Competition Abstracts

Poster 1

Developing GPR56-Targeted Antibody-Drug Conjugates for Triple-Negative Breast Cancer Treatment
Yueh-Ming Shyu^1,2, Joan Jacob³, Carla Godoy², Treena Chatterjee², Zhengdong Liang², Kendra S. Carmon²

¹The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX 77030; ²Center for Translational Cancer Research, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, Houston, TX 77030; ³Pediatrics Oncology-AICT, Baylor College of Medicine, Houston, TX 77030

Triple-negative breast cancer (TNBC) poses a significant challenge in oncology, comprising 10-15% of all breast cancer cases. Its aggressive nature, high metastatic potential, and lack of targetable receptors result in limited treatment options and poor prognosis. The emergence of chemotherapy resistance further complicates TNBC management, underscoring the urgent need for novel therapeutic strategies. Antibody-drug conjugates (ADCs) have emerged as a promising approach for targeted cancer therapy. These innovative drugs combine monoclonal antibodies (mAbs) specificity with potent cytotoxic payloads, enabling precise delivery of chemotherapy drugs to cancer cells. This targeted approach potentially enhances efficacy while minimizing damage to healthy tissues, making ADCs an attractive option for developing more effective TNBC treatments.

Our research identified G protein-coupled receptor 56 (GPR56) as a promising target for ADC development in TNBC. Analysis of The Cancer Genome Atlas (TCGA) data revealed that high GPR56 expression correlates with poor overall survival in breast cancer patients and is significantly elevated in TNBC compared to non-TNBC. Notably, GPR56 demonstrated a higher tumor-to-normal tissue expression ratio than TROP2, an approved ADC target. A BioID screen revealed that GPR56 associates with β1 integrins that mediate resistance and loss of GPR56 inhibited tumor growth. Therefore, we hypothesize that GPR56 is a potential target for treating TNBC. We previously developed an anti-GPR56 ADC (10C7-Duo) by conjugating duocarmycin payload to a GPR56-specific mAb, 10C7, which had significant antitumor efficacy in colorectal cancer models. Importantly, 10C7-Duo showed high potency in TNBC cell lines with high GPR56 expression, exhibiting target-dependent cytotoxicity. Combination therapy studies revealed synergistic effects between 10C7-Duo and AZD5305, a PARP1 inhibitor, in TNBC cells. Though 10C7-Duo has robust antitumor efficacy, 10C7 mAb showed agonist activity that may have long-term tumor promoting capabilities. Therefore, we are currently developing a new and improved ADCs incorporating different drug payloads and an anti-GPR56 mAb, 9E3, which does not exhibit agonist activity. We are also investigating GPR56-integrin interactions and their impact on FAK-Src signaling. Future work will focus on both ADC single-agent and combination therapies using TNBC cell lines, patient-derived samples, and xenograft mouse models.

This study aims to develop novel GPR56-targeted ADCs for TNBC, potentially enhancing efficacy, reducing side effects, and overcoming therapy resistance. By addressing the urgent need for effective TNBC treatments, this approach could significantly improve outcomes for TNBC patients.

Poster 2

Integrating Membrane Dynamics, Fragment-Based Drug Discovery and High Throughput Virtual Screening to identify novel inhibitors of Small GTPase Rheb

Chase M. Hutchins^1,2, Alemayehu A. Gorfe¹
¹McGovern Medical School, University of Texas Health Science Center at Houston, Department of Integrative Biology and Pharmacology, 6431 Fannin St., Houston, Texas 77030, USA;²UTHealth MD Anderson Cancer Center Graduate School of Biomedical Sciences, Houston, 6431 Fannin St., Texas 77030, USA

Background: Rheb is a small GTPase in the Ras superfamily that promotes cell growth and proliferation by directly activating mTORC1 and its dysregulation can worsen cancer initiation and progression. Although rapamycin and its analogs (rapalogs) inhibit mTORC1 and are approved for several cancers, prolonged treatment can lead to off-target inhibition of mTORC2. Consequently, selectively inhibiting mTORC1 via Rheb represents an attractive alternative therapeutic strategy; however there currently are no clinically approved inhibitors targeting Rheb. Recent studies in Ras proteins emphasize the critical role of membrane orientation dynamics and its potential as a therapeutically targetable process but the role of membrane reorientation in function and druggability in other small GTPases, including Rheb, remains largely unexplored. In this study, we explored the membrane orientation dynamics of Rheb and identify how orientation dynamics can be targeted to yield more clinically viable inhibitors.

Methods: To investigate how membrane orientation influences Rheb function and druggability, we employed a multi-tiered, integrated computational strategy. First, we performed microsecond-scale molecular dynamics (MD) simulations to map Rheb’s orientation dynamics at the atomic resolution. Next, probe-based MD simulations were used to identify novel binding pockets and orientation‐dependent changes in Rheb’s allosteric druggable surface. Finally we targeted the most druggable binding pockets identified by probe-based simulations to conduct a high‐throughput virtual screen of large, pocket-tailored chemical libraries to discover novel inhibitors targeting Rheb.

Results: We identified three meta-stable membrane orientations of Rheb, with each orientation state differing in allosteric druggable surface, pocket geometry and druggabiltiy rating. Leveraging these insights, we then selected the most druggable pocket conformations and virtually screened a pocket-tailored library of ~2 million molecules targeting the most druggable conformational states of Rheb from our probe-based MD simulations.

Conclusions: Our findings reveal that Rheb’s membrane orientation dynamics influence its druggability and present an untapped avenue for inhibitor development. Targeting Rheb directly could offer a more selective approach to modulating mTORC1 activity in cancer, potentially reducing off-target effects associated with long-term rapalog therapy.

Poster 3

Targeting Colorectal Cancer Stem Cells with an Antibody-Drug Conjugate-Based Combination Therapy to Overcome Relapse

Shraddha Subramanian^1,2, Zhengdong Liang¹, Peyton High^1,2, Adela Aldana¹, and Kendra S. Carmon^1,2
¹Center for Translational Cancer Research, The University of Texas Health Science Center, Houston, TX, USA;²The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX, USA

A significant hurdle in colorectal cancer (CRC) treatment is the relapse of residual disease, which can be attributed to cancer stem cells (CSCs). CSCs potentiate metastatic progression by exploiting their capacity to self-renew and differentiate. Furthermore, this immortal cell population exhibits plasticity, allowing cells to alter their phenotype in response to environmental cues that bolster inherent drug resistance. As a bona fide CSC marker, LGR5 is a promising drug target. We generated anti-LGR5 antibody-drug conjugates (ADCs) that inhibited tumor growth yet failed to prevent relapse following treatment completion. Follow-up studies indicate recurrent tumors evade therapy by converting into a drug-resistant LGR5-negative (LGR5^–) state and, at least in part, depend on MET-STAT3 cascade activation for survival.

We demonstrated the coupling of LGR5 to IQGAP1, a scaffolding protein that promotes poor disease prognosis in multiple cancer subtypes. Interestingly, our pull-down assays revealed that LGR5 knockdown (KD) enhances IQGAP1 interaction with MET and STAT3, highlighting a potential role for IQGAP1 in mediating MET-STAT3 activation. To eliminate drug-resistant LGR5^– cells, we generated MET-targeted ADCs by conjugating a highly selective MET monoclonal antibody (mAb) backbone to the DNA-crosslinking payload pyrrolobenzodiazepine via a site-specific conjugation methodology. The MET ADC was evaluated for cancer cell-killing efficacy in parallel with MET mAb, non-targeting control mAb (cmAb), and control ADC (cADC). MET ADC demonstrated high efficacy and potency in MET-expressing CRC cells in vitro, while unconjugated MET mAb, cmAb, and cADC exhibited minimal effects. Additionally, MET ADC had no impact on MET KD CRC cells, demonstrating its specificity. Safety studies in immunocompetent mice showed the MET ADC was well-tolerated with minimal toxicity. Moreover, when evaluated in CRC cell line and patient derived xenografts, the MET ADC induced significant tumor regression and prolonged overall survival. However, MET ADC monotherapy alone did not effectively prevent tumor relapse. Thus, an ADC combination strategy dual-targeting LGR5^– and LGR5⁺ cells is a more promising approach to eliminating heterogeneous tumors. Pilot cytotoxicity assays evaluating the combination of MET ADCs with next-generation LGR5 ADCs showed moderate synergistic effects in vitro. The improved iteration of our LGR5 ADC was produced by attaching a topoisomerase I inhibitor derivative to a previously characterized LGR5 mAb backbone in a site-specific manner.

Future work involves investigating the efficacy of combining MET ADCs and LGR5 ADCs in patient-derived xenograft (PDX) models of CRC. These findings present a novel mechanism underpinning CRC plasticity and a rationale for an ADC-based treatment strategy to overcome colorectal tumor resistance and heterogeneity.

Poster 4

Evaluation of EGFR and LGR5 dual-targeting therapeutic strategies for the improved treatment of colorectal cancer

Peyton High^1,2, Zhengdong Liang², Cara Guernsey-Biddle^{1, 2}, Adela Aldana², Yukimatsu Toh², and Kendra S. Carmon²
¹The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX, 77030; ²Center for Translational Cancer Research, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, TX, 77030

Despite extensive efforts to develop novel therapies, colorectal cancer (CRC) remains the second-leading cause of cancer-associated death in the United States. One of the primary challenges in treating CRC is therapy resistance mediated by cancer stem cells (CSCs), a subpopulation of cancer cells with infinite replicative potential that can differentiate to drive tumorigenesis and relapse. Thus, targeting CSCs has become an attractive therapeutic strategy.

Antibody-drug conjugates (ADCs) are a rapidly expanding class of anticancer drugs that utilize monoclonal antibody (mAb) specificity to hone cytotoxic payloads to cancer cells. We previously generated ADCs targeting leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a CSC biomarker that is highly overexpressed in CRC. LGR5 ADCs demonstrated high specificity and efficacy in CRC cells and xenograft models without notable toxicity, although tumors eventually relapsed following treatment cessation. Interestingly, FDA-approved therapies targeting epidermal growth factor receptor (EGFR) have been shown to increase LGR5 expression in patient-derived models of CRC. This study aims to determine the mechanisms of EGFR-mediated regulation of LGR5 expression and evaluate the therapeutic efficacy of EGFR-targeting therapies in combination with LGR5 ADCs for the improved treatment of CRC.

We have demonstrated that cetuximab (CTX), an EGFR-targeting mAb approved for KRAS^WT metastatic CRC (mCRC), increases LGR5 protein levels independent of KRAS/PIK3CA mutational status in CRC cell lines, tumor organoids, and cell line-derived xenografts. Additionally, we identified a novel EGFR-LGR5 interaction that is enhanced by CTX treatment. Furthermore, we have generated a camptothecin-based LGR5 ADC (8E11-CPT2) utilizing site-specific conjugation technology that demonstrates target- and dose-dependent cytotoxicity in CRC cells and was well-tolerated up to 20 mg/kg. Importantly, combination treatment of 8E11-CPT2 with CTX significantly reduced tumor burden with notable regression in some mice and extended survival compared to LGR5 ADC and CTX monotherapies in two RAS^MUTCRC patient-derived xenograft models. To evaluate alternative EGFR and LGR5 dual-targeting therapeutic strategies, we generated an EGFR:LGR5 bispecific antibody (bsAb) that binds its cognate receptors, internalizes to the lysosome, and promotes EGFR and LGR5 degradation. Future work will involve the development of an EGFR:LGR5 bsADC that will be evaluated in vivo against LGR5 ADC combination therapy with CTX. Taken together, these results demonstrate that dual-targeting of EGFR and LGR5 may prove more effective than LGR5- and EGFR-monotargeted approaches in treating CRC, providing an improved treatment option for RAS^MUT and PIK3CA^MUT mCRCs.

Poster 5

The promise of multi-targeted ADC therapy in colorectal cancer: epiregulin and amphiregulin

Cara Guernsey-Biddle^{1, 2}, Joan Jacob³, Zhengdong Liang², Kendra Carmon²
MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, 77030 ¹; Center for Translational Cancer Research, The Brown Foundation Institute for Molecular Medicine, The University of Texas Health Science Center, Houston, TX, 77030 ²

Pediatrics Oncology-AICT, Baylor College of Medicine, Houston, TX 77030 ³

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States with a 14% five-year survival rate when metastasized. Current treatment options for metastatic CRC (mCRC) can be limited by toxic side effects and drug resistance, so new targeted therapies are necessary to improve mCRC patient outcomes. Antibody-drug-conjugates (ADCs) act as guided missiles where a monoclonal antibody (mAb) conjugated to cytotoxic payloads binds its tumor-specific target, internalizes, and traffics to lysosomes for payload release, inducing tumor cell death while sparing normal tissues. Previous studies and our analyses of RNA-seq expression data from The Cancer Genome Atlas (TCGA) colorectal adenocarcinoma cohort show that the epidermal growth factor receptor (EGFR) ligand epiregulin (EREG) is frequently overexpressed in CRC tumors compared to normal tissues. Thus, we developed EREG-targeted ADCs that reduced CRC patient-derived xenograft tumor growth, yet regrowth was observed after treatment termination. Importantly, ADC efficacy depends, in part, on tumor cell-surface levels of the target antigen. Our TCGA analyses and previous studies show EGFR ligand amphiregulin (AREG) is often co-overexpressed with EREG in CRC patients. Therefore, we hypothesize that co-targeting EREG and AREG will enhance ADC efficacy by increasing the number of target antigens for ADC binding and internalization. We have cloned and purified human AREG-targeted mAbs to develop an AREG-targeted ADC as well as generated AREG overexpression cell lines for mAb characterization. Our preliminary data demonstrate AREG mAb specificity and binding, which promotes internalization and lysosomal trafficking in CRC cells. These data suggest AREG to be a suitable ADC target, and future work will test whether co-targeting EREG and AREG provides additional therapeutic benefit.

Poster 6

oHSV and Radiation Therapies Sensitize Glioblastoma to IGF1R-Targeted Therapy and Synergize in Triple Combination

Alexandra A. Miller^1,2*, Min Hye Noh^1*, Jin Muk Kang^1,3, Grace Nguyen¹, Min Xin Huang¹, Ji Yeon Kim¹, AmandaSKouaho¹, Yoshihiro Otani^1,4, Hiroshi Nakashima⁵, E. Antonio Chiocca⁵, Tae Jin Lee^1,2#, and Ji Young Yoo^1,2#

¹Department of Neurosurgery, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, 77030, USA. ²MD Anderson Cancer Center UTHealth Graduate School of Biomedical Science, Houston, TX 77225, USA. ³Department of Pediatric Hematology & Oncology, University Hospitals Cleveland Medical Center, Cleveland, OH, 44106, USA. ⁴Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8558, Japan. ⁵Department of Neurosurgery, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, 02115, USA.

^*These authors contributed equally.

Introduction: We recently uncovered a novel mechanism of resistant to oncolytic herpes simplex virus-1 (oHSV) therapy, where infection induces secretion of Insulin-like growth factor 2 (IGF2) and subsequent Insulin-like Growth Factor-1 Receptor (IGF1R) activation. IGF1R activation similarly contributes to radiotherapy (RTx) resistance and is a key marker of poor RTx response. In this study, we investigated the therapeutic potential of combining IGF1R blockade with oHSV and RT to overcome this shared mechanism of resistance.
Methods: Western blotting, MTT assay, colony formation assay, and flow cytometry were performed to investigate the mechanism of IGF1R activation and its impact on cytotoxicity. Viral replication was assessed using Cytation imaging and plaque formation assays. The synergistic interaction of the triple combination therapy, including oHSV, RTx, and IGF1R blockade (e.g., OSI-906 or PPP), was quantified using SynergyFinder+. Orthotopic mouse models were utilized to evaluate antitumor efficacy, with survival outcomes analyzed using Kaplan-Meier curves and two-sided log-rank test.
Results: oHSV treatment significantly activated IGF1R signaling both in vitro and in vivo, sensitizing tumors to IGF1R blockade. While IGF1R-targeting therapies show limited efficacy as monotherapy, their combination with oHSV significantly enhanced cytotoxicity in all tested breast cancer (BC) and glioma cell lines, and primary GBM cells. This effect was accompanied by a dose-dependent increase in PARP cleavage and caspase-3 activity. Additionally, RTx-induced IGF1 secretion further amplified IGF1R activation in combination with oHSV, leading to increased phosphorylation of eIF2α, and upregulation of YAP1 in combination with oHSV. Triple combination of oHSV, RTx, and IGF1R blockade exhibited strong synergy in BC and GBM cells in vitro and enhanced anti-tumor efficacy in orthotopic tumor models of BC and GBM in vivo.
Conclusion: Our study provides preclinical evidence of the efficacy of combined oHSV, RTx, and IGF1R blockade, supporting future clinical evaluation.

Poster 7

Dissecting 3D Chromatin Dynamics in Li-Fraumeni Syndrome

Mo-Fan Huang^1,2, Chuangye Qi³, Ruiying Zhao¹, Dung-Fang Lee^1,2
¹Department of Integrative Biology and Pharmacology, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA;²The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX 77030, USA;³Huffington Center on Aging, Baylor College of Medicine, Houston, TX77030, USA

The advancement of induced pluripotent stem cell (iPSC) technology has empowered the in vitro modeling of human diseases, particularly in cancer predisposition disorders like Li-Fraumeni syndrome (LFS) marked by germline p53 mutations. LFS patients are at an increased risk of developing multiple cancers throughout their lifetime, with a particular susceptibility to breast cancer, gliomas, sarcoma, and osteosarcoma. In this study, we utilized LFS iPSC-derived osteoblasts, the cells of origin for osteosarcoma, as a unique platform to delve into the intricate role of mutant p53 in osteosarcoma development, focusing specifically on 3D chromatin architecture.

Application of chromosome conformation capture (3C) and its derivative High-throughput chromosome conformation capture (Hi-C) have emerged as invaluable tools, offering researchers insights into the spatial organization of the genome within the nucleus. This technological progress enhances our comprehension of vital biological processes, including gene transcription regulation through physical chromatin interaction and chromatin 3D structure reshaping. The tumor suppressor p53, a key player in maintaining genomic stability, orchestrates cellular responses to genotoxic stress by controlling cell-cycle checkpoints and apoptosis. However, the full understanding of how a p53 mutation potentiates osteosarcoma progression, particularly the oncogenic property (gain-of-function) of mutant p53, is not yet complete. Therefore, we employed LFS iPSC-osteoblasts as a distinctive model to explore the complex involvement of mutant p53 in osteosarcoma formation, with a particular emphasis on the 3D chromatin structure. We investigated changes in A/B compartments, topologically associated domains (TADs), and chromatin loops in LFS and healthy family iPSC-derived osteoblasts and integrated with mRNA-seq as well as ChIP-seq for comprehensive investigation of functional oncogenes during osteosarcomagenesis. In summary, our findings provide insights into the potential role of mutant p53 in modulating 3D chromatin structure, including compartment switching, dynamics of TAD borders, and rewiring of loops, ultimately influencing aberrant oncogenic gene expression in LFS patients.

Poster 8

Regulation of gene transcription through a large intrinsically disordered region within Mediator complex

Hsiang-Ching Tseng^1,2, Hui-Chi Tang², Leon Palao III³, Tao Li², Shin-Fu Chen², Kenji Murakami³, Ti-Chun Chao², and Kuang-Lei Tsai^1,2*
¹The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX 77030; ²Department of Biochemistry and Molecular Biology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX 77030; ³ Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104

In eukaryotes, the Mediator complex is a highly conserved multi-subunit regulator of RNA Polymerase II (Pol II)-dependent transcription, serving as a critical bridge between transcriptional regulators and the transcription machinery. It consists of the Core Mediator (cMed) (~1 MDa) and a dissociable Cdk8 kinase module (CKM) (~0.5 MDa), which includes Med12, Med13, Cdk8, and Cyclin C (CycC). CKM is known to reversibly associate with cMed and modulate its activity, yet the molecular mechanisms governing this interaction remain poorly understood.

Here, we identified an intrinsically disordered region (IDR) within Med13 that is responsible for mediating CKM-cMed interaction and demonstrated its essential role in transcriptional repression. Biochemical and genetic analyses revealed that deletion or mutations within the conserved regions of Med13 IDR weakens CKM-cMed binding, resulting in growth defects, temperature sensitivity, and flocculation phenotypes, highlighting the crucial role of Med13 IDR in maintaining cellular homeostasis.

To further explore the molecular mechanism of Med13 IDR-mediated transcriptional repression, we performed competition binding assays and found that Med13-IDR directly antagonizes Pol II C-terminal domain (CTD) binding to cMed, suggesting a blocking mechanism that prevents Pol II recruitment. Additionally, in vitro transcription assays revealed that WT Med13 IDR effectively repressed activator-dependent transcription, underscoring the requirement of Med13-IDR in CKM-mediated transcriptional inhibition.

Collectively, our findings provide novel insights into the molecular basis of CKM-mediated transcriptional repression through Med13-IDR. These discoveries have broad implications for transcriptional control mechanisms in eukaryotic gene expression and their potential links to disease states where Mediator dysfunction is implicated.

Acknowledgements: This work is supported by the US National Institutes of Health grants R01 GM143587 (K.L.T.) and the Welch Foundation (AU-2050-20200401).

Poster 9

An Artificial Intelligence Optimized Hepatic Differentiation Unveils NR5A and AP-1 Transcriptional Regulation in Hepatic Maturation

Chih-Wei Chu^1,2, Zijun Huo¹, Wei-Lei Yang³, Mo-Fan Huang^1,2, An Xu¹, Megan E. Fisher^1,2, Yu-Wen Huang¹, Trinh T. T. Phan¹, Rachel Shoemake^1,2, Dung-Fang Lee^1,2, and Ruiying Zhao¹
¹Department of Integrative Biology and Pharmacology, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA; ²The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX 77030, USA; ³AIxMed Inc. Santa Clara, CA 95054, USA

Deriving hepatocyte-like cells (HLCs) from human pluripotent stem cells (hPSCs) holds significant promise for applications in cancer research, drug discovery, hepatotoxicity testing, liver regeneration, and cell-based therapies. However, current hepatic differentiation protocols often lack efficiency and consistency. A deeper understanding of the regulatory mechanisms governing hepatic development is crucial for optimizing differentiation strategies and improving success rates. To address these challenges, we developed a machine learning-based artificial intelligence (AI) tool that analyzes phase-contrast images of hepatic progenitor cells (HPCs), a key intermediate in HLCs generation. This approach enhances differentiation efficiency without the need for additional immunostaining or lineage tracing, enabling a highly reproducible protocol that consistently yields 90-95% pure HLCs. To further explore the molecular mechanisms underlying hepatic differentiation, we performed transcriptomic and epigenomic analyses, identifying NR5A2 and AP-1 as key transcription factors in hepatocyte maturation. Single-cell transcriptomics analysis confirmed the upregulation of these factors during differentiation, underscoring their role in guiding the transition from progenitor cells to functional hepatocytes. Functional assays, including mutagenesis and tumorigenesis studies, validated the safety of the optimized protocol, ensuring that the resulting HLCs are both highly pure and free of tumorigenic risk. This study presents an effective and scalable strategy for generating functional hepatocytes with high efficiency, providing a reliable cell source for liver disease modeling, toxicity screening, and therapeutic applications. By refining differentiation protocols and identifying critical regulatory factors, our findings advance the understanding of hepatic development and pave the way for further innovations in stem cell-based liver therapies.

Poster 10

TREM2 mediates microglial phagocytosis and neuroimmune fitness in glioma

Mekenzie Peshoff^1,2,3, Jiaying Zheng², Pravesh Gupta³, Shivangi Oberai³, Rakesh Trivedi³, Krishna Bhat^1,3, Long-Jun Wu^1,2
¹The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX 77030;²Center for Neuroimmunology and Glial Biology, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030;³Department of Translational Molecular Pathology, Brain Tumor Center, MD Anderson Cancer Center, Houston, TX 77030

TREM2 is a phagocytic receptor highly expressed on microglia, aiding these brain-resident immune cells in the detection and engulfment of pathogens. In neurodegenerative disorders, TREM2+ microglia play a protective role in resolving inflammation by clearing dead neurons and aggregated plaques. TREM2 has recently been implicated in the innate immune response to cancer; however, it governs pro-tumor immunosuppression. Due to these contrasting functions in neurological disorders and cancer, our goal is to understand what role TREM2 serves in glioma, the most common primary brain tumor affecting adults.

To investigate TREM2’s function in gliomas, we use resected tumors from human patients and global knockout mouse models. By employing cutting-edge techniques including multicolor spectral flow cytometry, single cell RNA-sequencing, and in vivo 2-photon imaging, we have uncovered a novel anti-tumor role of TREM2 in gliomas. Our research indicates that TREM2 is associated with phagocytosis and antigen presentation across species. In human brain tumors, TREM2 is positively correlated with macrophage scavenger receptor and phagolysosome expression but negatively correlated with immunosuppressive transcripts, supporting its role in activating the neuroimmune response to cancer. We used orthotopic transplants of murine glioma cell lines and found that mice genetically lacking TREM2 displayed increased glioma volume compared to wildtype controls. Probe-based RNA sequencing of these tumors revealed dysfunction of multiple pathways in the absence of TREM2, including microglia, astrocyte, neuron, and innate and adaptive immune responses. Therefore, we investigated the interactions between glioma cells and microglia in vivo; TREM2-deficient microglia exhibited impaired phagocytosis of cancer cells, providing a mechanism by which TREM2 exerts an anti-glioma function in the tumor microenvironment. Finally, we show that this is dependent on spleen tyrosine kinase (Syk) signaling downstream of TREM2 and that TREM2 deficiency further exacerbates the impaired adaptive immune response to cancer via downregulation of MHC class II cross-presentation to CD4+ T cells.

In conclusion, our research supports a role of TREM2 in boosting the microglial response against glioma progression and regulating homeostasis of multiple neural cell types during neoplastic challenge. Future studies include determining specific myeloid subsets and disease stages where TREM2 enacts the most phagocytic potential. We ultimately aim to determine if TREM2-boosting strategies can slow glioma progression in preclinical models.

Poster 11

Neural correlates of associative learning in nonhuman primates

Madison Shyer^1,2, Ally Grace McConnell^1,2, Mitchell Slapik^1,2, Melissa Franch³, Valentin Dragoi²;¹UTHealth MD Anderson Cancer Center Graduate School of Biomedical Sciences, Houston, TX 77030, USA; ²Department of Neurosurgery, Houston Methodist Research Institute, Houston, TX 77030, USA;
³Department of Neurosurgery, Baylor College of Medicine, Houston, TX 77030, USA

Background: Associative learning is crucial to the evolutionary survival of species. Arbitrary associations for survival can range from simple to complex; for example, humans and animals alike have learned to associate different fruits’ colors with their ripeness or nutritional value. However, how the primate brain integrates different information and signals across relevant areas of the brain during the learning process in naturalistic settings remains poorly understood. In this study, we investigate the integration and communication between sensory and executive brain regions during associative learning: specifically, how visual information is processed and transmitted to higher-order brain areas. We hypothesize that during associative learning, visual and executive areas will specialize in encoding task variables and decision-making processes, respectively, and communicate more efficiently with each other.

Methods: We trained nonhuman primates in a novel freely moving foraging paradigm where they learn to associate color cues with reward availability. In the task, animals learn to press a lever and receive a reward only when the correct color cue is presented on a monitor in the arena. Neural data is recorded simultaneously from the dorsolateral prefrontal cortex and visual area V4 using wireless microelectrode arrays, synchronized with eye-tracking data.

Results: As the animal learns to associate the stimulus with the reward, their response behavior decreases to only respond at the stimulus presentation. Furthermore, using support vector machine decoders and subspace communication analyses, we note an increase in encoding of task variables and communication within/between the two brain regions, respectively, during the course of learning, suggesting a more efficient encoding of task parameters and greater regional communication as learning occurs.

Conclusions: Examining behavioral performance synchronized to neural and eye-tracking data allows us to explore the activity and interaction of different brain regions during the learning process, aiming to understand how (1) behavior changes during learning, (2) specific brain regions encode task components, and (3) communication between these regions evolves with learning. The findings provide insights into the brain’s ability to integrate information across multiple regions to form new associations, as is expected in real-life learning scenarios, and highlight the importance of studying learning in naturalistic settings. By understanding the neural pathways involved in associative learning, this research could inform interventions for conditions with maladaptive association components, such as anxiety, PTSD, and substance use disorders.

Poster 12

Switching it on: An electrostatic switch underlies activation of the Rcs stress response

Hitt SJ ^1,2, Seonghoon Kim³, Wonpil Im³ Konovalova A ^1,2

¹Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston (UTHealth), Houston, TX, 77030, USA; ²The University of Texas MD Anderson UTHealth Graduate School of Biomedical Sciences, Houston, TX, 77030, USA; ³ Lehigh University, Department of Computer Science and Engineering, Bethlehem, PA, 18015, USA.

Corresponding author: Anna Konovalova, Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston (UTHealth), Houston, TX, 77030, USA [email protected].

Background: The Regulator of Capsule Synthesis (Rcs) envelope stress response is highly conserved in Enterobacteriaceae. Rcs is activated by multiple host immune factors and antibiotics, which target the bacterial cell envelope. Rcs regulates expression of many genes to prevent or mitigate cell envelope damage, and as such, Rcs is essential for survival in the host, virulence, and antibiotic resistance. Despite its importance, how Rcs detects envelope damage remains unknown. Rcs is a signal transduction pathway consisting of six components, including the sensor protein RcsF. RcsF forms a complex with several outer membrane proteins (OMP), which allows RcsF to co-localize with LPS at the cell surface. The overall goal of my project is to identify a molecular signal and the mechanism of RcsF activation. Our hypothesis is that RcsF monitors perturbations in LPS ionic interactions through direct interaction with LPS.
Methods: I have combined in vitro biochemical assays with genetic analysis to study how RcsF/LPS interactions control Rcs signaling. These include fluorescent binding assay as well as mutational analysis with reporter gene fusion readouts.
Results: Using a dansyl-fluorescence assay, I demonstrated that the NTD peptide binds LPS in vitro in a charge-dependent manner. Moreover, polymyxin B competes with the NTD for LPS binding, suggesting that a mechanism for RcsF activation may involve RcsF displacement from LPS. Consistent with this hypothesis, when LPS charge is reduced in vivo by the deletion in the LPS kinase gene waaP, it strongly activates Rcs. Interestingly, I also demonstrated that NTD charge is important for signaling as charge mutants were defective in PMB sensing and Rcs activity in the ΔwaaP background. To provide a molecular explanation for this phenotype we performed MD simulations of the RcsF/OMP complex in the activated conformation. They revealed that the positively charged residues previously found to be important for interactions with LPS are now interacting electrostatically with negatively charged residues in the OMP lumen. To further understand the regulatory function of the NTD, I performed genetic screens to identify gain- and loss-of-function mutations in the NTD. I am currently characterizing these mutants to determine the molecular basis for their phenotypes, specifically in relation to LPS binding and RcsF/OMP conformational changes.
Conclusions: Based on my results, we propose that an electrostatic switch-like mechanism initiates conformational changes in the RcsF/OMP complex, activating Rcs signaling. This model offers a mechanistic understanding of how enteric bacteria detect antibiotic binding to their cell surface and how they utilize phosphorylation-independent mechanisms to transmit signals across the cell envelope.
Acknowledgments.
This research is supported by the National Institute of General Medical Sciences R01GM133904, the Molecular Basis of Infectious Diseases Training Grant Fellowship T32AI055449 and the Welch Foundation Research Grant AU-1998.

Poster 13

The Therapeutic Role of Estrogen-Related Receptors in Muscle Regeneration and Duchenne Muscular Dystrophy

Huang Sophia^1,2, Nguyen Thi Thu Hao², Poliakova Svitlana², Sopariwala Danesh², Narkar Vihang^1,2

¹The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX 77030; ²Center for Metabolic and Degenerative Diseases, Institute of Molecular Medicine, McGovern Medical School at The University of Texas Health Science Center (UTHealth), Houston, TX 77030

Estrogen-related receptors (ERRs) belong to the nuclear receptor superfamily of evolutionarily conserved transcription factors. These receptors have characteristic ligand binding pockets that can be targeted with small molecules. The ERRα isoform is ubiquitously expressed in skeletal muscle and has emerged as the master regulator of mitochondrial biogenesis and angiogenesis in adult skeletal muscle. However, its role in muscle regeneration and myopathies is poorly understood. Duchenne Muscular Dystrophy (DMD) is a progressive muscle degenerative disease caused by the loss of dystrophin, resulting in chronic muscle damage and impaired regeneration. We have found that ERRα and its target mitochondrial and angiogenic gene expression are repressed in the skeletal muscles of mdx mice, a pre-clinical model for DMD. Transgenic overexpression of ERRα, specifically in mdx skeletal muscle, reduces muscle damage marked by decreased serum creatine kinase, improved muscle histology, and restored metabolic and angiogenic targets of the receptor. ERRα overexpression also enhanced muscle regeneration in real-time after BaCl₂ injury in mdx mice. Biomarkers of muscle regeneration, such as eMyHC, showed an increased expression at 4 days after injury (dpi) and an increase in muscle fiber size at 7 and 15 dpi, demonstrating that ERRα can boost muscle regeneration in dystrophic muscle. Strikingly, muscle stem cells isolated from mdx mice display decreased expression of ERRα and its target metabolic and angiogenic genes, coinciding with decreased cellular proliferation and differentiation. Activation of the ERRα pathway in dystrophic muscle stem cells restores its myogenic capacity. Our data demonstrate a deficiency in a key energy homeostasis and growth pathway regulated by ERRα in dystrophic skeletal muscles, likely originating in stem cells of dystrophic mice. Restoration of ERRα mitigates muscle damage in mdx mice by stimulating regeneration and could be a potential therapeutic target for the treatment and management of DMD.

Poster 14

Sex Matters: Deletion of fibroblast Sine Oculis Homeobox Homolog 1 (SIX1) protects against pulmonary fibrosis

Sarah Shin^1,2, Scott Collum¹, René Girard¹, Weizhen Bi¹, Eleanor Valenzi⁴ Danielle Wu^2,3and Harry Karmouty-Quintana^1,5

¹Department of Biochemistry and Molecular Biology, McGovern Medical School; ²Department of Diagnostic and Biomedical Sciences at UTHealth Houston School of Dentistry; ³Department of Bioengineering, George R. Brown School of Engineering, Rice University, Houston, TX 77005; ⁴Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, University of Pittsburgh, Pittsburg, PA 15260; ⁵Divisions of Critical Care, Pulmonary and Sleep Medicine, Department of Internal Medicine, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX 77030
Background: Systemic sclerosis (SSc) is a rare autoimmune disease that involves multiorgan fibrosis, including lung and skin. Interstitial lung disease (ILD) associated with SSc is a common manifestation. SSc has a higher prevalence in women than men; however, men are more likely to develop SSc-ILD and experience a higher mortality. Our lab has previously published that sine oculis homeobox homolog 1 (SIX1) was increased in the alveolar epithelium in pulmonary fibrosis. SIX1 is a developmental transcription factor, which is upregulated during embryogenesis, but then downregulated in mature lungs. Preliminary scRNA-seq data shows that there is increased SIX1, its co-regulators, and SIX1 target gene Cyclin A1 (CCNA1), in female SSc-ILD fibroblasts. Increased mediators cyclin A1 and Eya2 levels in SSc-ILD lung tissue have also been observed. However, the role of SIX1 and Cyclin A1 in SSC-ILD is not fully understood.
Hypothesis: We hypothesize that increased SIX1 levels in female fibroblasts promotes pulmonary fibrosis via transcription of cyclin A1.
Results: This project uses tamoxifen-inducible SIX1^fl/fl/Col1a2^cre mice to show that selective deletion of SIX1 in fibroblasts protects female mice but not male mice from bleomycin-induced pulmonary fibrosis. Lung function analysis using the FlexiVent shows significant improvements in compliance, tissue dampening and elastance in bleomycin-challenged female SIX1-knockout mice only. Fibroblasts were isolated from these mice to evaluate the extent of myofibroblast activation, and immunocytochemistry showed decreased alpha-smooth muscle actin-positive cells in the SIX1-knockout mice, suggesting reduced myofibroblast activation.
Conclusions: This study demonstrates that the deletion of SIX1 in fibroblasts selectively protects female mice against bleomycin-induced pulmonary fibrosis. This investigation proposes a mechanism for the sex bias in ILDs such as SSc-ILD via the reactivation of developmental genes. These findings further highlight the importance of sex as a biological variable and possible diagnostic and therapeutic targets for this detrimental disease.

Poster 15

The Effects of Alcohol on Cerebral Vascular Remodeling in The Aging Brain

J. Tran^{1, 2}, C. Walss-Bass³, H. Karmouty-Quintana¹
¹McGovern Medical School, University of Texas Health Science Center at Houston, Department of Integrative Biology and Pharmacology, 6431 Fannin St., Houston, Texas 77030, USA;²UTHealth MD Anderson Cancer Center Graduate School of Biomedical Sciences, Houston, 6431 Fannin St., Texas 77030, USA;³McGovern Medical School, University of Texas Health Science Center at Houston, Department of Psychiatry and Behavioral Sciences, 1941 East Rd., Houston, Texas 77054, USA

Although vascular remodeling has been well documented in other organ systems regarding alcohol abuse, it is less understood in the vasculature of the brain. Our research aims to elucidate the role of alcoholism in cerebral vascular smooth muscle cells (cVSMC). Previous studies have shown that exposure of ethanol to cVSMCs can lead to a disruption in ion balance resulting in cerebrovasospasms, ischemia, and rupturing of cerebral blood vessels. This damage impairs the brain ability to deliver oxygen and nutrients properly, leading to cellular death, brain atrophy, and in severe cases, memory loss. While cVSMCs are well studied in a neurodegenerative context, it is incompletely understood in alcohol-induced brain damage.

Vascular remodeling is a process in which a blood vessel restructures itself due to a physical or chemical injury. In particular, cVSMCs are sensitive to these changes and will alter the vessel structure by either thickening or thinning the medial layer to control for blood flow. A mechanism in which these vascular changes can occur is through alternative polyadenylation (APA), a mechanism involved in the mRNA 3’UTR isoform selection process. Previously in our lab, this process has been linked to vascular remodeling in pulmonary arterial hypertension. Therefore, our hypothesis is that alcohol consumption leads to cerebral vascular remodeling through aberrant APA.

Utilizing a local biobank, a differential gene expression and gene ontology analysis was performed on bulk RNAseq data from autopsied alcohol use disorder (AUD) and control human brains. The top three pathways that were found to be of significance were: cell-cell signaling, inflammation, and angiogenesis which are all involved in vascular remodeling. To test the involvement of vascular remodeling in AUD, we subjected human-derived cVSMCs to a model of chronic alcohol exposure for seven days. Our results were consistent with previous data where we found increased proliferation of cVSMCs when exposed to a chronic dose of alcohol. This increase in proliferation could be an indicator of the initiation of vascular remodeling.

Second, we determined the role of APA in chronic exposure to alcohol by assessing APA mediators such as NUDT21 and CPSF6. Thus far, our results indicate that a chronic dose to cVSMCs results in changes to the APA machinery such as a significant decrease in CPSF6 expression – which is heavily involved in the regulation of APA via pre-mRNA 3′-end processing and formation.

Lastly, to verify the association of vascular remodeling in subjects with AUD, we stained post-mortem human brain tissue of subjects with AUD in the prefrontal cortex for α-smooth muscle actin, which identifies vascular smooth muscle cells. To our surprise, we found that these vessels were thinned – which could indicate that vessels are more prone to collapsing or dying, leading to a loss of blood and nutrients to the surrounding area for neurons, glia, and other cells. This phenomenon could suggest a differential role for alcohol, APA, and vascular remodeling in regards to brain region and length of exposure. Future directions will be aimed at elucidating the bi-directional nature of vascular remodeling in cVSMCs and ultimately establish APA as a novel lens in which to view cerebrovascular changes in AUD.

Poster 16

TRMT1L-catalyzed m²₂G27 on tyrosine tRNA is required for efficient mRNA translation and cell survival under oxidative stress.

Sseu-Pei Hwang¹^,²^,³, Han Liao³, Katherine Barondeau³, Xinyi Han³, Hunter McConie³, Jeffrey T Chang³, and Catherine Denicourt¹^,³

¹ MD Anderson UTHealth Graduate School of Biomedical Sciences, Houston, TX 77030 2. The UTHealth Biomedical Informatics, Genomics and Translational Cancer Research Training; ²Program (BIG-TCR), Houston, TX 77030 3. Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center, Houston, TX 77030; ³ Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center, Houston, TX 77030

Recent advances in epitranscriptomic studies have revealed an intricate layer of gene regulation through RNA modifications, which confer RNA molecules with diverse characteristics and functions. As one of the most extensively modified RNAs, tRNAs undergo numerous modifications that significantly affect their biogenesis and functions, including decoding, folding, and stability. Dysregulation of tRNA modifications can disrupt translation programs and contribute to the development of diseases, including cancer.

While the enzymes responsible for tRNA modifications are well-studied in prokaryotes and yeast, their functions in vertebrates and human disease remain largely unexplored. Human TRMT1L, a vertebrate paralog of the well-studied RNA methyltransferase TRMT1, is dysregulated in various cancers, such as colon and breast cancer, indicating its potential role in cancer development. However, the specific molecular function and substrate of TRMT1L remain understudied. To characterize human TRMT1L, we first applied eCLIP-Seq to identify its potential substrates, revealing that TRMT1L is associated with a specific subset of tRNAs, mainly tyrosine tRNA (tRNA-Tyr) and cysteine tRNA (tRNA-Cys). Using nanopore-based direct RNA sequencing and RNA LC-MS we further identified that TRMT1L catalyzes N2,N2-dimethyl guanosine (m²₂G) modification on tRNA-Tyr and also facilitates the deposition of other modifications, 3-(3-amino-3-carboxypropyl)uridine (acp³U) and dihydrouridine (D), on the D loop structure of tRNA-Tyr-GUA, tRNA-Cys-GCA, and tRNA-Ala-CGC, which are typically catalyzed by other modification enzymes. Moreover, we observed that TRMT1L depletion reduces the level of tRNA-Tyr-GUA and the global translation rates in colon cancer cells while increasing the sensitivity of the cells to oxidative stress.

Together, our findings demonstrate the critical role of TRMT1L in tRNA modification, governing translational regulation and supporting cancer cells in coping with oxidative stress. This provides insights into the significant role of TRMT1L in connecting epitranscriptomic regulation with potential implications for cancer progression.

Poster 17

Variations in the 3’UTR Landscape Dictate Idiopathic and Post-Viral Lung Fibrosis

René A. Girard^1,2, Scott Collum¹, Sarah Shin^1,2, Jamie Tran^1,2, Weizhen Bi¹, Rahat Hussain³, Manish Patel³, Harry Karmouty-Quintana²

¹The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX 77030; ²Department of Biochemistry and Molecular Biology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX 77030; ³Center for Advanced Cardiopulmonary Therapies & Transplantation, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX 77030.

Background: Idiopathic pulmonary fibrosis (IPF), a chronic, progressive interstitial lung disease of unknown etiology affecting individuals at 65-70 yrs of age, is commonly underdiagnosed, and the current pharmacologic options (nintedanib and pirfenidone) only reduce rather than reverse or halt the rate of fibrosis. Delays in diagnosis further worsen adverse patient outcomes. The only effective treatment for IPF, is lung transplantation 3-5 years after diagnosis. Another fibrotic disease resulting in fibrotic lung injury necessitating lung transplantation is non-resolvable COVID-19 (NR-COVID-19). Unlike IPF, NR-COVID-19 patients average ~40 yrs of age, affects majority Hispanic individuals, and lung transplantation is required within 120 days after diagnosis. NR-COVID-19 presents a challenge for physicians since patients need the transplant so quickly after diagnosis, compared to the 3-5 years needed in IPF. Mechanisms responsible for the differences, specifically the aggressive rate of fibrosis, between these fibrotic pathologies are unknown and need to be studied. We found that expression of Nudix Hydrolase 21 (Nudt21/CPSF5) is a master regulator of alternative polyadenylation (APA), a process that modifies the 3’UTR of mRNAs to regulate expression of specific proteins, is decreased in IPF. This decrease of CPSF5 led to global 3’UTR shortening. The shorter 3’UTRs and increased expression of pro-fibrotic factors like TGFBR1 were present in both IPF and models of lung fibrosis. However, the role of APA in regulation of pro-fibrotic proteins in NR-COVID-19 has not been studied.

Hypothesis: We hypothesize that there are unique 3’UTR landscapes in IPF and NR-COVID-19 due to alterations in APA mediators, and these altered 3’UTR landscapes play a significant role in the pathogenesis of lung fibrosis.

Methods: Lung samples from control and transplant patients with NR-COVID-19 or IPF were used for Poly-A-Click sequencing. This data was then analyzed using the PolyA-miner algorithm. Western blot, qPCR, and immunohistochemistry were used to determine the expression of the key fibrotic, extracellular matrix (ECM), and APA transcripts.

Results: Western blot revealed that there were decreased levels of CPSF5, CPSF6, and CPSF7 in IPF whereas their levels were increased in NR-COVID-19. These differences in APA mediators caused the changes in the 3’UTR regions of ECM proteins. Poly-A-Click Seq data has shown that there are unique 3’ untranslated region (UTR) landscapes between IPF and NR-COVID-19. In total there were 362 unique UTR signatures in IPF, 1120 in NR-COVID-19. There were 509 significant 3’UTR signatures shared between the two diseases. These changes in the 3’UTR coincided with increase protein expression of the ECM genes like MMP7 in IPF or Serpine1 and MMP14 in NR-COVID-19.

Conclusions: We have demonstrated that there are changes in 3’UTR region in both IPF and NR-COVID-19. These changes may explain the differences in expression of extracellular matrix proteins leading to the rapid progression of fibrosis in NR-COVID-19 compared to IPF. The APA modifications to the 3’UTR may be used to identify therapeutic targets or risk factors for aggressive fibrosis like in NR-COVID-19.

Poster 18

Potential mitigative role of estrogen-related receptors in cancer cachexia

Anna DeBruine^1,2, Svitlana Poliakova², Mikhail Kolonin², Ashok Kumar³, Vihang Narkar^1,2

¹The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX 77030, USA; ²Center for Metabolic and Degenerative Diseases, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, TX 77030, USA; ³Institute of Muscle Biology and Cachexia, University of Houston College of Pharmacy, Houston, TX 77204, USA.

Cancer cachexia, primarily a muscle wasting disease, is a leading cause of morbidity and mortality in various cancers. Despite advances in cancer therapy, there are currently no effective treatments for cancer cachexia or related muscle wasting. A breakthrough for cachexia and other myopathies may come from studying skeletal muscle fitness pathways, especially the ones that are easily druggable. Estrogen-related receptors (ERRα, γ, β), belonging to the family of nuclear hormone receptors, have emerged as master-regulators of energy homeostasis. Our lab and others have shown that skeletal muscles highly express ERRα and γ (ERRα/γ). We have shown that ERRα/γ transcriptionally activates mitochondrial biogenesis, fatty acid oxidation, muscle vascularization, and muscle repair pathways that improve muscle endurance. Notably, these receptors have not been investigated for their role in cancer cachexia. To explore the role of ERRs in cancer cachexia, we measured gene/protein expression of ERRα/γ in skeletal muscles of pancreatic and lung cancer cachexia models – KPC and LLC xenographic mice. Expression of both receptors is suppressed in skeletal muscles of these cachectic mice. Additionally, ERRα/γ expression in cultured muscle cells was repressed when the myotubes were exposed to conditioned media from various cachexia-inducing cancer cell lines. Strikingly, in in vivo and in vitro models, expression of both metabolic and angiogenic target genes of ERRs are repressed demonstrating suppression of this hormone receptor pathway. Subsequently, we have found that the atrophic effect of KPC and LLC culture condition media on fully differentiated C2C12 myotubes is reversed by overexpression of adeno-ERR ERRα, but worsened by co-treatment with ERRα antagonist. Our experiments show that ERRα/γ expression is repressed in cachectic muscle along with its target metabolic and angiogenic genes, raising the likelihood that loss of ERRα/γ expression is linked to worse outcome in cancer cachexia. Moreover, reactivation of ERR pathway mitigates cancer cachexia in in vitro models, warranting further study of these receptors as therapeutic targets in cancer-associated muscle wasting.

Poster 19

Rab4 Spatially and Functionally Converges with Rab7 in The Degradative Endolysosomal Network Across Species

Haoyi Yang¹, Stephen M Farmer^1,2,3, Shiyu Xu¹, Yue Yu^1,2, Xin Ye¹, Jing Cai^1,2, Beatriz Rios^1,2, Amanda Solbach^1,2,4, Daniela Covarrubias^1,5, Lili Ye¹, Vicky Chuong^2,6, Qingchun Tong^1,2,7, Erin Furr Stimming⁸, Travis Moore^3,9, Sheng Zhang ^1,2,4,6*

¹Center for Metabolic and Degenerative Diseases, The Brown Foundation Institute of Molecular Medicine, McGovern Medical School at the University of Texas Health Science Center at Houston (UTHealth), Houston, Texas, United States of America;²Program in Neuroscience, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences (MD Anderson UTHealth GSBS), Houston, Texas, United States of America; ³Program in Molecular and Translational Biology, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences (MD Anderson UTHealth GSBS), Houston, Texas, United States of America; ⁴Program in Genetics and Epigenetics, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences (MD Anderson UTHealth GSBS), Houston, Texas, United States of America; ⁵Program in Biosciences, Rice University, Houston, TX, United States of America; ⁶Department of Neurobiology and Anatomy, McGovern Medical School at the University of Texas Health Science Center at Houston (UTHealth), Houston, Texas, United States of America; ⁷Center for Neuroimmunology and Glial Biology, The Brown Foundation Institute of Molecular Medicine, McGovern Medical School at the University of Texas Health Science Center at Houston (UTHealth), Houston, Texas, United States of America; ⁸Department of Neurology, HDSA Center of Excellence, McGovern Medical School at the University of Texas Health Science Center at Houston, Houston, Texas, United States of America; ⁹Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, TX, United States of America

Small Rab GTPases are key regulators of endosomal trafficking that are essential for all physiological functions in eukaryotes, and their dysfunctions are linked to diseases from cancer to neurodegeneration. In the current paradigm for endosomal pathways, which is established mostly from cell-based studies, Rab4 primarily resides on early endosomes to control fast endosomal recycling, while Rab7 localizes on spatially distinct late endosomes to direct degradative endolysosomal pathways. Here, we show that in Drosophila, extensive colocalization exists between endogenous Rab4 and Rab7 on endosomal compartments across different tissues and developmental stages. However, Rab4 and Rab7 are recruited to endosomal membranes through distinct mechanisms and have opposing effects on the sizes of late endosomes and lysosomes: Rab4 overexpression or Rab7 impairment causes enlarged endolysosomes, whereas loss of Rab4 or constitutive active Rab7 reduces them. Further, Rab4 deficiency suppresses the enlarged endolysosomes caused by Rab7 impairment. Conversely, the swelling endolysosome phenotype induced by Rab4 overexpression is mitigated by constitutive active Rab7. Interestingly, loss of Rab4 compromises the survival of rab7 deficient flies but not that of rab5 or rab11 mutants, suggesting a functional overlap between Rab4 and Rab7. Moreover, the levels of endogenous β1-Integrin, a cargo known to be recycled by Rab4 or Rab11 and degraded through Rab7-mediated endolysosomal pathway, are increased in the absence of Rab4 but decreased with Rab4 overexpression. Lastly, Rab4 and Rab7 also colocalize in cultured mammalian cells and mouse brains, and live imaging analyses reveal the dynamic trafficking of β1-Integrin between Rab4- and Rab7-positive endosomes. Together, our findings support that in addition to its role in endosomal recycling, Rab4 also functions in the degradative pathway by directing its cargos, such as β1 integrin, into Rab7-mediated late endolysosomes for degradation. Our study provides novel insight into Rab4’s physiological function and suggests the need for revising the current models of endosomal pathways and additional investigation of Rab4 and other endosomal regulators in vivo.

Poster 20

Effect of anti-microbial immunizations in the progression of amyloid pathology in a mouse model of Alzheimer’s disease

Catalina Valdes¹, Avram S. Bukhbinder^1,2, Yaobin Ling³, Kristofer Harris¹, Paul E. Schulz¹, Rodrigo Morales¹

¹Department of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, USA. ².Division of Pediatric Neurology, Massachusetts General Hospital, Boston, MA, USA. ³.McWilliams School of Biomedical Informatics, The University of Texas Health Science Center at Houston, Houston, TX, USA.

Abstract:

The main pathological contributors in Alzheimer’s disease (AD) include cerebral Aβ and tau deposition, and neuroinflammation. The role of the peripheral immune system in AD has also been described as its activation may induce detrimental events on the brain, including neuroinflammation. Along this line, peripheral microbial infections have been linked to increased risks to AD patients, as well as exacerbate pathological outcomes in animal models. The role of antimicrobial immunizations in AD have been recently explored. Recently, epidemiological studies proposed antimicrobial vaccinations as a novel protective factor for AD. Whether vaccinations can prevent the progression of the pathogenic markers of the disease in the brain is highly relevant as they may help to identify novel protective neuroimmune mechanisms. To asses this question, a mouse model of cerebral amyloid-β (Aβ) amyloidosis (APP/PS1) was chosen to study the role of vaccinations in the progression of AD pathology. Considering that the elderly population is particularly susceptible to respiratory infections, we included vaccines against SARS-CoV-2, influenza and pneumococcal bacteria. Importantly, the selected vaccines use different types of technologies: mRNA in the Pfizer COVID-19 vaccine, inactivated viruses in the Fluarix (flu) vaccine, and polysaccharide subunits in the Pneumovax23 (pneumococcal) vaccine. Controls included the inoculation of either PBS or lipopolysaccharide (LPS). All mice were treated once per month (5 times in total), from 90 days of age. Injectates were administered intramuscularly. Brains were collected at 220 days of age for histological and biochemical analyses. Serum was also collected to compare the inflammatory profiles in the brain and periphery of experimental and control subjects. All mice were evaluated for their cognitive abilities 10 days before sacrificing. Interestingly, the cognitive performance of mice treated with the Pfizer and Fluarix vaccines were considerable better compared to all other groups. However, no differences in amyloid load were identified between the groups, suggesting that cognitive changes were independent of this pathological hallmark (preliminary data). Interestingly, significant changes were identified in the peripheral immune profile, suggesting that the variable cognitive properties in these mice was due to changes at this level. The findings collected in serum samples will be compare with that collected from brains at biochemical and histological levels. In summary, our preliminary results demonstrate that some antimicrobial immunizations alleviate cognitive decline in an amyloid-independent fashion. Future studies will confirm/discard these preliminary findings and evaluate potential mechanisms mediating these responses.

Poster 21

Lipopolysaccharide Remodeling Confers Resistance to Bam Complex Inhibitors

Teresa Sullivan^1,2, Anna Konovalova^1,2
¹The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX 77030; ²Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center at Houston, Houston, TX 77030

Gram-negative bacteria present a significant problem for antimicrobial therapies due to the presence of an outer membrane. The outer membrane restricts the entry of many compounds, including antibiotics. One strategy to overcome the outer membrane permeability barrier is to develop inhibitors that target essential proteins on the bacterial surface. One such protein is BamA, an essential component of the β-barrel assembly machinery (Bam) complex that folds and inserts all outer membrane proteins. BamA emerged as an attractive target, and its small molecule, peptide, and antibody inhibitors have been reported by several companies and academic labs.

MRL-494 is a small molecule inhibitor of BamA, but mechanisms of action and resistance remain poorly understood. We isolated several novel MRL-494 resistant mutations outside the Bam complex target. These mutations caused structure modification to Lipopolysaccharides (LPS) either by directly targeting an LPS biosynthesis enzyme LpxM or regulatory PmrAB and QseBC pathways that control dynamic LPS modifications The PmrAB pathway has been well-studied for controlling enzymes that modify the lipid A portion of LPS, reducing its overall negative charge and conferring high resistance to polymyxin antibiotics. Through genetic analysis, I showed that resistance to MRL-494 is due to Lipid A modifying enzymes, EptA and ArnT. By characterizing the gain-of-function pmrA* mutant, I uncovered that LPS modifications do not improve Bam complex function during MRL-494 treatment. Instead; LPS modifications improve cell survival when Bam complex activity is limited. Moreover, I have shown that this resistance mechanism is not MRL-494 specific. It applies more broadly to Bam complex inhibiting conditions and promotes resistance to another BamA inhibitor, darobactin.

LPS modifications, including those caused by mcr-1 plasmid, that encodes an eptA homolog, have been a major threat for clinical resistance to the last-resort antibiotics like polymyxin B and colicin. The observation that the same LPS modifications confer cross-resistance to Bam complex inhibitors presents significant concerns and highlights the urgent need to address resistance mechanisms in order to develop effective antimicrobial therapies.

Acknowledgments: This research is supported by the National Institutes of Health (NIH) and the Welch Foundation.

Poster 22

Unraveling BrainAge in Bipolar Disorder: The Machine, The Model, The Mind, and The Meds

Natasha Topolski^1,7,8, Ercole J Barsotti², Andrea Boscutti¹, MD; Emese H C Kovacs¹⁰, Gail IS Harmata^3,4, PhD; Marie E Gaine^5,6, PhD, Gabriel R. Fries^1,7,8, PhD, Benson Mwangi^1,8, PhD, John A Wemmie^4,5,9, MD, PhD, Vincent A Magnotta, PhD^3,4, Jair C. Soares^1,7,8, MD, PhD
¹Translational Psychiatry Program, Faillace Department of Psychiatry and Behavioral Sciences, McGovern Medical School, University of Texas Health Science Center at Houston, (UTHealth), 77054, Houston, TX, USA ²Department of Epidemiology, University of Iowa, Iowa City, IA, USA ³Department of Radiology, University of Iowa, Iowa City, IA, USA ⁴Department of Psychiatry, University of Iowa, Iowa City, IA, USA ⁵Iowa Neuroscience Institute, The University of Iowa, 169 Newton Rd, 52242, Iowa City, IA, USA ⁶Pharmaceutical Sciences and Experimental Therapeutics, The University of Iowa, 180 South Grand Ave, 52242, Iowa City, IA, USA ⁷Neuroscience Graduate Program, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, 77054, Houston, TX, USA ⁸Center of Excellence in Mood Disorders, Faillace Department of Psychiatry and Behavioral Sciences, McGovern Medical School, The University of Texas Health Science Center at Houston, 1941 East Rd, 77054, Houston, TX, USA ⁹Department of Veterans Affairs Medical Center, Iowa City, IA, USA ¹⁰Department of Neuroscience and Pharmacology, The University of Iowa, 51 Newton Rd, 52242, Iowa City, IA, USA
Background: Studies of BrainAge, a prediction of age based on neuroanatomy, in bipolar disorder (BD) have been limited and few have investigated how factors such as the BrainAge model and medication use affect the discrepancy between BrainAge and chronological age known as BrainAgeGap.
Aims: This study investigates how clinical and technical factors affect different BrainAge models.
Methods: Previously developed BrainAge models trained on T1-weighted MRI scans from controls were utilized including a ridge regression (ENIGMA), ExtraTrees regression odel (PyBrainAge), and convolutional neural network (SFCN-reg). These models were tested on 336 BD (17% with current lithium use and 83% without) and 350 control subjects. Linear mixed-effects models were used to assess BrainAgeGap differences.
Results: The BrainAgeGap for controls was -1.16 (ENIGMA), -1.04 (PyBrainAge), and -0.409 (SFCN-reg) and 1.67 (ENIGMA), 1.97(PyBrainAge), and 0.482 (SFCN-reg) for BD. Diagnosis and scan quality significantly influenced the ENIGMA (ß=2.78, p<0.001, ß=2.4, p<0.01, ) and PyBrainAge (ß=3.03, p<0.001, ß=2.3, p<0.05) models, but not the SFCN-reg model (ß=1.03, p=0.07, ß=0.09, p=0.89).
Across all three Brain Age models, BD subjects not taking lithium showed significantly higher BrainAgeGap relative to controls (ENIGMA: t-value=4.68, SE=0.61, p<0.0001****, PyBrainAge: t-value=5.12, SE=0.65, p<0.0001****, Pyment: t-value=3.56, SE=0.43, p<0.001***). Conversely, none of the models showed a significant difference between BrainAgeGap in BD currently taking lithium and controls (ENIGMA: t-value=0.53, SE=1.10, p<0.59, PyBrainAge: t-value=0.44, SE=1.17, p<0.65, Pyment: t-value=0.22, SE=0.77, p<0.82).
Conclusions: Our findings suggest that choice of BrainAge model impacts the estimation of BrainAgeGap in BD. Moreover, Lithium use is associated with lower BrainAgeGap across models, adding to evidence supporting its neuroprotective properties. Further research should explore how model features, medication duration, illness severity, and environmental factors affect BrainAge variability.

Poster 23

LS^MC4RNeurons Integrate Anterior Cingulate Cortex (ACC) and Hypothalamic Inputs to Regulate Feeding Behaviors and Body Weight

Yuhan Cao^1,2, Yuanzhong Xu², and Qingchun Tong^1,2*
¹MD Anderson Cancer Center & UTHealth Houston Graduate School for Biomedical Sciences, University of Texas Health Science at Houston, TX 77030; ²Center for Neuroimmunology and Glial Biology, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Science Center at Houston, Texas, 77030

Background: Obesity is a growing global health issue with significant public health risks. The melanocortin 4 receptor (MC4R), activated by α-melanocyte-stimulating hormone (α-MSH) from pro-opiomelanocortin (POMC) neurons, reduces food intake, increases energy expenditure, and suppresses hunger. While MC4R is broadly expressed in the brain, its abundance in the paraventricular hypothalamus (PVH) highlights its role in regulating energy balance and homeostatic feeding. However, restoring MC4R specifically in the PVH does not fully reverse obesity and hyperphagia in MC4R-null mice. As an information processing center in the brain, the lateral septum (LS), traditionally linked to emotion, social interactions, and cognition, has recently been shown to play a key role in feeding behaviors. The LS also expresses MC4R, though the specific functions of MC4R-expressing neurons in the LS (LS^MC4R) remain poorly understood.

Aims: To explore the role of LS^MC4Rneurons in regulating feeding behaviors and body weight.

Methods: To investigate the role of LS^MC4R neurons, we injected AAV-DIO-Kir2.1, an inhibitory hyperpolarizing potassium channel, into the LS of MC4R-Cre mice for long-term silencing. Additionally, GCaMP6s was used to record neuronal activity in freely moving mice via fiber photometry, and channelrhodopsin-2 was injected to enable optogenetic activation during behavioral testing. Pseudotyped rabies tracing identified upstream inputs to LS^MC4R neurons.

Results: Long-term inhibition of LS^MC4R neurons promotes obesity in mice fed a high-fat diet (HFD) but does not affect body weight on a regular chow diet. LS^MC4Rneuronal activity decreases as mice approach or consume food, with the level of reduction varying based on the mice’s energy status and the food’s palatability. The specifically activation of LS^MC4Rneurons after fasting overnight reduced the refeeding without causing a place aversion. And the pseudotyped rabies indicate LS^MC4R neurons receive projections from the anterior cingulate cortex (ACC), a region involved in cognition and decision-making, as well as the hypothalamus, a center for energy regulation.

Conclusions: LS^MC4R neurons integrate cognitive inputs from the ACC and energy signals from the hypothalamus to regulate eating behaviors and body weight. Future studies will focus on deciphering how these signals are integrated within the lateral septum (LS).

Poster 24

WIPI2B/ATG-18 phosphorylation regulates neuronal autophagy in vivo
Heather Tsong^1,2, Andrea Stavoe^1,2
¹Department of Neurobiology and Anatomy, University of Texas Health Science Center McGovern Medical School, Houston, TX, USA; ²University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX, USA

With today’s aging population, the incidence of age-related diseases, such as neurodegenerative diseases (NDDs), is increasing. As such, there is a growing need to understand the cellular mechanisms contributing to neuronal aging. One of the hallmarks of aging is the loss of proteostasis, which can lead to the build-up of misfolded proteins and aggregates. Autophagy is a highly conserved cellular degradation pathway that neurons utilize to clear such waste and is essential for maintaining neuronal health. However, autophagy function decreases with age, and dysregulated autophagy is implicated in many NDDs. Previously, we found that in aged primary mouse neurons, there was a decrease in autophagosome biogenesis that could be rescued by the overexpression of WIPI2B, a key player in autophagosome biogenesis. Importantly, this rescue was contingent upon the phosphorylation state of WIPI2B. These data suggest that WIPI2B phosphorylation regulates its function in autophagosome biogenesis and that manipulating WIPI2B phosphorylation may modulate rates of autophagosome biogenesis. To test this hypothesis, we first verified the role of WIPI2B phosphorylation in neuronal autophagy in vivo in Caenorhabditis elegans. We generated C. elegans strains endogenously expressing phospho-mimetic or phospho-dead ATG-18 (the WIPI2B ortholog). We previously showed that autophagy regulates PVD axon outgrowth cell autonomously. Using PVD axon length as a simple, visual readout of neuronal autophagy, we determined that worms with phospho-mimetic ATG-18 had longer axons, suggesting defective autophagy. In contrast, worms with phospho-dead ATG-18 had wild-type axon lengths, suggesting functional autophagy. Further analysis of autophagosome biogenesis will determine how ATG-18 phosphorylation affects neuronal autophagy. Due to the importance of this phospho-site in autophagy, we aim to identify the kinase and phosphatase that regulate ATG-18/WIPI2B phosphorylation in neurons. Using a C. elegans genetic screen, we identified PP2A (protein phosphatase 2A) and CDK16 as a candidate phosphatase and kinase, respectively. Epistatic analysis also suggests that PP2A, CDK16, and ATG-18 function in the same pathway to modulate neuronal autophagy. Given the genetic evidence, we verified CDK16 enzymatic activity on WIPI2B through in vitro phosphorylation assays and showed co-localization of CDK16 with autophagosomes in cultured primary mouse neurons. These data support CDK16 as a kinase for WIPI2B in neurons. Ultimately, a better understanding of how neurons regulate autophagy will uncover novel targets for modulating neuronal autophagy, which may provide a therapeutic strategy to delay signs of aging and ameliorate symptoms of NDDs.

Poster 25

Untangling the Complex Regulatory Networks Controlling Urease Activity and Pathogenesis in MRSA CAUTI

Jana Gomez^1,2, Jennifer N Walker¹

Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center, Houston, TX^1;The University of Texas Health Houston MD Anderson Cancer Center Graduate School of Biomedical Sciences, Houston, TX²

Catheter associated urinary tract infections (CAUTI) are common, costly, and caused by a broad range of uropathogens. Additionally, long term urinary catheters (UCs) are asymptomatically colonized with understudied uropathogens, which increases the risk of symptomatic infection with those microbes and serves as a reservoir for antimicrobial resistance. Of these uropathogens, Staphylococcus aureus is particularly problematic as it frequently disseminates to bacteremia, resulting in severe disease and death. S. aureus is also commonly methicillin resistant, making it difficult to treat. Furthermore, S. aureus produces the enzyme urease, which promotes the accumulation of crystals on the UC, resulting in encrustations and further antibiotic recalcitrance. Our previous work shows that S. aureus isolates persistently colonizing long term UCs exhibit temporal increases in urease activity, suggesting the enzyme contributes to chronicity. Thus, we aim to understand how S. aureus urease functions on a molecular level to explore how the pathogen is adapting to the chronically catheterized bladder.

Studies with another uropathogen, Proteus mirabilis, suggest that urease is important for colonization of the bladder and dissemination to the kidneys during CAUTI. Our analysis using a S. aureus CAUTI model suggests that urease is important for kidney dissemination in early infection and that urease promotes inflammation in the kidneys and spleen. To further investigate the importance of urease in a urinary tract-like environment, we analyzed the expression levels of urease under various conditions. We have shown that urease is highly expressed in artificial urine media compared to nutrient rich growth media, suggesting that signals in the urinary tract promote urease expression.

Additional reports suggest urease expression is controlled by the CodY, CcpA, and Agr regulators. Using bioinformatics analyses, we identified potential binding sites for CcpA and CodY, but not AgrA, in the upstream regulatory region of the urease operon. Further, we identified potential binding sites of two other regulators –SigB and SrrA– upstream of the urease operon. Mutations in codY and in srrA exhibited increased urease activity compared to wildtype, suggesting that CodY and SrrA are negative regulators of urease. Conversely, sigB, ccpA and agr mutants exhibited lower urease activity, suggesting these regulators are activators of urease activity. Together, these data suggest CodY, SrrA, SigB, and CcpA directly regulate urease activity, while Agr regulates urease through an indirect mechanism. These data provide further insight into the molecular mechanism of S. aureus urease expression and activity. This work also expands our knowledge of how urease exacerbates inflammation and contributes to CAUTI pathogenesis. Future work will continue to investigate this mechanism and the genomic changes that select for increased S. aureus urease activity in individuals with long term UCs and how urease may contribute to long-term pathogenesis.

Poster 26

Imaging microglia activation by TREM2-ImmunoPET

Taylor Brinson^{1, 2}, Evanta Kabir², Onder Otlu², Ronnie The Phong Trinh⁴, Wei Xiong³, Yaima L. Lightfoot⁴, Wiliam J. Ray⁴, Zhiqiang An^{1, 3}, Ningyan Zhang³, and Federica Pisaneschi^{1, 2}
¹UTHealth MD Anderson Cancer Center Graduate School of Biomedical Sciences, Houston, TX, USA; ²Center for Translational Cancer Research, Brown Foundation Institute of Molecular Medicine, UTHealth Science Center, Houston, TX, USA; ³Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, UTHealth Science Center, Houston, TX, USA; ⁴Neurodegeneration Consortium, UT MD Anderson Cancer Center, Houston, TX, USA

Neuroinflammation, an inflammatory response located in the central nervous system and exhibited in many neurological diseases, can be observed by evaluating the activity of immune-associated glial cells, like microglia. TREM2 (triggering receptor expressed on myeloid cells-2) has become a compelling target in developing treatments for diseases such as Alzheimer’s, via its use as a marker of microglial activation, association with neuroinflammatory and neurodegenerative pathways, and role in tumor progression. Herein, we aim to detect neuroinflammation by ImmunoPET (Positron Emission Tomography), an antibody-based non-invasive nuclear imaging technique. ⁸⁹Zr-TREM2-Tfr_BsAb was engineered and analyzed for its specificity toward TREM2 in cellular models and its TREM2-mediated uptake in a murine model of neuroinflammation.

Zirconium-89 (⁸⁹Zr) was utilized to radiolabel a TREM2 bispecific agonist antibody (TREM2-Tfr_BsAb), created to also target the transferrin receptor (TfR) that facilitates blood-brain barrier penetration. The TREM2 antibody and isotype control (IgG) were conjugated with deferoxamine isothiocyanate (DFO-CNS) in DMSO for 2 hours at 37°C. Radiolabeling was performed by incubating both antibodies with ⁸⁹Zr-oxalate in sodium carbonate and PBS 1X (pH=7) at 37°C for 30 to 60 minutes. Reaction completion and purity were verified through radio-iTLC analysis. The specificity of TREM2 binding was evaluated using wild-type (WT) and TREM2-overexpressing (TREM-2 OE) HEK 293 cell lines. Biodistribution of the tracer was assessed in vivo by PET/CT imaging at 4, 24, and 48 hours post-injection (p.i.) in healthy immunocompetent mice. To induce neuroinflammation, lipopolysaccharide (LPS) was administered intraperitoneally at 2 mg/kg immediately before PET tracer injection.

⁸⁹Zr-TREM2-Tfr_BsAb and the IgG control were obtained at 74±3% (n=6) and 76±3% (n=6) activity yield. Both expressed a radiochemical purity higher than 99% and were stable in PBS at 4°C for up to 48 hours. TREM-2 OE cells showed 3.3- and 3.6-fold higher ⁸⁹Zr-TREM2-Tfr_BsAb uptake compared to WT cells, at 30 and 60 minutes. Compared to the control isotype, ⁸⁹Zr-IgG, ⁸⁹Zr-TREM2-Tfr_BsAb had more than 6-fold higher uptake in TREM-2 OE cells at both time points. In healthy animals, after 48h post-injection, the brain uptake of ⁸⁹Zr-TREM2-Tfr_BsAb was more obvious compared to ⁸⁹Zr-IgG, revealed by PET imaging. In the LPS-neuroinflammation model, uptake of ⁸⁹Zr-TREM2-Tfr_BsAb was also 2-fold higher in the brains of LPS-inflamed mice compared to the unthreaded control, both at 24 and 48 hours p.i.

In conclusion, we successfully developed a TREM2 specific imaging agent, ⁸⁹Zr-TREM2-Tfr_BsAb, for ImmunoPET of microglia activation in a model of neuroinflammation. We also demonstrated TREM2-mediated uptake in cells. In vivo, ⁸⁹Zr-TREM2-Tfr_BsAb uptake was higher in the brain of LPS-inflamed mice, compared to all controls, indicating a potentially valuable tool for neuroinflammatory screening.

Poster 27

The Potential Therapeutic Effect of the Ketogenic Diet in ARDS

Robert Lwanga^1,2, Victor Guaregua², Synthea Horton², Ragini Nair², Katherine Figarella², Jieun Kim², Thu Tran², Holger Eltzschig², Xiaoyi Yuan²
¹The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX 77030; ²McGovern Medical School, University of Texas Health Science Center at Houston, Department of Anesthesiology, 6431 Fannin St., Houston, Texas 77030

Background: Acute respiratory distress syndrome (ARDS) is marked by severe pulmonary edema and concomitant hypoxia. This is a critical condition affecting thousands of people a year, especially those in critical care conditions or undergoing high-risk surgeries, with estimates of 567,125 cases per year and 226,850 deaths per year in the USA (AHA Hospital Statistics. 2017). There is a crucial need to find treatment options. Previous studies have implicated that the ketogenic diet, which is a high-fat and low-carbohydrate diet, is generally anti-inflammatory and safe in the critically ill population. However, the impact of the ketogenic diet on the lung microenvironment or ARDS outcomes is unknown. Thus, we hypothesize that the ketogenic diet provides lung protection via the reduction of lung inflammation.

Method: To test our hypothesis, we started mice on a ketogenic diet for 7 days and then induced ARDS via lipopolysaccharide (LPS) injection, a model of endotoxin-induced lung injury. Afterward, we harvested the lungs, blood serum, and bronchoalveolar lavage fluid (BALF) to test various hallmarks of ARDS to see the effect of the ketogenic diet on ARDS outcome compared to a coco butter-based control diet. To investigate the impact of the ketogenic diet on the lung’s inflammatory microenvironment during ARDS, I performed various protein-based and RNA-based cytokine and chemokine array panels and carried out mass immune cytometry (IMC) to better understand the detailed cellular mechanisms.

Results: Mice maintained under the ketogenic diet retained more weight, have decreased levels of total protein and albumin in their BALF, have decreased total cell and neutrophil infiltration in the bronchial alveolar lavage, as well as have decreased acute lung injury score compared to the mice on control diet during LPS induced lung injury. Protein-based cytokine chemokine array in the bronchial alveolar lavage fluid and RNA-based panels in the BAL leukocytes showed the overall reduction of proinflammatory cytokines and chemokines in the mice under the ketogenic diet compared to the mice under control diet. The IMC data showed a significant decrease in neutrophils, macrophages, and antigen-presenting cells in the ketogenic mice injected with LPS compared to the control diet mice injected with LPS.

Conclusion: Our findings reveal that the ketogenic diet provides lung protection during endotoxin induced lung injury. Future studies will investigate the detailed cellular/molecular mechanism as well as the effect of the ketogenic diet in murine ARDS models representing different etiologies observed in human ARDS, including injurious mechanical ventilation, bacterial pneumonia, or viral pneumonia.

Poster 28

Staphylococcus aureus persists in individuals with long-term urinary catheters despite antimicrobial treatment and catheter changes.

Duran Ramirez, JM¹, Blake Hanson², Walker, JN^1,2
¹Department of Microbiology and Molecular Genetics, UTHealth;²Department of Epidemiology, Human Genetics & Environmental Sciences, Center for Infectious Diseases, School of Public Health, UTHealth, Houston, Texas, USA

Urinary catheterization (UC) is a common procedure among the elderly and increases the risk of developing symptomatic catheter-associated urinary tract infections (CAUTIs), as well as asymptomatic bacteriuria (ASB). Notably, ASB and CAUTI are caused by a wide range of uropathogens, including Gram positives such as Staphylococcus aureus. Additionally, CAUTI and ASB causing pathogens have a high prevalence of antimicrobial resistance. Furthermore, ASB in individuals with long-term UC can persist for more weeks and months. Yet, how these microbes persist or whether antibiotics or catheter exchanges disrupt these communities remain understudied. Thus, to explore how uropathogens persist within the catheterized bladder, we used whole genome sequencing and bioinformatics analyses to track one of the most frequent causes of ASB, S. aureus, within individuals with long term UC over time. Our strain collection consists of 142 S. aureus isolates from 21 participants, with an average of 7 isolates per person (ranging from 1-26). Genomic analyses indicate the most common sequence type (ST) was ST5 (10/21) and these isolates carried an average of 12 antibiotic resistance genes, including ~50% that were methicillin resistant. Furthermore, phylogenetic analyses showed the isolates clustered first by STs and then by participant, with most participants (19/21) maintaining the same ST throughout the study. Finally, whole genome analyses of single nucleotide polymorphisms (SNPs) revealed most strains of the same ST and from within participants had fewer than 30 SNPs despite being isolated weeks or months apart, suggesting that the same strain is persisting over time. Notably, almost half of the patients (10/21) received antibiotic treatment, and all had at least one catheter change during the collection period. Together, our results suggest S. aureus persists within the urinary tract of long-term UC individuals, despite antimicrobial exposure and/or catheter changes.

Poster 29

Neural dynamics of unfairness during cooperation in freely moving monkeys

Alexandra McConnell^1,2, Melissa Franch^1,4, Valentin Dragoi^1,2,3
¹Neurobio. and Anat., Univ. of Texas Hlth. Sci. Ctr. at Houston, Houston, TX; ²Neurosurg., Houston Methodist Res. Inst., Houston, TX; ³Electrical and Computer Engin., Rice Univ., Houston, TX

Advanced forms of cooperation, such as sensitivity to fairness, have been observed in macaques through experiments involving unequal rewards. Macaques sometimes adjust their cooperative behavior when experiencing unfair outcomes, yet the underlying neural mechanisms—particularly how these social variables are encoded and communicated across sensory and executive brain regions—remain poorly understood. Fairness perception involves at least two distinct types: self-disadvantageous unfairness (receiving less than a partner) and partner-disadvantageous unfairness (receiving more). While past research has primarily focused on self-disadvantageous unfairness, it remains unclear whether primates process these two conditions differently or if fairness sensitivity extends beyond self-interest.

To address this, we examine how unfairness influences behavior, attention, neural encoding, and inter-area communication during cooperation. We first analyze how coordination behavior changes under fair and unfair conditions, followed by an investigation of fixation patterns to assess visual attention during inequity. At the neural level, we use decoding analysis to determine when V4 and dlPFC differentiate between fairness conditions, and subspace communication analysis to assess how these regions interact over time. Additionally, we apply cross-correlogram (CCG) analysis of push timing to measure whether unfairness disrupts coordination between partners. By integrating these approaches, this study provides insight into how the primate brain processes fairness and how these perceptions shape cooperative decision-making.

Poster 30

Developing an X-ray triggered nanoscintillator complex for drug delivery to deep-seated tumors

Gabrielle Krouse^1,2, Onur Sahin¹, Yuri Mackeyev¹, Geraldine V. Vijay¹, Sunil Krishnan^1,2
¹Vivian L. Smith Department of Neurosurgery, The University of Texas Health Science Center, Houston, TX, USA; ²The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX, USA

Light-enabled therapies are an evolving field of research for the treatment of specific types of cancers. Photothermal and photodynamic therapy work particularly well for cancers near the surface or those that are endoscopically accessible, such as skin cancer and esophageal cancer. However, these therapies have limited utility for deep-seated tumors, such as pancreatic cancer, because of the poor penetration depth even of near infrared light. Furthermore, light-enabled therapies are operator-dependent, inconsistent, non-uniform, and often involve invasive procedures.

Nanoscintillators are nanomaterials capable of luminescence when excited by ionizing X-ray radiation. These are being explored as attractive internal sources of light for light-enabled therapies because X-rays can go any depth in tissues to access deep-seated tumors. X-ray excited photodynamic therapy (XPDT) is a form of light-enabled therapy where X-rays activate a nanoscintillator which emits light that is absorbed by a photosensitizer (PS) that generates reactive oxygen species (ROS). The spatiotemporal control and penetration depth gained from this therapy is promising; however practical applicability hinges on high quantum yields of luminescence with clinically relevant doses and qualities of radiation. Our lab has synthesized and characterized a novel XPDT agent, a PEGylated nanoshell containing Y₂O₃ doped with europium at its core and with a silica epilayer impregnated with the PS, methylene blue (pYSM). A single dose of 10 Gy X-ray irradiation triggers a high fluence of X-ray-excited luminescence at 611nm which activates the PS to then generate a high yield of singlet oxygen species. When delivered intravenously, pYSMs accumulate passively in pancreatic tumors and significantly sensitized these tumors to radiation therapy. Since we observed increased T cell infiltration of pYSM + radiation treated tumors, we coupled this therapy with anti-PD1 antibody therapy in syngeneic tumor models with bilateral thigh xenografts and noted that pYSM + radiation + anti-PD1 caused significant regression of tumors in both ipsilateral irradiated tumors and contralateral unirradiated tumors.

As an extension of these studies showing therapeutic efficacy of a scintillator coupled with a PS, we propose a new approach using a similar scintillator coupled to a caged cytotoxic drug via a photocleavable linker. For this, we need a scintillator that luminesces at a wavelength not typically encountered in clinical environments, forcing us to pivot away from Y₂O₃:Eu. We isolated YTaO₄ to be our new scintillator due to its high light yield, stability, and emission spectra peak at 330nm. Our first fabrication of YTaO₄ yielded particles close to the micron range, making these too larger for our nanocomplex. We have changed the synthetic pathway by moving from a solid-state synthesis to a wet synthesis method to obtain smaller particles. There is promising evidence that we could isolate particles with a size around 70nm. Our initial focus on Wortmannin as the desired toxin was not as fruitful due to its limited toxicity towards pancreatic cancer cells. Other toxic payload designs and syntheses are ongoing. Upon successful design, we will have created an on-demand, extrinsically triggered, spatially controlled drug delivery platform that homes highly toxic payloads to tumors for precise and efficient eradication of cancer cells while minimizing off-target toxicities.

Poster 31

A Potential Link Between Phospholipid Conjugation and Unconventional Protein Secretion

Thi Thu Trang Luu^{1, 2}, Dakai Zhang¹, Zhiping Wu³, Junmin Peng³ and Guangwei Du^1,2
¹ Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, TX 77030; ² Molecular and Translational Biology Program, MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX 77030, USA;
³ Center for Proteomics and Metabolomics, St. Jude Children’s Research Hospital, Memphis, TN, 38105-3678 USA

Protein lipidation, the covalent attachment of lipid moieties to proteins, enhances their hydrophobicity and targets them to specific membrane compartments. Phospholipid conjugation is a unique lipid modification found only in ATG8 family members and ubiquitin, which all belong to the ubiquitin superfamily. This modification in ATG8 proteins plays a crucial role in autophagy and protein secretion through the unconventional protein secretion (UPS) pathway. Despite extensive studies on other lipid modifications, phospholipid conjugation remains poorly understood, primarily due to technical limitations. To address this, we developed a novel method to analyze phospholipid-conjugated proteins at the whole-proteome level. This robust approach, combining click chemistry and mass spectrometry, led to the identification of 15 previously unrecognized phospholipid-conjugated proteins spanning multiple protein families. These include cytokines involved in inflammation, subunits of the vacuolar ATPase, which facilitate the acidification of eukaryotic intracellular organelles, and proteins from diverse families such as heat shock proteins and calcium-binding proteins. Notably, most of these proteins are secretory, with more than half lacking a signal peptide.

One of the major challenges in understanding UPS is how secretory proteins are loaded into vesicles for secretion without the signal peptide. Recent evidence suggests that certain cytosolic proteins may enter secretory compartments via translocation through protein channels. However, the precise mechanisms governing the recruitment of most leaderless proteins for secretion remain unclear. Considering the essential role of phospholipid conjugation in the membrane attachment and functionality of ATG8 proteins, our findings suggest that this modification may facilitate the recruitment of specific leaderless cytosolic proteins to membranes, enabling their subsequent translocation into secretory or intermediate vesicles, or directly across the plasma membrane.

This study expands our understanding of protein trafficking and post-translational modifications, emphasizing the potential role of phospholipid conjugation in UPS and its broader implications for disease mechanisms and therapeutic strategies targeting UPS-related disorders.

.Poster 32