Mechanism-based Biomarkers Research Articles

Identification and In Vivo Validation of Unique Anti-Oncogenic Mechanisms Involving Protein Kinase Signaling and Autophagy Mediated by the Investigational Agent PV-10.

PV-10 is a 10% formulation of rose bengal sodium that has potent immunotherapeutic and anti-cancer activity against various tumors, including metastatic melanoma and refractory neuroblastoma. Currently, PV-10 is undergoing clinical testing for refractory metastatic neuroendocrine cancer and melanomas. However, preclinical investigation of PV-10 activity and its mechanisms against phenotypically and molecularly diverse adult solid tumors had not been conducted. In a panel of human cell lines derived from breast, colorectal, head and neck, and testicular cancers, we demonstrated that PV-10 induces cytotoxicity by apoptotic and autophagic pathways involving caspase-mediated PARP cleavage, downregulation of SQSTM1/p62, and upregulation of beclin-1. Treatment with PV-10 also consistently reduced phosphorylation of WNK1, which has been implicated in cancer cell migration and autophagy inhibition. By wound healing assay, PV-10 treatment inhibited the migration of cancer cells. Finally, significant inhibition of tumor growth was also noted in tumor-bearing mice treated with PV-10 by intralesional or systemic administration. In addition to known PV-10-mediated tumor-specific cytotoxic effects, we identified the mechanisms of PV-10 and provide new insights into its effect on autophagy and metastasis. Our data provide essential mechanism-based evidence and biomarkers of activity to formulate clinical studies of PV-10 in the future.

Abstract 1015: Potential of kava in reducing lung cancer risk and associated disparities: Mechanism-based biomarker discovery and analysis

Abstract Background: Lung cancer is the leading cause of cancer-related deaths. Tobacco smoke is well-recognized as the dominant risk factor. Stress, lung inflammation, air pollution and other factors also contribute to the risk of developing lung cancer Racial/ethnic disparities in lung cancer risks exit as well. The quantitative contributions of different these risk factors to lung cancer and associated disparities, however, have not been systematically characterized. One major barrier is the lack of tools to non-invasively and objectively quantify these risk factors, which precludes the identification of high-risk individuals and the development of effective prevention. Data from our group, particularly results from a pilot clinical trial, suggest that kava, traditionally consumed in the South Pacific Islands as a beverage to reduce stress and promote relaxation, may holistically reduce lung cancer risk by impacting multiple factors that contribute to lung cancer risk. Aim: This study aims to discover mechanism-based non-invasive biomarkers reflective of different lung cancer risk factors and explore their potential roles in lung cancer risk disparities. Such biomarkers are also employed to characterize the holistic effects of kava in reducing lung cancer risk and its potential in mitigating associated disparities. Results and Analysis: One-week of kava significantly reduced the level of nicotine exposure among smokers, quantified by the Total Nicotine Equivalents (TNE) in the 24-h urine sample. Excitingly the reduction in TNE appeared to be higher among African American smokers in comparison to participants of other race/ethnicity. Biomarkers for stress (plasma cortisol and urinary total cortisol equivalents (TCE)) were significantly reduced upon one-week kava use, which may mechanistically contribute to the reductions in tobacco use. The reduction in plasma cortisol and TCE were higher among African American smokers as well. One-week kava exposure also increased urinary excretion of total NNAL and reduced urinary 3-methyladenine (3-mA) in participants, suggestive of its ability to reduce the carcinogenicity of nicotine-derived nitrosamine ketone (NNK). Kava effects on NNAL and 3mA were again more pronounced among African American smokers. Conclusion and future directions: These results demonstrate the potential of mechanism-based biomarkers in investigating lung cancer risks and associated disparities. Such biomarkers may also facilitate the translational development of effective and targeted interventions, such as kava, which may not only reduce lung cancer risk but also mitigate the associated disparities. Additional mechanism-based biomarkers on other tobacco carcinogens and inflammation are currently under development. Two clinical trials are ongoing that the current clinical samples will help validate these discoveries. Citation Format: Chengguo Xing, Breanne Freeman, Jessica Mamallapalli, Allison Lynch, Naomi Fujioka, Ramzi G. Salloum, John Malaty, Frank Orlando, Zhiguang Huo. Potential of kava in reducing lung cancer risk and associated disparities: Mechanism-based biomarker discovery and analysis [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 1015.

Immunoaffinity proteomics for kidney injury biomarkers in male beagle dogs.

Drug-induced kidney injury (DIKI) is a cause of drug development failure. Dogs represent a common non-rodent animal model in pre-clinical safety studies; however, biomarker assays for detecting nephrotoxicity in dogs are limited. To identify novel proteins and gain insight into the molecular mechanisms involved in DIKI, we developed an assay to evaluate proteomic changes associated with DIKI in male beagle dogs that received nephrotoxic doses of tobramycin for 10 consecutive days. Label-free quantitative discovery proteomics analysis on representative kidney cortex tissues collected on Day 11 showed that the tobramycin-induced kidney injury led to a significant differential regulation of 94 proteins mostly associated with mechanisms of nephrotoxicity such as oxidative stress and proteasome degradation. For verification of the proteomic results, we developed a multiplex peptide-centric immunoaffinity liquid chromatography tandem mass spectrometry assay (IA LC-MS/MS) to evaluate the association of eight DIKI protein biomarker candidates using kidney cortices collected on Day 11 and urine samples collected on Days -4, 1, 3, 7 and 10. The results showed that most biomarkers evaluated were detected in the kidney cortices and their expression profile in tissue aligned with the label-free data. Cystatin C was the most consistent marker regardless of the magnitude of the renal injury while fatty acid-binding protein-4 (FABP4) and kidney injury molecule-1 (KIM-1) were the most affected biomarkers in response to moderate proximal tubular injury in absence of changes in serum-based concentrations of blood urea nitrogen or creatinine. In the urine, clusterin is considered the most consistent biomarker regardless of the magnitude and time of the renal injury. To our knowledge, this is the most comprehensive multiplex assay for the quantitative analysis of mechanism-based proximal tubular injury biomarkers in dogs.

Biomarkers based on multiplatform comprehensive analysis: A systematic analysis of Geng-Nian-Shu in perimenopausal syndrome.

Although Geng-Nian-Shu has been shown to be clinically effective in perimenopausal syndrome, its active components and mechanism have not yet been elucidated. To demonstrate the mechanism-based biomarkers of Geng-Nian-Shu in treating perimenopausal syndrome, a total of 135 chemical constituents including 52 prototype blood constituents were identified via high-performance liquid chromatography-quadrupole-time of flight/mass spectrometry. Then, network pharmacology showed significant enrichment for the PhosphoInositide-3 Kinase/Akt pathway, suggesting that it may be the main regulatory pathway for the Geng-Nian-Shu treatment of the perimenopausal syndrome. Subsequently, multivariate analysis was performed between the Geng-Nian-Shu sham-treated and Geng-Nian-Shu ovariectomy-treated groups and further screened out 18 prototype blood constituents by correlation analysis with plasma estrogen levels to identify potential biomarkers associated with Geng-Nian-Shu treat the ovariectomy-induced perimenopausal syndrome. Finally, the results of pharmacological experimental verification and Pearson correlation analysis indicated that catalpol, ligustilide, paeoniflorin, and gallic acid were selected as biomarkers of Geng-Nian-Shu which were strongly and positively correlated with PhosphoInositide-3 Kinase/Akt signaling pathway. In this study, based on high-performance liquid chromatography-quadrupole-time of flight/mass spectrometry combined with pharmacodynamics, network pharmacology, pharmacology, and other disciplines, we explored the effects and mechanisms of Geng-Nian-Shu in the treatment of perimenopausal syndrome at multiple levels. Using multiplatform technology to investigate the role of Geng-Nian-Shu represents a new strategy for the selection and verification of biomarkers of Geng-Nian-Shu and provides a basis for further development and utilization of Geng-Nian-Shu.

Mechanism-based peripheral blood biomarkers to evaluate anticancer effects of the plinabulin/docetaxel combination (plin/doc) vs. doc alone in second/third line EGFR wild-type (WT) NSCLC.

e21017 Background: Pretreatment neutrophil to lymphocyte (L) (NLR), platelet to L (PLR), and L to monocyte (LMR) levels are established prognostic biomarkers for advanced NSCLC. Plinabulin (Plin) is a well-tolerated, differentiated tubulin-binding agent being developed for neutropenia prevention as a monotherapy and in combination, and for improving the efficacy of anticancer agents. DUBLIN-3 (NCT02504489) was a randomized, single-blinded (pts only), active-controlled Ph3 study in 2nd/3rd line stage IIIB/IV, EGFR WT NSCLC with a measurable lung lesion (RECIST 1.1). Pts (N = 559) were randomized 1:1 to Plin/Doc or Doc (21-day (D) cycle (C)). Doc (75 mg/m2) on D1 and Plin (30 mg/m2) on D1/D8 were given by IV infusion with steroid premedication (8 mg PO BID x 3D). Differential WBCs and platelet counts were collected at baseline (pre-steroid), D1, and D8. We analyzed the posttreatment effects of Plin on NLR, PLR, and LMR. Methods: All evaluable Plin/Doc vs Doc pts were included. NLR/PLR/LMR ratios and their respective change from baseline (cNLR/cPLR/cLMR) were obtained from blood draws taken at predose, C1D8, and/or C2D8. For intra/inter-arm overall survival (OS) stratification, baseline median NLR (3.2), PLR (177), and LMR (3.5) values were selected as thresholds. By examining responders & long-term survivors in both arms, posttreatment NLR/LMR and cNLR/cLMR thresholds were established for C1D8 and C2D8. Finally, PFS/OS benefits were stratified to determine the statistical significances of Plin/Doc vs Doc. Results: In agreement with literature, baseline threshold levels for NLR = 3.2, PLR = 177, LMR = 3.5 were statistically significantly predictive of OS in both the Plin/Doc and Doc arms (p values ranging from 0.05 to 0.0001). Plin prevents Doc-induced neutropenia, and post-Doc neutrophil counts were higher on D8 with Plin/Doc vs Plin (p < 0.001). Despite Doc-induced myelosuppression, Plin/Doc responding pts maintained similar NLR/LMR values as baseline at C1D8 and C2D8. After a single Plin dose at C1D8, lower NLR (cutoff = 3.0; 1.2x median value of all pts) and higher LMR (cutoff = 4.5; median value of all pts) thresholds independently predicted PFS/OS benefits of Plin/Doc vs Doc. Further, a higher cNLR threshold (cutoff = 0.25) at C2D8, best predicted Plin/Doc vs Doc in PFS and OS (p < 0.0001 and p = 0.0035 respectively). Conclusions: Bone marrow suppression remains a common serious risk of classical/non-classical cancer drugs. Under these conditions, the addition of Plin offers rapid protection of GMP lineage cells which can be captured by simple blood cell counts. Incorporation of blood-based biomarkers may help enrich NSCLC pts with better sensitivity to Plin/Doc combination than Doc alone. Clinical trial information: NCT02504489 .

Opportunities and Challenges of Kava in Lung Cancer Prevention.

Lung cancer is the leading cause of cancer-related deaths due to its high incidence, late diagnosis, and limited success in clinical treatment. Prevention therefore is critical to help improve lung cancer management. Although tobacco control and tobacco cessation are effective strategies for lung cancer prevention, the numbers of current and former smokers in the USA and globally are not expected to decrease significantly in the near future. Chemoprevention and interception are needed to help high-risk individuals reduce their lung cancer risk or delay lung cancer development. This article will review the epidemiological data, pre-clinical animal data, and limited clinical data that support the potential of kava in reducing human lung cancer risk via its holistic polypharmacological effects. To facilitate its future clinical translation, advanced knowledge is needed with respect to its mechanisms of action and the development of mechanism-based non-invasive biomarkers in addition to safety and efficacy in more clinically relevant animal models.

Prospective evaluation of the efficacy, safety, and optimal biomarker enrichment strategy for nangibotide, a TREM-1 inhibitor, in patients with septic shock (ASTONISH): a double-blind, randomised, controlled, phase 2b trial

Activation of the triggering receptor expressed on myeloid cells-1 (TREM-1) pathway is associated with septic shock outcomes. Data suggest that modulation of this pathway in patients with activated TREM-1 might improve survival. Soluble TREM-1 (sTREM-1), a potential mechanism-based biomarker, might facilitate enrichment of patient selection in clinical trials of nangibotide, a TREM-1 modulator. In this phase 2b trial, we aimed to confirm the hypothesis that TREM1 inhibition might improve outcomes in patients with septic shock. This double-blind, randomised, placebo-controlled, phase 2b trial assessed the efficacy and safety of two different doses of nangibotide compared with placebo, and aimed to identify the optimum treatment population, in patients across 42 hospitals with medical, surgical, or mixed intensive care units (ICUs) in seven countries. Non-COVID-19 patients (18-85 years) meeting the standard definition of septic shock, with documented or suspected infection (lung, abdominal, or urinary [in patients ≥65 years]), were eligible within 24 h of vasopressor initiation for the treatment of septic shock. Patients were randomly assigned in a 1:1:1 ratio to intravenous nangibotide 0·3 mg/kg per h (low-dose group), nangibotide 1·0 mg/kg per h (high-dose group), or matched placebo, using a computer-generated block randomisation scheme (block size 3). Patients and investigators were masked to treatment allocation. Patients were grouped according to sTREM-1 concentrations at baseline (established from sepsis observational studies and from phase 2a change to data) into high sTREM-1 (≥ 400 pg/mL). The primary outcome was the mean difference in total Sequential Organ Failure Assessment (SOFA) score from baseline to day 5 in the low-dose and high-dose groups compared with placebo, measured in the predefined high sTREM-1 (≥ 400 pg/mL) population and in the overall modified intention-to-treat population. Secondary endpoints included all-cause 28-day mortality, safety, pharmacokinetics, and evaluation of the relationship between TREM-1 activation and treatment response. This study is registered with EudraCT, 2018-004827-36, and Clinicaltrials.gov, NCT04055909. Between Nov 14, 2019, and April 11, 2022, of 402 patients screened, 355 were included in the main analysis (116 in the placebo group, 118 in the low-dose group, and 121 in the high-dose group). In the preliminary high sTREM-1 population (total 253 [71%] of 355; placebo 75 [65%] of 116; low-dose 90 [76%] of 118; high-dose 88 [73%] of 121), the mean difference in SOFA score from baseline to day 5 was 0·21 (95% CI -1·45 to 1·87, p=0·80) in the low-dose group and 1·39 (-0·28 to 3·06, p=0·104) in the high-dose group versus placebo. In the overall population, the difference in SOFA score from baseline to day 5 between the placebo group and low-dose group was 0·20 (-1·09 to 1·50; p=0·76),and between the placebo group and the high-dose group was 1·06 (-0·23 to 2·35, p=0·108). In the predefined high sTREM-1 cutoff population, 23 (31%) patients in the placebo group, 35 (39%) in the low-dose group, and 25 (28%) in the high-dose group had died by day 28. In the overall population, 29 (25%) patients in the placebo, 38 (32%) in the low-dose, and 30 (25%) in the high-dose group had died by day 28. The number of treatment-emergent adverse events (111 [96%] patients in the placebo group, 113 [96%] in the low-dose group, and 115 [95%] in the high-dose group) and serious treatment-emergent adverse events (28 [24%], 26 [22%], and 31 [26%]) was similar between all three groups. High-dose nangibotide led to a clinically relevant improvement in SOFA score (of two points or more) from baseline to day 5 over placebo in those with higher cutoff concentrations (≥532 pg/mL) of sTREM-1 at baseline. Low dose nangibotide displayed a similar pattern with lower magnitude of effect across all cutoff values. This trial did not achieve the primary outcome of improvement in SOFA score at the predefined sTREM-1 value. Future studies are needed to confirm the benefit of nangibotide at higher concentrations of TREM-1 activation. Inotrem.

Circulating indian hedgehog is a marker of the hepatocyte-TAZ pathway in experimental NASH and is elevated in humans with NASH

Non-alcoholic steatohepatitis (NASH)-induced liver fibrosis is emerging as the most common cause of liver disease. For evaluation of therapies, there is a pressing need to identify non-invasive, mechanism-based biomarkers. A pro-fibrotic process relevant to human NASH involves a pathway in which a transcriptional regulator called TAZ (WWTR1) in hepatocytes induces the secretion of pro-fibrotic Indian hedgehog (IHH). We therefore reasoned that circulating IHH may be a useful mechanism-based marker to assess changes in NASH fibrosis. Circulating IHH was assessed in wild-type and hepatocyte-TAZ-silenced NASH mice and in three separate cohorts of patients with mild-moderate NASH. Circulating IHH was elevated in mice with diet-induced NASH compared with chow-fed mice or with NASH mice in which hepatocyte TAZ was silenced, which is an effective means to decrease NASH fibrosis. In patients with fatty liver disease with or without NASH, NASH fibrosis was associated with increased concentrations of circulating IHH. The results of these analyses support further investigation to determine whether circulating IHH may be useful as a mechanism-based indicator of target engagement in anticipated future clinical trials testing NASH fibrosis therapies that block the IHH pathway. Non-alcoholic steatohepatitis (NASH)-induced liver fibrosis is a common cause of liver disease. Circulating biomarkers that reflect liver fibrosis in NASH would be very useful to evaluate therapies. One mechanism of NASH fibrosis with potential as a therapeutic target involves a liver-secreted protein called Indian hedgehog (IHH). We report that circulating levels of IHH in experimental and human NASH associates with NASH and NASH-associated liver fibrosis, providing the premise for further investigation into using circulating IHH to evaluate anticipated future NASH therapies that block the IHH pathway in liver.

Bridging the gaps towards precision psychiatry: Mechanistic biomarkers for early detection and intervention

Early detection and intervention in schizophrenia, improving prognosis, requires mechanism-based biomarkers that capture circuitry dysfunction, allowing optimized patient stratification, disease monitoring and treatment.Dr. Do's translational research, bridging basic neuroscience and clinical psychiatry, tackles an urgent need to develop effective treatments that target mechanisms underlying cognitive deficits, a critical dimension of schizophrenia, currently not well treated. By adopting a reverse translation of validated circuitry relevant human endpoints, her research brought new insights in mechanism-based biomarker guided treatment of patients in early stages of psychosis. She showed that oxidative stress/redox dysregulation, in reciprocal interaction with dopamine imbalance, NMDAR hypofunction, neuroinflammation and mitochondrial bioenergetic dysfunction, may represent a "hub" on which both genetic and environmental risk factors converge during neurodevelopment. This leads to impairments of structural and functional connectivity in microcircuits, involving impaired parvalbumin fast-spiking GABA neurons, and macrocircuits, impacting myelination of fiber tracts, at the basis of neural synchronization abnormalities, as well as sensory and cognitive deficits. These unique insights led to successful proof-of-concept clinical trials, targeting oxidative stress through antioxidant-based strategies in patients at various disease stages, paving the way for precision medicine in psychiatry.

Reducing tobacco-associated lung cancer risk: a study protocol for a randomized clinical trial of AB-free kava

BackgroundTobacco use is the leading cause of many preventable diseases, resulting in premature death or disease. Given that the majority of adult who smoke want to stop, this health burden could be significantly reduced if the success rate of tobacco cessation can be improved. In addition, most adults planning to quit were interested in trying complementary approaches to facilitating tobacco cessation, which is currently lacking. Therefore, there is an unmet and urgent need for novel interventions to improve the success of tobacco cessation. If such an intervention can reduce tobacco-associated lung carcinogenesis, that will be more desirable. The goal of this project is to develop a safe and effective kava-based intervention to enable tobacco cessation and reduce lung cancer risk, which will improve the health of smokers.MethodsA randomized controlled trial will enroll 80 adults who currently smoke at least 10 cigarettes daily and randomize 1:1 into the placebo and AB-free kava arms, being exposed for 4 weeks, with a total of six visits (weeks 0, 1, 2, 4, 8, and 12) to evaluate the compliance and potential issues of AB-free kava use among the participants, explore the potential effect of the AB-free kava intervention on tobacco dependence, tobacco use, and lung carcinogenesis biomarkers. Participants will be enrolled during their primary care clinic visit.DiscussionPrimary care settings play a critical role in tobacco-related disease screening, counseling, and early intervention, as the majority of adults who smoke visit their physicians annually. Building upon our promising pilot human trial results in conjunction with ample compelling lab animal results, and consistent with evidence of kava’s benefits from epidemiological data, this trial will evaluate the compliance of AB-free kava among adults who currently smoke with no intention to quit. The other exploratory aims include (1) whether AB-free kava intervention can reduce tobacco use and tobacco dependence; (2) whether AB-free kava use suppresses tobacco-induced carcinogenesis; and (3) the potential of the mechanism-based noninvasive biomarkers in precision AB-free kava intervention. The positive results from this study are expected to provide a great opportunity to effectively reduce smoking rates and tobacco-related diseases.Trial registrationClinicalTrials.gov with the identifier: NCT05081882. Registered on October 18, 2021.

Integrated proteomics identifies PARP inhibitor-induced prosurvival signaling changes as potential vulnerabilities in ovarian cancer

BRCA1/2-deficient ovarian carcinoma (OC) has been shown to be particularly sensitive to poly (ADP-ribose) polymerase inhibitors (PARPis). Furthermore, BRCA1/2 mutation status is currently used as a predictive biomarker for PARPi therapy. Despite providing a major clinical benefit to the majority of patients, a significant proportion of BRCA1/2-deficient OC tumors do not respond to PARPis for reasons that are incompletely understood. Using an integrated chemical, phospho- and ADP-ribosylation proteomics approach, we sought here to develop additional mechanism-based biomarker candidates for PARPi therapy in OC and identify new targets for combination therapy to overcome primary resistance. Using chemical proteomics with PARPi baits in a BRCA1-isogenic OC cell line pair, as well as patient-derived BRCA1-proficient and BRCA1-deficient tumor samples, and subsequent validation by coimmunoprecipitation, we showed differential PARP1 and PARP2 protein complex composition in PARPi-sensitive, BRCA1-deficient UWB1.289 (UWB) cells compared to PARPi-insensitive, BRCA1-reconstituted UWB1.289+BRCA1 (UWB+B) cells. In addition, global phosphoproteomics and ADP-ribosylation proteomics furthermore revealed that the PARPi rucaparib induced the cell cycle pathway and nonhomologous end joining (NHEJ) pathway in UWB cells but downregulated ErbB signaling in UWB+B cells. In addition, we observed AKT PARylation and prosurvival AKT-mTOR signaling in UWB+B cells after PARPi treatment. Consistently, we found the synergy of PARPis with DNAPK or AKT inhibitors was more pronounced in UWB+B cells, highlighting these pathways as actionable vulnerabilities. In conclusion, we demonstrate the combination of chemical proteomics, phosphoproteomics, and ADP-ribosylation proteomics can identify differential PARP1/2 complexes and diverse, but actionable, drug compensatory signaling in OC.

A Patient-to-Model-to-Patient (PMP) Cancer Drug Discovery Protocol for Identifying and Validating Therapeutic Agents Targeting Tumor Regulatory Architecture.

The current Achilles heel of cancer drug discovery is the inability to forge precise and predictive connections among mechanistic drivers of the cancer cell state, therapeutically significant molecular targets, effective drugs, and responsive patient subgroups. Although advances in molecular biology have helped identify molecular markers and stratify patients into molecular subtypes, these associational strategies typically fail to provide a mechanistic rationale to identify cancer vulnerabilities. Recently, integrative systems biology methodologies have been used to reverse engineer cellular networks and identify master regulators (MRs), proteins whose activity is both necessary and sufficient to implement phenotypic states under physiological and pathological conditions, which are organized into highly interconnected regulatory modules called tumor checkpoints. Because of their functional relevance, MRs represent ideal pharmacological targets and biomarkers. Here, we present a six-step patient-to-model-to-patient protocol that employs computational and experimental methodologies to reconstruct and interrogate the regulatory logic of human cancer cells for identifying and therapeutically targeting the tumor checkpoint with novel as well as existing pharmacological agents. This protocol systematically identifies, from specific patient tumor samples, the MRs that comprise the tumor checkpoint. Then, it identifies in vitro and in vivo models that, by recapitulating the patient's tumor checkpoint, constitute the appropriate cell lines and xenografts to further elucidate the tissue context-specific drug mechanism of action (MOA) and permit precise, biomarker-based preclinical validations of drug efficacy. The combination of determination of a drug's context-specific MOA and precise identification of patients' tumor checkpoints provides a personalized, mechanism-based biomarker to enrich prospective clinical trials with patients likely to respond. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.

Abstract 1121: Identification and in vivo validation of unique anti-oncogenic properties and mechanisms involving protein kinase signalling and autophagy mediated by the investigational new agent PV-10

Abstract Introduction: PV-10 (10% Rose Bengal) is a small molecule agent previously shown to have potent immunotherapeutic and anti-tumor activities against a number of tumors including metastatic melanoma and refractory neuroblastoma, and is currently undergoing clinical testing as a single-agent for refractory metastatic neuroendocrine cancer (NCT02693067) and in combination with checkpoint inhibitors for metastatic melanoma (NCT02557321) and metastatic uveal melanoma (NCT00986661). We have previously determined that PV-10 induces cell death at pharmacologically relevant concentrations in a panel of phenotypically diverse adult solid tumor cell lines. However, the molecular consequences of this phenomenon have not yet been fully elucidated. In this study, we investigated the target validation and modulation of PV-10 on protein kinase signalling and their associated impact on specific oncogenic pathways in these tumor cells. Methods: A panel of human tumor cell lines derived from breast (MCF-7, T-47D, MDA-MB-231), colorectal (LoVo, T-84), head and neck (CAL-27, Detroit-562, FaDu, UM-SCC-1), and testicular (NCC-IT, NTERA-2, TCAM-2) tissues were treated with PV-10 and their cytotoxic and pro-apoptotic effects were described. Protein kinase profiling was performed using the human phospho-kinase antibody array (#ARY003C, R&D Systems). Western blotting was used to investigate autophagic markers and protein kinase activity. Cell migration inhibition was determined by wound healing assay following treatment with a sublethal dose of PV-10 or pan-WNK inhibitor WNK463. Tumor xenograft studies were carried out according to established protocols. Results: Treatment with PV-10 leads to consistent inhibition of WNK lysine deficient protein kinase 1 (WNK1) phosphorylation based on protein kinase profiling of drug-treated cancer cells when compared with vehicle-treated cells. WNK1 has been implicated as an inhibitor of autophagy and a promoter of cell migration and invasion in several cancers. Western blot analysis showed that PV-10 leads to the downregulation of SQSTM1/p62, upregulation of Beclin-1, and conversion of LC3B-I into LC3B-II, indicating activation of autophagy. PV-10 was also found to significantly inhibit the migration of cancer cells similar to the inhibitor WNK463. Significant tumor regression and target modulation was noted in tumor-bearing animals treated with PV-10. Conclusion: In addition to the known activity of PV-10 to mediate tumor-specific immune responses and cytotoxic effects in neoplasms, we have identified novel therapeutic targets for PV-10, such as WNK1, and provide new insights into its effect on autophagy and metastasis. Our data provide essential mechanism-based evidence and biomarkers of activity to formulate effective PV-10 backbone clinical studies in the future. Citation Format: Son Tran, Satbir Thakur, Mohit Jain, Chunfen Zhang, Aru Narendran. Identification and in vivo validation of unique anti-oncogenic properties and mechanisms involving protein kinase signalling and autophagy mediated by the investigational new agent PV-10 [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 1121.

Development of a mechanism-based biomarker for Dioscorea bulbifera L. exposure and hepatotoxicity in rats

BackgroundDioscorea bulbifera L. (DBL) is a common herbal medicine where furanoterpenoid diosbulbin B (DSB) is a major component responsible for its hepatotoxicity. The metabolic oxidation of the furan moiety of DSB, resulting in covalent binding to hepatic protein, is considered to initiate its liver injury. PurposeWe aimed to develop a mechanism-based plasma protein adduction-based biomarker to determine DBL exposure and to predict the onset of hepatotoxicity induced by DBL. MethodsRats were intragastrically treated with DBL extract, and the plasma samples were collected. Plasma ALT and AST were measured with commercial kits. Plasma protein modification was determined by immunoblot assay. Assessment of DSB-induced protein adduction was achieved by LC-MS/MS analysis of complete proteolytic digestion of adducted protein to pyrroline derivative A4 using pronase enzyme. The structure of the resulting pyrroline derivatives was confirmed by NMR. ResultsPlasma protein of rats treated with DBL extract was covalently modified by the metabolite of DSB. Pyrroline derivative A4 was detected in proteolytic digestion of plasma obtained from rats administered DBL extract. The protein adduction elevated with the increase in the dosage of DBL extract. A detectable level of plasma was observed 10 days after withdrawal of DBL extract post 30-day continuous administration. In addition, the elevation trend of plasma ALT was found to be proportional to the accumulation trend of pyrroline derivative A4. ConclusionDSB-derived plasma protein adduction correlated well with the exposure of DBL in rats. The protein adduction may be used as a good biomarker for diagnosis of DBL-induced liver injury and a useful indicator for DBL medication plans.

IgM anti-ACE2 autoantibodies in severe COVID-19 activate complement and perturb vascular endothelial function.

BackgroundSome clinical features of severe COVID-19 represent blood vessel damage induced by activation of host immune responses initiated by the coronavirus SARS-CoV-2. We hypothesized autoantibodies against angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor expressed on vascular endothelium, are generated during COVID-19 and are of mechanistic importance.MethodsIn an opportunity sample of 118 COVID-19 inpatients, autoantibodies recognizing ACE2 were detected by ELISA. Binding properties of anti-ACE2 IgM were analyzed via biolayer interferometry. Effects of anti-ACE2 IgM on complement activation and endothelial function were demonstrated in a tissue-engineered pulmonary microvessel model.ResultsAnti-ACE2 IgM (not IgG) autoantibodies were associated with severe COVID-19 and found in 18/66 (27.2%) patients with severe disease compared with 2/52 (3.8%) of patients with moderate disease (OR 9.38, 95% CI 2.38–42.0; P = 0.0009). Anti-ACE2 IgM autoantibodies were rare (2/50) in non-COVID-19 ventilated patients with acute respiratory distress syndrome. Unexpectedly, ACE2-reactive IgM autoantibodies in COVID-19 did not undergo class-switching to IgG and had apparent KD values of 5.6–21.7 nM, indicating they are T cell independent. Anti-ACE2 IgMs activated complement and initiated complement-binding and functional changes in endothelial cells in microvessels, suggesting they contribute to the angiocentric pathology of COVID-19.ConclusionWe identify anti-ACE2 IgM as a mechanism-based biomarker strongly associated with severe clinical outcomes in SARS-CoV-2 infection, which has therapeutic implications.FUNDINGBill & Melinda Gates Foundation, Gates Philanthropy Partners, Donald B. and Dorothy L. Stabler Foundation, and Jerome L. Greene Foundation; NIH R01 AR073208, R01 AR069569, Institutional Research and Academic Career Development Award (5K12GM123914-03), National Heart, Lung, and Blood Institute R21HL145216, and Division of Intramural Research, National Institute of Allergy and Infectious Diseases; National Science Foundation Graduate Research Fellowship (DGE1746891)

New Insights into the Treatment of Glomerular Diseases: When Mechanisms Become Vivid.

Treatment for glomerular diseases has been extrapolated from the experience of other autoimmune disorders while the underlying pathogenic mechanisms were still not well understood. As the classification of glomerular diseases was based on patterns of juries instead of mechanisms, treatments were typically the art of try and error. With the advancement of molecular biology, the role of the immune agent in glomerular diseases is becoming more evident. The four-hit theory based on the discovery of gd-IgA1 gives a more transparent outline of the pathogenesis of IgA nephropathy (IgAN), and dysregulation of Treg plays a crucial role in the pathogenesis of minimal change disease (MCD). An epoch-making breakthrough is the discovery of PLA2R antibodies in the primary membranous nephropathy (pMN). This is the first biomarker applied for precision medicine in kidney disease. Understanding the immune system’s role in glomerular diseases allows the use of various immunosuppressants or other novel treatments, such as complement inhibitors, to treat glomerular diseases more reasonable. In this era of advocating personalized medicine, it is inevitable to develop precision medicine with mechanism-based novel biomarkers and novel therapies in kidney disease.

Improper Proteostasis: Can It Serve as Biomarkers for Neurodegenerative Diseases?

Cells synthesize new proteins after multiple molecular decisions. Damage of existing proteins, accumulation of abnormal proteins, and basic requirement of new proteins trigger protein quality control (PQC)-based alternative strategies to cope against proteostasis imbalance. Accumulation of misfolded proteins is linked with various neurodegenerative disorders. However, how deregulated components of this quality control system and their lack of general mechanism-based long-term changes can serve as biomarkers for neurodegeneration remains largely unexplored. Here, our article summarizes the chief findings, which may facilitate the search of novel and relevant proteostasis mechanism-based biomarkers associated with neuronal disorders. Understanding the abnormalities of PQC coupled molecules as possible biomarkers can help to determine neuronal fate and their contribution to the aetiology of several nervous system disorders.

Abstract P018: Integrative proteomics of PARP1 protein complexes and post-translational modifications implicates DDR and AKT-mTOR signaling in mediating response or primary resistance of ovarian carcinoma cells to PARP1 inhibitors

Abstract Background: About 40-50% of epithelial ovarian cancers (EOC) show defects in DNA repair by homologous recombination (HR), which are mostly associated with BRCA1/2 loss-of-function mutations. The PARP inhibitors (PARPis) olaparib, niraparib and rucaparib were recently approved for treatment of ovarian cancer patients with platinum sensitivity and recurrent ovarian cancer who carry inactivating BRCA1/2 mutations. These targeted drugs produce significant response rates ranging from 40-60% in patients with BRCA-linked advanced EOCs, but resistance is a continuing challenge. Whereas several studies have reported various mechanisms of acquired resistance to PARPis, the mechanisms of primary resistance are still poorly understood. Our goal is to develop predictors of PARPi response and to identify new targets for combination therapy to overcome primary resistance. We apply a novel integrated proteomics approach to develop mechanism-based biomarkers of response or primary resistance and to identify new therapeutic targets for rational combination approaches that can overcome resistance to single agent PARPi therapy. METHODS: The isogenic EOC cell line pair UWB1.289 with BRCA1 deletion (parental; UWB) and reconstituted with ectopic BRCA1 (UWB+B) was used. The effects of FDA approved PARPis on BRCA1-null and BRCA1-reconstituted UWB1.289 cells regarding short- and long-term cell viability were determined by CellTiterGlo and crystal violet assays. Chemical proteomics, global phosphoproteomics and ADP-ribosylation proteomics were used to identify the components of PARP1-based multiprotein complexes as well as protein post-translational modifications in the DNA damage signaling network in BRCA1/2-linked EOC cells. Specific PARP1-engaged protein complexes were further determined by immunoblotting. Frozen BRCA1-proficient and deficient ovarian cancer patient tumor samples collected at the time of debulking were also characterized by chemical proteomics. RESULTS: Cell viability assays confirmed the expected correlation between PARPi response and BRCA1/2 status. Chemical proteomics followed by validation with co-immunoprecipitation revealed differential composition of the PARP1/2-Ku70/Ku80 protein complexes in PARPi-sensitive UWB compared to UWB+B cells. Global phosphoproteomics and ADP-ribosylation proteomics further indicated that rucaparib induced the cell cycle and c-NHEJ pathways in UWB cells, but down-regulated the MAPK pathway in UWB+B cells. In addition, our results showed that inhibition of AKT PARylation and AKT-mTOR signaling may help to preserve cell viability in UWB+B cells after rucaparib treatment. Consistently, synergy with DNAPKi and AKTi was more pronounced in UWB+B cells. CONCLUSION: Ovarian cancers that do not respond to PARPi displayed significant changes in PARPi-engaged protein complexes as well as post-translational protein modifications. The combination of chemical, phospho- and ADP-ribosylation proteomics can generate a systems view of PARP1 complexes and diverse drug compensatory signaling in EOC. Citation Format: Ou Deng, Sweta Dash, Thales Nepomuceno, Bin Fang, Douglas Marchion, John Koomen, Alvaro N. Monteiro, Uwe Rix. Integrative proteomics of PARP1 protein complexes and post-translational modifications implicates DDR and AKT-mTOR signaling in mediating response or primary resistance of ovarian carcinoma cells to PARP1 inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P018.

Mechanism-Based Biomarker Prediction for Low-Grade Inflammation in Liver and Adipose Tissue.

Metabolic disorders, such as obesity and type 2 diabetes have a large impact on global health, especially in industrialized countries. Tissue-specific chronic low-grade inflammation is a key contributor to complications in metabolic disorders. To support therapeutic approaches to these complications, it is crucial to gain a deeper understanding of the inflammatory dynamics and to monitor them on the individual level. To this end, blood-based biomarkers reflecting the tissue-specific inflammatory dynamics would be of great value. Here, we describe an in silico approach to select candidate biomarkers for tissue-specific inflammation by using a priori mechanistic knowledge from pathways and tissue-derived molecules. The workflow resulted in a list of candidate markers, in part consisting of literature confirmed biomarkers as well as a set of novel, more innovative biomarkers that reflect inflammation in the liver and adipose tissue. The first step of biomarker verification was on murine tissue gene-level by inducing hepatic inflammation and adipose tissue inflammation through a high-fat diet. Our data showed that in silico predicted hepatic markers had a strong correlation to hepatic inflammation in the absence of a relation to adipose tissue inflammation, while others had a strong correlation to adipose tissue inflammation in the absence of a relation to liver inflammation. Secondly, we evaluated the human translational value by performing a curation step in the literature using studies that describe the regulation of the markers in human, which identified 9 hepatic (such as Serum Amyloid A, Haptoglobin, and Interleukin 18 Binding Protein) and 2 adipose (Resistin and MMP-9) inflammatory biomarkers at the highest level of confirmation. Here, we identified and pre-clinically verified a set of in silico predicted biomarkers for liver and adipose tissue inflammation which can be of great value to study future development of therapeutic/lifestyle interventions to combat metabolic inflammatory complications.

Mitochondrial, exosomal miR137-COX6A2 and gamma synchrony as biomarkers of parvalbumin interneurons, psychopathology, and neurocognition in schizophrenia

Early detection and intervention in schizophrenia requires mechanism-based biomarkers that capture neural circuitry dysfunction, allowing better patient stratification, monitoring of disease progression and treatment. In prefrontal cortex and blood of redox dysregulated mice (Gclm-KO ± GBR), oxidative stress induces miR-137 upregulation, leading to decreased COX6A2 and mitophagy markers (NIX, Fundc1, and LC3B) and to accumulation of damaged mitochondria, further exacerbating oxidative stress and parvalbumin interneurons (PVI) impairment. MitoQ, a mitochondria-targeted antioxidant, rescued all these processes. Translating to early psychosis patients (EPP), blood exosomal miR-137 increases and COX6A2 decreases, combined with mitophagy markers alterations, suggest that observations made centrally and peripherally in animal model were reflected in patients’ blood. Higher exosomal miR-137 and lower COX6A2 levels were associated with a reduction of ASSR gamma oscillations in EEG. As ASSR requires proper PVI-related networks, alterations in miR-137/COX6A2 plasma exosome levels may represent a proxy marker of PVI cortical microcircuit impairment. EPP can be stratified in two subgroups: (a) a patients’ group with mitochondrial dysfunction “Psy-D”, having high miR-137 and low COX6A2 levels in exosomes, and (b) a “Psy-ND” subgroup with no/low mitochondrial impairment, including patients having miR-137 and COX6A2 levels in the range of controls. Psy-D patients exhibited more impaired ASSR responses in association with worse psychopathological status, neurocognitive performance, and global and social functioning, suggesting that impairment of PVI mitochondria leads to more severe disease profiles. This stratification would allow, with high selectivity and specificity, the selection of patients for treatments targeting brain mitochondria dysregulation and capture the clinical and functional efficacy of future clinical trials.