Competition Abstracts

Poster 1

A Novel MET Antibody-Drug Conjugate-Based Combination Therapy to Overcome Colorectal Cancer Plasticity and Drug Resistance

Shraddha Subramanian^1,2, Tressie Posey^1,2, Joan Jacob^1,2, Adela Aldana¹ and Kendra S. Carmon^1,2

¹Center for Translational Cancer Research, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX, USA;²University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX, USA

Despite therapeutic advancements, colorectal cancer (CRC) remains the second deadliest malignancy in the United States. Challenges encountered in CRC treatment may be attributed to cancer stem cells (CSC), an immortal tumor cell population. CSCs potentiate tumor relapse by exploiting its infinite replicative potential and inherent drug resistance. Moreover, CSCs exhibit plasticity, allowing them to transition between differentiated and undifferentiated states in response to environmental cues to evade therapy and drive metastatic progression. MET is a receptor tyrosine kinase frequently upregulated in CRCs. We previously demonstrated that treatment with chemotherapies or antibody-drug conjugates (ADCs) targeting the CSC marker LGR5 led to the loss of LGR5 expression with concomitant MET-STAT3 pathway activation in therapy-resistant CRC cells. Further, we showed LGR5 couples to the scaffold protein IQGAP1, which correlates with poor prognosis in different cancer types. Interestingly, our new data shows that LGR5 knockdown enhances IQGAP1 interaction with MET and STAT3 via co-immunoprecipitation assays and decreases phosphorylation of SHP-2, a negative regulator of STAT3. These observations suggest a role for IQGAP1 and SHP-2 in regulating MET-STAT3 activation and plasticity of LGR5⁺ CRC cells.  To overcome therapy-induced plasticity, we generated MET-targeting ADCs conjugated to DNA-crosslinking payloads. First, we cloned and produced MET monoclonal antibodies (mAbs) and evaluated them for specificity and binding in a panel of CRC cell lines expressing different MET protein levels. MET mAbs demonstrated high selectivity for MET-expressing CRC cells and internalized to lysosomes, which is necessary for ADC payload release. To generate MET ADCs, we employed a site-specific conjugation methodology to attach cleavable peptide linker-drug moieties to the highest affinity MET mAb. MET ADCs were evaluated for cancer cell-killing efficacy in vitro in parallel with MET mAb, non-targeting control mAb (cmAb), and control ADC (cADC). MET ADC demonstrated high potency and efficacy in MET-expressing CRC cells, whereas MET mAb, cmAb, and cADC had minimal effects. MET ADCs were then tested in combination with chemotherapies or other targeted therapies.  The combination of MET ADCs with 5-fluorouracil showed a synergistic effect in vitro. Future work involves investigating the safety and efficacy of MET ADCs alone and combined with chemotherapies in xenograft models of CRC. These findings present a mechanism underpinning CRC plasticity and the rationale for a novel treatment modality to potentially overcome CRC resistance and relapse.

Poster 2

EGFR inhibitors increase LGR5 expression and enhance potency of LGR5 antibody-drug conjugates targeting colorectal cancer stem cells

Peyton High^1,2, 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

Colorectal cancer (CRC) is the second leading cause of cancer-associated death in the United States with an urgent need for improved therapeutic options. One of the primary challenges in treating CRC is therapy resistance thought to be mediated by cancer stem cells (CSCs), a subpopulation of cancer cells with infinite replicative potential that can differentiate to drive tumorigenesis and disease relapse. Thus, targeting CSCs has become an attractive therapeutic strategy.

Antibody-drug conjugates (ADCs) are amongst the fastest growing classes of anticancer drugs and utilize the specificity of monoclonal antibodies (mAbs) to hone cytotoxic payloads to cancer cells. We generated ADCs directed against leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a well-defined biomarker of CSCs that is highly overexpressed in CRCs. LGR5 ADCs demonstrated high specificity and efficacy in CRC cells and xenograft models, though tumor relapse after treatment withdrawal was a major obstacle. Interestingly, FDA-approved therapy targeting epidermal growth factor receptor (EGFR) has been shown to increase LGR5 mRNA expression levels in patient-derived models of CRC. This study aims to determine the mechanism of EGFR-mediated regulation of LGR5 expression and evaluate the therapeutic efficacy of combination treatments targeting EGFR and LGR5 for the improved treatment of CRC.

To analyze the effect of EGFR inhibition on LGR5 expression, a panel of CRC cell lines of different KRAS and PI3CKA mutational statuses were treated with FDA-approved EGFR- and HER2-targeted mAbs or small molecule inhibitors. LGR5 protein expression was shown to be upregulated in a dose- and time-dependent manner, independent of mutation status. We also observed increased LGR5 protein levels in patient-derived colorectal tumor organoids treated with EGFR-targeting mAb cetuximab (CTX) and in LoVo CRC cell line xenografts treated either with CTX or HER2-targeting mAb trastuzumab (TTZ). Notably, treatment of CRC cell lines with EGF, MEK1/2 inhibitor trametinib, and EGFR-directed siRNA all resulted in concomitant reduction in EGFR and LGR5 protein levels, suggesting EGFR and LGR5 are co-degraded. Co-immunoprecipitation and immunocytochemistry experiments identified a novel EGFR-LGR5 interaction and co-internalization mechanism in CRC cells, respectively. Furthermore, we showed EGFR-LGR5 interaction is enhanced by treatment with CTX. To evaluate the efficacy of combination therapies targeting EGFR and LGR5, CRC cells and a patient-derived tumor xenograft model were treated with LGR5 ADCs with or without CTX. Importantly, we showed CTX significantly enhanced the potency of LGR5 ADCs incorporating different classes of cytotoxic payloads in vitro and in vivo. These results suggest combining LGR5 ADCs with EGFR inhibitors may be a more effective approach for the treatment of CRC and eliminating CSCs to overcome resistance and relapse.

Poster 3

Membrane Dynamics and Druggability of Rheb and RhoA

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

Ras superfamily small GTPases are membrane bound molecular switches that regulate a wide variety of cellular processes including growth and proliferation, trafficking and cytoskeleton organization. Dysregulation of small GTPases can result in the development of a variety of diseases including cancer as famously illustrated by mutations Ras proteins. Small GTPases are thus attractive targets for cancer therapeutics, however, few FDA approved drugs for small GTPases exist today.

Ras proteins have been shown to dynamically reorient around the membrane, with some orientations with respect to the membrane showing differential signaling potential. Targeting reorientation dynamics have been proposed as a new therapeutic strategy, with a small molecule having been shown to stabilize a signaling deficient orientation.. While this phenomenon is well studied in Ras, little is known about orientation dynamics of other small GTPases. The goal of this research is to better understand the relationship between membrane orientation of small GTPases and signaling function by characterizing membrane dynamics and membrane-dependent druggability of two small GTPases: Rheb and RhoA. Rheb and RhoA, two small GTPases from the Ras and Rho families, respectively, play distinct roles in cancer. Rheb regulates growth and proliferation through direct activation of mTORC1, while RhoA modulates cytoskeleton actin organization and cell motility. Overexpression of Rheb has been observed in several cancer types and is correlated with poor outcomes, while overexpression of RhoA has been known to increase metastasis in gastric cancer.

To map membrane orientation dynamics of Rheb and RhoA, we simulated the full length and truncated lipid anchor of Rheb and RhoA using molecular dynamics (MD) simulations in bilayers modeling the native membrane targets of the proteins. We found that like previous studies of Ras, Rheb is highly dynamic and primarily samples 3 orientation states with respect to the membrane. In contrast, RhoA samples a much narrower distribution of orientation states, with only one primary orientation state. Rheb’s membrane reorientation profile to Ras implies a similar functional role. The lack of reorientation of RhoA implies that the primary orientation may be functionally necessary. To better understand the role of membrane organization in druggability of Rheb and RhoA, we utilized probe based molecular dynamics simulations specialized for membrane proteins (pMD-membrane) to compare the druggable surfaces of membrane-bound Rheb and RhoA with soluble forms. pMD-membrane is a fragment-based drug discovery method that can map the allosteric druggable surface of proteins by simulating the protein in a solvent containing common drug molecule fragments. We found that there is a significant difference in druggable surface between membrane bound small GTPases and soluble forms. Because the soluble forms of small GTPases are most commonly used for drug discovery studies, this finding could lead to significant improvement in future drug discovery for small GTPases.

Poster 4

Voices in my head: activation of auditory cortex during silent reading

Kathryn Snyder,^1,2 Kiefer Forseth,^1,2 Oscar Woolnough,^1,2 Nitin Tandon^1,2,3

¹Vivian L. Smith Department of Neurosurgery, McGovern Medical School at UT Health Houston, Houston, TX USA;²Texas Institute for Restorative Neurotechnologies, The University of Texas Health Science Center at Houston, Houston, TX, USA;³Memorial Hermann Hospital, Texas Medical Center, Houston, TX, USA

Spoken and written language are crucial to communication and likely recruit shared networks to map phonology and orthography to meaning. Models of comprehension for both domains suggest overlapping processes. The cortical instantiation of these models is critical to our understanding of comprehension, but the convergent neural mechanisms are unclear. We investigate the spatiotemporal dynamics of shared networks for listening and silent reading using ECoG.

Data were obtained from 64 patients who underwent ECoG and completed cued naming to matched spoken and written descriptions. We analyzed broadband gamma activity (BGA; 70-150Hz) and integrated group responses with mixed-effects multilevel analyses. We then analyzed BGA from depths along STG (n=37). Lastly, we applied direct cortical stimulation of STG during listening and silent reading (n=2).

For each spoken word, sustained activation of STG (128% BGA) was followed by activation of posterior middle temporal gyrus (pMTG; 41% BGA). For each written word, visual cortex activity was followed by activation of lateral occipitotemporal cortex, fusiform gyrus (Fus) intraparietal sulcus (IPS), STG (20% BGA), and pMTG (29% BGA). For both modalities, the last word triggered an identical lexical processing network (pMTG, Fus, IPS, pars triangularis). Activity from depths along STG (n=37) revealed a consistent anteroposterior gradient. Electrodes in Heschl’s gyrus (HG) showed sustained activation during listening consistent with acoustic processing and were quiescent during reading. Electrodes in planum temporale (PT) showed transient activity at speech onset and recurrent activity for written words. Stimulation of HG disrupted listening only, while stimulation of PT disrupted both listening and reading. Our prior work implicates PT in predictive encoding during production – this work suggests the same occurs in silent reading.

Using large-scale ECoG, we derived new insights into the neural basis of spoken and written language. Ultimately, we believe that this work would provide important insights to optimize the design of neural prosthetics for the treatment of reading-related disorders.

Poster 5

Detection of chronic wasting disease prions in processed meats and zoonotic potential 

Rebeca Benavente¹, Fraser Brydon² Francisca Bravo^1,3, Paulina Soto^1,3, J. Hunter Reed⁴, Mitch Lockwood⁴, Glenn Telling⁵, Marcelo Barria², Rodrigo Morales^1,3

¹Department of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston, Texas, USA; ²National CJD Research & Surveillance Unit Centre for Clinical Brain Sciences University of Edinburgh, Edinburgh, Scotland; ³Universidad Bernardo O’Higgins. Santiago, Chile; ⁴Texas Parks and Wildlife Department, Texas, USA; ⁵Prion Research Center, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO, USA

Background: Prion diseases are transmissible spongiform encephalopathies affecting several mammals including humans, cattle, ovine and cervids to name a few. These diseases are driven by conformational changes in the cellular prion protein (PrP^C) into a β-sheet rich structure prone to aggregation (PrP^Sc). Variant Creutzfeldt-Jakob’s disease (vCJD) occur due to the consumption of cow’s contaminated processed meats which has raised the concern if other prion diseases can infect humans. Chronic wasting disease (CWD) is a prion disease affecting cervids that has showed and exponential increase of cases in the U.S. both in wild and captive settings. The zoonotic potential of CWD remains unknown due to contradictory results. Worrisomely, some specific elk prion strains seem to induce the misfolding of human PrP^C in cell-free assays like protein misfolding cyclic amplification (PMCA), a technique has been used to assess the potential of CWD prions to propagate between and across species.
Aims: To identify the presence of CWD prions in processed meats derived from elk.
Materials and Methods: In this study, we analyzed different processed meats derived from a CWD-positive (pre-symptomatic) free-ranging elk. Products tested included filets, sausages, boneless steaks, burgers, seasoned chili meats, and spiced meats. The presence of CWD-prions in these samples was assessed by PMCA using white-tailed deer and elk substrates. The same analyses were performed in grilled and boiled meats to evaluate the resistance of the infectious agent to these procedures.
Results: Our results show positive prion detection in all the samples analyzed using deer and elk substrates. Surprisingly, cooked meats displayed increased seeding activities when analyzed with cervid substrate but no seeding was observed in human substrate. This data suggests that CWD-prions are available to people even after meats are processed and cooked. However, the species barrier of this particular prion isolate to humans seems to be strong.
Conclusions: These results suggest that CWD prions are accessible to humans through meats, event after processing and cooking. Considering the fact that these samples were collected from already processed specimens, the availability of CWD prions to humans is probably underestimated.
Funded by: NIH and USDA;Grant number: 1R01AI132695 and APP-20115 to RM
Acknowledgement: We would like to thank TPWD personnel for providing us with valuable samples

Poster 6

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 7

Elucidation Of The Molecular Signal for the Regulator Of Capsule Synthesis Stress Response

Hitt SJ ^1,2, 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.

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].

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. These include 1) cationic antimicrobial peptides, that disrupt the outer membrane by targeting polyanionic lipopolysaccharide (LPS), and 2) lysozyme and ß-lactam antibiotics that target the peptidoglycan (PG) cell wall. 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. Assembly of RcsF/OMP complex is required for RcsF signaling, but the underlying reasons have not been resolved. 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 packing through direct interaction with LPS. Mutations that alter LPS charge and structure induce Rcs in a Mg2+-dependent manner. I showed that increased expression of eptA, which modifies LPS and strengthens lateral interactions in a cation-independent manner, also causes a reduction in Rcs signaling in an LPS biosynthesis mutant, providing strong evidence for LPS lateral interactions as a potential Rcs signal. Moreover, the addition of divalent cations during PG synthesis inhibition by antibiotics such as A22 and mecillinam also leads to a significant reduction in Rcs activity. My result support two initial conclusions. First, RcsF seems to monitor LPS packing at the outer membrane and not the LPS structure itself. Second, stabilizing LPS packing alleviates Rcs signaling, not only when LPS is targeted but also when peptidoglycan biosynthesis is inhibited. Together, these findings suggest that disruptions to LPS packing, not cell wall, may be a direct and universal signal for Rc induction.

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 8

Identifying putative hyphal regulators of Candida albicans

Dakota Archambault^1,2, Michael Lorenz^1,2
1University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX;²University of Texas Health Science Center, McGovern Medical School, Department of Microbiology and Molecular Genetics, Houston, TX

Candida albicans is a normal resident of the human microbiota, but defects in the innate immune response can allow C. albicans to disseminate throughout the body, leading to multisystem organ failure and a patient mortality rate of ~40%. How C. albicans interacts with phagocytes is thus a relevant determinant for the development of infection. The interaction of C. albicans and host macrophages is complex and dynamic relationship with hyphal growth as a key feature that contributes to macrophage damage and escape from phagocytosis. Using transcriptomic data from several Candida species prior and post phagocytosis, we have identified two sets of uncharacterized genes as candidate virulence factors. The first set of genes are expressed at a significantly higher level in phagocytosed cells compared to controls. We have generated and screened a deletion library of 87 genes for host-relevant phenotypes in solid and liquid media conditions. We have identified twenty-five putative hyphal regulators and secondary screening has identified multiple genes of interest including, C6_02100W and C1_10240C. The second set of genes are more highly expressed in phagocytosed C. albicans cells compared to other less virulent species. This set is highly enriched for hyphal-associated genes and includes an uncharacterized homolog of the yeast Dig2 morphogenetic regulator. We are generating strains and tools to study this uncharacterized gene, C3_01800C, to test whether it is essential (as reported) and whether it impacts hyphal growth. In this study, we continue to probe these sets of novel genes for regulators of morphogenesis and virulence.

Poster 9

Defining the Role of SpxA1 and SpxA2 Function in Group A Streptococcus (GAS)

Gretchen Morrison^1,2, Luis Vega³, Anthony Flores³

¹University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX;²University of Texas Health Science Center, McGovern Medical School, Department of Microbiology and Molecular Genetics, Houston, TX; ³University of Texas Health Science Center, McGovern Medical School, Department of Pediatrics, Houston, TX

Spx is an oxidative stress response transcriptional regulator highly conserved across Bacillota. Studies of Spx proteins in Bacillus subtilis have shown degradation of Spx by the ClpXP protease regulates Spx activity and dysregulation of Spx was shown to affect growth. Spx proteins also possess a redox-sensitive CXXC motif that undergoes a thiol-disulfide switch required for transcriptional activation of specific genes in B. subtilis. In Group A Streptococcus (GAS), two Spx paralogs appear to independently contribute to oxidative stress tolerance (SpxA1) and virulence (SpxA2). I hypothesize that SpxA1 and SpxA2 have distinct regulatory roles in response to cell envelope and oxidative stresses. To begin testing this hypothesis, we will define how changes in SpxA1 and SpxA2 expression levels affect GAS growth under oxidative stress conditions (i.e., in atmospheric oxygen versus 5% carbon dioxide), as well as under nutrient starvation conditions. To that end, we have compared the growth of a WT strain to isogenic mutant strains lacking Spx homologs (∆spxA1, ∆spxA2) and strains overexpressing each paralog. We have also tested strains in which transcriptional activation by Spx paralogs is predicted to be disrupted by mutagenesis of the cysteine residues of the CXXC motif to alanines (SpxA1^C10A,C13A, SpxA2^C10A,C13A), and a strain lacking in the ClpXP protease (∆clpX) in which SpxA2 is also overexpressed. We have also tested tolerance to antimicrobial-induced cell envelope stress (bacitracin, polymyxin B) and oxidative stress (e.g., H₂O₂, paraquat, and diamide) of the aforementioned strains, as well as additional mutant strains in which the SpxA1 and SpxA2 promoters have been “swapped” (pA1::spxA2 and pA2::spxA1). Data shows that ∆spxA2 is affected by nutrient starvation (lower growth yield and longer lag phase), while ∆spxA1 is affected by atmospheric O₂ (lower growth yield, longer lag phase, and slower growth rate) and supplemented 5% CO₂ (longer lag phase). GAS is more susceptible bacitracin in the absence of SpxA2 (∆spxA2). Further, restoration of SpxA2 from the SpxA1 promoter rescues survival in the presence of bacitracin, however, overexpression of SpxA1 does not. Phenotypic differences between SpxA1 and SpxA2 mutant strains suggest that SpxA1 and SpxA2 control different sets of genes as transcriptional regulators. To investigate this, we performed RNA sequencing of WT and ∆spxA1 and ∆spxA2 grown in vitro to define the individual transcriptomes. Relative to the parental strain, differential expression of oxidative stress-related factors such as superoxide dismutase (sodA), NADH oxidase (nox.1), glutathione-S-transferase (gst) was associated with ∆spxA1 but not ∆spxA2. Conversely, differential expression of factors involved in countering immune cell signaling and activity (streptococcal pyrogenic exotoxin, speB; IgG-degrading protease, mac/ideS; arginine deiminase, arcA, streptokinase, ska) was associated with ∆spxA2 but not ∆spxA1. Understanding the role of Spx proteins in the GAS stress response will ultimately advance our knowledge of gene regulation contributing to disease caused by Gram-positive bacteria.

Poster 10

Prefrontal cortex neurons signal risky decision-making during a food-approaching vs. predator-threat avoidance test in rats

V. Chuong¹, G. Aquino-Miranda¹, X.O. Zhang¹, Victoria Albanese^1,2, F.H. Do Monte^1
1Department of Neurobiology & Anatomy, The University of Texas Health Science Center at Houston, Houston, TX, USA;²Department of Bioengineering, Rice University, Houston, TX, USA

 Neurons in the prelimbic (PL) subregion of the medial prefrontal cortex change their firing rates in response to fear- and reward-associated cues. PL activity is essential for the retrieval of both fear- and reward-related memories. However, it remains unknown how PL neurons respond during situations of conflict when reward cues and fear cues occur together in a fear-inducing context. To explore this question, male adult Long-Evans rats previously trained to press a lever for sucrose during the presentation of audiovisual cues were implanted with single-unit recording electrodes in the PL. Rats were then exposed to an approach-food vs. avoid-predator conflict model comprised of three phases: (i) reward phase, only food cues presented; (ii) cat odor phase, only a fear-inducing cat odor presented; and (iii) conflict phase, food cues concomitantly presented with cat odor. The next day, rats were returned to the same chamber and exposed to the food cues in the absence of the predator odor (contextual conflict test). During the approach-food vs. avoid-predator test, rats displayed increased defensive behaviors and reduced food-seeking responses during the conflict phase compared to the reward phase. During the contextual conflict test, cluster analysis of food-seeking responses revealed two distinct behavioral phenotypes: (i) risk-takers that displayed increased approach behaviors and (ii) risk-avoiders that showed increased avoidance behaviors. Moreover, risk-takers exhibited increased lever-press responses and reduced latency to lever press, whereas risk-avoiders displayed decreased lever-press responses and increased latency to lever press. Aligning PL activity from electrophysiological recordings to food cue onset during the approach-food vs. avoid-predator test revealed that a similar proportion of responsive neurons during the reward (~30%) and conflict (~35%) phases. However, only a fraction of cells (~10%) responded in both phases of the approach-food vs. avoid-predator conflict test, suggesting that distinct subpopulations of food cue responsive neurons are recruited during the conflict phase. During the contextual conflict test, aligning PL activity to food cues revealed differences between risk-takers and risk-avoiders. In risk-takers, the magnitude of food-cue responses was maintained in the same cells from early to late phases. In contrast, food-cue responses in risk-avoiders were attenuated from early to late phases. Both risk-takers and risk-avoiders showed the same number of excitatory and inhibitory food-cue responses during the early and late phases of the fear-inducing context. However, in risk-takers, a larger fraction of the cells responded to food cues during both early and late phases (~19%) when compared to risk-avoiders (~7%), suggesting that persistent food-cue responses in risk-takers may help to maintain lever presses constant across the session. Trial-by-trial analysis of PL food cue responses revealed an attenuation in the magnitude of the food-cue responses when rats did not press the lever (i.e., risk-avoiding trials) compared to when rats pressed it (i.e., risk-taking trials), which is consistent with our risk-takers vs. risk-avoiders food-cue comparison above. Together, these results demonstrate that modifications in food cue responses in PL neurons are associated with changes in behavioral choice during situations of conflict, suggesting a role for PL neurons in risky decision-making.

Grant Support: NIH-R01-MH120136, Rising STARs Award from the University of Texas System, and Brain & Behavior Research Foundation grant (NARSAD Young Investigator).

Poster 11

Endolysosomal trafficking controls yolk granule biogenesis in Drosophila oocytes: a robust tool for sensitive detection of Huntingtin’s conserved function in cellular homeostasis

Farmer SM^1,2, Yu Y^1,2, Chen D³, Xu S¹, Solbach A^1,2, Ye X¹,Ye L¹, Rios B^1,2, Covarrubias D⁴, Gao A⁵, Zhang S^1,2

¹The Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, Texas, USA;²The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, Texas, USA;³The College of Life Sciences, Anhui Normal University, Wuhu, Anhui, China;⁴Department of Biosciences, Rice University, Houston, TX, USA;⁵Department of Neuroscience, University of Texas at Austin, Austin, TX, USA

Endolysosomal trafficking is essential for nearly all aspects of physiological functions of eukaryotic cells, and converging evidence shows that defects in this crucial homeostasis process is a key mechanism for neurodegenerative diseases (NDs), including Huntington’s disease (HD). Importantly, Huntingtin (HTT), the enigmatic protein mutated in HD, is known to regulate endolysosomal trafficking, yet many of these findings are derived purely from in vitro studies with no validation in vivo. One valuable in vivo system to address this challenge is the vitellogenic oocyte in Drosophila, an organ that undergoes extensive endocytosis of Yolkless (Yl), a low-density lipoprotein receptor (LDLR) ortholog, to uptake extracellular lipoproteins into oocytes and package them into yolk granules, a specialized storage-lysosome. Yet, there is still a lack of sufficient understanding on the molecular and cellular processes that control yolk granule biogenesis, which is necessary to fully establish it as a reliable model for studying endolysosomal trafficking events in NDs such as HD. By creating a genome-tagging line for the Yl receptor and analyzing its distribution in oocytes together with Rab GTPases, the master regulators of endolysosomal trafficking, we found that Rab5 (early endosomes) and Rab11 (slow-recycling endosomes) but surprisingly not Rab7 (late endosomes), are essential for yolk granule biogenesis. To further assess whether yolk trafficking follows classical receptor-mediated endocytosis, we carried out a small-scale RNA interference (RNAi) screen on a set of reported Rab5-early endosome effectors, and indeed found that yolk granules transition from Rab5+ early endosomes to highly acidic Rab7+ late endosomes for proper yolk uptake. As a proof-of-principle, we further sought to determine whether this in vivo model faithfully re-capitulates the known function(s) of HTT in this important cellular homeostasis process, and found that endogenous HTT binds to Rab5+ early endosomes as previously reported in vitro. Most intriguingly, we found that HTT also interacts with Rab7+ late endosomes, a process that suggests a novel role for HTT in cargo degradation. Using stimulated emission depletion (STED) microscopy, we found that HTT-depletion causes Rab7+ late endosomes to become smaller, a phenotype that we found conserved in mammalian cells using structured illumination microscopy (SIM), establishing a conserved functional relationship between HTT and Rab7 in cargo trafficking. Lastly, using protein modeling, we found that HTT likely uses a structural basis to interact with Rab7+ endosomal membranes, establishing a promising structure-based therapeutic target for HD. This is the very first study showing HTT’s interaction on endosomes in vivo, including a novel interaction between HTT and Rab7, offering great potential for elucidating how these normal functions become altered in HD. Future work will explore the functional consequence of HTT on Rab7, for example whether HTT promotes or inhibits cargo degradation through Rab7+ endolysosomes and how this becomes altered in HD. Overall, our study provides novel insight into endolysosomal pathways and establishes yolk granules in Drosophila oocytes as an excellent in vivo model for dissecting conserved endolysosomal trafficking events relevant to human diseases including HD.

Poster 12

Determining the cellular and functional heterogeneity of schistosomes’ esophageal gland

Ryan Sloan^1,2,3, Pallavi Yadav², Sabona B. Simbassa^1,2, Jayhun Lee^1,2,4

¹ Microbiology and Infectious Diseases Program, MD Anderson UTHealth Houston Graduate School of Biomedical Sciences; ² Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030; ³ [email protected]; ⁴ [email protected]

Background: Schistosomes are parasitic flatworms that cause schistosomiasis, a major neglected tropical disease. They possess a digestive organ known as the esophageal gland (EG), which allows them to neutralize human immune components as they feed on blood. The development and maintenance of the EG is regulated by the tissue-specific transcription factor FoxA. RNAi-mediated depletion of foxA results in complete loss of the EG. Strikingly, this renders the parasites incapable of surviving in immunocompetent hosts, implicating the EG in a significant immune evasion mechanism. Preliminary whole-worm RNAseq of foxA knockdown parasites revealed ~40 significantly downregulated genes, which appear homogenously expressed in the EG when viewed using whole-mount in situ hybridization (WISH). However, in planarians and C. elegans, FoxA is responsible for development and maintenance of the pharynx, which contains tissues from all three germ layers, suggesting heterogeneity in cell types that FoxA regulates. Additionally, preliminary RNAseq data and existing single cell (sc)RNAseq data in schistosomes has revealed diversity among the genes expressed in the EG, including their functions and the tissues in which they are expressed. Together, these lead us to hypothesize that the EG is a heterogeneous tissue with regards to both the cell types that are present, as well as the functions of the gland-associated genes. Results: To define the genes and cell types of the EG and its associated tissues, we are taking a more focused approach using head enriched RNAseq and scRNAseq. This has revealed an additional ~60 genes which have expression patterns in and outside of the EG when viewed with WISH, including the anterior esophagus, gut, testes, and oral/ventral suckers, supporting the notion of heterogeneity among the EG and EG-associated genes. Additionally, pilot RNAi screens have shown that some of these genes may play a role in stem cell proliferation and/or differentiation, which may potentially be important for tissue maintenance and/or parasite survival. We plan to further define cell sub-types within the gland by performing double fluorescence in situ hybridization with known cell type markers, and to functionally characterize candidate genes using a series of RNAi-coupled in vitro and in vivo assays. Conclusions: Our results demonstrate potential cellular and functional heterogeneity of the EG and EG-associated tissues in schistosomes. Comprehensive characterization of the EG cell types and genes may identify factors that are vital for tissue homeostasis and function, with potential to be targeted for treatment of this disease. Acknowledgment: This work is supported by the Dean’s startup fund, the University of Texas Rising STAR award, and NIH/NIAID (R01AI175079) made available to J.L.

Poster 13

Elucidating the physiological function of evolutionarily conserved heat shock protein 110 in Drosophila

Rios B^1,2, Xu S¹, Yakubu UM^2,3, Ye X¹, Covarrubias D⁴, Farmer SM^1,2, Morano K³, and Zhang S^1,2

¹The Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston Texas, USA;
²Program in Neuroscience, The University of Texas MD Anderson Cancer Center UT Health Graduate School of Biomedical Sciences, Houston, Texas, USA;
³Department of Microbiology and Molecular Genetics, University of Texas McGovern Medical
School at Houston, Houston, Texas, USA; ⁴Department of Biosciences, Rice University, Houston, Texas, USA

Misfolded proteins are at an increased risk of aggregation and disrupted function, leaving cells vulnerable to degeneration. Chaperone proteins and physiologically essential substrates work in conjunction to establish and maintain the correct protein conformation, preserving protein homeostasis and overall cellular health. Previous research has firmly established the importance of heat shock proteins (HSPs) as chaperones and extensively delineated their biochemical functionality. In vitro experiments established HSP110 as a powerful “holdase”, when compared to other heat shock proteins, allowing it to sequester misfolded proteins but lacking the ability to refold them. Although HSP110’s biochemical role in the disaggregation machinery has been greatly explored through in vitro experiments and the unicellular eukaryote Saccharomyces cerevisiae, the characterization of its physiological functionality in higher eukaryotes is limited and requires further investigation, especially in postmitotic neurons that are non-renewable and require robust cellular homeostasis activity to maintain their longevity and function. Drosophila is an ideal model organism because the fly genome is amenable for easy genetic manipulation to elucidate mechanistic details of protein function. Also, there exists only a single Hsp110 gene (dHsp110) in Drosophila genome as compared to three Hsp110-like genes in both mouse and human genomes, therefore greatly simplifying its functional study. To facilitate the detection and manipulation of endogenous dHsp110 protein, we characterized and validated two genome trap lines. Using a hybrid pigP element, an artificial exon was introduced within the open reading frame of Hsp110, using a splicing acceptor and donor flanking and artificial open reading frame encoding a chimera flag-strep-venusYFP-strep (FSVS) tag, so it becomes part of the coding region and is transcribed with the native dHsp110. The tagged dHsp100 protein therefore can then be visualized through the inserted Venus-YFP tag. Importantly, in these two genome tagging lines, all dHsp110 proteins are expressed as dHsp110-FSVS fusion and the inserted chimera tag does not interfere with the normal function of dHsp110 protein. Examining larvae containing the FSVS genome trap line, we found dHsp110 was ubiquitously expressed in neuronal tissue and had a cytoplasmic localization. Leveraging this tool, future work will explore the functional consequence of gain and loss of Hsp110 function, specifically in neurons, and the chaperone’s function in the clearance mechanisms employed to remove protein aggregates in neuronal tissues in models of different neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases.

Poster 14

Exploring the mechanism of host immune-modulated esophageal gland-dependent schistosome development

Sabona B. Simbassa^1,2,3, Pallavi Yadav², Ryan Sloan^1,2, Jayhun Lee^1,2,4

¹ Microbiology and Infectious Diseases Program, MD Anderson UTHealth Houston Graduate School of Biomedical Sciences; ² Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030; ³ [email protected]; ⁴ [email protected]

Background: Schistosomes, parasitic flatworms and the etiological agent of schistosomiasis, have a fascinating ability to neutralize immune components ingested from continuous blood feeding. Intriguingly, previous work has shown that their intra-mammalian growth depends on the host immunity, in which their development is delayed in immunocompromised hosts. Meanwhile, their ability to block/degrade ingested leukocytes from blood feeding via the esophageal gland (EG), a digestive organ, appears essential for their survival. This creates a dichotomy where leukocytes are necessary for proper parasite development, but must be processed by the EG for survival. These lead us to hypothesize that the EG plays an important role in leukocyte-mediated parasite development. Results: To determine the nature of immune-modulated parasite development, we re-examined their growth in immunocompromised Rag1^-/- mice. Interestingly, the onset of developmental delay (~14 days post-infection) in Rag1^-/- mice coincided with a stage when parasites begin to feed on blood. To determine if feeding-mediated parasite growth is EG-dependent, we fed whole blood cells (that includes leukocytes) to schistosomula (larval stage) with (control RNAi) or without the EG (via foxA knockdown). Intriguingly, we found that while control schistosomula grew in their size after blood cell feeding, EG-lacking schistosomula failed to do so in a stem cell-dependent manner, indicating that processing of whole blood cells by the EG signals stem cells that promote proliferation and differentiation, leading to parasite development. We hypothesized that genes involved in leukocyte-EG-stem cell signaling would be upregulated in control RNAi schistosomula upon leukocyte co-culture but are unchanged or downregulated in foxA knockdown upon leukocyte co-culture. To this end, we performed comparative RNA-seq and identified 22 candidate genes that are upregulated in response to blood cell feeding that remain unchanged in foxA knockdown schistosomula. Further characterization of candidate genes revealed that many of the candidate genes are either involved in signaling or are transcriptional regulators, and are expressed in various non-EG cells such as neurons, muscles, and parenchyma. We plan to perform functional screen using RNAi coupled with EdU labeling and in situ hybridization, to identify key players and further dissect their roles in leukocyte-EG-dependent parasite development. Conclusions: Our results demonstrate the important role of the EG in leukocyte-feeding-mediated cell-to-cell communication that regulate stem cell-driven parasite development. We expect to unveil potential factors regulating developmental mechanisms deployed by schistosomes that may be exploited for targeting these parasites. Acknowledgment: This work is supported by the Dean’s startup fund, the University of Texas Rising STAR award, and NIH/NIAID (R01AI175079) made available to J.L.

Poster 15

Deciphering the role of IL-4 in colon repair after acute colitis

Nicolas Moreno¹, Yang Yang¹, Yankai Wen¹, Jie Zhao¹, Junda Gao¹, Yuanyuan Fan¹, and Cynthia Ju¹

¹McGovern Medical School, Department of Anesthesiology, University of Texas Health Science Center, Houston, TX, USA

Introduction: Therapies that promote epithelial repair and homeostasis would be major advancements in the treatment of inflammatory bowel disease (IBD), encompassing Crohn’s disease (CD) and ulcerative colitis (UC). IBD is characterized by relapsing and remitting inflammation and dysbiosis, often exacerbated by incomplete mucosal healing and leading to severe complications like colorectal cancer. Existing therapies have high failure and non-response rates, failing to control relapse effectively. Thus, there is a critical need to explore mechanisms that restore homeostasis after IBD flares. This project aims to characterize the interactions of Interleukin-4 (IL4) with immune cells and the microbiome and their contribution to colon homeostasis after colitis.

Methods: Genetically modified mice including IL-4 and IL4R⍺ deficient mice, neutrophil specific IL4R⍺ deficient mice, Il4/eGFP-enhanced transcript (4get) mice, and appropriate controls were treated with 3% dextran sulfate sodium (DSS) for 5 days to induce colon injury. Mice were observed during DSS treatment and until day 21, recording daily weight changes and qualitative assessment of colitis symptoms. Flow cytometry was used to assess cellular dynamics during and after colitis. Bone marrow macrophages were derived by culture of bone marrow mononuclear cells with L929 media for 7 days, and IL-4 stimulation with 20ng/mL of recombinant IL-4 was performed. Transcript-level changes were measured by qPCR.

Results and Discussion:

After treatment of 4get mice with DSS, flow cytometry revealed an influx of IL-4 producing eosinophils into the colon on days 8 and 14. These GFP⁺ eosinophils accounted for  >90% of IL-4-producing cells on day 8 and >75% on day 14, suggesting that eosinophils enter the colon during acute colitis, and produce IL-4 during acute injury and recovery phases.

After DSS treatment, IL-4 deficient mice experienced significantly more severe weight loss and delayed weight recovery from day 6-21 when compared with control mice treated in separate cages. On day 21, IL-4 deficient mice had significantly shorter colon length compared to controls. However, when co-housed IL-4 deficient and littermate control mice were treated with DSS, there were no phenotypic differences, suggesting a likely role for the microbiome in regulating colon inflammation after DSS.

After DSS treatment, neutrophil-specific IL4R⍺ deletion mice demonstrated premature mortality compared with littermate controls, suggesting that IL4 signaling through neutrophils may be important for controlling inflammation during acute colitis. Interestingly, IL-4 stimulation of bone-marrow-derived macrophages in vitro revealed 20-fold increases in transcription of HB-EGF, an EGFR ligand that has demonstrated protective effects during TNF⍺ and DSS-induced acute colitis in mice. IL-4 stimulation of bone marrow macrophages generated from IL4R⍺ deletion mice did not upregulate HB-EGF.

Future direction: To analyze the impact of IL-4 deficiency on the microbiome, 16S microbiome sequencing of naïve and DSS-treated IL4 deficient and control mice will be performed. Then, we will perform microbiota normalization by antibiotic treatment and reciprocal fecal material transfer between the IL-4 deficient and C57BL/6J mice, followed by DSS treatment to understand if the protective role of IL-4 during colitis can be shared by the microbiome. To analyze the impact of neutrophil-specific IL4R⍺ deletion on neutrophil activation and migration during colitis, we will perform DSS treatment followed by immunofluorescence staining to identify neutrophil presence and location using whole-body and neutrophil-specific IL4R⍺ deletion mice. To account for the direct action of IL-4 and macrophage-derived HB-EGF on the colon epithelium, we will culture colon organoids and treat them with DSS in-vitro followed by the introduction of IL-4 and/or IL-4 stimulated macrophages and assess cellular proliferation and migration kinetics.

Poster 16

Exploring the Impact of Substance Abuse on Wound Healing and Vascular Gene Expression

Phillip J. Urbanczyk^1,2, Laura Stertz^1,2, Benjamin Snyder¹, Maria Torres Carrizalez², Darrion Mouton^1,2, Bruno Kluwe-Schiavon^1,2, Consuelo Walss-Bass^1,2

¹Louis A. Faillace, MD Department of Psychiatry and Behavioral Sciences, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA; ²University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX, USA

Studies have shown that exposure to common substances of abuse such as alcohol and opioids can impact wound healing and alter the rate at which healing occurs. Previous work by our group has demonstrated the differential expression of several angiogenesis- and cardiovascular-related genes in postmortem brain tissue and in fibroblasts of patients with either Opioid Use Disorder (OUD) or Alcohol Use Disorder (AUD) within a model of both acute and chronic exposure, including of Vascular Endothelial Growth Factor (VEGF), Asporin (ASPN), Early Growth Response 1 (EGR1), Neuregulin 1 (NRG1) and Decorin (DCN). We sought to understand whether chronic alcohol and morphine exposure altered the rate at which patient-derived fibroblasts healed after wounding and to confirm the expression changes of angiogenesis genes. Postmortem-derived fibroblasts from OUD patients (n=6) and controls (n=8) were exposed to 10µM of morphine for 7 days. Separately, fibroblasts from AUD patients (n=4) and controls (n=4) were exposed to 50mM ethanol (EtOH, a 0.2 g/dL blood alcohol level equivalence) for 7 days. After treatment, wound healing assay was performed, and cells were monitored for 36hs. Images at 0h, 12hs, 24hs and 36hs were analyzed to measure the gap size and its rate of closure over time and gene expression was measured by qPCR.

Analysis was performed using a linear mixed model. Group and time were included as fixed effects and the sample replicates as random effects, while the outcome was the difference in the percentage of gap closing between the different time points (e.g., time points 2 [12h] minus time point 1 [0h]). Exploratory pairwise comparisons revealed that between 0h and 12h, and 0h and 24h control treated with morphine healed faster than controls with no treatment (12h-0h: t[40]=6.81, p<.0001; 24h-0h:t[40]=3.23,p=.014) and OUD with (12h-0h: t[79]=-3.87, p=.001; 24h-0h: t[76.8]=-2.97,p=.023) and without morphine treatment (12h-0h: t[79]=7.48, p<.0001; 24h-0h: t[76.8]=4.98,p<.0001). Additionally, between 0h and 36h control treated with morphine healed faster than controls and OUD without treatment (t[40]=3.33, p<.011; t[59.8]=4.11, p<.001), but not than OUD with treatment (t[59.8]=-2.44, p=.103). No main effects for group and time were found for ethanol treatment. Altogether, these findings suggest that when treated with morphine, postmortem-derived fibroblasts from OUD patients healed similarly as postmortem-derived fibroblasts from controls with no treatment.

The effect, if any, of opioid and alcohol exposure on gene expression is being determined. OUD, AUD, Depression, and cardiovascular disease have been shown to be significantly comorbid with each other to varying degrees. Understanding the role alcohol and opioid exposure plays in vascular changes and tissue remodeling may help clarify the underlaying etiology amongst these comorbidities and provide avenues for therapeutic intervention.

Poster 17

Ubiquitination-Targeted Therapies to Improve Dystrophin Stability in Becker Muscular Dystrophy (BMD)

Muchen Liu^1,3, Jianbo Wu¹, Chunru Lin² and Radbod Darabi³

¹UTHealth McGovern Medical School Institute of Molecular Medicine, Center for Stem Cell and Regenerative Medicine, Houston, TX, USA;²University of Texas MD Anderson Cancer Center, Houston, TX, USA;³ College of Pharmacy, University of Houston, Houston, TX, USA

Background: Muscular dystrophies are heterogeneous groups of inherited diseases leading to progressive muscular weakness and degeneration. In the case of Becker muscular dystrophy (BMD), non-disrupting mutations of the DMD gene reading frame is the causative defect. By the age of 30s, about 60 to 70% of BMD patients develop cardiomyopathy, which is often lethal. Unfortunately, there is currently no cure for BMD. Meanwhile, improvement of dystrophin stability by repressing polyubiquitination is a promising strategy. This study aims to test the efficacy of selected drugs interfering with the dystrophin polyubiquitination using BMD iPSCs as the model system. The hypothesis is that dystrophin expression of the BMD iPSC-derived myogenic progenitors can be improved after polyubiquitination inhibition. Methods: The in vitro studies include evaluation of myogenic cell proliferation and differentiation using BMD and control iPSCs to identify affected phenotypes. These will be done by measuring proliferation rate, myogenic marker quantification, cell response to stress, gene expression profiling, and cell cycle analysis.  The in vivo studies will be done by transplanting BMD iPSC-derived myogenic progenitors into immune-deficient NSG mice with daily treatment of selected polyubiquitination inhibitors. Muscle cryosections will be studied for quantification of donor cell engraftment and dystrophin expression. Results & Progress: The in vitro data indicates reduced myogenic potential of BMD-iPSCs, as well as defective cell cycle, proliferation, and differentiation, when compared to healthy iPSCs. In addition, Muscle sections from the BMD-iPSC engrafted mice treated with the selected ubiquitination inhibitors demonstrated significantly higher percentage of human cell engraftment, as well as enhanced dystrophin expression, indicating the efficacy of the treatment. Conclusions: Inhibition of ubiquitination significantly improves the BMD iPSC-derived myogenic engraftment with improved dystrophin expression. The study provides a new therapeutic strategy that utilizes selective agents to enhance dystrophin expression and longevity in BMD patients.

Poster 18

Dietary Fatty Acids Fine-Tune PIEZO2 Activity

Luis O. Romero^a,b,c, Rebecca Caires^a, Jungsoo Lee^a, Manisha Bade^a,c,d, Julio F. Cordero-Morales^a,c, and Valeria Vasquez^a,c

^a Department of Physiology, College of Medicine, University of Tennessee Health Science Center, Memphis, TN, 38103, USA;^b Integrated Biomedical Sciences Graduate Program, College of Graduate Health Sciences, Memphis, TN, 38163, USA;^c Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, TX 77030, USA;^d MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030, USA.

PIEZO2 is a mechanosensitive ion channel essential for touch discrimination, vibration, intero- and proprioception, and mechanical allodynia. Mechanosensitive ion channels rely on membrane composition to transduce physical stimuli into electrical signals. Dietary fatty acids are among the membrane lipid components that dynamically regulate ion channel function. We have previously demonstrated that PIEZO1, PIEZO2 close homolog, function is fine-tuned by dietary fatty acids¹. Here, we use electrophysiology and lipidomics to demonstrate that PIEZO2 is also modulated by dietary fatty acids. We found that the saturated fatty acid margaric acid inhibits PIEZO2 currents by increasing its mechanical threshold for activation², whereas polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic, and linoleic acids modify its inactivation^3,4. Moreover, we found that dietary interventions improve PIEZO2-associated behaviors in mouse models of neurogenetic diseases where PIEZO2 function is either down- (Angelman syndrome)⁴ or up-regulated (distal arthrogryposis type 5)³. Finally, we show that topical lotions can be used to alter touch responses of wild-type mice in vivo. Overall, our findings demonstrate that saturated and polyunsaturated fatty acids enriched in the plasma membrane modulate mechanical responses mediated by PIEZO2 in vitro, ex vivo, and in vivo.

References

¹ Romero, Massey, Mata-Daboin, et al. 2019. Nat. Commun. 10, 1200.
² Romero, Caires, Nickolls, et al. 2020. Nat. Commun. 11, 2997.
³ Ma, Dubin, Romero, et al. 2023 .Science 379,201-206.
⁴ Romero, Caires, Victor, et al. 2023. Nat. Commun. 14, 1167.

Poster 19

3’UTR Landscapes and Their Effects on Fibrotic Lung Diseases

René A. Girard^1,2, Scott Collum¹, Sarah Shin^1,2, Andrew Peters¹, Jamie Tran^1,2, Harry Karmouty-Quintana¹

¹McGovern Medical School, University of Texas Health Science Center at Houston, Department of Biochemistry and Molecular Biology, 6431 Fannin St., Houston, Texas 77030, USA; ²UTHealth MD Anderson Cancer Center Graduate School of Biomedical Sciences, Houston, 6431 Fannin St., Texas 77030, USA

Idiopathic pulmonary fibrosis (IPF) is a chronic, interstitial lung disease which has no curative treatments and is terminal without a transplant. IPF is often misdiagnosed, and this delay in diagnosis further worsens the outcome. Patients need a lung transplant 3-5 years after diagnosis and have no other recourse. Another fibrotic disease that develops end-stage fibrosis is non-resolvable COVID-19 (NR-COVID-19). This condition, like IPF, presents a fibrotic injury to the lungs and necessitates a lung transplant. However, NR-COVID-19 is unique in that patients are much younger (averaging 40 years in age), affects majority Hispanic individuals, and the injury is so severe that a lung transplant is needed within 120 days. Recent studies have shown that there are unique 3’ untranslated region (UTR) landscapes between IPF and NR-COVID-19. These landscapes are due to a process known as alternative polyadenylation (APA) which modifies the 3’UTR to form unique isoforms of mRNA to further regulate mRNA. Nudix Hydrolase 21 (Nudt21/CPSF5) is a master regulator of APA and is down in IPF and increased in NR-COVID-19, possibly explaining the differences in the 3’UTR landscape. How these 3’ UTR landscapes affected by aberrant alternative polyadenylation promote lung fibrosis is not known. Therefore, I 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. I will elucidate which APA mediators are altered and promote changes to the 3’UTR landscapes in fibrotic lung diseases. Using preliminary PAC-seq data, I have determined shortened and lengthened 3’UTR transcripts of key fibrotic, extra cellular matrix, and APA transcripts. Transcripts with shortened 3’UTRs show increased protein expression, while transcripts with lengthened 3’UTRs show decreased protein expression. Differences in APA mediators like CPSF5 and CPSF6 transcript expression may explain the differences in alternative polyadenylation between two similar fibrotic presenting diseases and possibly explain the rapid progression of NR-COVID-19 fibrosis compared to IPF.

Poster 20

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. Therefore, our research seeks to examine vascular remodeling in the context of alcohol use disorder (AUD). Previously, a differential gene expression and gene ontology analysis was performed on bulk RNAseq data from autopsied AUD and control human brains. The top three pathways that were found to be of significance were: cell-cell signaling, inflammation, and angiogenesis. To further test the involvement of angiogenesis in AUD, we exposed human-derived cVSMCs to moderate and heavy doses of alcohol both acutely and chronically. Our results were consistent with previous data where we found increased proliferation of cVSMCs when exposed to heavy, chronic alcohol use. This increase in proliferation could be an indicator of the beginnings of vascular remodeling. Second, we determined if alternative polyadenylation (APA), a mechanism involved in the mRNA 3’ isoform selection process, is altered in both acute and chronic exposure to alcohol by utilizing loss and gain of function approaches on these cVSMCs. So far, our results have shown that both moderate and heavy doses to cVSMCs have resulted in changes to the APA machinery such as NUDT21 and CPSF1 – complexes that are heavily involved in the regulation of APA via pre-mRNA 3′-end processing and formation. These complexes had opposing transcript levels when exposed to moderate vs. heavy doses of alcohol. Ultimately, the results of our research have established APA as a novel lens in which to view cerebrovascular changes in AUD.

Poster 21

Elucidating the role(s) of ubiquitin-mediated proteolysis factors on SKN-1 activity during pathogen infection of C. elegans

Larissa Tavizón^1,2, Carolaing Gabaldon², Melissa R. Cruz², Danielle A. Garsin^1,2

_{¹University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA;
²University of Texas Health Science Center, McGovern Medical School, Department of Microbiology and Molecular Genetics, Houston, TX, USA}

Bacterial pathogens appear to have an infinite capacity for developing resistance to antimicrobials and the result of their overuse is an alarming rise in untreatable infections. One strategy being explored to mitigate this serious public health problem is the harnessing of the immune response to fight infection rather than targeting the pathogen. In mammals, one protective immune component is Nrf, which belongs to the nuclear factor erythroid – related factor/CNC (Cap’n’collar) family of transcriptional regulators that counteracts oxidative damage. Our model organism, Caenorhabditis elegans, contains a functional ortholog of Nrf proteins called SKN-1. In response to pathogens, our laboratory previously showed that C. elegans activates SKN-1 by triggering the ROS-producing NADPH oxidase, BLI-3. Importantly, SKN-1 activity is protective against microbial infection. Several regulatory mechanisms that control SKN-1 activity were previously identified, however, most of these regulatory components were found by exposing animals to oxidants or ROS generating chemicals. What remains unknown, is knowledge of how SKN-1 activity is regulated in the presence of pathogen and whether the same or different factors are involved. WDR-23, a WD repeat protein, interacts with a ubiquitin ligase complex to target proteins for proteasomal degradation. LIN-23, a F-box protein, is a component of the SCF complex involved in ubiquitin-mediated proteolysis. Both WDR-23 and LIN-23 have been shown to regulate SKN-1 activity negatively and positively during infection, respectively, however their exact mechanisms of regulation have not been identified. Since this transcription factor plays a significant role in activating a protective response it is important to better understand how Nrf/SKN-1 regulates the immune response during infection. Doing so will address the critical need for new strategies for treating drug-resistant infections.

Poster 22

Deciphering the Role of FtsA Structural States in Escherichia coli Divisome Activation and Assembly

Abbigale Perkins^1,2, Mwidy Mounange-Badimi², William Margolin^1,2

¹Microbiology and Infectious Diseases Program, MD Anderson UTHealth Houston Graduate School of Biomedical Sciences; ²Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030

Bacterial cytokinesis, essential for cell proliferation, involves a complex interplay of numerous proteins within a dynamic nanomachine (divisome), which is organized as a ring-like structure at the mid-cell by the tubulin-like FtsZ protein. Actin-like FtsA plays a crucial role during early FtsZ-ring formation by anchoring FtsZ to the inner cell membrane and facilitating the recruitment of other essential divisome proteins. Previous studies have shown in vitro, FtsA assembles into three distinct oligomeric states: a closed mini-ring that may inhibit premature divisome assembly, an open arc conformation that is hypothesized to initiate divisome activity, and a double-stranded (DS) antiparallel filament conformation proposed to be necessary for full activation of the divisome and synthesis of septal peptidoglycan. Moreover, genetic variants of FtsA, FtsA_R286W and FtsA_G50E, can bypass one or more of the early stages and mimic the arc and double-stranded oligomeric states, respectively. Although these distinct forms of FtsA have been identified, it remains unproven which distinct conformation is responsible for assembling and activating the divisome.  To address this, we used an in vivo crosslinking assay for FtsA DS filaments along with a dominant negative mutant FtsA_{M96E R153D} to investigate the roles of FtsA oligomeric states in assembly and activation. We show that FtsA_{M96E R153D} cannot form DS filaments in vivo, indicating it is stuck in a mini-ring or arc confirmation. Additionally, we found that FtsA_{M96E R153D} cannot complement when FtsA is depleted, but activation of the divisome through the ftsL* hyperfission allele rescues the ability of FtsA_{M96E R153D} to function. As ftsL*’s hyperfission activity requires the divisome to be assembled, our results suggest that FtsA DS filaments are needed to activate the divisome. Consistent with this model, DS filaments require divisome assembly and are most prevalent at late stages of cell division. Through these specific FtsA variants, we aim to conclusively determine the role of FtsA’s structural states in the divisome’s assembly and activation.

Poster 23

Intracranial Neural Dynamics of Selective Attention in Rapid Visual Recognition

Meredith McCarty^1,2,3, Oscar Woolnough^1,3, Elliot Murphy^1,3, Nitin Tandon^1,3,4

¹Vivian L. Smith Department of Neurosurgery, McGovern Medical School, Houston, TX, USA; ²University of Texas MD Anderson Cancer Center UT Health Houston Graduate School of Biomedical Sciences, Houston, TX, USA; ³Texas Institute for Restorative Neurotechnologies, University of Texas Health Science Center at Houston, Houston, TX; ⁴Memorial Hermann Hospital, Texas Medical Center, Houston, TX

The selection of relevant information from a busy visual environment is a difficult task the brain performs rapidly. In this process of selective attention, the brain flexibly directs neural resources towards behaviorally relevant stimuli. The neural substrate of attention is proposed to be oscillatory coupling between task relevant brain regions, but direct evidence of this process in humans remains to be quantified. In this study, we utilize intracranial electroencephalographic recordings in 25 patients (14 female) performing a task where visual attention is directly modulated. In this task, visual stimuli of different categories (Faces, Words, Scenes, or Animals) were presented. Across different trial blocks, patients tracked and responded to one specific feature: a color change of a central fixation point, repetition of a stimulus (i.e. a one-back task), or the category the stimulus belonged to (e.g., the category “fruit or vegetable words” for which an exemplar is “apple”). This task design enables the isolation of attentional dynamics when the same stimuli are presented, but task demands shift. Through implementing a d-prime selectivity analysis on broadband high gamma activity (BGA; 70-150Hz), we identified electrodes that exhibit significant category-selectivity within the ventral occipitotemporal cortex (vOTC). We found BGA dynamics within vOTC exhibited early and sustained modulation by stimulus category. In contrast, electrodes in the frontal cortex exhibited a sustained increase in BGA as task demand increased. BGA dynamics in the inferior frontal cortex showed significant early modulation by attentional condition, followed by a later modulation in intraparietal regions. Through quantifying the rapid spatial and temporal dynamics between frontal and ventral regions involved in this task, we identify a potential mechanism by which the frontal cortex modulates vOTC activity based on selective attention.

Poster 24

Deciphering the role of Dnmbp in kidney development: implications for CAKUT

Brandy L. Walker^1,2, Vanja Krneta-Stankic^2,3, and Rachel K. Miller^1,2,4,5

¹Program in Genetics and Epigenetics, MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, TX; ²Department of Pediatrics, Pediatric Research Center, UTHealth McGovern Medical School, TX; ³Department of Pulmonary Medicine, Division of Internal Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030 ⁴Department of Medicine, Ralph H. Johnson Veterans Affairs Medical Center, SC; ⁵Department of Genetics, University of Texas MD Anderson Cancer Center, TX

Congenital anomalies of the kidney and urinary tract (CAKUT) encompass a spectrum of genitourinary system malformations, including polycystic kidney disease, renal hypoplasia, and agenesis. CAKUT accounts for nearly one-fourth of all congenital anomalies and over 40% of pediatric end-stage renal disease, yet only 10-20% of cases have a recognized monogenetic cause. With an average of a three to five year wait time to receive a deceased donor kidney, the need to find alternative treatments to preserve renal function is essential.  Adult human kidneys contain approximately one million microscopic structures called nephrons, which are composed of a complex network of capillaries and epithelial tubules responsible for filtering the blood to remove waste in the form of urine. Disruption of nephron development is one of the many congenital anomalies associated with CAKUT, often resulting in severe renal disease requiring transplantation. Proper nephric tube formation depends heavily upon the precise structural organization of epithelial cells, which is determined by the morphological changes that result from processes such as cell polarization, adhesion, signaling and vesicle transport, among others. To identify genetic alterations influencing CAKUT and enhance treatments for congenital renal disabilities, a better understanding of the cellular mechanisms underlying kidney development is needed. Dynamin binding protein (DNMBP) is a multi-domain scaffolding protein involved in various cellular processes and regulatory pathways, including vesicle trafficking, cell polarity establishment, tissue morphogenesis, and cell-cell junction assembly. Our published work has leveraged the Xenopus embryonic kidney to demonstrate that Dnmbp is essential for renal tube development, however the role of DNMBP signaling activity in the kidney remains unclear. Here we evaluate how Dnmbp signaling influences the cellular processes required for nephrogenesis to gain a better understanding of developmental abnormalities in tubule morphology and cyst formation. Given that disruption of either the vesicle transport targeting protein Dnmbp or the Wnt/PCP formin Daam1 results in nephron malformation, we evaluate the dynamics of Dnmbp-mediated transport of Daam1 within the developing kidney. Here we propose a model in which Dnmbp functions as a critical regulator of epithelial tissue morphogenesis and provides a functional link between the dynamic processes of actin cytoskeleton regulation, intracellular adhesion, and vesicular transport. Completion of this research will determine whether Dnmbp interaction with Daam1 facilitates junctional actin assembly by directing Daam1 to forming cell contacts via Dnmbp-associated vesicle targeting and will enhance our understanding of the cellular mechanisms influencing tubule morphogenesis.

Poster 25

TRPV6 Mediates Alcohol-Induced Gut Barrier Dysfunction

Briar Bell^{1, 2}, Avtar Meena², Pradeep Shukla², Rebeca Caires², Carlos Fernández-Peña², Valeria Vásquez¹, Jonathan H. Jaggar², Julio F. Cordero-Morales¹, Radhakrishna (R.K.) Rao²

Department of Biochemistry and Molecular Biology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA¹; Department of Physiology, College of Medicine, University of Tennessee Health Science Center, Memphis, TN, USA²

Excessive alcohol intake can induce gut barrier dysfunction, leading to endotoxemia and systemic inflammation. The molecular mechanism resulting in the alcohol-induced Ca²⁺ increase responsible for gut barrier dysfunction has remained elusive. TRPV6, a member of the transient receptor potential vanilloid subfamily, is unique in its high selectivity for Ca²⁺ and involvement in maintaining Ca²⁺ homeostasis. Interestingly, TRPV6 is highly expressed in the apical membrane of the intestinal epithelium. Here, we identified TRPV6 as a target whereby alcohol disrupts gut barrier function. Using patch-clamp electrophysiology, we found that ethanol and its metabolite, acetaldehyde, elicit currents in TRPV6-transfected HEK293 cells. Caco-2 cells, which serve as a model of the intestinal epithelial barrier, display endogenous currents in response to ethanol and acetaldehyde, which can be inhibited by SOR-C13, a TRPV6 antagonist. Similarly, ex vivo colonoids from mice (which recapitulate the intestine) exhibit Ca²⁺-influx in response to ethanol and acetaldehyde exposure, causing barrier dysfunction, which can be inhibited by SOR-C13. Moreover, colonoids isolated from Trpv6 knockout mice are protected from alcohol-induced effects. Finally, azibutanol photoaffinity labeling, which covalently crosslinks a reactive alcohol species to a target molecule, as well as mutagenesis experiments, demonstrated that a conserved histidine within the ankyrin repeat domain of the TRPV6 channel is a part of the putative ethanol binding site. These results revealed that TRPV6 plays a role in mediating gut barrier dysfunction and is a potential target for ameliorating gut barrier defects associated with chronic alcoholism.

Poster 26

Targeting IGF2-IGF1R Signaling to Reprogram the Tumor Microenvironment for Enhanced Viro-Immunotherapy

Alexandra A. Miller^1,2*, Min Hye Noh^1*, Jin Muk Kang^1*, Grace Nguyen¹, Minxin Huang¹, Hiroshi Nakashima², E. Antonio Chiocca^2,3, Zhongming Zhao^3,4, 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 Neurosurgery, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, 02115, USA. ⁴Center for Precision Health, McWilliams School of Biomedical Informatics, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA.

The FDA approval of oncolytic herpes simplex-1 virus (oHSV) therapy for patients with melanoma underscores its therapeutic promise as a cancer immunotherapy. However, despite this promise only a small subset of patients respond favorably in the clinic. Oncolytic virotherapies work both through direct oncolysis of infected cancer cells and by inducing of inflammatory response and concurrent activation of anti-tumor immunity through the release of tumor antigens from the lysed cancer cells, a phenomenon referred to as viro-immunotherapy. However, the induction of an immunosuppressive tumor microenvironment (TME) by the tumor both before and shortly after therapy poses the greatest hurdle to lasting efficacy of viro-immunotherapy. Our work centers on understanding mechanisms of resistance to oHSV therapy and characterizing the impact of viro-immunotherapy on the TME to enhance viro-immunotherapy broadening their applications in the clinic.

RNA-Seq analysis of oHSV-infected glioblastoma (GBM) and breast cancer (BC) cells identified Insulin-like growth factor 2 (IGF2) as one of the top 10 secreted proteins following infection. Moreover, IGF2 expression was significantly upregulated in 10 out of 14 recurrent GBM patients treated with oHSV, rQNestin34.5v.2 (71.4%) (p=0.0020) (ClinicalTrials.gov, NCT03152318), highlighting its clinical relevance.

IGF2 is upregulated in various malignancies and its overexpression is associated with resistance to chemotherapy and radiation therapy, poor prognosis, anti-tumor immune suppression within the TME, and metastasis. In order to mitigate oHSV therapy-induced IGF2 and improve the therapeutic efficacy of oHSV, we designed a novel oHSV construct, oHSV-D11mt, which integrates a secretable modified IGF2R domain 11 into the parental oHSV genome that serves as an IGF2 decoy receptor. The secreted IGF2RD11mt selectively binds to IGF2, effectively blocked oHSV-induced IGF2-IGF1R signaling, which lead to enhanced tumor cell cytotoxicity, reduced oHSV-induced neutrophils/PMN-MDSCs infiltration, reduced secretion of immunosuppressive/proangiogenic cytokines, and increased Cytotoxic T lymphocytes (CTLs) infiltration. These effects resulted in enhanced survival of both GBM and BC brain metastasis (BCBM) tumor-bearing mice, abrogating the resistance conferred by IGF2 secretion. Collectively, our findings suggest that secretion of IGF2 plays a critical role in resistance to oHSV therapy and our novel viral construct represents a promising therapeutic for enhanced viro-immunotherapy.