Citations

Abstract High-grade gliomas (HGGs) are aggressive tumors with poor outcomes and limited treatment options. Here, we combined genomic and transcriptomic tumor profiling with drug testing in a patient-derived 3-dimensional culture model to identify individualized treatments and predictive biomarkers. Activity of single agents targeting frequently dysregulated glioma pathways was relatively poor ex vivo and generally reflected historical patient data. However, compounds targeting PI3K, epigenetic, and survival/senescence signaling were effective in some cases. Drug sensitivity correlated with transcriptional rather than genomic features and suggested heterogeneity as a resistance mechanism. Bromodomain and extraterminal domain inhibition was particularly effective in tumors enriched in the mesenchymal transcriptional subtype, promoted proneural transition, and was overcome by upregulated PI3K signaling. Notably, combinations were largely effective, with 6 strategies exhibiting stronger efficacy than corresponding single agents in most cases (58-77%). This study identifies HGG vulnerabilities and associated biomarkers, resistance mechanisms, and effective combination strategies that warrant further clinical validation. Graphical Abstract

Abstract Background: Diffuse pleural mesothelioma (DPM) is a rare and aggressive cancer arising from mesothelial cells of the pleura. Over 2, 500 new cases are diagnosed annually in the United States, mainly attributable to asbestos exposure. The prognosis is poor, and while nivolumab (PD-1 inhibitor) and ipilimumab (CTLA-4 inhibitor) have been approved for first-line treatment of unresectable DPM, the three-year survival rate is only 23%. DPM tumors express high levels of epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition (MET), but targeted therapies like erlotinib (EGFR tyrosine kinase inhibitors) have shown no clinical benefit. We hypothesized that amivantamab, a bispecific antibody that targets both EGFR and MET, may have activity against DPM through antibody-dependent cellular cytotoxicity (ADCC). Objective: The purpose of this study was to assess the potential of amivantamab to mediate ADCC in tissue culture experiments with DPM cell lines. Methods: We assessed DPM for the expression of EGFR and MET using available data from bulk RNA sequencing. We also examined protein expressions of EGFR and MET by Western blot analysis in five epithelial (EM-Meso, H2452, H2461, Mero-41 and Mero-95), three sarcomatoid (H2373, H2596 and ONE58), and one normal mesothelial (MeT-5A) cell lines. Co-culture experiments were performed with the DPM and normal cell lines and NK cells in the presence of amivantamab to evaluate ADCC. Results: We identified high levels of EGFR and MET expression in both epithelial and sarcomatoid cases of DPM from 183 patients. This finding was corroborated by Western blot analysis of all eight DPM cell lines. In vitro experiments demonstrated that amivantamab induced dose-dependent ADCC in DPM cell lines, with robust NK cell-mediated ADCC observed across all tested DPM lines. Notably, sarcomatoid-type DPM cells were more sensitive to amivantamab-induced ADCC than epithelial-type cells. These findings suggest that amivantamab may offer therapeutic benefits for DPM tumors with high EGFR and MET expression. Conclusion: This study provides preclinical evidence supporting the potential of amivantamab as a novel therapeutic option for DPM, especially in sarcomatoid tumors. Ongoing in vivo studies including in patient-derived xenograft (PDX) models will further evaluate the efficacy of amivantamab in DPM. Citation Format: Shinichiro Suzuki, Lin Yang, Jennifer Ayers-Ringler, Ezequiel J. Tolosa, Kuan-Li Wu, Janet L. Schaefer-Klein, Farhad Kosari, Aaron S. Mansfield. Antitumor activity of amivantamab in diffuse pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7331.

Despite ongoing efforts to employ structure-based methods to discover targeted protein degraders (TPD), the prevailing strategy remains synthesizing a focused set of heterobifunctional compounds and screening them for target protein degradation.

Abstract Antibody-drug conjugates (ADCs) are increasingly being used in clinic, and many novel ADCs are being tested in clinical trials. Most ADCs act by delivering cytotoxic payloads into target cells. The activity of most ADCs is predicated upon internalization to release the cytotoxic payloads intracellularly to act on the malignant cells. The degree of ADC internalization is incomplete or absent for many cell surface targets, limiting the activity of ADCs. PROTACs are heterobifunctional molecules that result in the polyubiquitylation of their targets by inducing proximity with E3 ligases. The proteasome then recognizes the poly-ubiquitin chains and degrades these targets. We hypothesized that PROTACs that act on the same targets as ADCs could improve the internalization and efficacy of these ADCs. Using multiple cell lines derived from breast, lung or pancreatic cancers that express the oncogenic cell surface proteins EGFR (ERBB1), HER2 (ERBB2) or MET (HGF), we measured antibody internalization with live cell imaging. Antibodies were labeled with a pH sensitive reagent that fluoresces red (640 nm) upon exposure to acidic environments. Fluorescence was measured over time to create an area under the curve which was compared between the cell lines that were treated with and without PROTACs. Antibody internalization was observed in all cell lines that expressed the targets recognized by the antibodies. The addition of PROTACs targeting the same oncogenic cell surface proteins as the antibodies significantly increased antibody internalization beyond that observed without PROTACs. Internalization of antibodies was confirmed by measuring intracellular immunoglobulins by Western blot. Antibody internalization with PROTACs was dose-dependent, increased over time and was blocked by either the clatharin inhibitor Dyngo4a or the proteasome inhibitor MG132. In the case of HER2 for which there are FDA-approved anti-HER2 ADCs, the cytotoxicity of trastuzumab emtansine (TDM1) significantly increased with the addition of a HER2-targeting PROTAC across a range of doses. PROTACs that target the same oncogenic cell surface proteins as antibodies improve antibody internalization and ADC cytotoxicity, in a process dependent upon clatharin and the proteasome. This novel application of PROTACs may impact the use of ADCs, and provides a rationale to combine these agents in clinical trials. Citation Format: Ezequiel J. Tolosa, Lin Yang, Jennifer Ayers-Ringler, Jayapal Mallareddy, Janet Schaefer-Klein, Farhad Kosari, Amar Natarajan, Aaron S. Mansfield. Proteolysis targeting chimera (PROTAC)-driven internalization of antibodies targeting oncogenic cell surface receptors [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 4518.

Abstract Sequencing efforts have identified key oncogenic drivers and transcriptomic subtypes of high-grade gliomas (HGGs), but this knowledge has not resulted in individualized therapy or improved patient outcomes. Here, we used a functional genomics approach combining integrated genomic/transcriptomic analyses with patient-derived 3D micro-cancers (mCancers) to rapidly identify individualized therapeutic vulnerabilities and determine predictive response biomarkers. 30 primary/recurrent HGGs, including 20 glioblastomas, 5 astrocytomas, and 5 oligodendrogliomas, were included for drug testing. mCancers recapitulated the structural and molecular alterations, cellularity, and heterogeneity, of parental patient tumors. A total of 119 single-agent drugs and 183 drug combinations were tested, while a pre-selected drug panel comprising 25 glioma-relevant treatment strategies was tested across all cases. In general, mCancer efficacy corresponded well to historical responses to previously tested agents (EGFR, HDAC inhibitors, etc.). Combined, strong efficacy (i.e. >70% growth inhibition at Cmax) of single treatments targeting deregulated receptor tyrosine kinase (RTK), p53/apoptosis, cell cycle, DNA damage repair, IDH, or telomere maintainance pathways, was observed in 14 out of 30 cases. Surprisingly, transcriptional signatures, but not specific genetic alterations, were found to stratify drug responses. For example, mCancers enriched for the most aggressive mesenchymal (MES) and glycolytic/plurimetabolic (GPM) transcriptional subtypes exhibited stronger response to the BET inhibitor JQ1 (and other clinical BET inhibitors) and the DNA-PKcs/mTOR dual inhibitor CC-115. This therapeutic vulnerability was associated with enrichment of YAP/TAZ signaling, and the glioblastoma stem cell G-STEM signature. Notably, individual combination strategies were effective across tumors, with strong responses ranging from 38% to 77% of all tumors tested. Overall, this study underscores the value of functional genomics in identifying novel vulnerabilities of HGGs and associated response biomarkers. It also highlights the need for further clinical validation of several novel therapies and especially drug combination strategies with strong efficacy in the ex vivo setting.

Background Immune checkpoint inhibitors (ICIs) are now a first-line treatment option for patients with pleural mesothelioma with the recent approval of ipilimumab and nivolumab. Mesothelioma has a low tumor mutation burden and no robust predictors of survival with ICI. Since ICIs enable adaptive antitumor immune responses, we investigated T-cell receptor (TCR) associations with survival in participants from two clinical trials treated with ICI. Methods We included patients with pleural mesothelioma who were treated with nivolumab (NivoMes, NCT02497508) or nivolumab and ipilimumab (INITIATE, NCT03048474) after first-line therapy. TCR sequencing was performed with the ImmunoSEQ assay in 49 and 39 pretreatment and post-treatment patient peripheral blood mononuclear cell (PBMC) samples. These data were integrated with TCR sequences found in bulk RNAseq data by TRUST4 program in 45 and 35 pretreatment and post-treatment tumor biopsy samples and TCR sequences from over 600 healthy controls. The TCR sequences were clustered into groups of shared antigen specificity using GIANA. Associations of TCR clusters with overall survival were determined by cox proportional hazard analysis. Results We identified 4.2 million and 12 thousand complementarity-determining region 3 (CDR3) sequences from PBMCs and tumors, respectively, in patients treated with ICI. These CDR3 sequences were integrated with 2.1 million publically available CDR3 sequences from healthy controls and clustered. ICI-enhanced T-cell infiltration and expanded T cell diversity in tumors. Cases with TCR clones in the top tertile in the pretreatment tissue or in circulation had significantly better survival than the bottom two tertiles (p<0.04). Furthermore, a high number of shared TCR clones between pretreatment tissue and in circulation was associated with improved survival (p=0.01). To potentially select antitumor clusters, we filtered for clusters that were (1) not found in healthy controls, (2) recurrent in multiple patients with mesothelioma, and (3) more prevalent in post-treatment than pretreatment samples. The detection of two-specific TCR clusters provided significant survival benefit compared with detection of 1 cluster (HR<0.001, p=0.026) or the detection of no TCR clusters (HR=0.10, p=0.002). These two clusters were not found in bulk tissue RNA-seq data and have not been reported in public CDR3 databases. Conclusions We identified two unique TCR clusters that were associated with survival on treatment with ICI in patients with pleural mesothelioma. These clusters may enable approaches for antigen discovery and inform future targets for design of adoptive T cell therapies.

Abstract Diffuse gliomas are aggressive brain tumors that lack effective therapies. Despite advances in the molecular characterization of gliomas, targeted treatments have failed to improve patient outcomes to date. Here, we employed a functional genomics approach to rapidly identify therapeutic vulnerabilities and provide a strategy to individualize patient treatment. Our process combined comprehensive genomic and transcriptomic tumor profiling, custom algorithms and visualization software for data integration, and preclinical 3-dimensional ex vivo models (mcancers) for drug screening to assess response to therapeutic agents targeting specific molecular alterations. A total of 32 cases, including 5 oligodendrogliomas (1 primary and 4 recurrent), 5 astrocytomas (1 primary and 4 recurrent), and 22 glioblastomas (5 primary and 17 recurrent), were included in the study. Immunohistochemistry and integrated genomics confirmed that mcancers maintain key molecular alterations, including mutations, chromosomal rearrangements and focal amplifications, cellularity, and heterogeneity, faithfully recapitulating the original tumor. A total of 119 single drugs and 183 combinations were tested, including a panel of 26-brain penetrant targeted agents (18 single drugs and 8 combinations) that was curated to target common glioma oncogenic pathways and was tested across all cases. A strong correlation was observed between therapeutic vulnerabilities of glioma mcancers to targeted agents (EGFR, MET, PDGFR, HDAC inhibitors, etc.) and clinical experience. Single agent activity of panel compounds in the ex vivo setting was generally poor, with only 35% mCancers exhibiting &gt; 70% growth inhibition at Cmax. In contrast, combination strategies, especially ones including epigenetic drugs, were significantly more effective (affecting 22 out of 32 mCancers), underscoring the molecular complexity of gliomas and the need for clinical trials testing combination treatments. Our data identified a number of novel combination treatments with significant efficacy across glioma subtypes and molecular profiles, suggesting that our functional genomics pipeline can guide treatment selection and inform clinical trial enrollment for gliomas.

Abstract Malignant pleural mesothelioma is a disease primarily associated with exposure to the carcinogen asbestos. Consistent with this carcinogenic exposure, cytogenetic analyses have identified multiple recurrent structural chromosomal abnormalities in this malignancy, but more recent high-throughput sequencing evaluations of point mutations suggest that there is a low mutational burden in mesothelioma. Since tumor mutational burden has been correlated with responses to treatment with immune checkpoint inhibitors such as nivolumab, it was not consistent that patients with mesothelioma and low mutation burdens would have similar response rates in clinical trials with immune checkpoint inhibitors as patients with non-small cell lung cancer which is associated with a high mutation burden. In order to reconcile these differences, and given the potential for an improved understanding of the molecular pathogenesis of mesothelioma to improve therapeutic options, we used mate-pair sequencing (MPseq) and RNA sequencing (RNAseq) to understand how structural variants affect the transcriptome. MPseq differs from standard next generation sequencing approaches by tiling the whole genome with larger fragments (2-5kb) to reliably detect structural variants such as insertions, deletions and rearrangements. Amongst 22 mesothelioma specimens there were 1535 chromosomal rearrangements (median 41, range 3-298 per specimen), that resulted in junctions or novel fusions of non-coding DNA or genes. Six-hundred thirty-seven of these rearrangements (median 22, range 5-103 range per specimen) resulted in novel fusions of genes. Many of these inter- or intra-chromosomal rearrangements were consistent with a pattern of chromoanagesis such as chromoplexy or chromothripsis. Chromosomal rearrangements detected by MPseq were used to guide analysis of RNAseq data and revealed that these chromosomal junctions resulted in the expression of 179 novel amino acid sequences (median 5, 0-51 range per specimen). To determine whether transcription of chromosomal rearrangement-related junctions have neoantigenic potential, we used in silico tools to determine whether any of the expressed junctions contained peptides that could be presented by patient-specific HLA molecules. The top candidate rearrangement-related peptides with neoantigenic potential bound patient-specific HLA molecules nearly as well or as well as a positive control in competitive binding assays. Our findings represent the discovery of potential neoantigen expression driven by structural chromosomal rearrangements. These results may have implications for the development of novel therapeutic strategies, the selection of patients to receive immunotherapy, and blood-based treatment monitoring strategies. Citation Format: Aaron S. Mansfield, Tobias Peikert, James B. Smadbeck, Julia B. Udell, Farhad Kosari, Stephen J. Murphy, Hongzheng Ren, Vishnu V. Serla, Janet L. Schaefer Klein, Giannoula Karagouga, Faye R. Harris, Carlos Sosa, Sarah H. Johnson, Wendy Nevala, Svetomir N. Markovic, Aaron O. Bungum, Eric S. Edell, Haidong Dong, John C. Cheville, Marie Christine Aubry, Jin Jen, George Vasmatzis. Rearrangement-related peptides with neoantigenic potential in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5726.