Barrett, Michael T.

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.

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 > 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.

BACKGROUND A patient-derived organoid (PDO) platform may serve as a promising tool for translational cancer research. In this study, we evaluated PDO’s ability to predict clinical response to gastrointestinal (GI) cancers. METHODS We generated PDOs from primary and metastatic lesions of patients with GI cancers, including pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and cholangiocarcinoma. We compared PDO response with the observed clinical response for donor patients to the same treatments. RESULTS We report an approximately 80% concordance rate between PDO and donor tumor response. Importantly, we found a profound influence of culture media on PDO phenotype, where we showed a significant difference in response to standard-of-care chemotherapies, distinct morphologies, and transcriptomes between media within the same PDO cultures. CONCLUSION While we demonstrate a high concordance rate between donor tumor and PDO, these studies also showed the important role of culture media when using PDOs to inform treatment selection and predict response across a spectrum of GI cancers. TRIAL REGISTRATION Not applicable. FUNDING The Joan F. & Richard A. Abdoo Family Fund in Colorectal Cancer Research, GI Cancer program of the Mayo Clinic Cancer Center, Mayo Clinic SPORE in Pancreatic Cancer, Center of Individualized Medicine (Mayo Clinic), Department of Laboratory Medicine and Pathology (Mayo Clinic), Incyte Pharmaceuticals and Mayo Clinic Hepatobiliary SPORE, University of Minnesota-Mayo Clinic Partnership, and the Early Therapeutic program (Department of Oncology, Mayo Clinic).

Background: Morreton virus (MORV) is an oncolytic Vesiculovirus , genetically distinct from vesicular stomatitis virus (VSV). Aim: To report that MORV induced potent cytopathic effects (CPEs) in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) in vitro models. Approach and Results: In preliminary safety analyses, high intranasal doses (up to 10 10 50% tissue culture infectious dose [TCID 50 ]) of MORV were not associated with significant adverse effects in immune competent, non–tumor‐bearing mice. MORV was shown to be efficacious in a Hep3B hepatocellular cancer xenograft model but not in a CCA xenograft HuCCT1 model. In an immune competent, syngeneic murine CCA model, single intratumoral treatments with MORV (1 × 10 7 TCID 50 ) triggered a robust antitumor immune response leading to substantial tumor regression and disease control at a dose 10‐fold lower than VSV (1 × 10 8 TCID 50 ). MORV led to increased CD8 + cytotoxic T cells without compensatory increases in tumor‐associated macrophages and granulocytic or monocytic myeloid‐derived suppressor cells. Conclusions: Our findings indicate that wild‐type MORV is safe and can induce potent tumor regression via immune‐mediated and immune‐independent mechanisms in HCC and CCA animal models without dose limiting adverse events. These data warrant further development and clinical translation of MORV as an oncolytic virotherapy platform.

PURPOSE This investigation sought to evaluate the prognostic value of pretreatment of circulating tumor DNA (ctDNA) in metastatic biliary tract cancers (BTCs) treated with platinum-based first-line chemotherapy treatment. MATERIALS AND METHODS We performed a retrospective analysis of 67 patients who underwent ctDNA testing before platinum-based chemotherapy for first-line treatment for metastatic BTC. For analysis, we considered the detected gene with highest variant allele frequency as the dominant clone allele frequency (DCAF). Results of ctDNA analysis were correlated with patients' demographics, progression-free survival (PFS), and overall survival (OS). RESULTS The median age of patients was 67 (27-90) years. Fifty-four (80.6%) of 67 patients evaluated had intrahepatic cholangiocarcinoma; seven had extrahepatic cholangiocarcinoma, and six gallbladder cancers. Forty-six (68.6%) of the patients were treated with cisplatin plus gemcitabine, and 16.4% of patients received gemcitabine and other platinum (carboplatin or oxaliplatin) combinations, whereas 15% of patients were treated on a clinical trial with gemcitabine and cisplatin plus additional agents (CX4945, PEGPH20, or nab-paclitaxel). TP53, KRAS, FGFR2, ARID1A, STK11, and IDH1 were the genes with highest frequency as DCAF. The median DCAF was 3% (0%-97%). DCAF > 3% was associated with worse OS (median OS: 10.8 v 18.8 months, P = .032). Stratifying DCAF in quartiles, DCAF > 10% was significantly related to worse PFS (median PFS: 3 months, P = .014) and worse OS (median OS: 7.0 months, P = .001). Each 1% increase in ctDNA was associated with a hazard ratio of 13.1 in OS when adjusting for subtypes, metastatic sites, size of largest tumor, age, sex, and CA19-9. CONCLUSION DCAF at diagnosis of advanced BTC can stratify patients who have worse outcomes when treated with upfront platinum-based chemotherapy. Each increase in %ctDNA decreases survival probabilities.

Abstract Background and aims Biliary tract cancers (BTCs) are uncommon, but highly lethal, gastrointestinal malignancies. Gemcitabine/cisplatin is a standard‐of‐care systemic therapy, but has a modest impact on survival and harbors toxicities, including myelosuppression, nephropathy, neuropathy, and ototoxicity. Whereas BTCs are characterized by aberrations activating the cyclinD1/cyclin‐dependent kinase (CDK)4/6/CDK inhibitor 2a/retinoblastoma pathway, clinical use of CDK4/6 inhibitors as monotherapy is limited by lack of validated biomarkers, diffident preclinical efficacy, and development of acquired drug resistance. Emerging studies have explored therapeutic strategies to enhance the antitumor efficacy of CDK4/6 inhibitors by the combination with chemotherapy regimens, but their mechanism of action remains elusive. Approach and results Here, we report in vitro and in vivo synergy in BTC models, showing enhanced efficacy, reduced toxicity, and better survival with a combination comprising gemcitabine/cisplatin and CDK4/6 inhibitors. Furthermore, we demonstrated that abemaciclib monotherapy had only modest efficacy attributable to autophagy‐induced resistance. Notably, triplet therapy was able to potentiate efficacy through elimination of the autophagic flux. Correspondingly, abemaciclib potentiated ribonucleotide reductase catalytic subunit M1 reduction, resulting in sensitization to gemcitabine. Conclusions As such, these data provide robust preclinical mechanistic evidence of synergy between gemcitabine/cisplatin and CDK4/6 inhibitors and delineate a path forward for translation of these findings to preliminary clinical studies in advanced BTC patients.

Abstract Adenosquamous cancer of the pancreas (ASCP) is a subtype of pancreatic cancer that has a worse prognosis and greater metastatic potential than the more common pancreatic ductal adenocarcinoma (PDAC) subtype. To distinguish the genomic landscape of ASCP and identify actionable targets for this lethal cancer, we applied DNA content flow cytometry to a series of 15 tumor samples including five patient-derived xenografts (PDX). We interrogated purified sorted tumor fractions from these samples with whole-genome copy-number variant (CNV), whole-exome sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analyses. These identified a variety of somatic genomic lesions targeting chromatin regulators in ASCP genomes that were superimposed on well-characterized genomic lesions including mutations in TP53 (87%) and KRAS (73%), amplification of MYC (47%), and homozygous deletion of CDKN2A (40%) that are common in PDACs. Furthermore, a comparison of ATAC-seq profiles of three ASCP and three PDAC genomes using flow-sorted PDX models identified genes with accessible chromatin unique to the ASCP genomes, including the lysine methyltransferase SMYD2 and the pancreatic cancer stem cell regulator RORC in all three ASCPs, and a FGFR1-ERLIN2 fusion associated with focal CNVs in both genes in a single ASCP. Finally, we demonstrate significant activity of a pan FGFR inhibitor against organoids derived from the FGFR1-ERLIN2 fusion–positive ASCP PDX model. Our results suggest that the genomic and epigenomic landscape of ASCP provide new strategies for targeting this aggressive subtype of pancreatic cancer. Significance: These data provide a unique description of the ASCP genomic and epigenomic landscape and identify candidate therapeutic targets for this dismal cancer.

Abstract Background: Amplification of chromosome 9p24.1 targeting PD-L1, PD-L2, and JAK2 (PDJ amplicon) is present in ~15% of TNBCs and is associated with activation of the JAK/STAT pathway and poor clinical outcomes. In preclinical studies, PD-L1 expression is markedly and rapidly inducible by low-dose IFN-γ in a PDJ amplicon copy-number dependent manner, mimicking an in situ inflammatory response. This suggests PDJ amplification could identify those TNBCs that would respond to ICIs and/or JAK2 targeted-therapies. The goals in this study were to determine the PDJ status of a well annotated cohort of TNBC PDXs with a 3 color FISH assay and to further define the genomes and transcriptomes of PDJ+ and PDJ- TNBCs. Methods: Tissue microarrays (TMAs) of 49 cores from 27 breast cancer PDX samples, including 21 TNBCs were obtained from the Baylor College of Medicine. The TMAs were interrogated with a 3 color FISH assay consisting of three probes that simultaneously target the PDJ amplicon, 9q22, and 9q34.1. The ratios of PDJ to 9q22 and PDJ to 9q34.1 were calculated to distinguish PDJ amplification from polyploidy. In each case, 50 cells were scored. Samples with PDJ signal >4.0, PDJ/9q22 ≥2 and/or PDJ/9q34.1 ≥2 were called positive by FISH. A subset of PDJ+ PDXs was then flow sorted by DNA content and interrogated with 400k whole genome CNV arrays. These results were integrated with existing clinical, exome, and transcriptome data for the TMAs (pdxportal.research.bcm.edu). Results: PDJ amplification was identified in 6/27 PDX samples including 3/21 (14%) TNBCs. Notably the number of PDJ+ cells in each sample ranged from 2-18. Single cells with ≥10 copies were observed in each PDJ+ sample as well as two PDJ- TNBCs. PDJ+ samples had increased expression of PD-L1 and JAK2 in their RNAseq data. Whole genome CNV analysis of flow sorted PDJ+ samples confirmed the presence of the PDJ amplicon and identified co-occurring amplifications and deletions. A similar pattern of PDJ amplicon heterogeneity was seen in a Mayo Clinic Arizona cohort of 70 surgically resected TNBCs. Previous whole genome CNV analysis of flow sorted samples identified 13/70 (18%) PDJ+ cases. FISH analysis of 14 TNBCs from this cohort (PDJ+ and PDJ- cases) validated these results and identified PDJ+ tumors with cells containing >20 copies of the amplicon. Conclusion: Our multicolor FISH assay of PDX and primary resected samples confirms a prevalence of ~15% PDJ+ TNBCs. Previous studies have reported an enrichment of JAK2 amplification in neoadjuvant treated TNBCs. Given the heterogeneous FISH patterns in both PDJ+ and PDJ- samples in our cohorts, we hypothesize that neoadjuvant therapy could enrich the presence of PDJ+ cells and thereby sensitize TNBCs to ICIs and JAK2 targeted therapies. Future work will explore the role of PDJ amplification and the presence of PDJ+ cells in TNBCs in response to neoadjuvant therapy. Citation Format: Alexander S. Roesler, Smriti Malasi, Elizabeth Lenkiewicz, Barbara A. Pockaj, Michael T. Lewis, Lacey E. Dobrolecki, Lori Koslosky, Peter Hartmayer, Karen S. Anderson, Michael T. Barrett. PDJ amplicon heterogeneity in triple negative breast cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1790.