Background: Extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E) are a growing public health concern. This study investigates antibiotic resistance patterns for ESBL-E (Escherichia coli and Klebsiella species) cases in Maury and surrounding counties in Tennessee as part of the Emerging Infection Program’s Multi-site Gram-negative Surveillance Initiative. Methods: ESBL-E cases are defined as extended-spectrum cephalosporin-resistant E. coli, Klebsiella pneumoniae, Klebsiella variicola, or Klebsiella oxytoca isolated from urine or normally sterile sites from residents of the surveillance area. Cases from July 2019 through December 2024 were included. Susceptibility data were collected from clinical laboratory reports. E. coli isolate resistance was compared to aggregated Klebsiella species isolate resistance using χ or fisher’s exact tests. Organism specific yearly resistance trends excluded the incomplete 2019 collection year and were examined via simple linear regression. Analyses were performed in SAS 9.4. Results: We identified 2,206 isolates including 1,915 E. coli isolates, of which 1,868 (97.5%) were from urine, and 291 Klebsiella isolates, of which 281 (96.6%) were from urine. Overall percent resistance for selected antibiotics were as follows: ciprofloxacin: 69.0%, levofloxacin: 64.3%, cefepime: 81.1%, cefazolin: 98.7%, cefotaxime: 85.9%, ceftazidime: 53.2%, ceftriaxone: 92.8%, piperacillin-tazobactam: 2.6%, nitrofurantoin: 9.0%, trimethoprim-sulfamethoxazole: 57.8%. During the study period, E. coli isolates displayed increased resistance to cefepime (3.0% per year, P<0.001), ceftriaxone (1.3% per year, P:P: 0.02); Klebsiella isolates demonstrated increased resistance to levofloxacin (5.9% per year, P: 0.002) and cefepime (5.7% per year, P: 0.003). Over the study period, E. coli isolates displayed higher rates of resistance than Klebsiella isolates to ciprofloxacin (74.0% vs. 36.4%, P<0.001), levofloxacin (71.4% vs. 19.0%, P<0.001), cefotaxime (87.0% vs. 79.0%, P<0.001) and ceftriaxone (93.5% vs. 87.9%, P<0.001). Klebsiella isolates had higher resistance to ceftazidime (59.9% vs. 52.1%, P: 0.01), piperacillin-tazobactam (8.9% vs. 1.6%, P<0.001), nitrofurantoin (18.6% vs. 7.6%, P<0.001), and trimethoprim-sulfamethoxazole (68.6% vs. 56.1%, P<0.001). There were no significant differences in resistance to cefepime or cefazolin between groups. Conclusion: In our data, ESBL-E pathogens demonstrated trends of increasing resistance to certain antibiotics over time. There were significant differences in resistance between species within different classes of antibiotics. These data support the idea that organism type is important for determining the best antibiotic for empiric treatment. Continued monitoring of antibiotic resistance in ESBL-E is imperative to understanding changes in antibiotic resistance patterns. The increasing trends in proportion of antimicrobial resistant strains warrants strengthening antimicrobial stewardship and infection prevention programs within the surveillance area.

Background: Extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E) are a growing public health concern. This study investigates antibiotic resistance patterns for ESBL-E (Escherichia coli and Klebsiella species) cases in Maury and surrounding counties in Tennessee as part of the Emerging Infection Program’s Multi-site Gram-negative Surveillance Initiative. Methods: ESBL-E cases are defined as extended-spectrum cephalosporin-resistant E. coli, Klebsiella pneumoniae, Klebsiella variicola, or Klebsiella oxytoca isolated from urine or normally sterile sites from residents of the surveillance area. Cases from July 2019 through December 2024 were included. Susceptibility data were collected from clinical laboratory reports. E. coli isolate resistance was compared to aggregated Klebsiella species isolate resistance using χ or fisher’s exact tests. Organism specific yearly resistance trends excluded the incomplete 2019 collection year and were examined via simple linear regression. Analyses were performed in SAS 9.4. Results: We identified 2,206 isolates including 1,915 E. coli isolates, of which 1,868 (97.5%) were from urine, and 291 Klebsiella isolates, of which 281 (96.6%) were from urine. Overall percent resistance for selected antibiotics were as follows: ciprofloxacin: 69.0%, levofloxacin: 64.3%, cefepime: 81.1%, cefazolin: 98.7%, cefotaxime: 85.9%, ceftazidime: 53.2%, ceftriaxone: 92.8%, piperacillin-tazobactam: 2.6%, nitrofurantoin: 9.0%, trimethoprim-sulfamethoxazole: 57.8%. During the study period, E. coli isolates displayed increased resistance to cefepime (3.0% per year, P<0.001), ceftriaxone (1.3% per year, P:P: 0.02); Klebsiella isolates demonstrated increased resistance to levofloxacin (5.9% per year, P: 0.002) and cefepime (5.7% per year, P: 0.003). Over the study period, E. coli isolates displayed higher rates of resistance than Klebsiella isolates to ciprofloxacin (74.0% vs. 36.4%, P<0.001), levofloxacin (71.4% vs. 19.0%, P<0.001), cefotaxime (87.0% vs. 79.0%, P<0.001) and ceftriaxone (93.5% vs. 87.9%, P<0.001). Klebsiella isolates had higher resistance to ceftazidime (59.9% vs. 52.1%, P: 0.01), piperacillin-tazobactam (8.9% vs. 1.6%, P<0.001), nitrofurantoin (18.6% vs. 7.6%, P<0.001), and trimethoprim-sulfamethoxazole (68.6% vs. 56.1%, P<0.001). There were no significant differences in resistance to cefepime or cefazolin between groups. Conclusion: In our data, ESBL-E pathogens demonstrated trends of increasing resistance to certain antibiotics over time. There were significant differences in resistance between species within different classes of antibiotics. These data support the idea that organism type is important for determining the best antibiotic for empiric treatment. Continued monitoring of antibiotic resistance in ESBL-E is imperative to understanding changes in antibiotic resistance patterns. The increasing trends in proportion of antimicrobial resistant strains warrants strengthening antimicrobial stewardship and infection prevention programs within the surveillance area.

Background: The National Healthcare Safety Network (NHSN) Standardized Antimicrobial Administration Ratio (SAAR) is a widely used metric for benchmarking antimicrobial use and supporting antimicrobial stewardship efforts. The Centers for Disease Control and Prevention (CDC) Priority Core Elements of Hospital Antibiotic Stewardship Programs offer a complementary, framework-based approach to evaluating stewardship infrastructure and practices. Understanding the relationship between implementation of Priority Core Elements and SAAR performance may help clarify how structural stewardship components translate into measurable antimicrobial use outcomes. Tennessee Department of Health explored the association between NHSN SAARs and Priority Core Elements to better inform stewardship program evaluation and improvement strategies. Methods: Using 2023 NHSN data, TDH evaluated the relationship between all-antibacterial SAARs by overall Priority Core Element and by interventions that comprise each element. Aggregate SAAR data from qualifying adult units was utilized to create a hospital wide SAAR. Specific Priority Core Element components—including leadership, accountability, pharmacy expertise, action and reporting were analyzed. The tracking priority was excluded as participating hospitals had to submit antimicrobial use data to generate a SAAR. Associations between SAAR performance and presence of interventions for each included Priority Core Element were analyzed using t-tests in SAS 9.4. Results: Mean all-antibacterial SAAR declined with greater attainment of priority Core Elements (1.13, 1.07, and 1.01 for 0–2, 3–4, and 5 elements, respectively), though the difference was not statistically significant (p=0.31). Hospital leadership commitment was not associated with SAAR differences. Hospitals meeting the Accountability Core Element demonstrated significantly lower SAARs than those that did not (1.02 vs 1.14; p=0.035). Hospitals with physicians who completed infectious diseases fellowships (0.96 vs 1.10; p=0.004) or pharmacists who completed stewardship certificate programs (1.00 vs 1.11; p=0.027) demonstrated lower SAARs. Action and Reporting Core Elements showed trends toward lower SAARs. Facilities implementing treatment recommendations with both prospective audit and feedback and preauthorization had significantly lower SAARs (1.00 vs 1.13; p=0.014). Conclusion: Implementation of accountability and targeted stewardship training was associated with lower all-antibacterial SAARs, suggesting that specific structural and personnel-focused interventions may improve antibiotic use. Action and Reporting Core Elements demonstrated trends toward lower SAARs, but these were not statistically significant. Findings underscore the potential value of Priority Core Elements in guiding stewardship strategies; however, limited sample size may have reduced the ability to detect additional associations. Larger, multistate studies are needed to confirm these relationships and further elucidate how comprehensive Core Element implementation impacts antimicrobial use outcomes.

List of Differentially expressed genes in the subtypes of mCRPC in comparison with AR+/NE- tumors

Objective:To describe antimicrobial use in Tennessee from 2017 to 2023. Design:Retrospective analysis of antimicrobial use using data from the National Healthcare Safety Network (NHSN) Antimicrobial Use (AU) Option. Setting:Acute care and critical access facilities in Tennessee. Results:From 2017 to 2023, 97 facilities in Tennessee submitted data to the NHSN AU Option. The number of reporting facilities increased from 25 to 95. During this time, the statewide average antimicrobial use significantly rose from 593 days of therapy (DOT)/1000 days present (DP) to 621 DOT/1000 DP (P = .0478). The All-Antibacterial Standardized Antimicrobial Administration Ratio (SAAR) values remained near 1.0, indicating overall use was as predicted. However, the All-Antibacterial SAAR values, particularly in small facilities, revealed that they utilized antibiotic agents more than predicted during the study period. Additionally, the SAAR trends varied by patient care locations, with the oncology unit (ONC) experiencing a significant increase from 0.73 to 1.12 (P-value<.0001). West Tennessee had the highest antimicrobial use rate at 736 DOT per 1000 DP, and an All-Antibacterial SAAR of 1.21. The top antimicrobial agents-vancomycin, ceftriaxone, piperacillin/tazobactam, cefepime, and cefazolin-accounted for 54% of the total antimicrobial use. Conclusions:This statewide analysis of AU and SAAR trends identifies areas where additional antimicrobial stewardship efforts may be targeted to improve antimicrobial use. Facilities of different sizes and geographic locations have unique demographics that can affect antimicrobial use, requiring specialized antimicrobial stewardship techniques.

The androgen receptor inhibitor enzalutamide is one of the principal treatments for metastatic prostate cancer. Most patients respond. However, a subset is primary refractory. Seeking to understand enzalutamide extreme non-response (ENR), we analyzed RNA-sequencing in biopsies from men treated prospectively on an enzalutamide clinical trial. We focused on those with ENR (progression within 3 months) vs. long-term response (progression after 24 months). We identified an ENR program linked to proliferation, epithelial-to-mesenchymal transition, and stemness. High expression of this program in additional datasets was independently linked to poor tumor control with AR targeting but favorable tumor control with docetaxel, another standard treatment. CDK2 was implicated in the ENR program. CDK2 suppression reduced the ENR program and viability of ENR program-high prostate cancer models. The ENR gene program is predictive of non-response to AR targeting. Patients whose tumors harbor this program may be good candidates for docetaxel or CDK2 inhibitor clinical trials.

Background: The National Healthcare Safety Network (NHSN) Antibiotic Use (AU) Option aids hospital antimicrobial stewardship programs (ASPs) by facilitating tracking and reporting of AU data. In 2021, the Tennessee Department of Health (TDH) launched an AU data quality project to improve reporting accuracy. Quarterly reports are generated, assessing data across 15 quality flags, such as reporting antimicrobial days when days present (DP) are zero or drug-route mismatches. Flags also highlight significant outliers, including DP or AU rates outside the median ±2 interquartile ranges compared to the prior year. Reporting facilities receive actionable solutions for flagged concerns. Method: Data from AU quality flag reports generated by the NHSN AU Option for Tennessee facilities (2021–2023) were analyzed in this cross-sectional study. The analysis summarized the frequency and distribution of flagged issues across facilities and time. Archived data were utilized, excluding updates facilities made after quarterly reports. Quarterly flags per category were calculated for each facility, with total flags compiled annually to determine category frequency and percentage. Additionally, the number of distinct facilities contributing to the annual flag count was evaluated, providing insights into data quality trends across the study period. Result: From 2021 to 2023, 97 facilities submitted data to the NHSN AU Option, resulting in 7336 flags identified in the AU quality reports (Figure 1). The most frequent flag was “location-level AU rate greater than outlying upper boundary” (n=1677, 22.9%), reported by 67 facilities and the highest reported in 2023 (n=722, 23.8%). The second was “location-level DP greater than outlying upper boundary” (n=1588, 21.6%), reported by 68 facilities and highest in 2021 (n=547, 23.5%). The most frequent non-outlier-based quality issue was “antimicrobial days reported for any drug when DP were reported as zero” (n=439, 6.0%) followed by “antimicrobial days for a single drug greater than DP” (n=48) Conclusion: The study reveals data quality concerns in AU reporting among Tennessee facilities. Flags with changes in “Location-Level Days Present” and “AU Rate” outliers being prominent across the study period. These findings underscore the need for continuous monitoring and targeted feedback to enhance data accuracy, as well as a need for antimicrobial stewardship personnel to be able to identify and address changes in prescribing patterns and patient populations efficiently within their facilities. Addressing recurring challenges identified can improve AU data reliability, supporting more effective antimicrobial stewardship and better patient care outcomes.

Oregon Health & Science University, Portland, Oregon Funding/Support: None reported. Financial Disclosures: None reported. Correspondence: Christopher Evan, M.D., 3181 SW Sam Jackson Park Rd. Portland, OR 97239. Mail code L-475, Portland, Oregon 97239. Email: [email protected]

We describe the epidemiology and incidence of infections following gender-affirming vaginoplasty. Urinary tract and surgical site infections were the most common infections with incidences of 17.5% and 5.5%, respectively. We also identified a significant gap in human immunodeficiency virus screening and prescription of preexposure prophylaxis.

Background: Tracking antimicrobial use (AU) is a Core Element of Hospital Antimicrobial Stewardship Programs and important to help curb the public health threat of antimicrobial resistance. The National Healthcare Safety Network’s (NHSN) AU Option serves as a way for facilities, healthcare systems, and health departments to track and report AU rates within their jurisdictions. Many analyses at the state and national levels do not assess unit-level AU rates. This study investigates AU rates among patient care units in Tennessee reporting facilities from 2015 to 2023 and the most frequently used antimicrobial agents based on AU rates within select unit types. Methods: A retrospective analysis was conducted utilizing data obtained from the NHSN AU Option for inpatient units in Tennessee acute care hospitals. Units were defined as critical care (including neonatal), ward, oncology ward, stepdown, operating room (OR), and mixed acuity and specialty care areas, termed ‘other’. Unit types with fewer than five facilities represented were excluded. AU rates were determined by Antimicrobial Days of Therapy (DOT) per 1000 Days Present (DP). Statistical analyses , including descriptive statistics and comparison among the units by ANOVA test , were calculated using SAS Version 9.4. Results: Eighty-three facilities reported at least one month of data into the NHSN AU Option between 2015–2023. Among 70 facilities reporting inpatient units, the highest AU rate was observed in oncology ward units (n=12, 1114.6 DOT/1000 DP). A significant difference in AU rates was observed between oncology ward units compared to different unit types (p < 0 .001). Vancomycin, ceftriaxone, and piperacillin/tazobactam were the most used antimicrobials with AU rates of 83, 76, and 65 DOT/1000 DP, respectively. Vancomycin AU rates were significantly higher in oncology ward units compared to stepdown, ward, other, and OR units (p < 0 .0001). Ceftriaxone AU rate was significantly higher in stepdown units compared to oncology ward, other, and OR units (p < 0 .0001). Piperacillin/tazobactam AU rate was significantly higher in critical care units compared to different unit types (p < 0 .0001). Conclusion: During the study period, the AU rate varied across hospital inpatient units in Tennessee, with the highest AU rate in oncology wards. This unit-specific approach is critical to address the diverse antimicrobial prescribing behaviors observed, indicating that interventions should be customized to each unit’s distinct antimicrobial usage patterns for successful stewardship efforts. Targeted strategies focused on individual wards rather than facility-wide initiatives appear essential for effective reduction in antibiotic usage.

Background: Secondary malignancy is a long-term risk of radiation. External beam radiation therapy (EBRT) for prostate cancer treatment has been associated with later development of bladder cancer and worse bladder cancer features. Objective: We sought to provide an updated comparison of the long-term risk of bladder cancer after different localized prostate cancer treatments. Design, setting, and participants: Using the Surveillance, Epidemiology, and End Results (SEER) cancer registry, we compared an age-matched subset of patients who underwent radical prostatectomy (RP) with those who underwent EBRT, brachytherapy (BT), EBRT + BT, and RP followed by EBRT (RPtoEBRT) between 2000 and 2018. Our final cohort included 261 609 patients with a median follow-up of 11.6 yr. Outcome measurements and statistical analysis: Our primary outcomes were time to bladder cancer diagnosis, muscle-invasive bladder cancer diagnosis, and bladder cancer death. We used cause-specific hazard models considering death as a competing event. A similar analysis was performed on lung cancer, as a surrogate marker for smoking. We also compared proportions of variant histology, high-grade, and invasive disease among bladder cancers that occurred after radiation versus RP using chi-square testing. Results and limitations: All radiation groups were associated with bladder cancer diagnosis; hazard ratios (HRs) were 1.72, 1.85, 1.80, and 1.53 for EBRT, BT, EBRT + BT, and RPtoEBRT, respectively, using RP as a referent (all p < 0.001). HRs for bladder cancer death were even higher: 2.39, 2.57, and 3.02 for EBRT, BT, and EBRT + BT, respectively (all p < 0.001), except for RPtoEBRT (HR 1.43, p = 0.28). Lung cancer diagnosis was also associated with radiation but at lower HRs-1.63, 1.32, 1.42, and 1.30 for EBRT, BT, EBRT + BT, and RPtoEBRT, respectively (all p < 0.001). There were a higher proportion of >= T2, >= T3, and sarcomatoid variant bladder cancers after radiation (all p < 0.01) Conclusions: The risk of developing and dying from bladder cancer is increased in patients treated with radiation compared with those treated with RP. The risk was similar for BT and EBRT. Bladder cancers after radiation are more likely to be sarcomatoid variant and present as muscle invasive.

169 Background: AR/AR-variants and AKR1C3 play critical roles in prostate cancer progression and driving resistance to antiandrogens, and are attractive targets for therapeutic intervention for advanced prostate cancer. However, there are currently no clinically available therapies that simultaneously target both AR/AR variants and AKR1C3. We have developed the novel small molecule inhibitors, LX-1 and its analog LX-1S, which target both the AR-V7 splice variant and the enzyme AKR1C3. Methods: A library of the LX compounds was designed and synthesized according to structure based computer modeling. The effects of the lead LXs on the expression and activity of AR/AR-variants and AKR1C3 were evaluated. RNA-seq was performed on the lead LXs. Resistant cell sublines generated from C4-2B cells resistant to enzalutamide (MDVR), apalutamide (ApalR), darolutamide (DaroR), or abiraterone (AbiR) were treated with LX-1 or their respective antiandrogen. Mice bearing VCaP xenograft tumors and LuCaP35CR PDX tumors were treated with LX-1/LX-1s and effects on tumor growth were assessed. Results: Molecular docking studies and in vitro experiments demonstrated that LX compounds effectively bind to the AKR1C3 active site and inhibit AKR1C3 enzymatic activity. LX compounds were also shown to reduce AR/AR-V7 expression and inhibit their target gene signaling, induce G0/G1 arrest in anti-androgen resistant cell lines. LX-1/LX-1S treatment resulted in reduced tumor volumes and decreased intratumoral testosterone in both xenograft tumor and PDX models. LX-1 effectively inhibited the conversion of androstenedione into testosterone in tumor-based ex vivo enzyme assays. Moreover, LX-1 synergized with enzalutamide and abiraterone as well as docetaxel, suggesting its potential to enhance the anti-tumor activity of these standard therapies in resistant prostate cancer. Furthermore, LX-1/LX-1S improved enzalutamide treatment in resistant prostate cancer tumor models. Notably, LX-1S demonstrates significantly better bioavailability and efficacy than LX-1 in reducing resistant cell growth in both in vitro and in vivo animal models. Conclusions: Our study unveils the potential of LX compounds, especially LX-1 and its analog LX-1S, as promising therapeutic agents for advanced prostate cancer. These compounds not only inhibit AR and AKR1C3 signaling but also suppress resistant tumor growth and synergize with antiandrogens to enhance their therapeutic efficacy.

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Abstract Background: Neuroendocrine prostate cancer (NEPC) is the most aggressive type of prostate cancer with no effective treatments. Therefore, there is an urgent need to develop new treatment strategies. N-Myc plays a key role in driving NEPC progression, and Aurora kinase A (AURKA) prevents N-Myc degradation by interact with N-Myc and disallowing the intervention of ubiquitin ligases. The AURKA inhibitor Alisertib inhibits NEPC tumor growth by disrupting N-Myc signaling, but it is failed in a phase II clinical trial. In this study, we tested the synergy of novel HSP70 allosteric inhibitor JG231 and Alisertib in NEPC cells, organoids, and patient derived xenograft (PDX) models, which will provide new therapeutic strategies for the NEPC treatment. Methods: RT-PCR, western blotting, or immunohistochemistry (IHC) were used to determine the expression of N-Myc, neuroendocrine maker (NSE, SYP, CHGA, etc) in different prostate cancer cell lines and PDX tumors. After the knockdown of HSP70 and AURKA by siRNA transfection, cell proliferation and N-Myc expression were assessed by using cell viability assay, RT-PCR and Western blotting respectively. The co-immunoprecipitation assay was performed to detect the N-Myc ubiquitination. RNA-sequencing, gene set enrichment analysis was employed to determine the changes of JG231 on gene programs. The effects of JG231 and Alisertib on the proliferation were examined in NEPC cell lines and PDX organoids models. Results: We determined that PC3, CWR22Rv1, H660 cell lines, LuCaP49, and LuCaP93 PDX tumors had significantly increased the expression of N-Myc and neuroendocrine makers. Knockdown of HSP70 or using the HSP70 inhibitor, JG231, significantly inhibit the growth of H660 and CWR22Rv1 cells and leads to a synergistic effect with Alisertib (p<0.001). JG231 combined with Alisertib also inhibits growth and induces the death of LuCaP93 and H660 tumor derived organoids in a dose-dependent manner (p<0.001). Mechanistically, JG231 inhibits the protein expression of N-Myc through the ubiquitin-proteasome system and promote STUB1 to enter the nucleus to bind to N-Myc. Conclusions: JG231, a novel HSP70 inhibitor, can improves the treatment efficacy of AURKA inhibitor Alisertib against NEPC through the regulation of N-Myc protein homeostasis. Citation Format: Pengfei Xu, Joy C. Yang, Bo Chen, Shu Ning, Leyi Wang, Christopher Nip, Christopher P. Evans, Marc A. Dall'Era, Allen C. Gao, Jason Gestwicki, Chengfei Liu. Dual targeting of HSP70 and AURKA improves treatment in neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4567.

<p>XLS file - 56K, Variable Importance in Projection (VIP) data for Tissue, Serum and Urine</p>

INTRODUCTION AND OBJECTIVES Androgen receptor (AR) blockade using antiandrogens is a mainstay for the treatment of castration-resistant prostate cancer (CRPC). However, resistance to antiandrogens occurs unavoidably due to the escaping mechanisms of tumor cells that are not completely understood. Upregulation of non-canonical Wnt ligand Wnt5a is identified from circulating tumor cells from CRPC patients after being treated with enzalutamide. FZD2, the cognate frizzled receptor for Wnt5a, is the most commonly co-upregulated non-canonical Wnt signaling molecule in prostate cancer. In this study, we determined the role of non-canonical Wnt5a/FZD2 to enzalutamide resistance and neural lineage plasticity, and explore the potential of targeting Wnt5a/FZD2 to overcome resistance in advanced prostate cancer. METHODS Wnt5a and FZD2 expression was determined in enzalutamide-resistant C4-2B MDVR cells. Wnt5a and FZD2 expressions were modulated using specific siRNAs. Cell growth, colony formation, and migration were studied in vitro. Transcriptomic analysis was performed on C4-2B MDVR cells treated with FZD2 knocked down; gene program of non-canonical Wnt signaling, hallmark androgen response and AR-V7 associated genes, and neural lineage pathways were analyzed. A novel tRNA bioengineered Wnt5a siRNA was developed to target Wnt5a/FZD2 signaling. The effect of tRNA-siWnt5a on tumor growth, sensitivity to enzalutamide treatment, and neural lineage plasticity was evaluated in vitro and in vivo. RESULTS Wnt5a and FZD2 are highly upregulated in castration-resistant prostate cancer patients, which is verified overexpression in enzalutamide-resistant C4-2B MDVR cells. Knocking down Wnt5a and FZD2 diminishes the enrichment of the non-canonical Wnt signaling pathway. Downregulating Wnt5a and FZD2 expression by specific siRNAs suppresses prostate cancer cell proliferation and resensitized C4-2B MDVR cells to enzalutamide treatment. Suppressing Wnt5a and FZD2 abrogated the enrichment of gene programs regulating cancer cell survival/proliferation, and neural lineage pathways including neuron differentiation and embryonic stem cell markers. Using the bioengineered tRNA-Wnt5a siRNA effectively inhibited the growth of enzalutamide-resistant prostate cancer cells and resensitized tumor cells to enzalutamide treatment in vitro, and resistant CRPC PDX LuCaP35CR tumor growth in vivo. Conclusions Our results suggest that activation of noncanonical Wnt5a/FZD2 signaling confers to enzalutamide resistance and neural lineage plasticity. Targeting the Wnt5a signal could provide benefits for CRPC patients with tumors expressing a high level of Wnt5a, FZD2, and lineage markers, not only enhancing the anti-tumor effects of enzalutamide but suppressing the potential of neuroendocrine differentiation in advanced prostate cancer patients. Citation Format: Shu Ning, Wei Lou, Chengfei Liu, Alan Paul Lombard, Leandro S. D' Abronzo, Neelu Batra, Aiming Yu, Christopher P. Evans, Allen Gao. Targeting noncanonical Wnt5a signaling suppresses the neural lineage network and overcomes enzalutamide resistance in advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B023.

<p>XLS file - 56K, Variable Importance in Projection (VIP) data for Tissue, Serum and Urine</p>

Supplementary Figure 3. Recruitment of hnRNPA1 and hnRNPA2 to splice sites for AR variants other than AR-V7

Supplementary Figure 6. Expression levels of U2AF65 and ASF/SF2 in human clinical PCa samples