Prostaglandin Reductase Research Articles

Identification of novel molecules and pathways associated with fascin actin‑bundling protein 1 in laryngeal squamous cell carcinoma through comprehensive transcriptome analysis.

Laryngeal squamous cell carcinoma (LSCC) is a common malignant tumor with a poor prognosis. Fascin actin‑bundling protein 1 (FSCN1) has been reported to play a crucial role in the development and progression of LSCC; however, the underlying molecular mechanisms remain unknown. Herein, a whole transcriptome microarray analysis was performed to screen for differentially expressed genes (DEGs) in cells in which FSCN1 was knocked down. Atotal of 462 up and 601 downregulated mRNA transcripts were identified. Functional annotation analysis revealed that these DEGs were involved in multiple biological functions, such as transcriptional regulation, response to radiation, focal adhesion, extracellular matrix‑receptor interaction, steroid biosynthesis and others. Through co‑expression and protein‑protein interaction analysis, FSCN1 was linked to novel functions, including defense response to virus and steroid biosynthesis. Furthermore, crosstalk analysis with FSCN1‑interacting proteins revealed seven DEGs, identified as FSCN1‑interacting partners, in LSCC cells, three of which were selected for further validation. Co‑immunoprecipitation validation confirmed that FSCN1 interacted with prostaglandin reductase 1 and 24‑dehydrocholesterol reductase (DHCR24). Of note, DHCR24 is a key enzyme involved in cholesterol biosynthesis, and its overexpression promotes the proliferation and migration of LSCC cells. These findings suggest that DHCR24 is a novel molecule associated with FSCN1 in LSCC, and that the FSCN1‑DHCR24 interaction may promote LSCC progression by regulating cholesterol metabolism‑related signaling pathways.

The human PTGR1 gene expression is controlled by TE-derived Z-DNA forming sequence cooperating with miR-6867-5p.

Z-DNA, a well-known non-canonical form of DNA involved in gene regulation, is often found in gene promoters. Transposable elements (TEs), which make up 45% of the human genome, can move from one location to another within the genome. TEs play various biological roles in host organisms, and like Z-DNA, can influence transcriptional regulation near promoter regions. MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that play a critical role in the regulation of gene expression. Although TEs can generate Z-DNA and miRNAs can bind to Z-DNA, how these factors affect gene transcription has yet to be elucidated. Here, we identified potential Z-DNA forming sequence (ZFS), including TE-derived ZFS, in the promoter of prostaglandin reductase 1 (PTGR1) by data analysis. The transcriptional activity of these ZFS in PTGR1 was confirmed using dual-luciferase reporter assays. In addition, we discovered a novel ZFS-binding miRNA (miR-6867-5p) that suppressed PTGR1 expression by targeting to ZFS. In conclusion, these findings suggest that ZFS, including TE-derived ZFS, can regulate PTGR1 gene expression and that miR-6867-5p can suppress PTGR1 by interacting with ZFS.

RNA interference confirmed a close association of CYP, ApD, and UCH with the function of CpG ODNs in Litopenaeus vannamei

CpG oligonucleotides (CpG ODNs) have been demonstrated to enhance the innate immune response in various organisms, including Litopenaeus vannamei, a species of significant economic importance. However, the precise mechanism or the genetic factors of CpG ODNs underlying function in L. vannamei remained elusive. Our previous experiment has identified five potential marker genes, including cytochrome P450 (CYP), gamma-butyrobetaine dioxygenase (γ-BDO), apolipoprotein D (ApD), prostaglandin reductase 1 (PGR1), and ubiquitin carboxyl-terminal hydrolase (UCH). In this study, RNA interference (RNAi) was employed to validate the relevance of these five genes in responding the stimulation of CpG ODNs. Briefly, shrimps were fed CpG ODNs and subsequently injected dsRNA targeting these five genes for interference. Finally, the non-specific immune parameters of shrimps, including the expression of immune-related genes and antioxidant capacity of hepatopancreas, as well as the mortality of shrimps after pathogen attack were assessed. The tissue distribution analysis revealed a high abundance of CYP in the hepatopancreas, γ-BDO in the hemocytes, ApD in the eyestalk, PGR1 in the hemocytes, and UCH in the gonad. After blocking CYP, ApD, and UCH through RNAi, a significant down-regulation of the mRNA expression levels of immune-related genes, including ALF, TNF-α, Toll, and TOR, was observed. Additionally, a notable decline in the activity of antioxidant enzymes, such as CAT, GPX, GST, and SOD, was also observed. Moreover, the mortality rates of shrimps in the CYP, ApD, and UCH inhibited groups significantly increased compared to other treatment groups following exposure to Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VpAHPND) and white spot syndrome virus (WSSV). These results collectively suggest a close association of CYP, ApD, and UCH with the function of CpG ODNs in shrimps which will provide significant theoretical insights into comprehending the mechanism of CpG ODNs and optimizing their practical application in shrimp's aquaculture.

NUCB2 promotes hepatocellular carcinoma cell growth and metastasis by activating the E2F4/PTGR1 axis.

Background: The important role of nucleobindin 2 (NUCB2) in various cancers has been recently recognized. However, its biological functions and regulatory mechanisms in hepatocellular carcinoma (HCC) remain unclear. Methods: The expression level of NUCB2 in HCC was assessed using public databases, immunohistochemistry, and Western blotting. The effects of NUCB2 on cell proliferation and metastasis were investigated using colony formation, EdU, Transwell assays, and an in vivo mouse xenograft model. Regulation of E2F4 by NUCB2 was identified by protein half-life and in vivo ubiquitylation assays. The relationship between E2F4 and prostaglandin reductase 1 (PTGR1) was investigated by qRT-PCR, RT-PCR, and chromatin immunoprecipitation assays. Results: This study found that NUCB2 expression was significantly higher in HCC tissues than in normal liver tissues, and patients with high expression displayed shorter survival rates. Inhibition of NUCB2 reduced the proliferation and metastatic potential of HCC cells in vitro and in vivo. NUCB2 depletion reduced PTGR1 expression, which reduced cell proliferation and migration. Our findings suggested that NUCB2 suppressed E2F4 degradation by interacting with E2F4. Additionally, increased E2F4 levels facilitated PTGR1 transcription by directly binding to the PTGR1 promoter. Conclusion: This study demonstrated the oncogenic properties of NUCB2 in HCC and suggested that NUCB2 facilitates hepatocellular carcinoma progression by activating the E2F4/PTGR1 axis.

Serum proteomic profiling reveals potential inflammatory biomarkers in long-COVID patients: a comparative analysis with healthy controls.

The highly transmissible severe acute respiratory syndrome-Coronavirus-2 was responsible for the 2020 COVID-19 pandemic. COVID-19 mostly affects the respiratory system; however, this infection also affects several other organs. In addition, the sequelae of this disease affect patients for several months after recovery, resulting in long-COVID syndrome. In order to characterize the differences between healthy control individuals and long-COVID patients, proteomic profiling of the serum of both groups was performed by mass spectrometry. The obtained data were analyzed with multivariate and univariate statistical analyses. Initially, performing a partial latent square discriminant analysis (PLS-DA) made it possible to identify thirty-three proteins of interest, which were then subjected to a receiver operating characteristic (ROC) analysis. Four proteins were identified as potential stand-alone biomarkers: Sirtuin 1, Natriuretic Peptide B, Hemopexin, and Arachidonate 5-Lipoxygenase. Moreover, a multivariate ROC analysis identified a panel of biomarkers composed of Natriuretic Peptide B, Anterior Gradient 2 Protein, Adiponectin, Endothelin Converting Enzyme 1, Interferon Induced Transmembrane Protein 1, Mannose Binding Lectin 2, Prostaglandin-Endoperoxide Synthase 2, Pirin, Prostaglandin Reductase 1 and Cystatin C. The identified biomarkers are associated with inflammatory processes, corroborating literature evidence that long-COVID patients develop an inflammatory state that damages many tissues. Nevertheless, these data should be validated in a larger cohort.

Nucleotide excision repair deficiency is a targetable therapeutic vulnerability in clear cell renal cell carcinoma

Due to a demonstrated lack of DNA repair deficiencies, clear cell renal cell carcinoma (ccRCC) has not benefitted from targeted synthetic lethality-based therapies. We investigated whether nucleotide excision repair (NER) deficiency is present in an identifiable subset of ccRCC cases that would render those tumors sensitive to therapy targeting this specific DNA repair pathway aberration. We used functional assays that detect UV-induced 6–4 pyrimidine-pyrimidone photoproducts to quantify NER deficiency in ccRCC cell lines. We also measured sensitivity to irofulven, an experimental cancer therapeutic agent that specifically targets cells with inactivated transcription-coupled nucleotide excision repair (TC-NER). In order to detect NER deficiency in clinical biopsies, we assessed whole exome sequencing data for the presence of an NER deficiency associated mutational signature previously identified in ERCC2 mutant bladder cancer. Functional assays showed NER deficiency in ccRCC cells. Some cell lines showed irofulven sensitivity at a concentration that is well tolerated by patients. Prostaglandin reductase 1 (PTGR1), which activates irofulven, was also associated with this sensitivity. Next generation sequencing data of the cell lines showed NER deficiency-associated mutational signatures. A significant subset of ccRCC patients had the same signature and high PTGR1 expression. ccRCC cell line-based analysis showed that NER deficiency is likely present in this cancer type. Approximately 10% of ccRCC patients in the TCGA cohort showed mutational signatures consistent with ERCC2 inactivation associated NER deficiency and also substantial levels of PTGR1 expression. These patients may be responsive to irofulven, a previously abandoned anticancer agent that has minimal activity in NER-proficient cells.

Molecular Docking and Dynamics Simulations of Ammi visnaga L. Constituents as Antimelanogenic, Anti-Inflammatory and Anticoagulant Agents.

In this study, anti-melanogenic, anti-inflammatory and anti-coagulant potentials of eighteen selected constituents of Ammi visnaga L. were investigated by Induced Fit Docking (IFD) and molecular dynamic simulation with Schrödinger software. The binding free energies of the selected natural compounds were computed by means of ΔG MM-GBSA studies. Anti-melanogetic activity of the constituent against agaricus bisporus tyrosinase, Priestia megaterium tyrosinase and Homo sapiens tyrosinase were evaluated. The result showed that apiumetin had more negative binding free energy against three tyrosinase enzymes than cognate ligands, tropolone and kojic acid. Docking analysis was also performed to predict the constituents with anti-inflammatory activity against human Tumor necrosis factor, Cyclooxygenase-2, Prostaglandin D2 11-ketoreductase AKR1C3 and Prostaglandin reductase PTGR2. The results showed that pyranocoumarins (visnadin, dihydrosamidin, samidin) have more negative binding free energy against Cyclooxygenase-2 and Prostaglandin D2 11-ketoreductase receptors than cognate drugs, rofecoxib and indomethacin. In addition, docking analysis shows that pyranocoumarins, apiumetin and cimifugin have more negative binding free energy against Vitamin K epoxide reductase than S-warfarin drug, predicting that they have anticoagulant activity. Furthermore, the constituents and their cognate drugs were subjected to 100 ns MD Simulation to predict their stability at the active sites of the enzymes.

Conditional Dependency of LP-184 on Prostaglandin Reductase 1 is Synthetic Lethal in Pancreatic Cancers with DNA Damage Repair Deficiencies.

The greater efficacy of DNA-damaging drugs for pancreatic adenocarcinoma (PDAC) relies on targeting cancer-specific vulnerabilities while sparing normal organs and tissues due to their inherent toxicities. We tested LP-184, a novel acylfulvene analog, for its activity in preclinical models of PDAC carrying mutations in the DNA damage repair (DDR) pathways. Cytotoxicity of LP-184 is solely dependent on prostaglandin reductase 1 (PTGR1), so that PTGR1 expression robustly correlates with LP-184 cytotoxicity in vitro and in vivo. Low-passage patient-derived PDAC xenografts with DDR deficiencies treated ex vivo are more sensitive to LP-184 compared with DDR-proficient tumors. Additional in vivo testing of PDAC xenografts for their sensitivity to LP-184 demonstrates marked tumor growth inhibition in models harboring pathogenic mutations in ATR, BRCA1, and BRCA2. Depletion of PTGR1, however, completely abrogates the antitumor effect of LP-184. Testing combinatorial strategies for LP-184 aimed at deregulation of nucleotide excision repair proteins ERCC3 and ERCC4 established synergy. Our results provide valuable biomarkers for clinical testing of LP-184 in a large subset of genetically defined characterized refractory carcinomas. High PTGR1 expression and deleterious DDR mutations are present in approximately one third of PDAC making these patients ideal candidates for clinical trials of LP-184.

Metformin escape in prostate cancer by activating the PTGR1 transcriptional program through a novel super-enhancer

The therapeutic efficacy of metformin in prostate cancer (PCa) appears uncertain based on various clinical trials. Metformin treatment failure may be attributed to the high frequency of transcriptional dysregulation, which leads to drug resistance. However, the underlying mechanism is still unclear. In this study, we found evidences that metformin resistance in PCa cells may be linked to cell cycle reactivation. Super-enhancers (SEs), crucial regulatory elements, have been shown to be associated with drug resistance in various cancers. Our analysis of SEs in metformin-resistant (MetR) PCa cells revealed a correlation with Prostaglandin Reductase 1 (PTGR1) expression, which was identified as significantly increased in a cluster of cells with metformin resistance through single-cell transcriptome sequencing. Our functional experiments showed that PTGR1 overexpression accelerated cell cycle progression by promoting progression from the G0/G1 to the S and G2/M phases, resulting in reduced sensitivity to metformin. Additionally, we identified key transcription factors that significantly increase PTGR1 expression, such as SRF and RUNX3, providing potential new targets to address metformin resistance in PCa. In conclusion, our study sheds new light on the cellular mechanism underlying metformin resistance and the regulation of the SE-TFs-PTGR1 axis, offering potential avenues to enhance metformin’s therapeutic efficacy in PCa.

Exercise intervention during pregnancy induces DNA methylation alterations in maternal blood and cord blood.

Exercise intervention during pregnancy induces DNA methylation alterations in maternal blood and cord blood.

Tumor sensitization to PARP inhibitors by DNA damaging synthetically lethal acylfulvenes.

e15123 Background: 20-30% of solid tumors (ovarian, breast, pancreatic, prostate cancers) harbor some level of deficiency in DNA Damage Repair (DDR) pathways. Such cancers become dependent on alternative DDR pathways to maintain genome integrity, making them selectively vulnerable to DNA damaging agents. The concept of synthetic lethality has been leveraged to treat subsets of such tumors. Clinically, PARP inhibitors (PARPi) have been successful in Homologous Recombination Deficiency (HRD) cancers. LP-100 and LP-184 are acylfulvene (AF) class small molecule prodrugs that are metabolized to active compounds by prostaglandin reductase 1 (PTGR1) that is often elevated in solid tumors. AF-induced DNA lesions are likely selectively processed by repair pathways including BRCA1/2 mediated Homologous Recombination (HR). Methods: Cell Titer Glo based 10 day cell viability assays were used to generate drug IC50 values in vitro. Bliss synergy scores were computed with scores ≥ 10 reflecting synergy. Drug-induced DNA double strand breaks (DSB) were detected and quantified with STRIDE (SensiTive Recognition of Individual DNA Ends) assay in cancer cells. A subcutaneous tumor xenograft model was used to determine tumor volumes in control and drug treated mice. Results: In a BRCA2 mutant ovarian cancer cell line OVCAR3, LP-184 and olaparib co-treatment in vitro showed synergistic effects (Bliss score 10). Similarly in a BRCA2 mutant prostate cancer cell line 22Rv1, LP-100 combination with rucaparib was synergistic (Bliss score 18). 400 nM LP-184 showed a time dependent increase in DNA DSB signals in vitro, 1.7X more so in BRCA2 mutant vs wild type (WT) DLD-1 isogenic colon cancer cell lines up to 24 hours. To validate the hypothesis that AF-induced DSBs remain unresolved if exposed to and further synergize with PARPi in vivo, we tested LP-100 plus olaparib in a DU145 (BRCA2 mutant) prostate cancer xenograft model. LP-100 + olaparib treatment resulted in sustained, durable tumor regression indicating in vivo synergy whereas either agent alone did not produce tumor shrinkage. Day 42 tumor growth inhibition was 66% for 2.5 mg/kg LP-100 alone, 34% for 40 mg/kg olaparib alone and 108% for the combination. Moreover, LP-184 treatment resulted in early onset and complete tumor regression in 7 PDX models of PARPi resistant triple negative breast cancers (TNBC) harboring BRCA1/2 loss whereas olaparib/ niraparib remained entirely ineffective in these. Conclusions: AF response is influenced by tumor DNA repair competence. LP-100 and LP-184 are synthetically lethal resulting from their abilities to cause unresolvable DNA damage where tumor cells express high PTGR1 and are deficient in the HR pathway. LP-184 single agent was superior to PARPi in TNBC and LP-100 combination made an olaparib resistant tumor highly sensitive, thereby facilitating a path to extend the opportunities for both AFs and PARPis and enhance the clinical utilization of PARPi.

Abstract 6241: LP-184, an acylfulvene class small molecule therapeutic, is synthetically lethal in DNA damage repair deficient cancers

Abstract As many as one third of solid tumors (ovarian, breast, pancreatic, prostate cancers) harbor deficiency in some DNA Damage Repair (DDR) pathways. Such cancers become dependent on alternative DDR pathways to maintain genome integrity, making them selectively vulnerable to DDR inhibitors. The concept of synthetic lethality has been successfully applied to develop strategies to treat subsets of such tumors. Clinically, PARP inhibitors (PARPi) have been successful particularly in Homologous Recombination Deficiency (HRD) cancers. Other agents have also been designed to target these DDR deficiencies in cancer cells and have potential to be clinically beneficial. LP-184 is a next generation acylfulvene prodrug metabolized to an active compound by prostaglandin reductase 1 (PTGR1) that is often elevated in multiple tumors. LP-184-induced DNA lesions are likely repaired by the Nucleotide Excision Repair (NER) and Homologous Repair (HR) pathways. Reduced activities of multiple DDR-related pathways in solid tumor models correlated with increased sensitivities to LP-184 as indicated by Gene Set Enrichment Analysis. Chinese Hamster Ovary cells with NER mutations (ERCC1/2/6) were at least 4 fold more sensitive to LP-184 relative to otherwise isogenic parental cells. ERCC2 or BRCA2 depletion enhanced LP-184 sensitivity 5-8X in a metastatic prostate cancer cell line. LP-184 showed ~120 fold higher potency (IC50 77 nM) compared to Olaparib in HRD prostate cancer organoid model LuCaP 96 (BRCA2/CHEK2 inactivating mutations). Across 14 patient-derived pancreatic, lung and prostate tumor models harboring HR mutations (BRCA1/2, CHEK1/2, ATM/ATR, PALB2, PARP1/2, RAD51, FANCA/B), LP-184 showed ex vivo IC50s in the range of 30-300 nM, whereas Olaparib IC50s ranged from 1700-6900 nM. LP-184 treatment resulted in complete tumor regression (107-141% TGI) in 10/10 HR deficient triple negative breast cancer PDX models of which 7/10 were resistant Olaparib/Niraparib and to Doxorubicin/Cyclophosphamide. LP-184 treatment resulted in complete and durable tumor regression in HRD (BRCA1/ATR mutant) pancreatic cancer PDX models (112-140% TGI). In the ovarian cancer cell line OVCAR3 having HRD/NERD phenotype, LP-184 IC50 was 13 nM, Olaparib IC50 was 145 nM and their combination Bliss Synergy Score was 16 indicating synergy. LP-184 is synthetic lethal resulting from its abilities to cause unresolvable DNA damage if tumor cells express high PTGR1 and are deficient in NER/HR genes. Unlike PARPi, LP-184 has striking activity in both NERD as well as HRD cancers. These strong data have prompted the development of a soon to be launched clinical trial to translate the broad preclinical anticancer activity of LP-184 in solid tumors with HR/NER pathway defects, such as pancreatic, prostate, ovarian and breast cancers. Citation Format: Aditya Kulkarni, Jianli Zhou, Neha Biyani, Kishor Bhatia, Panna Sharma. LP-184, an acylfulvene class small molecule therapeutic, is synthetically lethal in DNA damage repair deficient cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6241.

Abstract P4-01-06: LP-184, an acylfulvene class small molecule therapeutic, is synthetically lethal in HR deficient and PARP inhibitor resistant triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer accounting for approximately 15% to 20% of all newly diagnosed breast cancer cases. TNBCs are defined by lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2).Due to its special molecular phenotype, it is not sensitive to endocrine therapy or targeted therapy. Therefore, chemotherapy such as anthracyclines or taxanes is the main systemic treatment, but the efficacy of conventional postoperative adjuvant chemoradiotherapy is poor. ~35% of TNBC tumors show abnormalities in the HR pathway, making them sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) and DNA-damaging agents. Although more than 40% BRCA1/2-deficient patients fail to respond to PARPi and a substantial proportion of patients acquire PARPi resistance over time. TNBCs heterogeneous nature, poor prognosis and limited treatment options presents the urgent need of development of novel agents targeting tumor specific alterations. Lantern Pharma is advancing LP-184, an acylfulvene-derived prodrug that is specifically activated in tumors that overexpress the oxidoreductase enzyme Prostaglandin Reductase 1 (PTGR1), for the treatment of solid tumor indications including TNBC. LP-184 has multiple mechanisms of action (MOA). While It is synthetically lethal in tumors harboring DNA damage repair defects including Homologous Recombination (HR) deficiencies, it can also interrupt transcription. LP-184 efficacy was tested in a panel of breast cancer cell lines and patient derived TNBC xenografts models, both sensitive as well as resistant to PARP inhibitors and anthracyclines. LP-184 demonstrated nanomolar potency in six NCI-60 breast cancer cell lines (median IC50 = 327 nM). Subcutaneous patient-derived TNBC xenograft mouse models were used to determine tumor volume responses to LP-184 treatment in vivo. Xenograft tumors were derived from 10 treatment-naive HR deficient (HRD score > 50) primary TNBC patients with known BRCA1/2 loss of heterozygosity (LOH), 7 of which subsequently progressed on PARP inhibitor Olaparib. LP-184 (4mg/kg i.v., (q2d x 5 then 7 days off)x2) led to complete and durable regression in all 10 TNBC HRD-PDX models tested as compared to control (p < 0.0001). A tumor growth inhibition range of 107-141% was achieved across all the 10 models. For LP-184, T/C at the control group end day was 0% in 10/10 models, whereas across the same models for Olaparib, T/C was 0% in 2/10 models and ranged from 15 - 90% in 8/10 models. Terminal body weight change was only a transient weight loss < 4% with LP-184 treatment across all models. LP-184 exhibited superior potency than olaparib in TNBC PDX models that carry HRD mutations including PARP resistant models and was well tolerated in mice. As acylfulvene-induced damage is primarily repaired by transcription-coupled nucleotide-excision repair (TC-NER) and HR pathways, response to LP-184 is influenced by tumor DNA damage repair pathway status. Recent data highlight an important role of super enhancer driven core transcription regulatory circuits in the pathogenesis of TNBCs. Our results support the superior efficacy of LP-184 in TNBCs, likely linked to the multiple MOAs, and establish LP-184 as a promising new agent for future clinical testing in TNBC patients. We finally propose that LP-184 may be broadly efficacious in solid tumors with HR and/or TC-NER pathway defects, such as pancreatic, prostate, ovarian and bladder cancers. Citation Format: Aditya Kulkarni, Neha Biyani, Laura Brullé-Soumaré, Stefano Cairo, Panna Sharma, Kishor Bhatia. LP-184, an acylfulvene class small molecule therapeutic, is synthetically lethal in HR deficient and PARP inhibitor resistant triple negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-01-06.

Identification and characterization of bioactive metabolites of 12-hydroxyheptadecatrienoic acid, a ligand for leukotriene B4 receptor 2.

12(S)-hydroxyheptadecatrienoic acid (12-HHT) is a bioactive fatty acid synthesized from arachidonic acid via the cyclooxygenase pathway and serves as an endogenous ligand for the low-affinity leukotriene B4 receptor 2 (BLT2). Although the 12-HHT/BLT2 axis contributes to the maintenance of epithelial homeostasis, 12-HHT metabolism under physiological conditions is unclear. In this study, 12-keto-heptadecatrienoic acid (12-KHT) and 10,11-dihydro-12-KHT (10,11dh-12-KHT) were detected as 12-HHT metabolites in the human megakaryocytic cell line MEG01s. We found that 12-KHT and 10,11dh-12-KHT are produced from 12-HHT by 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and prostaglandin reductase 1 (PTGR1), key enzymes in the degradation of prostaglandins, respectively. The 15-PGDH inhibitor SW033291 completely suppressed the production of 12-KHT and 10,11dh-12-KHT in MEG01s cells, resulting in a 9-fold accumulation of 12-HHT. 12-KHT and 10,11dh-12-KHT were produced in mouse skin wounds, and the levels were significantly suppressed by SW033291. Surprisingly, the agonistic activities of 12-KHT and 10,11dh-12-KHT on BLT2 were comparable to that of 12-HHT. Taken together, 12-HHT is metabolized into 12-KHT by 15-PGDH, and then 10,11dh-12-KHT by PTGR1 without losing the agonistic activity.

Abstract B033: LP-184, a tumor site activated small molecule therapeutic, is synthetically lethal in pancreatic cancers with DNA damage repair defects

Abstract Biomarker-based therapeutic strategies associated with increased efficacy, manageable toxicity and extended disease-free survival are urgently needed in pancreatic cancer (PaCa). 15-20% of PaCa carry mutations in DNA repair pathways (BRCA1/BRCA2/PALB2/RAD51/ATR/ATM/FANCD2). Additionally, mutations in nucleotide excision repair (NER) genes (ERCC2/3/4/5/6) have been reported in ~5% of PaCa. LP-184 is an acylfulvene analog that is converted to an active alkylating agent in the strict dependency on the oxidoreductase, prostaglandin reductase 1 (PTGR1). LP-184 induced tumor cell kill is enhanced in the presence of DNA damage repair (DDR) defects. Mutation or expression driven TC-NER and/or HR deficiency is expected to predispose PaCa cells to increased sensitivity to LP-184. To test the idea of LP-184 activity in DNA repair-deficient tumors, we evaluated the anti-tumor effects of LP-184 in mouse subcutaneous PDX models harboring pathogenic frameshift mutations in ATR (CTG-1522 model) or BRCA1 (CTG-1643 model) representing HR deficiency in PaCa. Two cycles of LP-184 treatment in CTG-1522 resulted in 140% tumor growth inhibition (TGI), relative to 80% TGI with gemcitabine in this FOLFIRONOX non-responder. Similarly, complete and durable tumor regression with 112% TGI was observed in CTG-1643 with single agent LP-184. There was <5% body weight loss and no significant hematological toxicity in both these models during or after LP-184 treatment. In selected PaCa models, we determined LP-184 efficacy to be synergistic in combination with multiple treatment modalities including standard of care agent gemcitabine, and ERCC3 degrader spironolactone in vitro, and with radiation therapy (RT) in vivo. Overall Bliss synergy scores (score > 10 indicates synergy) of 12.37 for LP-184 + gemcitabine in Capan-1, and 14.08, 16.47 and 15.52 for LP-184 + spironolactone in Capan-1 (BRCA2 loss), Hs766t (ATR mutant) and Panc03.27 cell lines respectively were achieved. Taking advantage of the transcriptional regulation underlying the NRF2 mediated anti-oxidant response pathway, we reasoned that induction of PTGR1 expression, a downstream NRF2 target gene, in irradiated PaCa tumors would thereafter poise them for LP-184 treatment. Indeed, we found that Panc03.27 xenograft tumors treated with 3 mg/kg i.p. LP-184 + 4 Gy RT once weekly for 3 weeks were significantly smaller than those receiving LP-184 alone or RT alone, thus capitalizing on the opportunity to increase PTGR1 expression (and linked LP-184 anti-tumor cytotoxicity) selectively in irradiated tumors. Increased PTGR1 expression has emerged as a promising biomarker for LP-184 cytotoxicity. Moreover, LP-184 can become synthetically lethal in multiple contexts when combined with genetic or pharmacologic DDR pathway aberrations, or with RT. Based on these properties, we anticipate that LP-184 will extend therapeutic opportunities to a large subset of PaCa patients. Citation Format: Aditya Kulkarni, Diana Restifo, Troy Dos Santos, Dusica Cvetkovic, Joseph McDermott, Kishor Bhatia, Panna Sharma, Charlie Ma, Igor Astsaturov. LP-184, a tumor site activated small molecule therapeutic, is synthetically lethal in pancreatic cancers with DNA damage repair defects [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B033.

Comparative Photoaffinity Profiling of Omega-3 Signaling Lipid Probes Reveals Prostaglandin Reductase 1 as a Metabolic Hub in Human Macrophages.

The fish oil constituent docosahexaenoic acid (DHA, 22:6 n-3) is a signaling lipid with anti-inflammatory properties. The molecular mechanisms underlying the biological effect of DHA are poorly understood. Here, we report the design, synthesis, and application of a complementary pair of bio-orthogonal, photoreactive probes based on the polyunsaturated scaffold DHA and its oxidative metabolite 17-hydroxydocosahexaenoic acid (17-HDHA). In these probes, an alkyne serves as a handle to introduce a fluorescent reporter group or a biotin-affinity tag via copper(I)-catalyzed azide-alkyne cycloaddition. This pair of chemical probes was used to map specific targets of the omega-3 signaling lipids in primary human macrophages. Prostaglandin reductase 1 (PTGR1) was identified as an interaction partner that metabolizes 17-oxo-DHA, an oxidative metabolite of 17-HDHA. 17-oxo-DHA reduced the formation of pro-inflammatory lipids 5-HETE and LTB4 in human macrophages and neutrophils. Our results demonstrate the potential of comparative photoaffinity protein profiling for the discovery of metabolic enzymes of bioactive lipids and highlight the power of chemical proteomics to uncover new biological insights.

MCL-319 LP-284 - A Highly Potent Small Molecule Targeting Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is a rare and aggressive form of B-cell non-Hodgkin lymphoma (NHL). Approximately 40%-50% of MCL patients carry inactivating mutations in the gene ataxia telangiectasia mutated (ATM), a core component of the cell's DNA damage response system. MCL cells with mutations in ATM are known to have increased chromosomal imbalance, copy number loss, and hypersensitivity to DNA-damaging agents. LP-184, a small-molecule DNA-damaging agent, is known to be synthetically lethal in tumors with DNA-repair deficiencies, including tumors with ATM mutations. However, DNA damage by LP-184 is dependent on activation by the oxidoreductase prostaglandin reductase 1 (PTGR1), which is expressed at low levels in many hematological cancers. We synthesized LP-284, an enantiomer of LP-184, which retains synthetic lethality in cancers with impaired DNA damage repair pathway genes (e.g., ERCC1, ERCC2, ERCC3, ERCC6), but LP-284's activity is independent of PTGR1 expression. We demonstrated that LP-284 has enhanced tumoricidal activity in multiple hematological cancer cell lines using a B-cell NHL panel. The IC50 values for all 6 MCL cell lines treated with LP-284 were among the lowest for all hematological cell lines tested with LP-284. These MCL cell lines included cell lines resistant to ibrutinib, zanubrutinib, venetoclax, and bortezomib. Additionally, among the 6 MCL cell lines tested, the 3 cell lines with mutations in the nucleotide excision repair (NER) or homologous recombination (HR) DNA damage repair pathways had lower LP-284 IC50 values than the 3 cell lines without NER or HR mutations. Because ATM orchestrates the activities of the NER and HR pathways, MCL patients with ATM deficiencies are likely to have better responses to LP-284 treatment. Collectively, these data indicate that LP-284 is a promising preclinical DNA-damaging agent that possesses nanomolar-range anti-tumor activities in multiple and diverse MCL cell lines, with the potential to treat MCL patients who are resistant to other therapies.

Abstract 5442: LP-184, a tumor site activated small molecule synthetic lethal therapeutic, is effective in central nervous system cancers

Abstract Blood-brain barrier (BBB) permeable agents effective against recurrent, chemotherapy-resistant central nervous system (CNS) tumors are urgently needed, particularly in Glioblastoma multiforme (GBM) and atypical teratoid/rhabdoid tumors (ATRT). These represent 2 extremely aggressive and lethal types of CNS malignancies sharing a median overall survival of 12-18 months and 5-year survival rate of 5 to 30% in the US. LP-184 has received orphan drug designation for the treatment of malignant gliomas. LP-184 is activated to a highly labile metabolite by Prostaglandin Reductase 1 (PTGR1) and has potential to be a potent therapeutic as a single agent for PTGR1-expressing malignancies. LP-184 was found to have nanomolar range potency (IC50 30-400 nM) against multiple human GBM cell lines and patient-derived neurospheres in vitro. Intravenous LP-184 induced complete and durable regression of pre-established subcutaneous U87 and M1123 GBM xenografts and prolonged survival of mice bearing orthotopic U87 and M1123 xenografts (p<0.0001). Activated LP-184 creates covalent DNA adducts (e.g. N3 of adenine) that are selectively repaired via ERRC-dependent nucleotide excision repair or by homologous recombination. Consistent with this, we observed 2-5X increase in cytotoxicity in U87, Mayo39, M1123 GBM cells treated with LP-184 in combination with the ERCC3 inhibitor Spironolactone relative to LP-184 alone. This supports LP-184’s function as a synthetic lethal agent in the presence of DNA repair deficiencies. > 50% of newly diagnosed GBMs fail to respond to standard of care temozolomide (TMZ) due to MGMT expression (i.e.unmethylated mgmt promoter) and essentially all GBMs ultimately develop TMZ resistance. Our in silico analyses and type of DNA adduct generated by LP-184 indicate that sensitivity to LP-184 is independent of MGMT expression and potentially higher in MGMT unmethylated cancers. This is supported by in vitro toxicity assays showing LP-184 IC50s of 46 nM in LN-18 (unmethylated mgmt) and 203 nM in U87 (hypermethylated mgmt) cell lines. Analysis of genes associated with LP-184 sensitivity in solid tumor cell lines revealed that expression of SMARCB1 is significantly anti-correlated to LP-184 sensitivity, suggesting that LP-184 will be effective in ATRTs and other tumors with SMARCB1 loss. LP-184 treatment induced tumor regression in mice implanted with SMARCB1 deficient CHLA06 ATRT subcutaneous xenografts, with 2 of 10 treated mice being virtually tumor-free after 2 cycles over 22 days. Pharmacokinetic analyses showed favorable in vivo BBB permeability (mouse brain/blood ratio=0.112) comparable to that reported for TMZ. LP-184 was also active in vitro against models of brain metastases from primary lung and breast cancers. These findings identify LP-184 as a promising new agent and support its further development for treating CNS tumors in adult and pediatric populations. Citation Format: Aditya Kulkarni. LP-184, a tumor site activated small molecule synthetic lethal therapeutic, is effective in central nervous system cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5442.

Comparative Transcriptome Analysis Reveals the Process of Ovarian Development and Nutrition Metabolism in Chinese Mitten Crab, Eriocheir Sinensis.

Ovarian development is a key physiological process that holds great significance in the reproduction of the Chinese mitten crab (Eriocheir sinensis), which is an economically important crab species for aquaculture. However, there is limited knowledge for the regulatory mechanisms of ovarian development. To study the molecular mechanisms of its ovarian development, transcriptome analysis was performed in the ovary and hepatopancreas of E. sinensis during ovarian stages I (oogonium proliferation), II (endogenous vitellogenesis), and III (exogenous vitellogenesis). The results showed that 5,520 and 226 genes were differentially expressed in the ovary and hepatopancreas, respectively. For KEGG enrichment analysis, the differentially expressed genes in the ovary were significantly clustered in phototransduction-fly, phagosome, and ECM-receptor interaction. Significantly enriched pathways in the hepatopancreas included fatty acid biosynthesis, fatty acid metabolism, and riboflavin metabolism. Further analysis showed that 25 genes and several pathways were mainly involved in oogenesis, including the ubiquitin-proteasome pathway, cyclic AMP-protein kinase A signaling pathway, and mitogen-activated protein kinase signaling pathway. Twenty-five candidate genes involved in vitellogenesis and endocrine regulation were identified, such as vitellogenin, vitellogenin receptor, estrogen sulfotransferase, ecdysone receptor, prostaglandin reductase 1, hematopoietic prostaglandin D synthase and juvenile hormone acid O-methyltransferase. Fifty-six genes related to nutritional metabolism were identified, such as fatty acid synthase, long-chain-fatty-acid-CoA ligase 4, 1-acyl-sn-glycerol-3-phosphate acyltransferase 4, fatty acid-binding protein, and glycerol-3-phosphate acyltransferase 1. These results highlight the genes involved in ovarian development and nutrition deposition, which enhance our understanding of the regulatory pathways and physiological processes of crustacean ovarian development.

Abstract PO-036: LP184, a novel alkylating agent, is highly effective in pancreatic cancers with DNA damage repair defects

Abstract Biomarker-based chemotherapy with increased efficacy and prolonged disease-free survival are urgently needed in pancreatic cancer (PDAC). A 15-20% subset of PDAC tumors carry mutations in DNA repair pathway (BRCA1/BRCA2/PALB2/RAD51/ATM/FANCD2). Additionally, mutations in nucleotide excision repair (NER) genes (ERCC2/3/4/5/6) have been reported in ~5% of PDAC. LP184 is a novel synthetic small molecule acylfulvene analog. Using precise synthetic chemistry, we determined that only a negative enantiomer of LP184 is converted to an active alkylating agent in the strict dependency on oxidoreductase, prostaglandin reductase 1 (PTGR1). By computational analyses, we demonstrate a strong positive correlation of LP184 sensitivity with PTGR1 transcript levels (r=0.89, p<10−15) in a broad panel of cancer cell lines. Once activated by PTGR1, highly reactive LP-184 nucleophile creates covalent DNA adducts that are selectively repaired via Nucleotide Excision Repair (NER) mechanism coupled to transcription (TC-NER) and/or homologous recombination (HR). We reasoned that mutation or expression driven TC-NER and HR deficiency would predispose PDAC cells to increased sensitivity to LP184. To test the idea of LP184 activity in DNA repair-deficient tumors, we evaluated LP184 chemosensitivity in genetically defined PDAC models in vitro, ex vivo, and in xenografts. Testing in six different pancreatic cancer cell lines (Capan-1, CFPAC-1, Panc1, MiaPaCa2, Panc03.27 and BxPC-3) resulted in very potent inhibition with LP184 IC50 values ranging from 114 to 182 nM. In this cell line panel, LP184 sensitivity correlated negatively with transcript levels of an NER pathway gene ERCC8 (r = -0.94). In comparison to these PDAC cell lines, a normal pancreatic epithelial cell line HPNE was 3-6 times less sensitive to LP184 (IC50 670 nM). Ex vivo cultures of 4 out of 5 low-passage patient-derived xenografts with HR deficiency showed nanomolar sensitivity to LP184 with IC50s ranging from 45 to 270 nM. These tumor graft models which were at least 6 times less sensitive to olaparib in the same assay. Depletion of ERCC4 enhanced sensitivity to LP184 about 2-fold relative to the parental cell line. To define PTGR1 as a biomarker for LP184 activity, we used CRISPR/Cas9-mediated gene editing to deplete PTGR1 expression. We found PTGR1-null Capan-1 cell line-derived xenografts were poorly sensitive to LP184, whereas PTGR1-expressing xenografts showed near complete tumor regression in all LP184 treated animals with 109% tumor growth inhibition relative to the control group in this study. Furthermore, PTGR1 depleted cells were completely resistant to LP184 in vitro. Our preclinical data demonstrate that PDAC models carrying a range of DNA repair pathway mutations are highly sensitive to LP-184 in vitro and in vivo. Increased PTGR1 expression is a validated biomarker for LP184 cytotoxicity, and is the exclusive convertase of LP184 to an active alkylator drug. We anticipate LP184 will extend the therapeutic opportunities to a large subset of PDAC patients carrying these genetic alterations. Citation Format: Diana Restifo, Aditya Kulkarni, Caleb Schimke, Joseph McDermott, Umesh Kathad, Kishor Bhatia, Panna Sharma, Igor Astsaturov. LP184, a novel alkylating agent, is highly effective in pancreatic cancers with DNA damage repair defects [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-036.