Karagouga, Giannoula

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.

Abstract Background: Mutations in RAS is reported in embryonal rhabdomyosarcoma (ERMS). Upregulation of IGF2/IGF1R axis and IGF2 overexpression is a common finding. Targeting RAS downstream is associated with high toxicity. Combination therapies targeting parallel pathways is necessary to overcome treatment resistance. Dual inhibition of RAS downstream and IGF2/IGF1R axis can be a potential mechanism of targeted treatment. The efficacy of this mechanism was studied using a microcancer 3D exvivo tumor cell viability assay in a KRAS mutant ERMS. Design: Tumor was collected for a high-risk pediatric ERMS patient. Comprehensive genomic analysis, for identification of targetable genomic alterations, was performed by integrating WES, MPseq and RNAseq. Tumor spheroids grown in 3D environment were tested against multiple single drugs and combinations. The tumor viability was assed employing the Cell Titer Glow Luminescent Cell Viability Assay. Results were expressed as nMATP and were shown as drug-response curve exhibiting the ATP concentration changes at Cmax. Results: Genomic findings included an oncogenic KRAS (G12D) mutation. BAY-293 has a partial cell killing response as a single agent. Given to the presence of IGF2 over-expression, IGF2/IGF1R pathway was targeted by linsitinib. Addition of BAY-293 to linsitinib led to enhanced effectiveness of this agent (Table 1). Conclusion: Combination of comprehensive genomic profiling and a laboratory-based 3D micro-cancer model using the patient’s own tumor is an effective way to determine drug efficacy. Targeting RAS pathway by single agents, increases the possibility of resistance. Dual targeting of downstream pathways by MEK and PI3K pathway inhibition is highly toxic, and the clinical usage of this combination is limited. Our results have indicated inhibition of IGF-1R/IGF2 axis in combination with other agents targeting RAS pathway, can be a potential method to address these limitations. ABEL 1. Summary of molecular alterations and targeting methods Molecular alteration Drug Target Kill% at Cmax KRAS (G12D) Mutation BAY-293 RAS-SOS1 interaction 55.3 Trametinib MEK1/2 88.3 Selumetinib MEK1/2 76 Paxalisib PI3K/mTOR 89.7 Samotolisib PI3K/mTOR 92.9 Copanlisib PI3K 95.2 Perifosine AKT 89.2 IGF2 Over-expression Linsitinib IGF-1R/InsR 84.1 BMS-754807 IGF-1R/InsR 67.4 NTRK2 mutation Larotrectinib NTRK 41.9 KMT2D mutation Panobinostat HDAC 85.3 Combinations Kill% at Cmax BAY-293+Selumetinib 100 BAY-293+Samotolisib 100 BAY-293+Paxalisib 99.7 BAY-293+Linsitinib 97.5 Selumetinib+Samotolisib 93.1 Citation Format: Dorsay Sadeghian, Wan-Hsin Lin, James Smadbeck, Alan Panheiter, Michael Barrett, Alexa F. Mccune, Giannoula Karagouga, Sarah Johnson, Ryan W. Feathers, Faye R. Harris, Lindsey Kinsella, Taylor Berry, John Cheville, Panos Anastasiadis, Candace Granberg, Patricio Gargollo, Vasmatzis George. Dual inhibition of RAS and the IGF2/IGF1R pathways: a potential targeting strategy for RAS-mutated embryonal rhabdomyosarcoma [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 3391.

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 Introduction/Objective High-grade endometrial cancer (EC) recurs in 50% of patients following surgery and first-line chemotherapy. There are no established blood-based biomarkers with sufficient specificity and sensitivity to monitor EC patients for residual disease and recurrence. The ability to detect and monitor EC currently relies on serial imaging based approaches. Liquid biopsies via blood-based tests for cell-free circulating tumor DNA (ctDNA) are becoming increasingly popular, but none have been FDA approved for EC and current approaches have not been validated in patients with uncommon tumor mutations. We hypothesized that personalized testing panels based on tumor-specific chromosomal rearrangements would allow ctDNA to be monitored and that ctDNA presence correlates with other clinical indicators of recurrence. Methods/Case Report Twelve patients with FIGO Stage I-IV and biopsy confirmed high-grade EC were prospectively enrolled and underwent primary tumor resection. DNA was isolated from tumor tissue and sequenced on Illumina NGS platforms [San Diego, CA]. BIMA algorithm and SVAtools identified junctions of somatic chromosomal rearrangements and 4-5 junctions were chosen per patient, based primarily on copy number/amplification level. Blood was collected before surgery, and longitudinally between 1 and 7 timepoints. Total cell-free DNA (cfDNA) was extracted from platelet poor plasma with the Qiagen CNA kit [Germany]. Personalized ctDNA qPCR Taqman [IDT, Coralville, Iowa] probe assays were developed with pooled pre-amplification to maximize sensitivity for the multiple junctions and normalized to input. Results (if a Case Study enter NA) ctDNA was detected in 9/12 and 5/12 cases pre- and post-surgery, respectively. The detection or absence of measurable ctDNA following surgery showed high correlation with cancer recurrence as detected by CT or MRI scans. Conclusion The detection of ctDNA in high-grade EC using personalized junction panels shows promise for a blood-based assay that may help physicians better monitor and treat patients.

AbstractA crucial mutational mechanism in malignancy is structural variation, in which chromosomal rearrangements alter gene functions that drive cancer progression. Herein, the presence and pattern of structural variations were investigated in twelve prospectively acquired treatment‐naïve pancreatic cancers specimens obtained via endoscopic ultrasound (EUS). In many patients, this diagnostic biopsy procedure and specimen is the only opportunity to identify somatic clinically relevant actionable alterations that may impact their care and outcome. Specialized mate pair sequencing (MPseq) provided genome‐wide structural variance analysis (SVA) with a view to identifying prognostic markers and possible therapeutic targets. MPseq was successfully performed on all specimens, identifying highly rearranged genomes with complete SVA on all specimens with > 20% tumour content. SVA identified chimeric fusion proteins and potentially immunogenic readthrough transcripts, change of function truncations, gains and losses of key genes linked to tumour progression. Complex localized rearrangements, termed chromoanagenesis, with broad pattern heterogeneity were observed in 10 (83%) specimens, impacting multiple genes with diverse cellular functions that could influence theragnostic evaluation and responsiveness to immunotherapy regimens. This study indicates that genome‐wide MPseq can be successfully performed on very limited clinically EUS obtained specimens for chromosomal rearrangement detection and potential theragnostic targets.

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.