Tang, Nanyun

Abstract The selectivity of the blood-brain-barrier (BBB) can prevent therapeutics from reaching the brain, and this may contribute to the high failure rate of new drugs for tumors like glioblastoma (GBM) and brain metastasis. The capacity to detect BBB penetrance in real-time in a noninvasive manner would arguably aid the rate of therapeutic development. Extracellular vesicles, which are membrane bound particles secreted by all cell types in the body, have potential as a neuro pharmacodynamic reporter given that their protein and RNA cargo reflect the originating cell population. We hypothesized that the exposure of GBM cells to therapy results in altered EV cargo, and that the detection of this shift can be leveraged as a liquid biopsy from patients during early clinical trials to determine BBB penetrance and tumor engagement. To identify cargo shifts indicative of treatment, we exposed four short-term patient derived glioma cell lines to arsenic trioxide (an idiopathic toxin), MLN4924 (a neddylation inhibitor), and the alkylating agent temozolomide, then harvested EVs for unbiased mass spectrometry and RNA sequencing. Interestingly, we identified conserved upregulated and downregulated protein and RNA species across cell lines and across drug treatments, despite the diverse mechanisms of action. Candidate proteins were further validated by mesoscale immunoassays and exoview evaluation of tetraspanin-captured EVs. RNA species were validated by qRT-PCR. Overall, we demonstrate that GBM-derived EV cargo changes in response to therapy, supporting the development of EVs as a reporter of drug delivery in GBM. Ongoing and future work is focused on identifying enrichment handles in order to selectively evaluate GBM-derived EV cargo from patient biofluids. Thus far, B7H3, PTPRZ1, EGFRvII and IL13Ra2 have been screened as putative handles. Success in this pursuit could substantially enhance findings and accelerate drug testing in early-stage clinical trials in brain tumor patients by reporting drug accessibility to the tumor and engagement with drug targets. Citation Format: Valerie DeLuca, Nanyun Tang, Nathaniel Hansen, Anjali Raju, Charles Shaffer, Yue Hao, Terry C. Burns, Jann N. Sarkaria, Ian Parney, Patrick Pirrotte, Kendall Van Keuren-Jensen, Michael E. Berens. Glioblastoma-derived extracellular vesicle cargo as a reporter of drug delivery and effect [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 2130.

Abstract Development of a non-invasive test that can measure drug delivery and target engagement in GBM represents an opportunity to enhance knowledge during early phase clinical trials of a new compound’s pharmacological suitability and potential efficacy. We present here data supporting that the cargo within extracellular vesicles (EV) can potentially serve as a liquid biopsy that reports on the pharmacodynamics of GBM therapeutics. We treated short-term PDX cultures with vehicle, temozolomide, MLN4924, or arsenic trioxide. EVs were isolated from culture supernatants using size exclusion chromatography and then analyzed by mass spectrometry (proteomics) and RNA sequencing to characterize and compare cargo profiles for each condition. We found that at the proteome level, FDFT1 and FARSB were detected only in temozolomide EVs across all cell lines. In addition, there were drug-agnostic indications of exposure, including decreased abundance in all treated EV conditions of SLC3A2, C3, PLG, and A1BG, and increased abundance of GSTP1. These cargo changes were further validated by western blotting and ExoView immunofluorescence on isolated EVs. At the EV-transcriptome level, depending on treatment, there were shared alterations in EV cargo between models in the expression of long non-coding RNAs, genes that are overrepresented in biological processes such as cytoplasmic translation and cellular macromolecule biosynthesis, and circular RNAs. Thus, these results indicate that EV cargo post-treatment may be a novel pharmacodynamic reporter in GBM. To allow immunoprecipitation of patient GBM-derived EVs with the selective interrogation, the potential biomarkers, including PTPRZ1, B7H3, IL13RA2, and EGFR were screened across isolated EVs from both pre-clinical and clinical patient samples. Future efforts are focused on evaluating immunoprecipitated GBM-EVs from longitudinally collected patient biofluids. Overall, we anticipate that the results of this study will lead to development of a clinical test that reflects BBB penetration and tumor response, which will likely aid in novel drug development efforts.

Abstract Glioblastoma (GBM) is a lethal brain tumor with limited treatment options, due in part to the low blood-brain-barrier (BBB) penetrance of many anti-cancer compounds. Development of a non-invasive test that can measure drug delivery and target engagement in GBM represents an opportunity to enhance knowledge during early phase clinical trials of a new compound’s pharmacological suitability and potential efficacy. We present here data supporting that the cargo within extracellular vesicles (EV) can potentially serve as a liquid biopsy that reports on the pharmacodynamics of GBM therapeutics. We treated short-term cell cultures established from four patient-derived xenografts with vehicle, temozolomide, MLN4924, or arsenic trioxide. EVs were isolated from culture supernatants using size exclusion chromatography and then analyzed by RNA sequencing and mass spectrometry (proteomics) to characterize and compare cargo profiles for each condition. We found that depending on treatment, there were shared alterations in EV cargo between models in the expression of long non-coding RNAs, genes that are overrepresented in biological processes such as cytoplasmic translation and cellular macromolecule biosynthesis, and circular RNAs. Mass spectrometry of protein cargo further demonstrated both upregulated and downregulated peptide abundances with treatment compared to vehicle. Thus, our preliminary results indicate that EV cargo post-treatment may be a novel pharmacodynamic reporter in GBM. To allow for the selective interrogation of patient GBM-derived EVs, we have simultaneously pursued the identification of biomarkers to allow for immunoprecipitation of EVs from patient biofluids. Potential biomarkers, including PTPRZ1, B7H3, and IL13RA2, were screened across isolated EVs from both pre-clinical and clinical patient samples. Future efforts are focused on evaluating immunoprecipitated GBM-EVs from longitudinally collected patient biofluids. Overall, we anticipate that the results of this study will lead to development of a clinical test that reflects BBB penetration and tumor response, which will likely aid in novel drug development efforts.

Abstract Glioblastoma (GBM), a lethal astrocytic brain tumor, has limited available treatment options. A major hindrance to the progress of new therapies is the selectivity of the blood brain barrier (BBB) which limits drug access to the tumor. Further, there are currently no minimally-invasive tests that can quickly and accurately determine whether novel therapeutics have crossed the BBB during clinical trials. Therefore, development of a non-invasive test that can identify drug delivery and target engagement in GBM represents an opportunity to enhance knowledge during early phase trials of a new compound’s pharmacological suitability and potential efficacy. This study proposes that cargo contained in GBM-derived extracellular vesicles (EVs), which are small membrane-bound particles released from cells, may serve as one such pharmacodynamic reporter. We analyzed RNA-seq data for EVs isolated from two GBM patient-derived xenograft models grown as short-term in vitro cultures, GBM10 and GBM120, following treatment with either temozolomide (TMZ) or pevonedistat (MLN). We assessed whether EV RNA payloads vary with different drug concentrations (EC50 vs EC80) and at different time points (4hr vs 24hr) post treatment. In the samples treated with MLN, a few long non-coding RNAs are commonly upregulated across the different time points and two models. Under different TMZ concentrations, differentially expressed genes are overrepresented in biological processes such as cytoplasmic translation and cellular macromolecule biosynthesis. We also found a negative correlation between the TMZ concentration and Y RNA expression level, which are functionally involved with DNA replication. Additionally, using CIRI2, we predicted the amount of circular RNAs in the EV cargo; we found that the ratio of circular RNAs to their linear counterparts (CLR) varies under different treatments. In GBM10, the percentage of circRNA with CLR > 1 decreases with increasing drug effect. Thus, our preliminary results indicate that EV cargo post-treatment may be a novel pharmacodynamic reporter in GBM. To apply such an assessment to patients, we have simultaneously pursued the development of an immunoprecipitation (IP) approach to enrich GBM-derived EVs that can then undergo cargo profiling. Potential biomarkers were screened across isolated GBM EVs from in vitro supernatant by western blot and ExoView analysis. B7H3 has emerged as a lead candidate for GBM-EV enrichment. To validate the ability of EV cargo to reflect drug distribution across the BBB in patients, current efforts are focused on applying IP enrichment of GBM-EVs in longitudinally-acquired cerebrospinal fluid samples from patients during their disease course. Overall, we anticipate that the results of this study will lead to development of a clinical test that reflects BBB penetration and tumor response, which will likely aid in novel drug development efforts. Citation Format: Valerie DeLuca, Nanyun Tang, Yue Hao, Anjali Raju, Charles Shaffer, Cecile Riviere-Cazaux, Terry C. Burns, Jann Sarkaria, Ian Parney, Patrick Pirrotte, Kendall Van Keuren-Jensen, Michael E. Berens. Extracellular vesicles as a pharmacodynamic reporter in glioblastoma [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 2788.

Abstract Triptolide, which was initially extracted from a Chinese medicinal herb, demonstrates potent antitumor, anti-inflammatory, and immunosuppressive properties, yet it is clinically limited due to its poor solubility, bioavailability, and toxicity. Minnelide, a water-soluble analog of triptolide, has potent antiproliferative activity. It is currently in Phase I/II clinical trials for Acute Myeloid Leukemia (AML) and 7 solid tumor indications, including advanced gastrointestinal tumors, pancreatic cancer, and colorectal cancer. To overcome the potential drug resistance and to improve the drug potency as well as to minimize the drug toxicity, we conducted a high throughput screening (HTS) for drug synergy, combining Minnelide with compounds from the LOPAC and/or Prestwick libraries in 3 different cancer cell lines: BxPC3 (pancreatic), HT29 (colon) and SNU5 (gastric). LOPAC and Prestwick libraries consist of drugs approved by FDA and/or drugs that have reached clinical trial stages, representing 2,274 unique compounds in total. After quality control evaluation and data normalization, hits were selected if greater than 20% inhibition was achieved when combined compared with Minnelide alone. A total of 43, 54, and 47 hits were identified for each cell line, representing an average hit rate of 2.1%. We further prioritized the hits by selecting those that were common in two or more cell lines, which resulted in 9 compounds from Prestwick library and 20 compounds from LOPAC library (Harmane is in common between the two libraries). Only 17 compounds were available for purchase for hits confirmation, which was conducted in a similar manner as in primary HTS, except that drugs were tested in a dose-dependent manner instead of a single concentration done in HTS, a duplicated biological run was performed, and a total of 9 cell lines were screened, with 3 cell lines for each tumor type (pancreatic, colon and gastric). IC50 and Combination Index (CI) values for each drug combination were calculated for each cell line. A fold change of 2 or more for the IC50 values and CI value < 1.0 were used for hit selection. In general, colon lines had the most synergistic combinations and gastric had the least. A total of 15 drug candidates were confirmed to have synergistic effect, representing a confirmation rate of 88%, among which 12 drugs demonstrated synergy in >50% of the cell lines tested, with Harmane and Harmine demonstrating synergy in all 9 cell lines tested. In addition, CGS-15943, Amiloride, Naphthalimide, and Idazoxan indicated good synergy with Minnelide in at least 6 out of 9 cell lines. Interestingly, Phenamil, an irreversible inhibitor of amiloride-sensitive Na+ channels, showed synergy in all cell lines tested for pancreatic and colon cancers, but not in gastric cancer cell lines. Overall, our study shed light on potential drug combinations with Minnlide for clinical use. Citation Format: Nanyun Tang, Chris Sereduk, Ashok Saluja, Mohana Velagapudi, Haiyong Han, Daniel D Von Hoff, Hongwei Holly Yin. Drug combination screen to identify novel synergy with Minnelide [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A142. doi:10.1158/1535-7163.TARG-19-A142

Abstract Purpose: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by loss of the SWI/SNF ATPase subunits SMARCA4 and SMARCA2. A great need exists for effective targeted therapies for SCCOHT. Experimental Design: To identify underlying therapeutic vulnerabilities in SCCOHT, we conducted high-throughput siRNA and drug screens. Complementary proteomics approaches profiled kinases inhibited by ponatinib. Ponatinib was tested for efficacy in two patient-derived xenograft (PDX) models and one cell-line xenograft model of SCCOHT. Results: The receptor tyrosine kinase (RTK) family was enriched in siRNA screen hits, with FGFRs and PDGFRs being overlapping hits between drug and siRNA screens. Of multiple potent drug classes in SCCOHT cell lines, RTK inhibitors were only one of two classes with selectivity in SCCOHT relative to three SWI/SNF wild-type ovarian cancer cell lines. We further identified ponatinib as the most effective clinically approved RTK inhibitor. Reexpression of SMARCA4 was shown to confer a 1.7-fold increase in resistance to ponatinib. Subsequent proteomic assessment of ponatinib target modulation in SCCOHT cell models confirmed inhibition of nine known ponatinib target kinases alongside 77 noncanonical ponatinib targets in SCCOHT. Finally, ponatinib delayed tumor doubling time 4-fold in SCCOHT-1 xenografts while reducing final tumor volumes in SCCOHT PDX models by 58.6% and 42.5%. Conclusions: Ponatinib is an effective agent for SMARCA4-mutant SCCOHT in both in vitro and in vivo preclinical models through its inhibition of multiple kinases. Clinical investigation of this FDA-approved oncology drug in SCCOHT is warranted. Clin Cancer Res; 24(8); 1932–43. ©2018 AACR.

Abstract Hepatocellular carcinoma (HCC), the primary malignancy of the liver, is the fourth leading cause of cancer deaths worldwide, with a 5-year survival rate of∼10%. Recently, a new multikinase inhibitor, sorafenib (Nexavar®), which mainly targets the VEGFR and the Raf/Mek/Erk pathways, has shown limited response rate and modest survival benefits in patients with advanced stage HCC. We hypothesize that there exist genetic alterations or contexts of vulnerability (COV) in cancer cells that dictate sensitivity to sorafenib. In order to identify specific COVs and obtain an increased understanding of drug's mechanism of action, we performed a custom chemosensitization high-throughput siRNA screen. We developed and optimized a sorafenib chemosensitization assay in 3 HCC cell lines, one of which, Hep3B, a AFP and HBsAg positive cell line, was selected for primary RNAi screening with a kinase siRNA library, which covers 700 genes (4 siRNA sequences per gene). Primary screen data was rigorously evaluated for multiple quality control metrics (transfection efficiency, CV values, accessing plate-to-plate and set-to-set variation) and found to exceed all expected performance parameters. The top sorefenib sensitizing hits were identified and supplemented with additional genes selected based on related biological concepts. This sensitization effect of the genes was further tested and compared in two additional HCC cell lines (HepG2 and PLC). Additionally, based on the sensitizer list from the RNAi screening, 11 chemical compounds were tested for synergistic combination with sorafenib. Notable synergism with sorafenib was observed in the combination with erlotinib or doxorubicin. More strikingly, we also observed synergy between sorafenib and multiple MEK inhibitors as well as gemcitabine in the selected HCC cell lines. Our data suggest that MEK inhibitors might improve the efficacy of sorafenib in HCC treatment in the clinic. Further mechanistic studies are being conducted for this particular combination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-285.