Gao, Huanyao

Endocrine therapy remains the cornerstone of treatment for estrogen receptor alpha (ERα)-positive breast cancer, yet the relationship between individual genetic background and molecular response to ERα-targeted therapy remains incompletely understood. Using a well-characterized panel of lymphoblastoid cell lines (LCLs), we performed genome-wide pharmacogenomic expression quantitative trait locus (PGx-eQTL) analysis to identify estradiol (E2)- and tamoxifen (TAM)-induced SNP-gene pairs. PGx-eQTL signals were integrated with previously published breast cancer genome-wide association study datasets to examine their association with clinically relevant breast cancer phenotypes. We identified two ER-mediated PGx-eQTL SNP-gene pairs associated with breast cancer prognosis post-treatment with E2 or TAM. Notable loci included E2-regulated MRPL15 and TAM-regulated SYCP3, which have effects in a genotype-dependent manner, with genotype-dependent survival outcomes, from worse to better relapse-free survival. Similar endocrine-therapy effects on patient survival and breast cancer risk were observed in SIK2, post TAM-treatment, and in LSM4 with E2-treatment. Moreover, qRT-PCR validation in an independent LCL panel confirmed the genotype-specificity of these signals. Overall, we identified ER-mediated PGx-eQTL SNP-gene pairs which represent potential pharmacogenomic tools for identifying patients likely to benefit from ERα-targeted endocrine therapy, offering a foundation for more genotype-informed individualized treatment decisions in breast cancer.

Abstract Mitochondrial dysfunction and lipid dysregulation are among the earliest abnormalities in Alzheimer’s disease (AD), yet their mechanistic interplay and therapeutic potential remain poorly understood. Here, we investigated whether restoration of mitochondrial function can reverse metabolic dysfunction and promote resilience in advanced-stage AD. Female APP/PS1 mice were treated with the brain-penetrant mitochondrial complex I (mtCI) modulator CP2 beginning at 19 months of age, when pathology and cognitive deficits were well established. To define the metabolic mechanisms underlying therapeutic response, we developed iMiceBrain , the first brain-specific genome-scale metabolic model of the mouse brain, and integrated transcriptomics, targeted metabolomics, lipidomics, and metabolic network analyses. CP2 treatment broadly reprogrammed AD-associated molecular signatures and restored pathways involved in mitochondrial function, glucose utilization, lipid metabolism, synaptic activity, and cellular stress responses. Metabolic modeling identified enhanced mitochondrial substrate flexibility, activation of fatty acid utilization, restoration of pyruvate dehydrogenase flux, and normalization of cholesterol metabolism as key features of the therapeutic response. Lipidomic analyses further demonstrated correction of disease-associated alterations in cholesteryl esters, phospholipids, and sphingolipids. Together, these findings demonstrate that mild mtCI modulation restores metabolic resilience by coordinating mitochondrial and lipid metabolism, establishing it as a disease-modifying therapeutic strategy for AD.

Abstract Background: Approximately 70% of breast cancers are estrogen receptor-positive (ER+), and endocrine therapies, including selective estrogen receptor modulators (SERMs) and aromatase inhibitors (AIs), are standard-of-care. However, interpatient variability in treatment response and relapse remains substantial, suggesting underlying germline pharmacogenomic factors that modify ER signaling. Here, we mapped ER pharmacogenomic eQTLs to identify germline variants that modulate transcriptional responses to estradiol (E2) and tamoxifen (Tam). Methods: We treated 30 genotyped lymphoblastoid cell lines (LCLs) with vehicle, E2, Tam, or E2+Tam and performed RNA-seq to quantify ligand-induced expression fold changes. Using MatrixEQTL, we identified SNP-gene PGx-eQTLs (cis, ±200 kb), requiring evidence of ER transcriptional specificity via (i) reversal by dual treatment, and (ii) overlap with ER ChIP-seq binding annotations. Clinical relevance was assessed by linkage to breast cancer GWAS datasets, including NSABP, MA.27, and M3. Chromatin regulatory states were annotated (ChromHMM), and transcription factor motif enrichment was performed from SNP-centered ±200 bp regions. Results: Distinct PGx-eQTL architectures were identified for E2 and Tam, with partial overlap in ligand-responsive loci. ER PGx-eQTL variants were predominantly enriched in enhancer chromatin and co-localized with canonical ER-binding motifs. Integrative analysis with GWAS of breast cancer outcomes highlighted multiple drug-response-associated loci, including rs311392-MRPL15 and rs7953325-SYCP3. High MRPL15 expression was linked to shorter relapse-free survival, whereas high SYCP3 expression correlated with improved outcomes. Additional loci derived from a GWAS catalog for cancer and hormone phenotypes included rs2850247-SIK2 and rs11673604-LSM4, where higher SIK2 expression was associated with improved relapse-free survival and higher LSM4 expression with poorer outcomes. Conclusions: We identified ligand-dependent ER PGx-eQTLs that mechanistically connect germline regulatory variants to transcriptional response heterogeneity and clinical endocrine therapy outcomes. These SNP-gene relationships represent potential biomarkers for precision endocrine therapy selection and may reveal endocrine-dependent therapeutic vulnerabilities. Citation Format: Martin Meng, Huanyao Gao, August John, Arnab Ghosh, Shreya Indulkar, Liewei Wang, . Estrogen receptor-mediated pharmacogenomic eQTLs as predictors of endocrine therapy response in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3610.

Abstract Background Early mechanisms of neuronal damage in Alzheimer's Disease (AD) include mitochondrial dysfunction and abnormal energy homeostasis. AD‐associated mitochondrial signaling was shown to occur upstream of amyloid precursor protein processing, Ab production, apolipoprotein E (APOE) expression, and tau pathology. However, whether alterations in mitochondrial function are sufficient to instigate AD remains unclear. Mitochondrial complex I (mtCI) is a rate‐limiting enzyme in the oxidative phosphorylation (OXPHOS) machinery. Changes in mtCI activity influence ATP and ROS production, cellular energy and redox homeostasis. We examined how reduced activity of mtCI due to a knockout of one of its accessory subunits Ndufs4 in mice affects transcriptional networks in the brain. We also determined whether reduced activity of mtCI abrogates neuroprotective properties of a small molecule CP2. Method RNA‐sequencing of the brain tissue was performed using Ndufs4 ‐/‐ and control mice ( n = 3–5 per group). The co‐expression modules of differentially expressed genes were compared to the AMP‐AD patient cohort data. Key mechanisms, including mitophagy signaling, were confirmed using ELISA and western blot analyses; the effect on mtCI activity was confirmed using functional assays. Results In Ndufs4 ‐/‐ mice, a residual activity of mtCI was ∼50% of the control. This loss of function led to a global disruption of mitochondrial homeostasis, energy metabolism, synaptic function, significant increase in mitophagy and a reduction in biogenesis. Transcriptomic signatures in brain tissue of male and female Ndufs4 ‐/‐ mice overlapped with changer reported for AD patients and AD mouse models with Ab and p ‐tau pathologies. These changes were partially rescued by a neuroprotective mitochondria‐targeting small molecule CP2. Consistent with studies in AD mice, CP2 treatment in Ndufs4 ‐/‐ mice augmented the expression of genes associated with mitochondrial biogenesis and turnover, synaptic activity, autophagy, redox balance, and reduced expression of genes related to inflammation. Sex‐specific differences included better adaptation to a reduced activity of mtCI and to CP2 treatment in female mice. Conclusion Reduced mtCI activity was sufficient to induce the AD‐like transcriptomic changes in brain. Residual mtCI function was sufficient to mediate neuroprotective effect of CP2, emphasizing a key role of mitochondria in AD development and treatment.

Abstract INTRODUCTION Mitochondrial dysfunction is implicated in Alzheimer's disease (AD), but whether it drives AD‐associated changes is unclear. We assessed transcriptomic alterations in the brains of Ndufs4 −/− mice, a model of mitochondrial complex I (mtCI) deficiency, and evaluated the therapeutic effects of the neuroprotective mtCI inhibitor CP2. METHODS Cortico‐hippocampal tissue from Ndufs4 −/− and wild‐type mice was subjected to transcriptomic analysis, followed by cross‐species comparisons to human late‐onset AD and familial AD mouse datasets. RESULTS Knockout of Ndufs4 ‐mediated mtCI deficiency disrupted mitochondrial homeostasis, energy metabolism, and synaptic gene expression, recapitulating transcriptomic signatures of AD. CP2 treatment partially reversed these changes, with female Ndufs4 −/− mice showing greater compensatory adaptations and treatment responses. DISCUSSION Loss of mtCI activity alone is sufficient to induce AD‐like molecular changes in the brain, independent of amyloid beta or phosphorylated tau. CP2‐mediated rescue highlights the potential of targeting mitochondria as a therapeutic strategy for AD. Sex‐specific responses suggest important considerations for personalized therapeutics. Highlights Activity of mitochondrial complex I (mtCI) affects broad mitochondrial and neuronal transcriptional networks. A reduction of mtCI activity is sufficient to induce transcriptomic changes reminiscent of those observed in late‐onset Alsheimer's disease (AD) patients and familial mouse models of AD. Pharmacological targeting of mtCI mediates neuroprotective signaling. Male and female mice have differential responses to the loss of mtCI activity and to the mitochondria‐targeted therapeutics. Mitochondria play a key role in AD development and treatment.

Background: Prostate cancer is the most common cancer in men in the United States with an estimated 288,300 new cases diagnosed in 2023. The treatment strategies include androgen deprivation therapies (ADTs) for metastatic prostate cancer (mPC) but 10-20% of patients eventually progress to castration-resistant prostate cancer (CRPC). However, nearly all patients develop primary and acquired resistance overtime, and developing therapies and identifying novel molecular targets are essential in enhancing the clinical outcomes of patients with prostate cancer. In this study, we report a small molecule that belongs to a class of tricyclic quinolones, QL47 that was previously screened among various other compounds in prostate PDX organoid models and showed a potent anti-proliferative activity. Methods: QL47 was tested on prostate cancer cell lines (22Rv1, 22RV1 abiraterone-resistant and DU145) for its anti-proliferative potential. Further, we used the biotinylated QL47 (QL47B) to pulldown its potential targets and we identified the targets through immunoprecipitation-mass spectrometry (IP-MS) technique followed by Western blot analysis. Cell cycle and cell death analyses were performed after staining the cells with propidium iodide and Annexin V followed by flow cytometry analysis. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was investigated using the Seahorse assay. Results: The initial screening of QL47 in prostate cancer cell lines showed that it inhibits the proliferation at an IC50 of 50-200 nM. Through IP-MS and Western blot analysis, we identified a member of the pyruvate dehydrogenase complex dihydrolipoamide S-acetyltransferase (DLAT) as a direct target of QL47. Mechanistically, we found that QL47 does not increase cell death or affect cell cycle, but inhibits metabolic function, with a decrease in both oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Conclusion: We have identified a novel mechanism of action of QL47 on the pyruvate dehydrogenase complex, and we plan to further validate the safety and efficacy for the treatment of prostate cancer. Citation Format: Grace Nirmala Jesuthankaraj, August John, Huanyao Gao, Arun Kanakkanthara, Richard Weinshilboum, Liewei Wang. Targeting metabolic dysfunction in prostate cancer: the anti-proliferative effects of ql47 through inhibition of dlat [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4297.

Abstract Background: Second generation androgen receptor signaling inhibitors (ARSIs), such as abiraterone, have demonstrated improved survival in metastatic castration-resistant prostate cancer (mCRPC) patients, however, acquired resistance to these agents often arises. Novel and alternative therapies are needed to combat the ARSI resistant tumor. In the last two decades patient-derived xenograft (PDX) and PDX organoids have become pivotal models in anticancer drug development and biomarker discovery due to their ability to faithfully recapitulate the complex and heterogeneous tumor biology. Methods: We analyzed RNAseq data from mCRPC patients and corresponding PDX models (PROMOTE study, NCT #01953640) and employed L1000 Chem Perturbation down/up signatures database to find candidate drugs to overcome abiraterone resistance by reverse signature search. We then performed proliferation assay using 49 compounds in three PROMOTE-PDX organoid models (2 abiraterone-resistant and 1 abiraterone-sensitive) using our semi high-throughput drug screening platform. Results: Overall, we identified a total of 32 compounds with IC50<10µM in at least one of the models, among which, 5 agents are active in all three models and 5 in at least two of the models. (Table 1). Within active compounds we find selective kinase inhibitors (BRAF, JAK2, AKT, FLT3) and histone deacetylase inhibitors (belinostat and pracinostat). Particularly interesting are two drugs that are only active in the abiraterone resistant setting but not the sensitive PDX. Conclusions: In summary, we leveraged RNAseq data from mCRPC patients and PDX models and performed a high-throughput drug screen in PDX-derived organoid models to identify potential drugs that could overcome abiraterone-resistance in lethal advanced prostate cancer. These data highlight potential drug combinations or subsequent therapeutic strategies for these patients. Citation Format: Irene Marín-Goñi, Huanyao Gao, Joachim L. Petit, Shreya Indulkar, Adam M. Kase, Cassandra N. Moore, Krishna R. Kalari, Michael T. Barrett, Richard Weinshilboum, Winston Tan, John A. Copland, Liewei Wang. Overcoming resistance in mCRPC: drug discovery using PDX models and high-throughput drug screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5658.

Abstract The TopBP1-ATR axis is critical for maintaining genomic stability during DNA replication stress, yet the precise regulation of TopBP1 in replication stress responses remains poorly understood. In this study, we identified PHD and Ring Finger Domains 1 (PHRF1) as an important ATR activator through its interaction with TopBP1. Our analysis revealed a correlation between PHRF1 and genomic stability in cancer patients. Mechanistically, PHRF1 is recruited to DNA lesions in a manner dependent on its PHD domain and histone methylation. Subsequently, PHRF1 mono-ubiquitinates TopBP1 at lysine 73, which enhances the TopBP1-ATR interaction and activates ATR. Depletion of PHRF1 disrupts ATR activation and sensitizes cells to replication stress-inducing agents. Furthermore, conditional knockout of Phrf1 in mice leads to early lethality and impaired ATR-Chk1 axis signaling. Collectively, our findings establish PHRF1 as a novel E3 ligase for TopBP1, coordinating the replication stress response by enhancing TopBP1-ATR signaling.

ABSTRACT Background Inclusion body myositis (IBM) is the most prevalent muscle disease in adults for which no current treatment exists. The pathogenesis of IBM remains poorly defined. In this study, we aimed to explore the interplay between inflammation and mitochondrial dysfunction in IBM. Methods The study population consisted of 38 IBM patients and 22 age‐ and sex‐matched controls without a myopathy. Mean age was 62.9 years (SD = 9) in IBM group and 59.7 (10) in controls. Bulk RNA sequencing, Meso Scale Discovery electrochemiluminescence (ECL), western blotting, histochemistry and immunohistochemistry were performed on frozen muscle samples from the study participants. Results We demonstrated activation of the NLRP3 inflammasome in IBM muscle samples, with the NLRP3 inflammasome being the most upregulated pathway on RNA sequencing, along with increased expression of NLRP3 and ASC proteins in IBM group. NLRP3 RNA levels most strongly correlated with TLR7 (correlation coefficient ρ = 0.91) and complement activation‐related genes, and inversely correlated with several mitochondria‐related genes among others. On muscle histopathology, there was increased NRLP3 immunoreactivity in both inflammatory cells and muscle fibres. Mitophagy is critical for removing damaged mitochondria and preventing the formation of a vicious cycle of mitochondrial dysfunction—NLRP3 inflammasome activation. Herein, we showed altered mitophagy, as witnessed by the elevated levels of p‐S65‐Ubiquitin, a mitophagy marker, in muscle lysates from IBM patients compared to controls (median of 114.3 vs. 81.25 ECL units, p = 0.005). The p‐S65‐Ubiquitin levels were most significantly elevated in IBM males compared to male controls (136 vs. 83.5 ECL units; p = 0.013), whereas IBM females had milder nonsignificant elevation compared to female controls (97.25 vs. 69 ECL units, p = 0.31). On muscle histopathology, p‐S65‐Ubiquitin aggregates accumulated in muscle fibres that were mostly Type 2 and devoid of cytochrome‐c‐oxidase reactivity. NLRP3 RNA levels correlated with p‐S65‐Ubiquitin levels in both sexes (males: ρ = 0.48, females: ρ = 0.54) but with loss of muscle strength, as reflected by the manual motor test score, only in males (males: ρ = 0.62, females: ρ = −0.14). Lastly, we identified sex‐specific molecular pathways in IBM. Females had upregulation of pathways related to response to stress, which could conceivably offset some of the pathomechanisms of IBM, while males had upregulation of pathways related to cell adhesion and migration. Conclusions There is activation of the NLRP3 inflammasome in IBM, along with altered mitophagy, particularly in males, which is of potential therapeutic significance. These findings suggest sex‐specific mechanisms in IBM that warrant further investigation.

Most patients with metastatic prostate cancer eventually develop resistance to primary androgen deprivation therapy. To identify predictive biomarker for Abiraterone acetate/prednisone resistance, we screened alternative splice variants between responders and non-responders from the PROMOTE clinical study and pinned down the most significant variant, CENPK–delta8. Through preclinical patient-derived mouse xenograft (PDX) and 3D organoids obtained from responders and non-responders, as well as in vitro models, aberrant CENPK–delta8 expression was determined to link to drug resistance via enhanced migration and proliferation. The FLNA and FLOT1 were observed to specifically bind to CENK–delta8 rather than wild-type CENPK, underscoring the role of CENPK–delta8 in cytoskeleton organization and cell migration. Our study, leveraging data from the PROMOTE study, TCGA, and TCGA SpliceReq databases, highlights the important function of alternative splice variants in drug response and their potential to be prognostic biomarkers for improving individual therapeutic outcomes in precision medicine.