22Rv1 Cell Lines Research Articles

Fine Mapping Regulatory Variants by Characterizing Native CpG Methylation with Nanopore Long-Read Sequencing.

5-methylcytosine (5mC) is the most common chemical modification occurring on the CpG sites across the human genome. Bisulfite conversion combined with short-read whole genome sequencing can capture and quantify the modification at single nucleotide resolution. However, the PCR amplification process could lead to duplicative methylation patterns and introduce 5mC detection bias. Additionally, the limited read length also restricts co-methylation analysis between distant CpG sites. The bisulfite conversion process presents a significant challenge for detecting variant-specific methylation due to the destruction of allele information in the sequencing reads. To address these issues, we sought to characterize the human methylation profiling with the nanopore long-read sequencing, aiming to demonstrate its potential for long-range co-methylation analysis with native modification call and intact allele information retained. In this regard, we first analyzed the nanopore demo data in the adaptive sampling sequencing run targeting all human CpG islands. We applied the linkage disequilibrium (LD) R2 to calculate the co-methylation in nanopore data, and further identified 27,875, 50,481, 26,542 and 51,189 methylation haplotype blocks (MHB) in COLO829, COLO829BL, HCC1395 and HCC1395BL cell lines, respectively. Interestingly, while we found that majority of the co-methylation were in a short range (≤200bp), a small portion (1~3%) showed long distance (≥1,000bp), suggesting potential remote regulatory mechanisms across the genome. To further characterize the epigenetic changes related to transcription factor binding, we profiled the 5mC percentage changes surrounding various motif sites in JASPAR collection and found that CTCF and KLF5 binding sites showed reduced methylation, while FOXE1 and ZNF354A sites showed increased methylation. To further investigate the allele-specific 5mCG in the prostate genome, we designed a target region covering methylation quantitative trait loci (mQTL) and genome-wide association study (GWAS) risk germline variants and generated long reads with adaptive sampling run in the 22Rv1 cell line. To identify the allele-specific methylation in the 22Rv1 cell line, we performed long-read based phasing and compared the 5mCG signals between the two haplotypes. As a result, we identified 6,390 haplotype-specific methylated regions in the 22Rv1 cell line (p-MWU ≤ 1e-5 and delta ≥ 50%). By examining haplotype-specific methylated regions near the phasing variants, we identified examples of allele-specific methylated regions that showed allelespecific accessibility in the ATAC-seq data. By further integrating the ATAC-seq data of 22Rv1, we found that methylation levels were negatively correlated with chromatin accessibility at the genome-wide scale. Our study has revealed native methylome profiling while preserving haplotype information, offering a novel approach to uncovering the regulatory mechanisms of the human prostate genome.

Design, synthesis and biological evaluation of dual inhibitors targeting AR/AR-Vs and PARP1 in castration resistant prostate cancer therapy

The combination of androgen signaling inhibitors and PARP inhibitors has shown promising results in clinical trials for the treatment of castration-resistant prostate cancer (CRPC). Multi-target inhibitors can inhibit tumors through different pathways, addressing the limitations of traditional single target inhibitors. We designed and synthesized dual inhibitors targeting AR/AR-Vs and PARP1 using a pharmacophore hybridization strategy. The most potent compound, II-3, inhibits AR/AR-Vs signaling and induces DNA damage by inhibiting PARP1. The IC50 values of II-3 in the castration-resistant prostate cancer cell lines 22RV1 and C4–2 are 4.38 ± 0.56 µM, and 3.44 ± 0.63 µM, respectively. II-3 not only suppresses the proliferation and migration of 22RV1 and C4–2 cells, but also promotes their apoptosis. Intraperitoneal injection of II-3 effectively inhibits tumor growth in 22RV1 xenograft nude mice without evident drug-induced toxicity. Overall, a series of novel dual inhibitors targeting AR/AR-Vs and PARP1 were designed and synthesized, and meanwhile the in vivo and in vitro effects were comprehensively explored, which provided a potential new therapeutic strategy for CRPC.

Design, Synthesis, and Biological Evaluation of Potent EZH2/LSD1 Dual Inhibitors for Prostate Cancer.

As histone modification enzymes, EZH2 mediates H3K27 trimethylation (H3K27me3), whereas LSD1 removes methyl groups from H3K4me1/2 and H3K9me1/2. Synergistic anticancer effects of combining inhibitors of these two enzymes are observed in leukemia and prostate cancer. Thus, a series of EZH2/LSD1 dual inhibitors are designed and synthesized to evaluate their anticancer activity. After the structure-activity study, one of the best compounds, ML234, displayed excellent antiproliferative capacity against prostate cancer cell lines LNCAP, PC3, and 22RV1. Enzymatic assays ascertained that the anticancer potency of ML234 was mediated through coinhibition of EZH2 and LSD1. Moreover, the accumulation of H3K4me2 and H3K9me2 and the decrease of H3K27me3 induced by ML234 were verified by Western blot analysis. More importantly, the compound remarkably suppressed the tumor growth and enhanced the therapeutic efficacy of clinical drug enzalutamide in the 22RV1 xenograft mouse model, indicating that it may have potential as an anticancer agent in prostate cancer.

Structurally Diverse Secondary Metabolites from a Deep-Sea Derived Cladosporium sp. SCSIO 41318and Their Biological Evaluation.

Four new compounds, including one drimane sesquiterpene lactone (1), one isocoumarin (2), one coumarin (3), and a new natural product (4), as well as fourteen known compounds were obtained from a deep-sea derived Cladosporium sp. SCSIO 41318. The structures of the new compounds were determined using extensive NMR and HRESIMS spectroscopic analysis, electronic circular dichroism calculations, and single-crystal X-ray diffraction measurements. Biological assays showed that compounds (1, 6, 7, 9-12, 14, 15, 17, 18) exhibited varying degrees of antimicrobial activity against the tested human pathogenic bacteria and plant pathogenic fungi. Besides, penicitrinone A (11) and penicitrinol A (12) displayed weak antitumor activities against the 22Rv1 cell line.

Multi-Omics Analysis of Primary Prostate Cancer Datasets Reveals Novel Biomarkers.

Prostate cancer (PCa) ranks second in cancer-related deaths in men. Current screenings used in the diagnosis are not sufficient enough in the early stages therefore, more diagnostic biomarker studies are needed. We performed a meta-analysis on the biomarker potential of miRNAs, mRNAs, and methylation for the early stages of PCa by searching available microarrays from the GEO dataset for PCa tissue and benign prostatic hyperplasia (BPH) or normal adjacent to PCa. Target genes of miRNAs were determined using the miRWalk and miRDB datasets. The results were visualized using network analysis. qPCR quantification of potential miRNA and genes was performed in human prostate epithelial cell line (RWPE-1) and human prostate carcinoma epithelial cell line (22RV1). Our meta-analysis of potential biomarkers for the diagnosis of PCa identified several candidates. It was shown that miR-7-5p is overexpressed. CAMKK2, TMEM97 expression were upregulated and CLIP1 expression was downregulated and these genes were shown to be targets of miR-7-5p. CAMKK2, TMEM97, and CLIP1 genes were found to be hypermethylated. Although the changes in the expression levels of miR-7-5p and CAMKK2, TMEM97, and CLIP1 in the two cell lines used in our study were not consistent with the significant expression differences observed in the meta-analysis, our meta-analysis results would be promising in human prostate tissue or different human tumor cell line studies. This highlights the importance of our meta-analysis results in prostate cancer biomarker research, given the difficulty of experimental validation of our large-scale data meta-analysis results using a specific cell line.

The polyunsaturated fatty acid docosahexaenoic affects mitochondrial function in prostate cancer cells

BackgroundProstate cancer (PCa) shows a rewired metabolism featuring increased fatty acid uptake and synthesis via de novo lipogenesis, both sharply related to mitochondrial physiology. The docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid (PUFA) that exerts its antitumoral properties via different mechanisms, but its specific action on mitochondria in PCa is not clear. Therefore, we investigated whether the DHA modulates mitochondrial function in PCa cell lines.MethodsHere, we evaluated mitochondrial function of non-malignant PNT1A and the castration-resistant (CRPC) prostate 22Rv1 and PC3 cell lines in response to DHA incubation. For this purpose, we used Seahorse extracellular flux assay to assess mitochondria function, [14C]-glucose to evaluate its oxidation as well as its contribution to fatty acid synthesis, 1H-NMR for metabolite profile determination, MitoSOX for superoxide anion production, JC-1 for mitochondrial membrane polarization, mass spectrometry for determination of phosphatidylglycerol levels and composition, staining with MitoTracker dye to assess mitochondrial morphology under super-resolution in addition to Transmission Electron Microscopy, In-Cell ELISA for COX-I and SDH-A protein expression and flow cytometry (Annexin V and 7-AAD) for cell death estimation.ResultsIn all cell lines DHA decreased basal respiratory activity, ATP production, and the spare capacity in mitochondria. Also, the omega-3 induced mitochondrial hyperpolarization, ROS overproduction and changes in membrane phosphatidylglycerol composition. In PNT1A, DHA led to mitochondrial fragmentation and it increased glycolysis while in cancer cells it stimulated glucose oxidation, but decreased de novo lipogenesis specifically in 22Rv1, indicating a metabolic shift. In all cell lines, DHA modulated several metabolites related to energy metabolism and it was incorporated in phosphatidylglycerol, a precursor of cardiolipin, increasing the unsaturation index in the mitochondrial membrane. Accordingly, DHA triggered cell death mainly in PNT1A and 22Rv1.ConclusionIn conclusion, mitochondrial metabolism is significantly affected by the PUFA supplementation to the point that cells are not able to proliferate or survive under DHA-enriched condition. Moreover, combination of DHA supplementation with inhibition of metabolism-related pathways, such as de novo lipogenesis, may be synergistic in castration-resistant prostate cancer.

Identify Regulatory eQTLs by Multiome Sequencing in Prostate Single Cells.

While genome-wide association studies and expression quantitative trait loci (eQTL) analysis have made significant progress in identifying noncoding variants associated with prostate cancer risk and bulk tissue transcriptome changes, the regulatory effect of these genetic elements on gene expression remains largely unknown. Recent developments in single-cell sequencing have made it possible to perform ATAC-seq and RNA-seq profiling simultaneously to capture functional associations between chromatin accessibility and gene expression. In this study, we tested our hypothesis that this multiome single-cell approach allows for mapping regulatory elements and their target genes at prostate cancer risk loci. We applied a 10X Multiome ATAC + Gene Expression platform to encapsulate Tn5 transposase-tagged nuclei from multiple prostate cell lines for a total of 65,501 high quality single cells from RWPE1, RWPE2, PrEC, BPH1, DU145, PC3, 22Rv1 and LNCaP cell lines. To address data sparsity commonly seen in the single-cell sequencing, we performed targeted sequencing to enrich sequencing data at prostate cancer risk loci involving 2,730 candidate germline variants and 273 associated genes. Although not increasing the number of captured cells, the targeted multiome data did improve eQTL gene expression abundance by about 20% and chromatin accessibility abundance by about 5%. Based on this multiomic profiling, we further associated RNA expression alterations with chromatin accessibility of germline variants at single cell levels. Cross validation analysis showed high overlaps between the multiome associations and the bulk eQTL findings from GTEx prostate cohort. We found that about 20% of GTEx eQTLs were covered within the significant multiome associations (p-value ≤ 0.05, gene abundance percentage ≥ 5%), and roughly 10% of the multiome associations could be identified by significant GTEx eQTLs. We also analyzed accessible regions with available heterozygous SNP reads and observed more frequent association in genomic regions with allelically accessible variants (p = 0.0055). Among these findings were previously reported regulatory variants including rs60464856-RUVBL1 (multiome p-value = 0.0099 in BPH1) and rs7247241-SPINT2 (multiome p-value = 0.0002- 0.0004 in 22Rv1). We also functionally validated a new regulatory SNP and its target gene rs2474694-VPS53 (multiome p-value = 0.00956 in BPH1 and 0.00625 in DU145) by reporter assay and SILAC proteomics sequencing. Taken together, our data demonstrated the feasibility of the multiome single-cell approach for identifying regulatory SNPs and their regulated genes.

Antitumor aspochalasin and antiviral benzofuran derivatives from a marine-derived fungus Aspergillus sp. SCSIO41032

Chemical investigation of the EtOAc extract of a deep-sea derived fungus Aspergillus sp. SCSIO41032 resulted in the isolation of ten known compounds, including eight aspochalasins. Their structures were elucidated by using extensive NMR spectroscopic, mass spectrometric and single crystal X-ray diffraction analysis. The detailed crystallographic data for structures 1, 2, and 4, along with the relative configurations of aspochalasin E (3) determined by its acetonide derivative were reported for the first time. The results of antitumor and antiviral activities showed that 3 displayed moderate antitumor activities against 22Rv1, PC-3, A549, and HCT-15 cell lines with IC50 values ranged from 5.9 ± 0.8 to 19.0 ± 7.7 μM, and 9 exhibited moderate antiviral activities against HSV-1/2 with EC50 values of 9.5 ± 0.5 and 5.4 ± 0.6 μM, respectively. Plate clone formation assays results indicated that 3 inhibited the 22Rv1, PC-3 cells growth in a dose-dependent manner.

Novel selective agents for the degradation of AR/AR-V7 to treat advanced prostate cancer

The androgen receptor AR antagonists, such as enzalutamide and apalutamide, are efficient therapeutics for the treatment of prostate cancer (PCa). Even though they are effective at first, resistance to both drugs occurs frequently. Resistance is mainly driven by aberrations of the AR signaling pathway including AR gene amplification and the expression of AR splice variants (e.g. AR-V7). This highlights the urgent need for alternative therapeutic strategies. Here, a total of 24 compounds were synthesized and biologically evaluated to disclose compound 20i, exhibiting potent AR antagonistic activities (IC50 = 172.85 ± 21.33 nM), promising AR/AR-V7 protein degradation potency, and dual targeting site of probably AR (ligand-binding domain, LBD and N-terminal domain, NTD). It potently inhibits cell growth with IC50 values of 4.87 ± 0.52 and 2.07 ± 0.34 μM in the LNCaP and 22RV1 cell lines, respectively, and exhibited effective tumor growth inhibition (TGI = 50.9 %) in the 22RV1 xenograft study. These data suggest that 20i has the potential for development as an AR/AR-V7 inhibitor with degradation ability to treat advanced prostate cancer.

Abstract LB161: Discovery of an oral, potent, and selective CDK9 molecular glue degrader SLX-3065 active in aggressive variant prostate cancers (AVPC)

Abstract Background: The use of new potent targeted therapies in prostate cancers has led to the resistance and development of highly aggressive variant prostate cancers (AVPC), including neuroendocrine prostate cancers (NEPC). N-MYC and C-MYC oncogenes overexpress in all aggressive prostate cancers (PCa). This overexpression is highly prevalent in these lethal subtypes of cancers, detected in 2% of all Prostate Cancers and over 10-17% of mCRPC patients. Transcriptional targets of CDK9 are MYCs, which act as an oncogenic driver, sufficient to transform human-derived prostate cancer cells to take on AVPC and NEPC phenotypic changes. Additionally, N-MYC and C-MYC play key roles in tumor drug resistance, and their downregulation, through CDK9 inhibition or degradation lowers tumor growth. Our efforts have demonstrated that targeting drivers of NEPC and mCRPC with highly selective CDK9 molecular glue degraders SLX-3064 SLX-3065 was designed from reversible SLX-3039 for therapeutic intervention of mCRPC, AVPC, and NEPCs. Materials and Methods: We employed our MolecuLernTM-trained molecular glue library design strategies and in vitro CDK9 kinase binding assays. Other experiments include cellular efficacies of single-agent Molecular Glue degraders and their effect in combination experiments on 22Rv1, LASCPC-01, C4-2, and C4-2B MYC expressed PCa cells. Kinase selectivity, Nano BRET, ADME-Tox, in vitro safety, secondary pharmacological assays, Cell engraftment mouse tumor mode, and PK studies were performed. Results: We designed and synthesized a series of novel small molecule CDK9 Molecular Glue degraders based on our initial CDK9 inhibitor lead SLX-3030 and SLX-3039. The SLX-3064 and SLX-3065 reversibly bind to CDK9 with an IC50 of 9 and 7 nM in kinase binding, in target engagement assays, elicit pharmacological responses in N-MYC, C-MYC, driven prostate cancer cells in vitro in LASCPC-01 (21, 35 nM), 22Rv1 (108, 236 nM), C4-2B (15, 12 nM), C4-2 (2.7, 1.5 nM) and VCap (86, 73 nM) cell lines. SLX-3064 and SLX-3065 exhibited potent activity degrading total, pCDK9, MYCs, Mcl-1, and its downstream RNA Pol II, Ser 2, 5, 7, and pAR Ser-81 in a dose-dependent manner. Additionally, we performed in vitro ADME-Tox, hERG, P450, safety panel screen, and in vivo PK experiments in rodent and beagle dog species. Conclusion: In summary, both SLX-3064 and SLX-3065 molecular glues display complete degradation of CDK9 and its downstream markers, and these degraders possess ideal developable criteria as oral agents. With these characteristics, SLX-3065 was selected as a candidate that had the appropriate developable properties and our plans for conducting additional IND-enabling studies and future clinical trial plans will also be discussed. Citation Format: Chenyu Lin, Zhaoliang Li, Kyle Medley, David J. Bearss, Hariprasad Vankayalapati. Discovery of an oral, potent, and selective CDK9 molecular glue degrader SLX-3065 active in aggressive variant prostate cancers (AVPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB161.

Abstract LB259: Cell context-dependent role of KDM5D in ATR and CHK1 inhibitor sensitivity in prostate cancer

Abstract Epigenetic regulation plays an important role in prostate cancer progression and metastasis. KDM5D is a male-specific histone-modifying enzyme encoded on the Y chromosome that demethylates di- and tri-methylated forms of lysine 4 in histone H3, resulting in transcriptional repression of certain genes. In this study, we investigated the role of KDM5D in resistance to inhibitors targeting the G2/M cell cycle checkpoint kinase CHK1. Loss of KDM5D expression was associated with reduced cell proliferation and resistance to the CHK1 inhibitor SRA737 in castration-resistant (CRPC) and neuroendocrine (NEPC) prostate cancer cell lines. In KDM5D expressing cells, depletion of KDM5D differentially impacted the proliferation of prostate cancer cell lines. The androgen-sensitive LNCaP cell line was particularly sensitive to the knockdown of KMD5D. On the other hand, in CRPC cell line 22Rv1, the knockdown of KDM5D caused resistance to both inhibitors, indicating a dual role of KDM5D in mediating ATR and CHK1 inhibitor sensitivity and cell proliferation in prostate cancer cells. Further analysis demonstrated a self-regulatory cycle between CHK1 and KDM5D in 22Rv1 cells, where depletion of KMD5D reduced CHK1 mRNA, while decreased CHK1 caused upregulation of KDM5D and overexpression of KDM5D resulted in increased CHK1 expression. The relevance of this regulatory mechanism to ATR and CHK1 inhibitor sensitivity is currently under investigation, along with several potential mediators of the differential responses to these inhibitors. Additional ATR and CHK1 inhibitors will also be examined in this context. This study may uncover novel biomarkers for ATR and CHK1 inhibitors that are currently in clinical trials for various cancers. KDM5D may be used to stratify prostate cancer patients receiving these highly potent anticancer agents. Citation Format: Wenxiao Zheng, Yuzhou Chen, Qiming J. Wang. Cell context-dependent role of KDM5D in ATR and CHK1 inhibitor sensitivity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB259.

Abstract LB177: Discovery of HC-4955, a novel AR-V7-targeting PROTAC for the treatment of mCRPC

Abstract Prostate cancer is the second most commonly diagnosed cancer and the second leading cause of cancer death among men worldwide. Androgens play an important role in the development and progression of prostate cancers and targeting androgen receptor (AR) signaling axis over decades has remained a mainstay of prostate cancer therapy. Next generation hormonal agents (NHAs) enzalutamide and abiraterone have brought clinical benefits for metastatic castrate-resistant prostate cancer (mCRPC) patients, AR axis-targeted therapy resistance is inevitable due to AR gene amplification or mutations. AR-V7 is the most clinically relevant AR splice variant associated with endocrine resistance and poor prognosis. AR-V7 constitutively activates AR signaling and lacks the ligand binding domain (LBD) where hormones and AR antagonists interact, resulting in resistance to AR signaling inhibitors (ARSi). Thus, AR-V7 is a promising therapeutic target to address ARSi resistance in mCRPC. We have discovered a highly potent AR-V7 proteolysis targeting chimera (PROTAC), HC-4955 that degrades both the wild-type AR (WT-AR) and AR-V7 mutant. HC-4955 degrades AR-V7 with DC50 < 5 nM. In silico molecular docking analysis predicts the binding of HC-4955 to the N-terminal domain of AR and then the essential amino acids contributed to HC-4955 binding are validated by mutational analysis studies. HC-4955 also potently degrades mutants of AR-LBD mutations including L702H and T878A and these mutations are also associated with ARSi resistance. HC-4955 strongly inhibits proliferation of ARSi-resistant prostate cancer cell lines with IC50 < 10 nM. HC-4955 shows a robust anti-tumor activity in the AR-V7-bearing 22Rv1 cell line derived tumor xenograft (CDX) and patient-derived xenograft (PDX) mouse models, with majority of the tumors completely regressed and > 90% AR-V7 expression decreased at 1-5 mg/kg (QD, PO) in these models. The pre-clinical results support the clinical development of HC-4955 for the treatment of mCRPC. Selected pre-clinical data along with the chemical structure of HC-4955 will be presented. Citation Format: Jing Li, Zhilin Tu, Wu Du, Xi Xiang, Zeyu Wen, Yang You, Lijuan Zhao, Yin Chen, Chengcheng Yang, Hongwei Yuan, Xinghai Li. Discovery of HC-4955, a novel AR-V7-targeting PROTAC for the treatment of mCRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB177.

Abstract 2824: Establishing prostate cancer models for orthotopic and bone metastatic growth using the 22Rv1cell line

Abstract Prostate cancer is the second most common cancer in men with new cases diagnosed in 1.4 million men each year. Luckily the 5-year survival rates in developed countries are high reaching over 90%. However, approximately one third will develop a metastatic disease. In metastatic patients, prostate cancer almost always progresses despite the androgen deprivation therapy and is then referred to as “castration-resistant (mCRPC)”. Bone is involved in 80% of patients with metastasis, and the bone lesions are typically osteoblastic. The 22Rv1 human prostate carcinoma cell line is androgen sensitive and shown to express androgen receptor (AR). They also express low levels of prostate-specific membrane antigen (PSMA). The aim of this study was to establish orthotopic and intratibial preclinical in vivo prostate cancer models that can be used in drug development when targeting cancer cells and their local environment. Male athymic nude mice (5-6 weeks old) and NOG mice (over 20 weeks old) were used in the study. 2.5 × 105 22Rv1 cells (ATCC) were inoculated orthotopically into the prostate of NOG mice, and 5 × 105 cells were inoculated intratibially into the bone marrow cavity of athymic nude mice. Tumor growth was followed by PSA measurements every second week using Human PSA ELISA assay. In addition, in the intratibial model, tumor-induced bone changes were monitored by X-ray imaging of the hind limbs. Prostates and hind limbs were collected at sacrifice, fixed in 10% NBF, and processed to paraffin blocks for further histological analysis. Sections of prostate were stained with hematoxylin-eosin (HE) and AR. Tumor-bearing and contralateral (healthy) tibias were stained with HE - OrangeG, MGT, TRAP, and AR. The observed tumor take rate was 92% in the orthotopic and 100% in the intratibial models. Confirmed by IHC, tumors formed in both the orthotopic and intratibial models expressed AR. 22Rv1 cells formed osteoblastic - lytic mixed bone lesions in the intratibial model. Based on lesion areas, randomization of the tumor bearing mice in the intratibial model can be done after 2 weeks from cancer cell inoculation. Taken together, the 22Rv1 prostate cancer models are relatively fast growing, express AR and present good take rates. Furthermore, the intratibial model represents robust tumor-induced changes in bone with strong osteoblastic component. Thus, these models have several advantages in efficacy studies of novel drug candidates for prostate cancer. Citation Format: Justyna Zdrojewska, Katja Fagerlund, Jenni H. Mäki-Jouppila, Mari I. Suominen. Establishing prostate cancer models for orthotopic and bone metastatic growth using the 22Rv1cell line [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 2824.

Abstract 7228: In-vitro efficacy of Dordaviprone/GSK126 combination therapy on castration resistant and neuroendocrine prostate cancer

Abstract Neuroendocrine prostate cancer (NEPC) is an aggressive histologic subtype associated with poor prognosis that commonly arises in later stages as a mechanism of treatment resistance. Activation of oncogenic drivers, in combination with epigenetic changes (such as EZH2 overexpression and DNA methylation) further promotes tumor proliferation and expression of downstream neuroendocrine lineage pathways (in part controlled by transcription factors, including SOX2, ASCL1, and BRN2). Importantly, EZH2 inhibitors (EZH2i) can restore AR expression in CRPC (Ku et al., 2017). Imipridones, including Dordaviprone (ONC201), show promise in treating neuroendocrine tumors by interacting with DRD2 and CLpP (Anderson et al., 2022). These results warrant investigation into the potential for synergism in imipridone/EZH2i combination therapies. In the PCa cell lines LNCap, PC3, 22Rv1, and DU145 the combination of ONC201 and EZH2i GSK126 was assessed. Cell viability data after treatment with ONC201 and GSK126 reveals several dose ranges with potential synergistic activity after 72h. Preliminary western blots after 72 hours of GSK126 treatment indicate modulation of proteins relevant to the mechanism of action of imipridones, specifically DR5 and CLpP, in LNCap, PC3, and 22Rv1. Notably, the decrease in DR5 expression warrants investigation into the effects of EZH2i on TRAIL sensitivity and whether imipridones may rescue DR5 expression to maintain TRAIL sensitivity. Subsequent experiments will investigate dosing and time point-based effects on viability and protein expression, in addition to assessing the synergy of other imipridones and EZH2i’s. Further, while the cell lines PC3, 22Rv1, and DU145 are castration-resistant, the neuroendocrine prostate cell line NCI-H660 will be used as a positive control as it expresses higher neuroendocrine features, including DRD2. Additionally, the modulation in DR5 expression warrants inquiry into the effects of combination therapy on tumor-immune cell interactions. Further, we will manipulate the expression of neuroendocrine transcription factors to assess changes in therapy sensitivity. Citation Format: Connor Purcell, Praveen Srinivasan, Maximilian Pinho-Schwermann, William MacDonald, Elizabeth C. Ding, Vida Tajiknia, Wafik El-Deiry. In-vitro efficacy of Dordaviprone/GSK126 combination therapy on castration resistant and 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 7228.

Abstract 3294: The GSPT1 molecular glue degrader MRT-2359 is active against prostate cancer

Abstract We have previously described our GSPT1 molecular glue degrader MRT-2359, which was optimized to achieve preferential antiproliferative activity in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) expressing high N-MYC or L-MYC mRNA. A clinical trial with this new drug candidate is ongoing (NCT05546268). Here, we present pre-clinical profiling of MRT-2359 across hundreds of cancer cell lines representing multiple cancer types, aiming at identifying other cancers where preferential sensitivity is associated with high expression of a MYC family member. These studies showed that prostate cancer cell lines clearly segregate into groups that are either sensitive or insensitive. Sensitive prostate cancer cell lines could be further divided into two subgroups: (1) androgen receptor positive cell lines displaying higher c-MYC mRNA expression levels than the other prostate cancer cell lines, (2) neuroendocrine prostate cancer cell lines with or without high N-MYC. The latter is in line with our previous observation that the neuroendocrine phenotype is generally associated with heightened sensitivity to MRT-2359 independently of MYC status. The insensitive cell lines were androgen receptor and neuroendocrine negative and displayed lower c-MYC mRNA levels. GSPT1 degradation in androgen receptor-positive cell lines led to rapid and deep loss of c-MYC protein as well as reduction of androgen receptor, including the splice variant AR-V7 that is associated with resistance to agents targeting androgen signaling. In androgen receptor-negative cell lines, the levels of c-MYC protein decreased only modestly upon GSPT1 degradation. Finally, in immunocompromised mice, xenografts of the AR-V7-positive cell line 22RV1 and the neuroendocrine prostate cell line NCI-H660, both of which are sensitive to MRT-2359 in vitro, were treated with several MRT-2359 dose regimens including continuous as well as intermittent (5 days on/9 days off) dosing. Most regimens led to marked tumor regression. Tumors of both models fully regressed after a 4-week course of 10 mg/kg MRT-2359 once daily, and no tumor regrowth could be detected after cessation of treatment. No significant in vivo response was observed for xenografts of the insensitive cell line PC-3. These data support the clinical investigation of MRT-2359 in prostate cancer. Citation Format: Ralph Tiedt, Martin Schillo, Arnaud Osmont, Débora Bonenfant, Rajiv Narayan, Owen Wallace, Markus Warmuth. The GSPT1 molecular glue degrader MRT-2359 is active against 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 3294.

Abstract 4750: The MNK inhibitor EB1 sensitizes AR-V7 positive castration-resistant prostate cancer to enzalutamide

Abstract Introduction: Resistance to androgen receptor signaling inhibitors (ARSI) like Enzalutamide is frequent in castration resistant prostate cancer (CRPC). The mechanisms driving drug resistance include the overexpression of androgen receptor (AR) and the presence of splice variants like androgen receptor splice variant 7 (AR-V7). Clinical studies (PROPHECY) have demonstrated that AR-V7 positive CRPC patients treated with Enzalutamide show poorer overall survival and thus lack an effective targeted line of treatment. Recently, AR signaling and the regulation of protein translation via the translation initiation factor 4E (eIF4E) and MAPK-interacting kinases (MNKs) have been shown to depend on each other and targeting eIF4E has been proposed to increase the efficacy of ARSI. Objectives: We have recently described EB1 as the first in class non-ATP competitive inhibitor of MNK1/2 (Bou-Petit et al. JMC, 2022) and have now tested its applicability in CRPC. In this work we describe the impact of EB1 on the efficacy of Enzalutamide treatment in CRPC. Methodology: Cell lines 22RV1 and LNCaP-95 were used as AR-V7 positive models of CRPC. Effects of EB1 and Enzalutamide alone and in combination on cell growth were studied through crystal violet assay. Drug combination Index was calculated following Chou-Talalay method using CompuSyn Software. Cell death was studied through propidium iodide (PI) staining. AR transcriptional activity was assessed through RT-qPCR of AR canonical target genes like PSA. A head-to-head study with eFT-508 (Tomivosertib, eFFECTOR Therapeutics, Inc.) as the most advanced Type I MNK inhibitor was performed. Lastly, spheroids obtained through centrifugation in ultra-low attachment plates were used for validating drug interaction in 3D growth and viability studies. Spheroid viability was assessed through PI and Calcein AM staining as well as MTT assay. Results: In vitro results demonstrate strong to moderate synergistic inhibition of cell growth through the induction of cell death by the combination of Enzalutamide and EB1 in AR-V7 positive CRPC cell lines, as well as reduction of AR transcriptional activity (PSA). Drug combination also reduced growth and viability of spheroids, indicating good tumor penetrating properties in 3D models and giving in vitro proof-of-concept for in vivo applications. Furthermore, drug synergy with Enzalutamide could only be observed with EB1 and not with Type-I MNK inhibitors like eFT-508, indicating that MNK1/2 functions beyond its kinase activity might be relevant for cancer progression. Conclusion: Here we demonstrate that the novel MNK inhibitor EB1 shows promising in vitro results, which cannot be obtained with Type-I MNK inhibitors. The combination of EB1 with the standard of care Enzalutamide provide a novel therapeutic opportunity for the treatment of CRPC, particularly in AR-V7 positive patients. Citation Format: Sara García-Ortega, Leticia Suárez-Cabrera, Joan Morote, Olga Méndez, Marta Cano-Galietero, Elena Muro-Blanc, Jose I. Borrell, Anna Santamaria, Santiago Ramón y Cajal, Stefan Hümmer. The MNK inhibitor EB1 sensitizes AR-V7 positive castration-resistant prostate cancer to enzalutamide [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 4750.

Abstract 4504: Discovery of CBPD-409 and CBPD-268 as highly potent and orally efficacious CBP/p300 PROTAC degraders for the treatment of castration-resistant prostate cancer

Abstract Prostate cancer remains the leading cause of cancer-related death globally in men. While current AR-targeted therapies are effective for the treatment of prostate cancer and improve patient survival, resistance in patients typically develops within 18 months. CBP/p300 are AR transcriptional co-activators and are promising therapeutic targets for the treatment of castration-resistant prostate cancer (CRPC). We developed a series of highly potent, selective, and orally efficacious CBP/p300 PROTAC degraders, with CBPD-409 being the best compound. CBPD-409 induces robust CBP/p300 degradation with DC50 0.2-0.4 nM and displays strong anti-proliferative effects with IC50 1.2-2.0 nM in VCaP, LNCaP and 22Rv1 AR+ cell lines. It has a favorable oral pharmacokinetic profile and achieves 50% of oral bioavailability in mice. A single oral administration of CBPD-409 at 1 mg/kg achieves >95% depletion of CBP/p300 proteins in the VCaP tumor tissue at 3 h and 24 h time points. CBPD-409 exhibits strong and dose/schedule-dependent tumor growth inhibition and is more potent and efficacious than two CBP/p300 inhibitors CCS1477 and GNE-049 and the AR antagonist Enzalutamide. To further improve the degradation potency and the oral bioavailability in rats, we developed a new class of CBP/p300 degraders based on our optimized CRBN ligand TX-16. Our efforts led to the discovery of CBPD-268 as an exceptionally potent, effective, and orally efficacious degrader of CBP/p300 proteins. In the VCaP, LNCaP and 22Rv1 cell lines, CBPD-268 induces consistent and robust CBP/p300 degradation with DC50 of ≤0.03 nM and Dmax >95%, leading to potent cell growth inhibition. CBPD-268 has excellent oral bioavailability in both mice and rats and has a good ADME profile. Oral administration of CBPD-268 at 0.3-3 mg/kg resulted in profound and persistent depletion of CBP and p300 proteins in tumor tissues and achieved strong antitumor activity in the VCaP and 22Rv1 xenograft tumor models in mice, including tumor regression in the VCaP tumor model. CBPD-268 was well tolerated in mice and rats and displayed a therapeutic index of >10. Taken together, CBPD-409 and CBPD-268 are highly promising CBP/p300 degraders for further extensive evaluations for the treatment of CRPC and other types of human cancers. Citation Format: Zhixiang Chen, Mi Wang, Dimin Wu, Lijie Zhao, Tianfeng Xu, Hoda Metwally, Yu Wang, Donna McEachern, Wei Jiang, Longchuan Bai, Jie Luo, Meilin Wang, Ruiting Li, John Takyi-Williams, Lu Wang, Qiuxia Li, Bo Wen, Duxin Sun, Arul M. Chinnaiyan, Shaomeng Wang. Discovery of CBPD-409 and CBPD-268 as highly potent and orally efficacious CBP/p300 PROTAC degraders for the treatment of castration-resistant 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 4504.

Abstract 4649: BLX-3030, a potent, selective, orally available small molecule CDK9 inhibitor for aggressive variant prostate cancers

Abstract Background: The use of novel, potent targeted therapies in prostate cancers has led to the development of highly aggressive variant cancers, including neuroendocrine prostate cancer (NEPC). Gene amplification of N-MYC and C-MYC oncogenes leads to its overexpression in NEPC and mCRPC patients, highly prevalent in these lethal subtypes of cancers detected in 2% of all Prostate Cancers (PCa) and over 10-17% of mCRPC patients. N-MYC is a transcriptional target of CDK9, which acts as an oncogenic driver sufficient to transform human-derived prostate cancer to take on NEPC phenotypic changes resulting in a more aggressive disease identified in late-stage human PCa patients. Additionally, N-MYC and C-MYC are required for tumor drug resistance, and their downregulation through CDK9 inhibition lowers tumor growth. Our efforts have demonstrated that targeting both N-MYC and C-MYC as a driver of NEPC and mCRPC with highly selective CDK9 inhibitor BLX-3030 is a viable approach for therapeutic intervention of mCRPC and NEPC. Materials and Methods: Our empirical data-driven AI/ML MolecuLernTM fragment library, workflows, LigEdit design strategies, and hit2-lead optimization methods are employed. In vitro experiments include kinase glo, cell titer glo, selectivity profiler, ADME-Tox, FACS/apoptosis, cell migration, live cell imaging, immunofluorescent, western blot, combination studies and in vivo experiments including PK, prostate tumor models, 7-day tox, BLX-3030 non (GMP) scaleup and safety pharmacology experiments conducted. Results: We synthesized a series of novel small molecule CDK9 inhibitors that reversibly bind to CDK9, competitively block the ATP site with an IC50 of 1.06 nM, and elicit pharmacological responses in N-MYC, C-MYC, and other prostate cancer cells in vitro and in vivo. We studied the CDK9 N-MYC expression detected in PC-3, DU-145, LNCaP, and in LASCPC-01, NCI-H660, and 22RV1 cell lines. BLX-3030 exhibited potent cellular efficacy with an IC50 of 22 to 590 nM in PC cell lines and in N-MYC expressed cells had an activity of 76, 470, and 132 nM respectively, and reduced pCDK9, C-MYC, Mcl-1, N-MYC and its downstream RNA Pol II (Ser-2) and pAR (Ser-81) dose-dependently. BLX-3030 exhibits in vivo antitumor activity in LASCPC-01, 22RV1, and C4-2 prostate cancer mouse models. Several ADME-Tox, MTD, dose range finding, hERG and other in vivo safety pharmacology experiments have advanced BLX-3030 to the IND enabling studies. Conclusions: BLX-3030 is currently being investigated in non-clinical toxicology, toxicokinetic, and core battery of safety pharmacology studies including the assessment of effects on cardiovascular, central nervous, and respiratory systems. Citation Format: Zhaoang li, Chenyu Lin, Kyle Medley, Hariprasad Vankayalapati, David J. Bearss. BLX-3030, a potent, selective, orally available small molecule CDK9 inhibitor for aggressive variant prostate cancers [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 4649.

Abstract 3036: Sensitivity of ONC201/TIC10 cancer therapeutic on neuroendocrine prostate cancer (NEPC) and neuroendocrine differentiation (NED)

Abstract Although androgen deprivation therapy and androgen receptor (AR) blockade are highly effective therapies for metastatic prostate cancer (PCa), most patients develop resistance, leading to castration-resistant prostate cancer (CRPC). A subset of CRPC shows neuroendocrine prostate cancer (NEPC) features, which are associated with poor prognosis and limited therapeutic strategies. NEPC is characterized by oncogenic driver activation and epigenetic changes, partly controlled by transcription factors like BRN2 and SOX2. AR has been shown to suppress BRN2 transcription, which is required for NEPC, and BRN2-dependent regulation of the NEPC marker SOX2 (Bishop et al., 2017). Dordaviprone (ONC201/TIC10) is a first-in-class small molecule that antagonizes DRD2, induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and activates the integrated stress response (ISR). Neuroendocrine tumors express significant DRD2 levels, suggesting ONC201 as a potential therapeutic strategy in NEPC or when drivers for disease progression are overexpressed (OE). PCa cell lines PC3, DU145, LNCaP, and 22RV1 are ONC201 sensitive, with IC50=2.87 μM, 3.18 μM, 1.31 μM, and 1.16 μM, respectively (Ding et al., 2023). To characterize the association of BRN2/SOX2 dysregulation with ONC201 sensitivity, we transiently OE BRN2/SOX2 in PCa. We hypothesized that markers of neuroendocrine differentiation (NED), activation of the ISR, TRAIL, and ClpP and dopamine receptor expression contribute to NEPC cell death and sensitivity to ONC201. We performed CellTiter-Glo experiments to assess viability after 96 hours ± BRN2 OE. DU145 cells with BRN2 OE increased their sensitivity to ONC201 (IC50=2.75 µM) compared to empty vector (IC50=3.03 µM). Colony formation assays were conducted in PCa and analyzed 7 days after ONC201 treatment to show ONC201 effect on PCa cell lines. DU145 was treated at 0.5, 1, and 2 µM of ONC201. There were 95 ± 3.22 colonies for the control group, 83 ± 5.22 colonies with 0.5 µM, 74 ± 3.64 colonies with 1 µM, and 8 ± 2.03 colonies with 2 µM ONC201. DU145 showed a significant reduction in colony-forming ability when treated with 1 and 2 uM ONC201 compared to the control, with P values of <0.05 and <0.0001, respectively. We OE BRN2 and observed changes in NED markers and ISR pathway. In PC3, FoxO1 and ATF4 levels increased. In DU145, FoxO1, ENO2, PGP9.5, CgA, and ATF4 levels decreased. We reveal that DU145 OE at 48 hours does not result in greater expression of NEPC markers. BRN2 OE significantly increased PCa cell sensitivity to ONC201 and was evident without an increase in common phenotypical markers used for NEPC assessment (FoxO1, ENO2, PGP9.5, CgA). Expression change was not shown when PCa had an integral AR pathway. Ongoing efforts are observing ONC201 treatment effects on NED markers and the TRAIL pathway due to BRN2 OE and showing increased sensitivity of PCa to ONC201 with BRN2 OE. Citation Format: Elizabeth C. Ding, Maximilian Pinho-Schwermann, Shengliang Zhang, Connor Purcell, Wafik S. El-Deiry. Sensitivity of ONC201/TIC10 cancer therapeutic on neuroendocrine prostate cancer (NEPC) and neuroendocrine differentiation (NED) [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 3036.

Abstract 3238: Therapeutic targeting of the genome guardian sirtuins in metastatic prostate cancer

Abstract Introduction: The effective treatment paradigm for the lethal form of metastatic prostate cancer (mPCa) remains challenging. Almost all mPCas eventually develops therapy resistance against androgen receptor (AR) inhibitors (e.g., Enzalutamide, Abiraterone) or to poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors (Olaparib, Niraparib, Talazoparib) (HRRd) when used as monotherapy. Although the patients in the ARSi+PARPi combination produced significantly longer radiologic progression-free survival (rPFS), a substantial number had attained therapy resistance by the end of the study tenure. So, a newer therapy is urgently needed. In recent years, a new targetable therapeutics development domain has been unfolding with an expanding understanding of the role of SIRTuins in prostate cancer cell survival, invasion, and progression to an aggressive metastatic state. Here, we will investigate the therapeutic efficacy of SIRT1/7 inhibitor (SIRT1/7i) and determine synergy with DNA-damaging PARP inhibitor combinations through a battery of cell and molecular assays using various prostate cancer cellular models. Methods: We have treated the mPCa cell lines 22Rv1, LAPC4, PC3 and LnCAP as well as prostate epithelial cell line RWPE1 with SIRT1 inhibitor SIRT1-IN-2 (MedChemExpress, HY-146689), SIRT7 inhibitor 97491 (MedChemExpress, HY-135899) and AR inhibitor Enzalutamide individually as well as in combination of PARP inhibitor Olaparib. The treatment of the SIRT1 and SIRT7 monotherapy arm was 72 hours long, and combination therapy continued for 7 days. The cell viability was assessed with CellTiter-Glo® 2.0 Cell Viability Assay (Promega). The cell cycle progression and proliferation analysis were done using flow cytometry through Propidium iodide staining and AnnexinV-FITC apoptosis detection chemistry. Cell survival was orthogonally assessed using colony counting assays. We adopt RAD51 foci counting assays and COMET assays for evaluating treatment-induced genome instability. Results: The preliminary result suggested that the SIRT1i+Olaparib and SIRT7i+Olaparib combination significantly sensitized the mPCa cell lines 22Rv1, PC3, and LnCAP for cell death compared to the prostate epithelial cell RWPE1. Upon the combination treatment, the mPCa cell lines showed elevated apoptotic cell death, reconfirmed through colony counting assay. Conclusion: Therapeutic combination treatment of SIRT1 and SIRT7 inhibition with Olaparib could be an effective treatment option for aggressive metastatic prostate cancer. Citation Format: Subhajit Giri. Therapeutic targeting of the genome guardian sirtuins in metastatic 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 3238.