Citations

Abstract Introduction: The standard of care for metastatic hormone-sensitive prostate cancer (mHSPC) has evolved with the expanded approval of androgen receptor pathway inhibitors (ARPIs) alongside androgen deprivation therapy (ADT). ADT alone has been shown to affect cognitive function. However, the cognitive impact of distinct treatment combinations remains unclear. We investigated the association between first-line systemic treatments and the trajectory of subjective cognitive function among mHSPC survivors in the International Registry for Men with Advanced Prostate Cancer (IRONMAN). Methods: This analysis included 2435 newly diagnosed mHSPC patients enrolled across 15 countries between July 2017 and August 2024. Treatments were reported by site personnel and physicians. For this analysis, we focused on first-line systemic treatment and classified treatment groups at enrollment as ADT monotherapy (referent), ADT + ARPIs, or ADT + chemotherapy. Subjective cognitive function was assessed via the EORTC QLQ-C30 Cognitive Function subscale, with patient-reported scores (range 0-100) collected at enrollment and every 3 months for up to 5 years; higher scores reflect better cognitive function. Associations were estimated using linear mixed effects models with clustering by study site and country to examine differences at enrollment and across trajectories of subjective cognitive function by first-line treatment, adjusting for demographic and clinical factors. Results: Median age at enrollment was 70 years, with a median follow-up of 1.7 years. 724 (30%) participants were treated with ADT monotherapy, 1317 (54%) with ADT + ARPIs, and 394 (16%) with ADT + chemotherapy. Mean cognitive function scores at enrollment were 85.4 (Standard Deviation [SD] 19.5) for participants treated with ADT monotherapy, 86.7 (SD 17.3) for ADT + ARPIs, and 86.7 (SD 18.0) for ADT + chemotherapy. Cognitive function scores decreased by -0.60 points (95% Confidence Interval [CI] -0.72, -0.48) per year among those treated with ADT monotherapy, with similar rates observed for those treated with ADT + chemotherapy. Participants treated with ADT + ARPIs had a slower decline in cognitive function, with scores decreasing by -0.36 points (95% CI -0.60, -0.08) per year. Conclusion: In this international cohort of mHSPC survivors, subjective cognitive function scores were generally high at enrollment but declined over time. There was no evidence of worse cognitive function in those treated with ADT + ARPIs nor ADT + chemotherapy over time compared to ADT monotherapy. Cognitive functioning is a key component of survivorship, and defining cognitive trajectories associated with first-line treatment can inform supportive interventions, patient treatment decision-making, and clinical guidelines. Citation Format: Michelle O. Sodipo, Alicia K. Morgans, Erica T. Warner, Emily M. Rencsok, Lauren E. Howard, Colleen B. McGrath, Anna Siefkas, Konrad H. Stopsack, Christopher Sauer, Robert Drecier, Emilio Esteban, Hassan M. Dogo, Ademola Popoola, Charles Waihenya, Anders Bjartell, Kim Chi, Sebastien Hotte, Richard Cathomas, Deborah Enting, Scott Tagawa, Michael Ong, David Lorente, Elisabeth Heath, Rana McKay, Stefanie Fischer, Heather H. Cheng, Young E. Whang, Frederic Pouliot, Simon Crabb, Monica Chatwal, Miguel A. Climent, Raymond McDermott, Ian D. Davis, Camille Ragin, Folakemi Odedina, Natalie Greaves, Simone Badal, Simon Anderson, Sharon Harrison, Karen Autio, Laurel Cannon, Marie Grant, Alyssa Chan-Cuzydlo, Travis Gerke, Hannah D. McManus, Philip W. Kantoff, Daniel George, Sebastien Haneuse, Lorelei A. Mucci, on behalf of the IRONMAN Registry. First-line therapies and subjective cognitive function trajectories among international metastatic hormone-sensitive prostate cancer survivors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3630.

Abstract HER2+ breast cancer is an aggressive subtype of cancer that often develops resistance to the current first-line therapy, Trastuzumab. In our previous work, we demonstrated that HER2 signaling activates mitochondrial creatine kinase 1 (MtCK1), which enhances mitochondrial energy metabolism for rapid proliferation of breast cancer cells. However, pharmacological blockage of MtCK1 with a creatine analog, Cyclo-Creatine (CCr), requires millimolar concentration to inhibit breast cancer cell proliferation and is associated with brain toxicity, limiting its clinical application. This brain toxicity may result from the inhibition of the creatine transporter SLC6A8 by CCr, which decreases to transport creatine into brain. In this study, we took a prodrug development approach to identify and optimize protection groups for CCr, designed to bypass the SLC6A8 transporter, enhancing bioavailability and reducing toxicity. Liquid chromatography-mass spectrometry (LC-MS) was utilized to analyze drug uptake and creatine metabolites in cells treated with the prodrugs. Prodrug C, which is our lead prodrug candidate, demonstrated a seven-fold increase in cellular uptake and more than a hundred-fold improvement in cell-killing effects as compared to CCr in an MtCK1 specific manner in breast cancer cells. This approach offers a promising therapeutic strategy to inhibit HER2+ breast cancer cell by disrupting mitochondrial energy metabolism. Future investigations will focus on validating this prodrug’s pharmacokinetics (PK) and pharmacodynamics (PD) through further preclinical studies. Citation Format: Ya Li, Yetong Huang, Puja Dey, Sadae Hitosugi, Sarah A. Buhrow, Joel M. Reid, Taro Hitosugi. Prodrug development of Cyclo-Creatine: Enhancing permeability and reducing toxicity for HER2+ breast cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4157.

Abstract Recent evidence has highlighted the critical requirement for “immune triads” of CD8+ T cells, CD4+ T cells and dendritic cells (DC) for tumor elimination in response to checkpoint inhibition (CPI) immunotherapy. However, the mechanistic underpinnings of this cross-talk are poorly understood. Immune triads are often located in peripherally-distributed DC-rich niches within the tumor stroma. We have hypothesized that stromal remodeling in response to tumor expansion generates homeostatic signals that trigger an adaptive immune response that may ultimately fail through effector exhaustion or apoptosis. In particular, stromal proteolysis of the large matrix proteoglycan versican (VCAN) has been associated with T-cell infiltration across multiple tumor types. The objective of this study was to interrogate a mechanistic link between stromal remodeling, immune triad formation and response to CPI. We used HALO image analysis to determine the spatial relationship of CD4+ and CD8+ T cells in a panel of 95 human non-small cell lung cancers. Each tumor was VCAN proteolysis-scored based on a binning system measuring staining intensity for a neoepitope exposed through site-specific VCAN proteolysis. Tumors displaying VCAN proteolysis intensity of zero (0) had an average of 0.122 CD8+ T cells located with 10um of a CD4+ T cell vs. 0.216 in tumors with any VCAN proteolysis (intensity 1, 2, 3 combined); p=0.04. Similar results were obtained when measuring proximity of CD4+ to CD8+ T cells (0.124 vs 0.223, p=0.02). These data demonstrate that remodeling stroma promotes CD4+:CD8+ crosstalk. Site-specific VCAN proteolysis releases a 441 N-terminal bioactive fragment, versikine. Versikine constitutes a classical “matrikine”, i.e., a bioactive matrix fragment possessing novel functions compared to its parent macromolecule. To determine the mechanisms by which versikine may promote immune triad niches, we generated several transplantable syngeneic tumor cell lines engineered to stably secrete versikine into the TME. One CPI-refractory tumor cell line (Lewis Lung Carcinoma, LLC) and two partially CPI-responsive transplantable tumor lines (CT26 and MC38 colorectal carcinoma) were extensively characterized through flow cytometry, spatial immunofluorescence and scRNAseq. Our results demonstrate that in all contexts, versikine results in expansion of Tfh-like CD4+ T cells expressing Cd40lg and Icos. This expansion is associated with specific patterns of CD8+ T cell exhaustion that will be discussed. Therapeutically, versikine reversed anti-PD-1 pathway refractoriness in the poorly-immunogenic LLC model and demonstrated superior synergy with CPI in the more immunogenic CT26 and MC38 models, leading to tumor eradication with resistance to tumor rechallenge. Our data bolster the mechanistic rationale for exploitation of stromal remodeling signals to promote CD4+T:CD8+T:DC cross-talk for enhanced CPI efficacy. Citation Format: Daniel Lagal, Duncan Hong, Athanasios Papadas, Alicia Gibbons, Yun Huang, Yaling Dou, Alexander Cicala, Elsa Molina, Kristina Matkowskyj, Dustin Deming, Fotis Asimakopoulos. Stromal remodeling promotes immune triad formation in the tumor microenvironment (TME) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2588.

Abstract Breast cancer (BC) is the most common cancer diagnosed in American women. The majority of breast cancers express estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER2). However, 15-20% lack expression of all three and are called triple-negative breast cancer (TNBC). TNBC accounts for a disproportionately large amount of morbidity and mortality due to its aggressive nature and the paucity of effective adjuvant maintenance therapy, driving an urgent need for novel therapeutics. Z-endoxifen (ENDX) is a potent tamoxifen metabolite that dually targets ERα and protein kinase C beta I (PKCβI). We recently described next-generation molecules, the first-in-class endoxifen-PROTAC (ENDX-PROTAC) compounds which incorporate ENDX as the warhead and cereblon (CRBN) as the E3 ubiquitin ligase recruitment moiety (Jayaraman 2024, SABCS). We used the PRISM screen of over 900 cancer cell lines to determine that ENDX-PROTACs have broad antitumor activity in both TNBC and ER+ breast cancer in addition to, leukemia, pancreatic, lymphoma, lung, melanoma, and ovarian cancer cells, among others. ENDX-PROTACs primarily target a key translation termination factor, G to S phase transition 1 (GSPT1). Here, we demonstrate for the first time that these compounds also cause marked DNA damage. The lead compounds of this series, EPrC-007 and EPrC-009 demonstrated potent anti-proliferative effects in MDA-MB-231, BT549, and MDA-MB-436 TNBC cell lines. EPrC-007 induced substantial DNA double strand breaks in MDA-MB-231 cells as assessed by γ-H2AX Western blotting. Interrogation of DNA repair pathways revealed activation of ATM/CHK2 and downregulation of ATR/CHK1, suggesting inhibition of DNA repair as a potential mechanism. Additionally, EPrC-007 also induced γ-H2AX foci in MDA-MB-231 and BT549 cells, but not in the MCF10A normal breast tissue cell line, suggesting selective anti-tumor activity. Since we suspect that EPrC-007 blocks DNA repair machinery, we next sought to determine if these molecules would synergize with DNA damaging agents or other DNA repair inhibitors. Promisingly, EPrC-007 demonstrated remarkable synergy with olaparib (poly-(ADP-ribose) polymerase (PARP) inhibitor), hydroxyurea (ribonucleotide reductase inhibitor), and cisplatin (direct DNA damaging agent), but not camptothecin (topoisomerase I inhibitor). Importantly, only olaparib co-treatment demonstrated synergy in the PARP- and PROTAC-resistant HCC1143 TNBC cell line. Furthermore, the combination of EPrC-007 with olaparib induced markedly more γ-H2AX foci than either agent alone, confirming synergy at the DNA damage level. Taken together, our studies demonstrate the remarkable potential of ENDX-PROTACs in the single-agent setting as well as in combination with PARP inhibitors. Citation Format: Aamod S. Dekhne, Swaathi Jayaraman, Lucy Rai Thulung, Becky Bruinsma, Baustin M. Welch, Samuel Wyatt, Esther P. Rodman, Sayantani Bhattacharya, Thomas R. Caulfield, John R. Hawse, Matthew J. Schellenberg, Matthew P. Goetz. Novel endoxifen-based proteolysis targeting chimera (PROTAC) small molecules synergize with olaparib and exhibit potent antitumor activity in triple negative breast cancer through induction of DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1666.

Abstract Introduction: We previously developed a urine liquid biopsy assay to measure urine tumor DNA (utDNA) levels in bladder cancer (BC) patients. Here, we extended this approach significantly by layering in cell-free DNA fragmentomic and cell-free RNA analysis, and extending the method to the full spectrum of genitourinary (GU) cancers, to enable both GU cancer detection and tumor tissue of origin prediction. Methods: A total of 155 GU cancer patients and 34 healthy adults were enrolled in this prospective study. Preoperative urine was collected from 94 bladder cancer (BC) patients, including 65 with muscle-invasive disease, all undergoing cystectomy. Additionally, urine samples were collected from 18 renal cell carcinoma (RCC) patients undergoing nephrectomy and 42 metastatic prostate cancer (mPC) patients. Urine cfDNA and cfRNA were isolated and sequenced, with cfRNA sequenced to 100 million reads per sample on an Illumina NovaSeq X and cfDNA sequenced at 5x genome-wide coverage on a Novoseq S4 flow cell. To identify malignant tissue of origin, we analyzed somatic copy number alterations (CNAs), genome-wide fragment length ratios, and 4-mer end motifs. Tumor fraction (TFx) was estimated using ichorCNA, while DELFI was employed for fragment length ratios in 5 megabase bins across the genome and chromosome arm-level z-scores. Individual logistic regression models were developed based on each fragmentomic feature, and validated using a training and held-out testing approach. Outputs from these models were combined into a single comprehensive model, also trained and held-out tested using the same approach. Urine cfRNA transcript isoform abundance was quantified using Salmon and normalized to Transcripts Per Million (TPM) to account for sequencing depth variations. Results: Bladder cancer (BC) showed the highest mean tumor fraction (TFx, 10.5%), followed by RCC (3.8%) and mPC (3.4%). Among 94 BC patients, 42% achieved a pCR, while 58% had residual disease and did not. Patients without pCR had significantly higher urine TFx compared to those with pCR (14.7% vs. 4.7%, p=0.0008). A machine learning model based on genome-wide fragment length ratios achieved an ROC AUC of 0.74, while models using genome-wide CNAs and end motifs achieved AUCs of 0.81 and 0.88, respectively. Combining all features into a single model yielded an AUC of 0.92, with classification accuracies of 89% for bladder cancer, 80% for RCC, and 60% for prostate cancer. Additionally, urine cfRNA analysis using the top 1, 000 coding transcripts achieved an AUC of 0.95, with classification accuracies of 100% for mPC, 87% for BC, and 78% for RCC. Conclusion: Fragmentomic features from urine cfDNA and transcriptomic features from urine cfRNA demonstrate strong potential for predicting tumor tissue-of-origin in GU cancers, underscoring the promise of non-invasive diagnosis across the spectrum of GU malignancies through urine liquid biopsy. Citation Format: Pradeep Singh Chauhan, Irfan Alahi, Jessica Linford, Arpit Panda, Nathan Colon, Ryan Mueller, Faridi Qaium, Peter K. Harris, John Sheng, Eric H. Kim, Melissa A. Reimers, Zachary L. Smith, Woodson W. Smelser, Aadel A. Chaudhuri. Predicting genitourinary cancer tissue-of-origin using urine cell-free DNA fragmentomics and cell-free RNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3258.

Tumor treating fields (TTFields), a noninvasive anticancer treatment modality, has received FDA approval for pleural mesothelioma, metastatic NSCLC following platinum-based therapy, newly diagnosed and recurrent glioblastoma multiforme. TTFields have also demonstrated potential benefits in other cancer types, including pancreatic ductal adenocarcinoma (PDAC). Although the PANOVA trial yielded positive results in PDAC patients, the mechanisms underlying these beneficial effects remain unclear. Severe desmoplasia is a hallmark characteristic of PDAC due to accumulation of excess extracellular matrix (ECM) caused by activated fibroblasts and cancer cells. This dense ECM contributes to an immunosuppressive tumor microenvironment (TME) and drug resistance, which ultimately leads to poor 5-year survival in PDAC patients. Our previous work with atomic force microscopy (AFM) showed that the nanomechanical properties of the ECM change dynamically based on its composition and the presence of PDAC cells, and vice versa. Herein, we prepared an ECM complex comprising of collagen, fibronectin and laminin at physiological concentrations. Following which, we investigated the alteration of stiffness of both PDAC cells and the ECM in presence of TTFields using an AFM. A frequency-dependent analysis of TTFields showed that a frequency of 150 KHz led to consistent decreases ECM stiffness at 48 and 72 hours. Next, we evaluated the influence of TTFields in PDAC cells alone. In Panc-1 cells exposed to TTFields for 48 and 72 hours, we observed a more than 6-fold increase in membrane stiffness compared to untreated controls. Additionally, AsPC-1 cells subjected to TTFields exhibited a greater than 2-fold increase in membrane stiffness at these time points. Furthermore, we have investigated influence of TTFields on PDAC cells and ECM components in a co-culture model. In this co-culture scenario for Panc-1 cells, TTFields increase membrane stiffness by 5-fold compared to untreated at these time points. Also, AsPC-1 cells demonstrated a 1.5-fold increase in membrane stiffness in the presence of TTFields compared to the untreated. Lastly, we also evaluated the influence of cells on the ECM stiffness in the presence of TTFields. It significantly increased the ECM stiffness in presence of Panc-1 co-culture by 1.3-folds compared to control. We also observed 3- and 4-fold increase in ECM matrix stiffness when co-cultured with AsPC-1 for 48 and 72 hours from TTFields, respectively compared to control. In conclusion, TTFields significantly modulate the stiffness of PDAC cells and the ECM, both individually and in co-culture. Citation Format: Tanmay Kulkarni, Hani Babiker, Debabrata Mukhopadhyay, Santanu Bhattacharya. Tumor treating fields (TTFields) alter nanomechanical attributes of cell-ECM complex in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5105.

Abstract Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors that regulate the function and activity of matrix metalloproteinases (MMPs), enzymes that are known for their involvement in cancer progression. MMP-9, also known as gelatinase B, plays a crucial role in tumorigenesis of several cancers including triple negative breast cancer (TNBC); tumor cell-produced MMP-9 promotes malignant progression and metastasis of basal-like TNBC. Here, we have used full-length TIMP-1 as a scaffold for engineering inhibitors that bind with higher selectivity to MMP-9, for potential therapeutic applications. We previously developed a yeast surface display library of human TIMP-1, with diversity incorporated at 17 residues within five loops; mutation burden was tuned to 3-5 mutations per clone. Here, we have screened the TIMP-1 library for affinity toward recombinant MMP-9 by using fluorescence-activated cell sorting (FACS). Yeast cells were incubated with an incrementally decreasing concentration of biotinylated MMP-9 (400 nM to 25 nM) in successive rounds of sorting. Samples were analyzed using flow cytometry and highest affinity populations were selected using FACS. Among the TIMP-1 variants with highest enrichment, we identified the TIMP-1-C15 variant that showed 12-fold enhanced binding with improved selectivity toward MMP-9 compared with wild type TIMP-1 (WT TIMP-1). We next expressed and purified soluble recombinant TIMP-1-C15 and conducted inhibition studies using a fluorogenic substrate to evaluate inhibitory potency towards MMP-9 activity. These experiments showed that TIMP-1-C15 inhibits MMP-9 activity with an inhibition constant (Ki) of 0.61 nM, more than 5-fold stronger than WT TIMP-1; these studies also corroborated the improved selectivity towards MMP-9 over other MMPs. Additionally, we further evaluated the functional impact of the TIMP-1-C15 variant on cancer cell invasiveness using TNBC cell lines in cellular invasion assays. The TIMP-1-C15 variant markedly reduced the invasive capabilities of TNBC cells to a greater extent than WT TIMP-1. Currently, we are working to isolate additional TIMP-1 variants from our library that exhibit enhanced selectivity for MMP-9, with MMP-8 included in the sorting process as a competitive target to ensure specificity. Selective inhibition of MMP-9 over MMP-8 is particularly important in breast cancer therapy, as MMP-8 has been shown to have anti-tumorigenic properties, and its inhibition could counteract therapeutic efforts aimed at reducing cancer invasiveness. Our findings suggest that engineered TIMP-1 variants could serve as potent inhibitors of MMP-9, offering a novel approach to reducing the invasiveness of breast cancer cells and potentially limiting the progression of metastatic disease. Citation Format: Alireza Shoari, Alexandra Hockla, Mathew A. Coban, Alexandra M. Dimesa, Derek C. Radisky, Evette S. Radisky. Engineered TIMP-1 for selective MMP-9 inhibition: A novel strategy to suppress breast cancer invasiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5115.

Abstract Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia among adults in the US. Despite significant improvement of current novel agent treatments, CLL remains incurable with many eventually experiencing disease relapse. Therefore, it remains of high interest to expand therapeutic options. Transcriptional chemical inducers of proximity (TCIPs) are a new class of anticancer drugs designed and developed by Stanford teams. These agents recruit cancer drivers or transcription factors to activate proapoptotic genes' expression and kill cancer cells. Current TCIPs are produced by covalently linking small molecules that bind B-cell lymphoma 6 (BCL6) to those that bind to transcriptional activators, such as bromodomain-containing protein 4 (BRD4) (BRD4-BCL6, TCIP1) or cyclin-dependent kinases 9 (CDK9) (CDK9-BCL6, TCIP2). In collaboration with the Stanford teams, we have initiated TCIPs cytotoxicity assessment in primary leukemic cells from untreated CLL patients and found heterogeneous but evident drug responsiveness with a panel of TCIPs. Our current work has shown that TCIP2 demonstrated remarkable killing ability of CLL B cells with IC50 of 9nM in 72-hours (h), while the IC50 of TCIP1 was 560nM. TCIPs showed time and dose-dependent drug-associated killing in primary CLL B cells over a 24-72h interval using a flow cytometry-based apoptosis assay. After 24, 48, and 72h exposure of CLL B cells to TCIPs, the IC50 was 1054, 820 and 555nM for TCIP1, and 9, 8, and 9nM for TCIP2, respectively. TCIP2 is designed and produced by covalently linking BI-3812 (BCL6 inhibitor) as a BCL6 binder and SNS-032 (CDK9 inhibitor) as a CDK9 binder with a rigid linker. Comparing TCIP2 to its negative controls (TCIP compounds with only one active binder, either BCL6 or CDK9), TCIP2 exhibited significant cytotoxicity in primary CLL B cells. TCIP2 also showed a significant increase in potency, compared to SNS-032 and BI-3812 alone at 72h. Normal human peripheral blood mononuclear cells (PBMCs) were 6-fold less sensitive to TCIP2 compared to CLL PBMCs with high percentage of CLL B cells. Immunoblotting data showed induction of BCL6 protein level after 20nM TCIP1 treatment in CLL B cells. In samples from relapsed/refractory (RR) CLL patients, TCIPs maintained good cytotoxicity with IC50 of 1805nM for TCIP1 and 84nM for TCIP2 in 24h using a cell viability assay. Our study indicates that TCIPs targeting BCL6 and BRD4/CDK9 have a strong capacity to induce high levels of cell death in primary CLL B cells. This notable killing effect may be due to its unique gain-of-function drug mechanism where BCL6 is induced. This is the first study of TCIPs ability to target CLL B cells from both untreated and RR patients. Further studies to more fully evaluate the preclinical activity of TCIPs and investigate the molecular mechanism(s) for TCIP mediated killing of leukemic B cells will help to define the potential role of TCIPs as a novel therapeutic approach in CLL. Citation Format: Weiguo Han, Aisharya Bhattacharya, Basel Karim, Wenzhi Ji, Roman C. Sarott, Meredith Nix, Sai Gourisankar, Andrey Krokhotin, Sutapa Sinha, Zhiquan Wang, Tait D. Shanafelt, Sameer A. Parikh, Nathanael S. Gray, Gerald R. Crabtree, Neil E. Kay. Rewiring cancer drivers to induce apoptosis in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5633.

Abstract Introduction: Cholangiocarcinoma (CCA) is an aggressive biliary malignancy with poor survival outcomes and multiple mechanisms of resistance to targeted therapies. We previously validated NXP800, a novel GCN2 small-molecule activator, as an effective anti-cancer drug in six CCA cell lines. In vitro testing identified upregulation of phosphorylated eIF2a, as well as upregulation of ATF4, in CCA cells treated with of NXP800 compared to vehicle, suggesting that treated cells led to stimulation of stress-induced genes and subsequently, apoptosis. Furthermore, patient derived xenografts (PDX) models treated with NXP800 were found to have decreased tumor size, particularly in models containing ARID1A, FGFR1, and ATM mutations. Here, we aimed to predict response to NXP800 using sensitivity and resistance signatures from our profiled PDX models. Methods: GCN2 activation in multiple previously profiled PDX models was evaluated. Whole Exome Sequencing (WES) was done on multiple PDX models to characterize the mutational signature and activity in CCA. Two additional CCA xenografts were expanded into the flank of NOD/SCID mice. Tumor bearing mice were treated with NXP800 (35 mg/kg) or vehicle via oral gavage for 28 days once tumors averaged 120 mm3. Tumor volume and animal weight were collected twice weekly. Median Efficacy Index (MEI) scores were generated as drug response rates based on tumor growth. Predicted sensitivity and resistance kinase signatures to predict drug response were produced using multi-omics. Results: Treatment with NXP800 was associated with a statistically significant decrease in tumor size in two additional PDX models, which included ARID1A and BRAF mutants. Preliminary multi-omics on the PDX models revealed that sensitivity mechanisms were upregulated most in EGFR signaling, while resistance signatures were focused around CSNK2A1 signaling. Conclusion: Multi-omics analysis revealed a sensitivity mechanism structured around EGFR signaling, providing insights into which patients should be selected for treatment with NXP800. A Phase 1b clinical trial investigating the effects of NXP800 use in patients with advanced CCA is open and currently accepting patients. Citation Format: Danielle Marie Carlson, Hendrien Kuipers, Amro Abdelrahman, Erik Jessen, Joshua Roldan Kalill, Jennifer Tomlinson, Jack Sample, Akhilesh Pandey, Mark Truty, Rory Smoot. Modeling response prediction to a novel GCN2 kinase activator (NXP800) in patient-derived xenograft cholangiocarcinoma models using a multi-omics approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5592.

Abstract Introduction: The binding of Programmed Death-Ligand 1 (PD-L1) on the surface of tumor cells to PD-1 on T cells prevents T-cell-mediated anti-cancer immune responses. Immune checkpoint blockades that target the PD-L1/PD-1 interaction have improved survival rates but have several critical limitations. Post-translational modifications (PTMs) of PD-L1, such as glycosylation, are crucial for its stability and engagement with PD-1. Thus, targeting PD-L1 PTMs may be a novel method to improve immune checkpoint blockade efficacy. The mevalonate pathway (MVP) generates various substances involved in PTMs process. Here, we explore the effect of MVP in altering the PTMs of PD-L1 and its impacts on cell surface PD-L1 function. Methods: The triple-negative human breast cancer (TNBC) cell lines MDA-MB-231 and BT549 were treated with the HMG-CoA reductase inhibitor lovastatin (LOV) with or without the addition of IFN-γ and/or MVP intermediates for 48 hours. PD-L1 transcription was evaluated by qPCR. Alterations in PD-L1 size were detected by Western blotting. PD-L1 surface levels were measured by flow cytometry. HMG-CoA reductase (HMGCR) knockdown was performed to validate the non-off-target effect of LOV. The binding of PD-1 protein to the cell surface PD-L1 was measured by PD-1/PD-L1 binding assay. PD-L1 glycosylation levels were measured by Concanavalin A (Con A) lectin binding assay with immunoprecipitated PD-L1 protein. Results: LOV treatment caused a significant molecular weight shift of PD-L1 from approximately 45 kDa to 40 kDa and reduced the cell surface PD-L1 levels. However, PD-L1 transcription was not affected by LOV. HMGCR knockdown produced similar changes, indicating LOV's impact on PD-L1 was not due to off-target effects. The addition of MVP intermediates mevalonate (MEV) or geranylgeraniol, but not farnesol prevented LOV-induced changes in PD-L1 molecular weight and surface levels. Interestingly, the addition of 25-hydroxycholesterol, but not dolichol, partially prevented LOV-induced decreases in PD-L1 surface levels. Geranylgeranyl transferase inhibitor GGTI-286, which prevents protein geranylgeranylation, also altered PD-L1 molecular weight and decreased surface levels. In addition, LOV impaired the binding of PD-1 protein to tumor cells' surface PD-L1, which was reversed by the addition of MVP substrate MEV. Con A lectin-binding of immunoprecipitated PD-L1 showed that the LOV did not reduce the N-glycosylation level of PD-L1. Conclusions: This study reveals the involvement of the MVP in regulating PD-L1 PTMs and highlights the MVP as a potential therapeutic target to modulate PD-L1’s molecular weight and function and improve the efficacy of immunotherapy interventions, presenting a promising avenue for further investigation. Citation Format: Ruoheng Zhang, Emily J. Koubek, Jessie L. Reed, Jeffrey D. Neighbors, Joel M. Reid, Raymond J. Hohl. The role of the mevalonate pathway in post-translational modifications of PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4265.