her2+ status, and reduced phosphorylation of p38 and its downstream effectors MK2 and Hsp27 in tissue samples from early lesions of human breast cancer. In addition, ectopic expression of Wip1 reduced phosphorylation of p38, MK2 and Hsp27 in cell models of breast cancer early lesions.We found that Wip1 promoted migration, invasion and EMT, while Wip1 knockdown abrogated the ability of Her2 to induce these disseminating phenotypes, in cell line models of early lesion breast cancer. Thus, Wip1 induction is both sufficient, and necessary for Her2, to promote migration, invasion and EMT in early lesion breast cancer cells. In an attempt to identify the Wip1 downstream effectors involved in disseminating phenotypes, we found that ectopic expression of the constitutively active mutants of p38 reversed the ability of Wip1 to suppress MK2 and Hsp27 phosphorylation and to induce migration, invasion and EMT in early lesion breast cancer cells. These results suggest that Wip1 promotes breast cancer early dissemination by suppressing p38. We investigate the mechanism by which Her2 induces Wip1 expression. Ectopic expression of Her2 did not alter the level of Wip1 mRNA or the Wip1 protein stability, suggesting that Her2 promote Wip1 expression by regulating its translation. We are currently investigating the mechanism by which Her2 induces Wip1 translation.Moreover, inhibitors of Her2, Wip1 and Skp2 can each reduce the disseminating phenotypes in cells models of breast cancer early lesions ectopically expressing Her2, Wip1 or Skp2. (d)Our findings identify the Her2-Wip1-p38-Mk2-Hsp27 cascade as a novel mechanism mediating breast cancer early dissemination and provide a basis for new therapies targeting early metastatic dissemination in Her2+ breast cancer using inhibitors for Her2, Wip1 or Skp2. Citation Format: Nan Wang. Her2 promotes early dissemination of breast cancer by suppressing the p38 pathway through upregulation of Wip1 [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 1568.

Enhancement of preference, catalytic activity and thermostability of polyphenol oxidase from Rosa Chinensis by semi-rational engineering

Improvement of activity and property in polyphenol oxidases (PPOs) by protein engineering may contribute to synthesis of theaflavin (TF1) in industrial processes. Here, the potential noncatalytic residues of PPO from Rosa chinensis (RcPPO) were selected by sequence alignment, structural analysis and surface residues prediction and the mutants were gained by semi-rational site-directed mutagenesis. Mutants L173F, A174K and N334I possessed 10.48, 9.74 and 7.13 times increases respectively in preference for diphenols over monophenols. Enzymatic activity, half-life at 40 and 50 °C of the most active mutant RcPPO-FKI increased to 2.67, 2.62 and 3.37 times respectively in comparison with wild type, implying that activity and thermostability of RcPPO through mutation were improved. The improvement of activity and thermostability in the mutant should be ascribed to more hydrogen bonds formed by the conserved catalytic histidine residues with substrate, reduced fluctuation of the loop regions and increased hydrogen network of the mutational amino acids, revealed by molecular dynamics simutation and molecular docking. Moreover, the yield of TF1 was improved by catalysis of mutant RcPPO-FKI with higher synthetic efficiency of TF1 in comparison with some reported catalysts and as a promising alternative to commercial mushroom tyrosinase. PPOs with enhanced performances provide the robust candidate for industrial application.

Abstract 188: Investigating the macrophage and microglia response to ferroptosis in glioblastoma

Abstract Glioblastoma (GBM) is the most common malignant primary brain tumor. The median survival is less than two years. The mesenchymal (MES) subtype of GBM is particularly associated with treatment resistance, poor prognosis, and enriched infiltration of immune cells, including tumor-associated microglia and/or macrophages (TAMs). Despite promising results in preclinical studies, clinical trials targeting TAMs have been unsuccessful. Necrosis is a hallmark of GBM and correlates with unfavorable outcomes, but how necrosis results in poor prognosis is not well understood. We previously showed that neutrophils promote tumor necrosis and GBM progression by inducing ferroptosis, a form of regulated cell death due to iron-dependent lipid peroxidation. Understanding the effects of GBM ferroptosis on TAMs can provide further insights into how tumor necrosis ultimately leads to poor prognosis in GBM. We developed an orthotopic xenograft GBM mouse model harboring a constitutively active mutant of the Hippo pathway transcriptional coactivator with PDZ-binding motif (TAZ). Tumors of this model displayed MES characteristics and developed necrosis to an extent similar to patients with GBM. Using this model, confocal microscopy and flow cytometry studies revealed the presence of immune cells positive for markers of neutrophils and macrophages/microglia in the necrotic region of the tumor. Although we have yet to establish an in vitro model to study these cells, we found that conditioned media (CM) derived from ferroptotic GBM cells induced more macrophage differentiation from bone marrow cells when compared to live GBM CM. This led us to hypothesize that ferroptosis in GBM is immunogenic and orchestrates the immune response mounted by TAMs. We conducted an RNA sequencing (RNA-seq) study of bone marrow cells cultured in ferroptotic CM. Compared to live CM, ferroptotic CM upregulated several pro-inflammatory cytokine signaling pathways. We validated the mRNA expression levels of the identified cytokines by RT-qPCR. RNA-seq analysis also revealed the activation of phagosome formation pathway. Through phagocytosis assay, we showed that macrophages preferentially engulfed ferroptotic GBM cells over necrotic or live cells. To identify transcriptional changes in GBM cells undergoing ferroptosis, we carried out an RNA-seq study showing that ferroptotic GBM cells also upregulated several pro-inflammatory cytokine signaling pathways, which were validated by RT-qPCR. We also found that ferroptotic GBM cells released ATP extracellularly, where such release temporally coincided with the increase in pro-inflammatory cytokines. Our results suggest that ferroptosis in GBM is immunogenic and induces a pro-inflammatory response in macrophages/microglia. We are conducting further in vitro and in vivo studies to better understand how these macrophages/microglia modulate other immune cells and play a role in GBM progression. Citation Format: Stephen Y. Chih, Miaolu Tang, Soo Yeon Kim, Tong Lu, Rachel Harter, Jessica Thorpe, Wei Li. Investigating the macrophage and microglia response to ferroptosis in glioblastoma [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 188.

The redox-sensitive GSK3β is a key regulator of glomerular podocyte injury in type 2 diabetic kidney disease

Emerging evidence suggests that GSK3β, a redox-sensitive transducer downstream of insulin signaling, acts as a convergent point for myriad pathways implicated in kidney injury, repair, and regeneration. However, its role in diabetic kidney disease remains controversial. In cultured glomerular podocytes, exposure to a milieu of type 2 diabetes elicited prominent signs of podocyte injury and degeneration, marked by loss of homeostatic marker proteins like synaptopodin, actin cytoskeleton disruption, oxidative stress, apoptosis, and stress-induced premature senescence, as shown by increased staining for senescence-associated β-galactosidase activity, amplified formation of γH2AX foci, and elevated expression of mediators of senescence signaling, like p21 and p16INK4A. These degenerative changes coincided with GSK3β hyperactivity, as evidenced by GSK3β overexpression and reduced inhibitory phosphorylation of GSK3β, and were averted by tideglusib, a highly-selective small molecule inhibitor of GSK3β. In agreement, post-hoc analysis of a publicly-available glomerular transcriptomics dataset from patients with type 2 diabetic nephropathy revealed that the curated diabetic nephropathy-related gene set was enriched in high GSK3β expression group. Mechanistically, GSK3β-modulated nuclear factor Nrf2 signaling is involved in diabetic podocytopathy, because GSK3β knockdown reinforced Nrf2 antioxidant response and suppressed oxidative stress, resulting in an improvement in podocyte injury and senescence. Conversely, ectopic expression of the constitutively active mutant of GSK3β impaired Nrf2 antioxidant response and augmented oxidative stress, culminating in an exacerbated diabetic podocyte injury and senescence. Moreover, IRS-1 was found to be a cognate substrate of GSK3β for phosphorylation at IRS-1S332, which negatively regulates IRS-1 activity. GSK3β hyperactivity promoted IRS-1 phosphorylation, denoting a desensitized insulin signaling. Consistently, in vivo in db/db mice with diabetic nephropathy, GSK3β was hyperactive in glomerular podocytes, associated with IRS-1 hyperphosphorylation, impaired Nrf2 response and premature senescence. Our finding suggests that GSK3β is likely a novel therapeutic target for treating type 2 diabetic glomerular injury.

Engineering the Propeptide of Microbial Transglutaminase Zymogen: Enabling Substrate-Dependent Activation for Bioconjugation Applications.

Microbial transglutaminase (MTG) from Streptomyces mobaraensis is a powerful biocatalytic glue for site-specific cross-linking of a range of biomolecules and synthetic molecules that have an MTG-reactive moiety. The preparation of active recombinant MTG requires post-translational proteolytic digestion of a propeptide that functions as an intramolecular chaperone to assist the correct folding of the MTG zymogen (MTGz) in the biosynthesis. Herein, we report engineered active zymogen of MTG (EzMTG) that is expressed in soluble form in the host Escherichia coli cytosol and exhibits cross-linking activity without limited proteolysis of the propeptide. We found that the saturation mutagenesis of residues K10 or Y12 in the propeptide domain generated several active MTGz mutants. In particular, the K10D/Y12G mutant exhibited catalytic activity comparable to that of mature MTG. However, the expression level was low, possibly because of decreased chaperone activity and/or the promiscuous substrate specificity of MTG, which is potentially harmful to the host cells. The K10R/Y12A mutant exhibited specific substrate-dependent reactivity toward peptidyl substrates. Quantitative analysis of the binding affinity of the mutated propeptides to the active site of MTG suggested an inverse relationship between the binding affinity and the catalytic activity of EzMTG. Our proof-of-concept study provides insights into the design of a new biocatalyst using the MTGz as a scaffold and a potential route to high-throughput screening of EzMTG mutants for bioconjugation applications.

Mechanism of Mutation-Induced Effects on the Catalytic Function of TEV Protease: A Molecular Dynamics Study.

Tobacco etch virus protease (TEVp) is wildly exploited for various biotechnological applications. These applications take advantage of TEVp's ability to cleave specific substrate sequences to study protein function and interactions. A major limitation of this enzyme is its relatively slow catalytic rate. In this study, MD simulations were conducted on TEV enzymes and known highly active mutants (eTEV and uTEV3) to explore the relationship between mutation, conformation, and catalytic function. The results suggest that mutations distant from the active site can influence the substrate-binding pocket through interaction networks. MD analysis of eTEV demonstrates that, by stabilizing the orientation of the substrate at the catalytic site, mutations that appropriately enlarge the substrate-binding pocket will be beneficial for Kcat, enhancing the catalytic efficiency of the enzyme. On the contrary, mutations in uTEV3 reduced the flexibility of the active pocket and increased the hydrogen bonding between the substrate and enzyme, resulting in higher affinity. At the same time, the MD simulation demonstrates that mutations outside of the active site residues could affect the dynamic movement of the binding pocket by altering residue networks and communication pathways, thereby having a profound impact on reactivity. These findings not only provide a molecular mechanistic explanation for the excellent mutants, but also serve as a guiding framework for rational computational design.

Backbone Modification Provides a Long-Acting Inverse Agonist of Pathogenic, Constitutively Active PTH1R Variants.

Parathyroid hormone 1 receptor (PTH1R) plays a key role in mediating calcium homeostasis and bone development, and aberrant PTH1R activity underlies several human diseases. Peptidic PTH1R antagonists and inverse agonists have therapeutic potential in treating these diseases, but their poor pharmacokinetics and pharmacodynamics undermine their in vivo efficacy. Herein, we report the use of a backbone-modification strategy to design a peptidic PTH1R inhibitor that displays prolonged activity as an antagonist of wild-type PTH1R and an inverse agonist of the constitutively active PTH1R-H223R mutant both in vitro and in vivo. This peptide may be of interest for the future development of therapeutic agents that ameliorate PTH1R malfunction.

NRF2 Activation in Trp53;p16-deficient Mice Drives Oral Squamous Cell Carcinoma.

Human squamous cancers frequently show constitutive NRF2 activation, associated with poorer outcomes and resistance to multiple therapies. Here, we report the first activated NRF2-driven and human-relevant mouse model of squamous cell carcinoma that develops in the background of p16 and p53 loss. The availability of this model will lead to a clearer understanding of how NRF2 contributes to the initiation, progression, and therapeutic response of OSCC.

Modulating the pH profile of vanillin dehydrogenase enzyme from extremophile Bacillus ligniniphilus L1 through computational guided site-directed mutagenesis

Vanillin dehydrogenase (VDH) has recently come forward as an important enzyme for the commercial production of vanillic acid from vanillin in a one-step enzymatic process. However, VDH with high alkaline tolerance and efficiency is desirable to meet the biorefinery requirements. In this study, computationally guided site-directed mutagenesis was performed by increasing the positive and negative charges on the surface and near the active site of the VDH from the alkaliphilic marine bacterium Bacillus ligniniphilus L1, respectively. In total, 20 residues including 15 from surface amino acids and 5 near active sites were selected based on computational analysis and were subjected to site-directed mutations. The optimum pH of the two screened mutants including I132R, and T235E from surface residue and near active site mutant was shifted to 9, and 8.6, with a 2.82- and 2.95-fold increase in their activity compared to wild enzyme at pH 9, respectively. A double mutant containing both these mutations i.e., I132R/T235E was produced which showed a shift in optimum pH of VDH from 7.4 to 9, with an increase of 74.91 % in enzyme activity. Therefore, the double mutant of VDH from the L1 strain (I132R/T235E) produced in this study represents a potential candidate for industrial applications.