Grinu Mathew

Background/Objectives: The stem cell transcription factor SOX2 is expressed in more than 20 types of cancer. SOX2 expression is also elevated by a wide range of clinically used drugs and has been linked causally to drug and radiation resistance. Hence, SOX2 is recognized as an important therapeutic target. This study set out to identify a small molecule inhibitor that reduces SOX2 in a wide range of tumor types. Methods: In this study, the WNT inhibitor, PRI-724 (Foscenvivint), was tested for its ability to downregulate SOX2 in ten tumor cell lines using western blot analysis. We also used western blot analysis to examine whether PRI-724 blocks the upregulation of SOX2 by clinically used drugs and radiation. Additionally, we examined the overall effects of PRI-724 on the transcriptome by performing RNA-Seq and STRING analysis. Results: PRI-724 not only downregulated SOX2 in ten different tumor cell lines representing eight different tumor types, but also blocked the upregulation of SOX2 induced by vemurafenib, gemcitabine and radiation. Besides downregulating SOX2 at the protein and RNA level, without altering the stability of SOX2 RNA, PRI-724 significantly modified the transcriptome. STRING analysis of the upregulated and downregulated genes indicates that PRI-724 altered the expression of genes involved in stress-response pathways, including endoplasmic reticulum (ER) stress. Conclusions: The WNT inhibitor PRI-724 downregulates SOX2 expression in a wide range of tumor cell types and blocks the upregulation of SOX2 by clinically used regimens. PRI-724 also modulates the expression of genes associated with stress-response pathways.

The receptor tyrosine kinase AXL has context-dependent roles in prostate cancer (PCa). Although AXL signaling has been implicated in PCa progression, it can also promote disseminated tumor cell dormancy. Paradoxically, AXL expression has been reported to be lost in metastatic PCa. This raises two critical questions: Is AXL loss associated with aggressive disease, and does it create therapeutically exploitable vulnerabilities? In this study, we comprehensively analyzed AXL expression across pan-cancer and PCa cohorts and found that AXL downregulation was significantly associated with treatment resistance, aggressive PCa phenotypes, and shorter overall survival. To investigate the functional consequences of AXL loss, we used CRISPR-Cas9 based gene editing to generate isogenic AXL ^KO cells and characterized their proliferative and migratory properties. To identify therapeutically exploitable vulnerabilities associated with AXL loss, we screened these cells against a library of 1,814 kinase inhibitors using the isogenic cell lines. We identified Torin-2 as a candidate therapeutic agent and validated its activity using dose-response assays, which demonstrated an ∼ 18-fold lower IC ₅₀ in AXL ^KO cells than in AXL ^WT cells. In parallel, PRISM/DepMap analysis provided orthogonal evidence of drug dependencies associated with low AXL expression and revealed a selective vulnerability of AXL ^KO cells to PI3K/AKT/mTOR pathway inhibition. Collectively, our findings link AXL downregulation to aggressive PCa phenotypes and identify mTOR inhibition as a potential therapeutic strategy for AXL-deficient PCa. Significance: AXL downregulation is associated with aggressive PCa, treatment resistance, and shorter overall survival, highlighting its potential clinical significance. By identifying a selective vulnerability of AXL-deficient cells to mTOR inhibition, this study reveals a potential therapeutic strategy for AXL-low PCa. Abstract Figure: Graphical abstract

Prostate cancer (PC) remains a significant health concern as the second leading cause of cancer-related death in American men. Androgen deprivation therapies represent the standard of care for patients with PC, regardless of disease stage. Gonadotropin-releasing hormone (GnRH) antagonists, such as Degarelix, are commonly used hormone therapies for PC and work by suppressing testosterone production. Previous studies indicate a prostate involution-regeneration cycle following surgical castration and testosterone addback. However, whether a similar response occurs following pharmacologic androgen deprivation with agents such as Degarelix remains poorly understood. In particular, the temporal dynamics of prostate involution and subsequent regeneration after chemical castration have not been systematically defined. Our study utilizes immunocompetent mouse models to evaluate the systemic and temporal effects of Degarelix treatment and determine its efficacy in suppressing PC tumor growth. We show that continuous Degarelix treatment significantly suppressed systemic testosterone levels in mice, whereas testosterone levels returned to physiological baseline following Degarelix withdrawal. Gross anatomical, H&E, and Trichrome analyses demonstrated that Degarelix induced castration resulted in prostate involution, characterized by reduced gland size and increased stromal remodeling. This phenotype was reversed after Degarelix withdrawal, highlighting the reversibility of pharmacological androgen deprivation. Immunofluorescence staining revealed changes in basal and luminal epithelial cell populations within the prostatic gland, indicating changes in prostate glandular architecture following Degarelix induced castration and subsequent regeneration. Next, we evaluated the impact of chemical castration on PC tumor growth. Although Degarelix initially suppressed tumor growth, this response was not sustained through the study endpoint, suggesting the emergence of acquired resistance to androgen deprivation. These findings establish this immunocompetent model as a physiologically relevant platform for dissecting the mechanisms that drive therapeutic resistance and tumor progression following androgen deprivation. Our findings highlight the effectiveness of chemical castration with Degarelix and establish a robust model to study both therapeutic responses and the emergence of castration resistance in PC.

Treatment-emergent neuroendocrine prostate cancer (NEPC) is an aggressive, therapy-resistant disease arising in up to 20% of castration resistant prostate cancers, yet robust biologically relevant preclinical models remain scarce. Here, we describe a technical blueprint for establishing an integrated platform of patient-derived models from visceral and bone metastases collected through a prostate cancer rapid autopsy program (PC RAP). We report the establishment and characterization of patient-derived xenograft (PDX) models from liver metastasis tissue, liver and bone metastasis-derived organoid lines (PDOs), and corresponding patient-derived organoid xenograft (PDOX) models. In addition, we established, to our knowledge, the first mesenchymal stem cell (MSC) cultures derived from neuroendocrine prostate cancer (NEPC) bone metastases. The PDOs preserved intratumoral heterogeneity, displaying both CRPC-NE and CRPC-adenocarcinoma features. These organoids retained neuroendocrine identity across multiple passages, with transcriptomic profiles concordant with the original patient tissue and matched PDX models generated at our institution and at the National Cancer Institute (NCI Patient-Derived Models Repository). To model the bone metastatic microenvironment, we generated novel organoid-based New Approach Methodologies (NAMs) by co-culturing PDOs with iPSC-derived bone marrow organoids, establishing a physiologically relevant vascularized organotypic model of PC bone metastasis. To extend our studies in vivo, we established preclinical models using the liver and bone metastasis-derived organoid models. The PDOX models were tumorigenic and developed spontaneous lymph node metastases, providing clinically relevant models for investigating lethal NEPC biology. Together, these complementary patient-derived models provide a robust and versatile platform for investigating NEPC biology, metastatic progression, and evaluating new therapeutic strategies.

SUMMARY: Treatment-emergent neuroendocrine prostate cancer (NEPC) is an aggressive, therapy-resistant disease arising in up to 20% of castration resistant prostate cancers, yet robust biologically relevant preclinical models remain scarce. Here, we describe a technical blueprint for establishing an integrated platform of patient-derived models from visceral and bone metastases collected through a prostate cancer rapid autopsy program (PC RAP). We report the establishment and characterization of patient-derived xenograft (PDX) models from liver metastasis tissue, liver and bone metastasis-derived organoid lines (PDOs), and corresponding patient-derived organoid xenograft (PDOX) models. In addition, we established, to our knowledge, the first mesenchymal stem cell (MSC) cultures derived from neuroendocrine prostate cancer (NEPC) bone metastases. The PDOs preserved intratumoral heterogeneity, displaying both CRPC-NE and CRPC-adenocarcinoma features. These organoids retained neuroendocrine identity across multiple passages, with transcriptomic profiles concordant with the original patient tissue and matched PDX models generated at our institution and at the National Cancer Institute (NCI Patient-Derived Models Repository). To model the bone metastatic microenvironment, we generated novel organoid-based New Approach Methodologies (NAMs) by co-culturing PDOs with iPSC-derived bone marrow organoids, establishing a physiologically relevant vascularized organotypic model of PC bone metastasis. To extend our studies in vivo, we established preclinical models using the liver and bone metastasis-derived organoid models. The PDOX models were tumorigenic and developed spontaneous lymph node metastases, providing clinically relevant models for investigating lethal NEPC biology. Together, these complementary patient-derived models provide a robust and versatile platform for investigating NEPC biology, metastatic progression, and evaluating new therapeutic strategies. Graphical Highlights: Novel preclinical models of visceral and bone metastases established from a prostate cancer rapid autopsy program. This study is the first to establish mesenchymal stem cell cultures from NEPC bone metastases. PDOs preserve heterogeneity, showing both CRPC-NE and CRPC-Adeno features, with transcriptomic profiles concordant with originator tissue and PDX models. PC RAP-derived organoids are tumorigenic in vivo and generate spontaneous lymph node metastases.

Compositional analysis of microbial communities following antibiotic ablation. A, LEfSe analysis demonstrating enrichment of specific bacterial taxa at the phylum level in antibiotic-receiving (Abx) leukemic mice and water-receiving (water) leukemic mice at 3 weeks after engraftment. B, Predicted microbial function pathways at the phylum level in antibiotic-receiving leukemic mice and water-receiving mice at 3 weeks after engraftment. LDA, linear discriminant analysis.

Figure S1. Confirmation of continuous antibiotic delivery in the antibiotic-mediated gut microflora ablation model.

Compositional analysis of microbial communities as CLL disease progresses. A, LEfSe analysis demonstrating enrichment of specific bacterial taxa at the phylum level in transgenic Eμ-TCL1 mice (n = 12) and WT B6 mice (n = 10) at 12 months of age. LDA, linear discriminant analysis. B, Predicted microbial function pathways at the phylum level in transgenic Eμ-TCL1 mice and WT B6 mice at 12 months based on 16S rRNA sequencing data analyzed using ggPICRUSt2. ECM, extracellular matrix.

CLL alters gut bacterial diversity and generates a dysbiotic microbiome compared with WT. A, Study schematic illustrating transgenic Eμ-TCL1 and WT B6 mice were monitored with serial blood and fecal pellet sample collection over 12 months. DNA isolated from fecal pellets was subject to 16S rRNA sequencing at the indicated time points. At study end, mice were sacrificed, and tissues were harvested (n = 7–13 mice/genotype). B, CLL disease burden was measured by the percentage of CD45+/CD19+/CD5+ cells in the peripheral blood via flow cytometric analysis. Asterisks denote the significance between Eμ-TCL1 mice over disease course (*, P < 0.05; **, P < 0.01). Hashtags denote the significance between WT B6 and Eμ-TCL1 mice at each time point (##, P < 0.01; ###, P < 0.001). Two-way ANOVA with Dunnett multiple comparisons was applied for testing. C, Alpha diversity measured by the Shannon diversity index in transgenic Eμ-TCL1 mice compared with age-matched WT B6 mice at 7, 10, and 12 months (*, P < 0.05). Data are presented as box-and-whisker plots, with box edges representing the 25th and 75th percentiles, the center line showing the median value, and whiskers extending to minimum and maximum values. Asterisks denote the significance between WT B6 and Eμ-TCL1 mice at each time point. Unpaired Welch t test was applied for testing. D, PCoA plot depicting Bray–Curtis distance as a measure of beta diversity between transgenic Eμ-TCL1 mice (blue) and age-matched WT B6 mice (red) at all time points (4 – 12 months). E, Alpha diversity measured by the Shannon diversity index in transgenic Eμ-TCL1 mice over time (4–12 months). Data are presented as box-and-whisker plots, with box edges representing the 25th and 75th percentiles, the center line showing the median value, and whiskers extending to minimum and maximum values. F, PCoA plot depicting Bray–Curtis distance as a measure of beta diversity in transgenic Eμ-TCL1 mice over time (4–12 months). G, Relative abundance bar plots of bacterial taxa present in the gut microbiome of Eμ-TCL1 and WT B6 mice over 12 months. Sequences with a quality score of Q28 and higher were assembled for paired-end sequencing using default parameters of Phredd33 encoding and included in the analysis. Per these parameters, a single 4-month WT B6 sample met this criterion and was retained during this step in the analysis. Additional WT B6 samples in the three subsequent time points (7, 10, and 12 months) passed this quality control check and as such were included. Remainder includes all remaining taxa present in the microbiome at decreased abundance.

Figure S4. Evidence of intestinal barrier disturbances in Eµ-TCL1 mice with advanced disease.