Patient-derived Orthotopic Xenograft Research Articles

Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma

Radiation is one of the standard therapies for pediatric high-grade glioma (pHGG), of which the prognosis remains poor. To gain an in-depth understanding of biological consequences beyond the classic DNA damage, we treated 9 patient-derived orthotopic xenograft (PDOX) models, including one with DNA mismatch repair (MMR) deficiency, with fractionated radiations (2 Gy/day x 5 days). Extension of survival time was noted in 5 PDOX models (P < 0.05) accompanied by γH2AX positivity in >95 % tumor cells in tumor core and >85 % in the invasive foci as well as ∼30 % apoptotic and mitotic catastrophic cell death. The model with DNA MMR (IC-1406HGG) was the most responsive to radiation with a reduction of Ki-67(+) cells. Altered metabolism, including mitochondria number elevation, COX IV activation and reactive oxygen species accumulation, were detected together with the enrichment of CD133+ tumor cells. The latter was caused by the entry of quiescent G0 cells into cell cycle and the activation of self-renewal (SOX2 and BMI1) and epithelial mesenchymal transition (fibronectin) genes. These novel insights about the cellular and molecular mechanisms of fractionated radiation in vivo should support the development of new radio-sensitizing therapies.

Abstract PO1-29-01: Single agent and combinatorial efficacy with imipridone ONC206 via inhibition of mitochondrial function in preclinical models of chemorefractory triple negative breast cancer

Abstract Breast cancer remains a major global health concern, with triple-negative breast cancer (TNBC) being one of the most aggressive subtypes associated with limited targeted therapies. Chemotherapy resistance in TNBC patients poses a significant clinical challenge and is associated with poor progression-free and overall survival. Thus, exploring novel approaches to improve chemotherapeutic efficacy is of paramount importance. We previously demonstrated a role for mitochondrial fusion in supporting oxidative phosphorylation (oxphos) activity and cell survival in residual TNBC (PMID: 30996079 and 36813854) cells refractory to chemotherapy treatments. Based on these observations, we sought to inhibit mitochondrial function to improve chemotherapeutic efficacy. We used ONC206, a novel agonist of mitochondrial serine protease, ClpP (Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit) to induce mitochondrial proteotoxic stress. ONC201, a parent molecule of ONC206, has shown efficacy in TNBC as a single agent or when combined with a MEK inhibitor in vitro (PMID: 34680527). We demonstrate treatment of TNBC cell lines with ONC206 results in reduced mitochondrial fusion and OXPHOS. Furthermore, ONC206 co-treatment enhanced chemo-sensitivity in vitro. Based on these promising findings, we tested this in orthotopic patient-derived xenograft (PDX) mouse preclinical trials. Three PDX models of TNBC were tested, BCM-2665, BCM-5471, and BCM-0002. ONC206 as a single agent (twice weekly for 4 weeks, oral gavage, 100mg/kg) significantly decreased tumor growth in all three PDX models. Remarkably, treatment ONC206 achieved nearly complete tumor regression as a single agent or when combined with carboplatin in BCM-2665. Regressed tumors did re-grow, but this was significantly delayed when combined with carboplatin. Treatment with ONC206 in BCM-5471 resulted in a modest but significant reduction in tumor growth rate as a single agent. However, ONC206 did not improve carboplatin (weekly, i.p., 50mg/kg) nor docetaxel (weekly, i.p., 20mg/kg) efficacy in this model. ONC206 treatment of BCM-0002 resulted in prolonged tumor stasis and a slight enhancement of docetaxel and carboplatin efficacy. Interestingly, the efficacy of ONC206 in these PDX models correlates with their relative mRNA expression of ClpP. The mice demonstrated tolerance to ONC206 as a single agent or when combined with chemotherapy during the course of treatment. Our study underscores the potentially crucial role of mitochondria in driving chemotherapy resistance in TNBC and provides novel insights into mitochondria-targeted therapeutic approaches. We report a novel targeting option for TNBC and anticipate that these findings will stimulate further research into innovative combination therapies for TNBC patients, and may lead us to a viable inhibitor. Citation Format: Lily Baek, Lacey Dobrolecki, Stefanie Faucher, Christina Sallas, Nia Griffith, Varun Prabhu, Bora Lim, Michael Lewis, Gloria Echeverria. Single agent and combinatorial efficacy with imipridone ONC206 via inhibition of mitochondrial function in preclinical models of chemorefractory triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-29-01.

Direct Implantation of Patient Brain Tumor Cells into Matching Locations in Mouse Brains for Patient-Derived Orthotopic Xenograft Model Development.

Background: Despite multimodality therapies, the prognosis of patients with malignant brain tumors remains extremely poor. One of the major obstacles that hinders development of effective therapies is the limited availability of clinically relevant and biologically accurate (CRBA) mouse models. Methods: We have developed a freehand surgical technique that allows for rapid and safe injection of fresh human brain tumor specimens directly into the matching locations (cerebrum, cerebellum, or brainstem) in the brains of SCID mice. Results: Using this technique, we successfully developed 188 PDOX models from 408 brain tumor patient samples (both high-and low-grade) with a success rate of 72.3% in high-grade glioma, 64.2% in medulloblastoma, 50% in ATRT, 33.8% in ependymoma, and 11.6% in low-grade gliomas. Detailed characterization confirmed their replication of the histopathological and genetic abnormalities of the original patient tumors. Conclusions: The protocol is easy to follow, without a sterotactic frame, in order to generate large cohorts of tumor-bearing mice to meet the needs of biological studies and preclinical drug testing.

Efficient CAR T cell targeting of the CA125 extracellular repeat domain of MUC16

BackgroundOvarian cancer (OC) is the leading cause of death from gynecologic malignancies in the Western world. Contributing factors include a high frequency of late-stage diagnosis, the development of chemoresistance, and...

Suppression of Glioblastoma Stem Cell Potency and Tumor Growth via LRRK2 Inhibition.

Leucine-rich repeat kinase 2 (LRRK2), a large GTP-regulated serine/threonine kinase, is well-known for its mutations causing late-onset Parkinson's disease. However, the role of LRRK2 in glioblastoma (GBM) carcinogenesis has not yet been fully elucidated. Here, we discovered that LRRK2 was overexpressed in 40% of GBM patients, according to tissue microarray analysis, and high LRRK2 expression correlated with poor prognosis in GBM patients. LRRK2 and stemness factors were highly expressed in various patient-derived GBM stem cells, which are responsible for GBM initiation. Canonical serum-induced differentiation decreased the expression of both LRRK2 and stemness factors. Given that LRRK2 is a key regulator of glioma stem cell (GSC) stemness, we developed DNK72, a novel LRRK2 kinase inhibitor that penetrates the blood-brain barrier. DNK72 binds to the phosphorylation sites of active LRRK2 and dramatically reduced cell proliferation and stemness factors expression in in vitro studies. Orthotopic patient-derived xenograft mouse models demonstrated that LRRK2 inhibition with DNK72 effectively reduced tumor growth and increased survival time. We propose that LRRK2 plays a significant role in regulating the stemness of GSCs and that suppression of LRRK2 kinase activity leads to reduced GBM malignancy and proliferation. In the near future, targeting LRRK2 in patients with high LRRK2-expressing GBM could offer a superior therapeutic strategy and potentially replace current clinical treatment methods.

Glioblastoma-instructed microglia transition to heterogeneous phenotypic states with phagocytic and dendritic cell-like features in patient tumors and patient-derived orthotopic xenografts

BackgroundA major contributing factor to glioblastoma (GBM) development and progression is its ability to evade the immune system by creating an immune-suppressive environment, where GBM-associated myeloid cells, including resident microglia and peripheral monocyte-derived macrophages, play critical pro-tumoral roles. However, it is unclear whether recruited myeloid cells are phenotypically and functionally identical in GBM patients and whether this heterogeneity is recapitulated in patient-derived orthotopic xenografts (PDOXs). A thorough understanding of the GBM ecosystem and its recapitulation in preclinical models is currently missing, leading to inaccurate results and failures of clinical trials.MethodsHere, we report systematic characterization of the tumor microenvironment (TME) in GBM PDOXs and patient tumors at the single-cell and spatial levels. We applied single-cell RNA sequencing, spatial transcriptomics, multicolor flow cytometry, immunohistochemistry, and functional studies to examine the heterogeneous TME instructed by GBM cells. GBM PDOXs representing different tumor phenotypes were compared to glioma mouse GL261 syngeneic model and patient tumors.ResultsWe show that GBM tumor cells reciprocally interact with host cells to create a GBM patient-specific TME in PDOXs. We detected the most prominent transcriptomic adaptations in myeloid cells, with brain-resident microglia representing the main population in the cellular tumor, while peripheral-derived myeloid cells infiltrated the brain at sites of blood–brain barrier disruption. More specifically, we show that GBM-educated microglia undergo transition to diverse phenotypic states across distinct GBM landscapes and tumor niches. GBM-educated microglia subsets display phagocytic and dendritic cell-like gene expression programs. Additionally, we found novel microglial states expressing cell cycle programs, astrocytic or endothelial markers. Lastly, we show that temozolomide treatment leads to transcriptomic plasticity and altered crosstalk between GBM tumor cells and adjacent TME components.ConclusionsOur data provide novel insights into the phenotypic adaptation of the heterogeneous TME instructed by GBM tumors. We show the key role of microglial phenotypic states in supporting GBM tumor growth and response to treatment. Our data place PDOXs as relevant models to assess the functionality of the TME and changes in the GBM ecosystem upon treatment.Graphical

Abstract 3537: Assessing machine learning models of drug response predictions using orthotopic patient-derived xenograft

Abstract Accurate prediction of drug response using machine learning (ML) remains a significant challenge in drug development as well as in personalized cancer therapy. Furthermore, there is a scarcity of rigorous external validation studies for evaluating drug response prediction models. Even when available, such validations are often confined to in-vitro studies. Orthotopic patient-derived xenograft (O-PDX) mice serve as essential preclinical models that closely replicate the human tumor microenvironment and assess the therapeutic response to cancer treatments. We present CertisAITM, a novel ensemble of machine learning models that have been trained on a vast array of experimental high-throughput screenings of both monotherapies and combination therapies, incorporating over 4500 investigational and FDA-approved drugs across 10 major cancer indications. The results of our ML drug response prediction models show an average R2 of 0.75 and RMSE of 0.62 (across internal 5-fold cross-validation) for all 10 cancer prediction models. Our evaluation of CertisAI's drug response predictions within O-PDX pharmacology studies has shown a notable overall correlation between actual observed tumor growth inhibition (TGI) and predicted TGI, with an r value of 0.45 across seven indications. The most accurate prediction model achieved an r value of 0.7 for colorectal cancer, encompassing 37 treatments in 6 O-PDX studies. Altogether, these results highlight CertisAI’s potential in enhancing pre-clinical model selection as well as personalized treatment strategies in oncology. Citation Format: Yuan-Hung Chien, Raffaella Pippa, Warren Andrews, Javier Rodriguez, Kristen Buck, Derrick Gorospe, Elizabeth Valencia, Bianca Carapia, Emily Eastwood, Jantzen Sperry, Jonathan Nakashima, Long Do. Assessing machine learning models of drug response predictions using orthotopic patient-derived xenograft [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 3537.

Abstract 4661: A dual degrader of HER2 and EGFR obliterates oncogenic signaling, overcomes therapy resistance, and inhibits metastatic lesions in HER2-positive breast cancer models

Abstract Background: HER2 is an oncogenic receptor tyrosine kinase (RTK) implicated in several types of human cancer. It is strongly expressed in about 20% of breast cancer (BC), known as HER2-positive BC (HER2+ BC), due to gene amplification. HER2 amplification or overexpression is a strong predictor of poor disease prognosis. HER2-targeted therapies are the linchpin of treating HER2+ BC. However, drug resistance is common. The main resistance mechanism is unknown. We exploited cell lines and mouse models (orthotopic patient-derived xenografts, and metastatic lesions in the brain and lung of therapy-resistant HER2+ BC) for experiments of genetic and pharmacologic depletion of HER2 and its family member EGFR, analysis of RTKs and no-RTKs, and evaluation of cell and tumor growth, to delineate the mechanism of HER2+ BC resistance to HER2 inhibitors and assess the therapeutic activity of PEPDG278D. PEPDG278D, a recombinant human protein, is the first-in-class dual degrader of HER2 and EGFR. Results: Trastuzumab-resistant HER2+ BC cells and tumors are highly vulnerable to targeted degradation of HER2 and EGFR. Trastuzumab is the most commonly used HER2 inhibitor in the clinic. Targeted degradation of HER2 and EGFR by PEPDG278D, which binds to their extracellular domains, causes extensive inhibition of oncogenic signaling proteins in HER2+ BC cells resistant to trastuzumab. This is accompanied by strong growth inhibition of cultured cells, orthotopic patient-derived xenografts, and metastatic lesions in the brain and lung of trastuzumab-resistant HER2+ BC. siRNA knockdown shows that eliminating both HER2 and EGFR is important to maximize therapeutic outcome. Collectively, our results reveal the key vulnerability of trastuzumab-resistant HER2+ BC and shows a promising strategy that overcomes trastuzumab resistance by targeting this vulnerability. Conclusions &amp; Therapeutic Relevance: This study identifies a key therapeutic vulnerability of drug-resistant HER2+ BC and shows that an agent that targets this vulnerability is highly effective in overcoming drug resistance in this disease. The findings provide new insights and innovations to advance treatment of drug-resistant HER2+ BC that remains an unmet problem. Citation Format: Lu Yang, Arup Bhattacharya, Darrell Peterson, Yun Li, Xiaozhuo Liu, Elisabetta Marangoni, Valentina Robila, Yuesheng Zhang. A dual degrader of HER2 and EGFR obliterates oncogenic signaling, overcomes therapy resistance, and inhibits metastatic lesions in HER2-positive breast cancer models [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 4661.

Abstract 143: Molecular profiling of tumor-initiation events in rhabdomyosarcoma uncovers mitochondrial vulnerabilities

Abstract Background: Alveolar Rhabdomyosarcoma (ARMS) is a very aggressive soft tissue tumor affecting children and adolescents. It is found that pediatric patients with RMS harboring chimeric PAX fusion transcription factors (TFs) exhibit a greater incidence of tumor relapse, metastasis, and poor survival outcome, thereby underscoring the urgent need to develop effective therapies to treat this subtype of childhood cancer. Methods: We generated a novel ARMS model by stable transduction of myogenic progenitor cells with a cassette encoding the exact PAX3/7-FOXO1 fusion protein found in ARMS tumors. We analyzed genome-wide expression levels with RNA-seq and utilized Cut&amp;Run to map the binding sites of PAX3/7-FOXO1, active histone marks, enhancers, and super-enhancers in our novel model. Hi-C and promoter capture HiC (pCHiC) were also performed to generate 3D chromatin conformation maps in cells expressing the oncogenic TFs. We compared these data with analogous datasets from orthotopic patient-derived xenografts (O-PDXs) of PAX3/7-FOXO1 fusion-positive ARMS tumors from St. Jude and established ARMS cell lines to validate whether our model system recapitulates ARMS tumors. CRISPRi was performed to silence key targets in two representative ARMS cell lines and proliferation was scored after silencing. Next, we found that ARMS tumors displayed anomalies in mitochondrial mass and numbers, metabolic parameters, and membrane potential by performing TEM, Seahorse assays, and FACS-based TMRM and mitotracker Red dye uptake in a panel of ARMS cells and PDX samples. We performed cell viability and colony forming assays in ARMS cells treated with single drugs and combinations of drugs targeting FGFR4 and mitochondrial metabolism. Results: Our data provide evidence to support muscle progenitors as potential cells-of-origin for ARMS. We showed that rewiring of chromatin architecture mediated by PAX3/7-FOXO1 TFs is evident across established cell lines, primary tumors as well as O-PDXs. By integrating PAX3/7-FOXO1 binding, RNA-seq, and pCHiC data, we identified key promoter-enhancer (P-En) interactions that are erased or established de novo at multiple loci of master TFs like MYOD, MYOG and MYCN. We also identified key regulators of proliferation, including FGFR4, and mitochondrial function that are essential for tumor growth and invasiveness. Our data indicated aberrant and enhanced mitochondrial metabolism in ARMS as well as selective sensitivity to mitochondrial inhibitors in RMS cells. Administration of these inhibitors preferentially inhibited cell viability and clonogenicity in ARMS, and prompted a pre-clinical phase II study to test these inhibitors at St. Jude Research Hospital. Conclusion: We present a novel treatment strategy leveraging the mitochondrial dependencies of fusion positive PAX-driven tumors which can be exploited for precision therapy in high-risk ARMS. Citation Format: Bhargab Kalita, Gerard Martinez-Cebrian, Justina McEvoy, Brittney Gordon, Kaley Blankenship, Asa Karlstorm, Elizabeth Stewart, Michael Dyer, Brian Dynlacht. Molecular profiling of tumor-initiation events in rhabdomyosarcoma uncovers mitochondrial vulnerabilities [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 143.

Abstract 5113: Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma

Abstract Radiation is one of the standard therapies for pediatric high-grade glioma (pHGG), of which the prognosis remains poor. To gain an in-depth understanding of the biological activities beyond the classic DNA damage, we treated 6 patient-derived orthotopic xenograft (PDOX) models, including one with DNA mismatch repair (MMR) deficiency, with fractionated radiations (2 Gy/day x 5 days). Extension of survival time was noted in 4/6 PDOX models (P&amp;lt;0.05) and widespread γH2AX positivity in &amp;gt;95% tumor cells in tumor core and &amp;gt;85% in the invasive foci, accompanied by ~30% apoptotic and mitotic catastrophic cell death. The model with DNA MMR (IC-1406HGG) was the most responsive to radiation with a reduction of Ki-67(+) cells. Altered metabolism, including mitochondria number elevation, COX IV activation and reactive oxygen species buildup, were detected together with the accumulation of CD133+ tumor cells. The latter was caused by the entry of quiescent G0 cells into cell cycle and the activation of self-renewal (SOX2 and BMI1) and epithelial mesenchymal transition (fibronectin) genes. These novel insights about the cellular and molecular mechanisms of fractionated radiation in vivo should support the development of new radio-sensitizing therapies. Citation Format: Zi-Lu Huang, Zhi-Gang Liu, Qi Lin, Ya-Lan Tao, Xinzhuoyun Li, Patricia Baxter, Jack MF Su, Adekunle M. Adesina, Chris Man, Murali Chintagumpala, Wan Yee Teo, Yu-Chen Du, Yun-Fei Xia, Xiao-Nan Li. Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma [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 5113.

Abstract 1660: Tumor pharmacokinetics and pharmacodynamics assessment of TEAD inhibitor VT03989 in patient-derived xenograft models of glioblastoma

Abstract Background: The Hippo-YAP/TAZ pathway regulates cell proliferation, cell growth and migration. The major effectors of the Hippo pathway are the TEAD transcription factors, which are deregulated in several cancers, including glioblastoma (GBM). In this study, we evaluated the pharmacokinetics and pharmacodynamics of VT03989, a highly potent TEAD inhibitor, in orthotopic patient-derived xenograft (PDX) models of GBM. Methods: Mice with intracranial PDX GBM tumors were randomized into two cohorts to receive 10 mg/kg (oral) VT03989 or placebo for 4 days. VT03989 levels in plasma, tumor, and contralateral brain tissue were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) at 2, 6, and 12 hours after the last treatment of VT03989. Unbound fractions were determined by equilibrium dialysis. Quantitative RT-PCR was performed to assess levels of TEAD target genes, CTGF and CYR61. Immunohistochemistry of Ki67 and cleaved caspase 3 (CC3) was performed. Results: The median unbound concentrations of VT3989 were 27 nM in plasma and 26 nmol/kg in the tumor. The median brain-to-plasma and tumor-to-plasma partition coefficients of unbound VT03989 were 0.65 and 0.99, respectively - indicating favorable brain and tumor penetration ability of VT03989. A decrease in CTGF and CYR61 indicated TEAD inhibition and was accompanied by an increase in CC3 (+) cells. Conclusion: VT03989 is well tolerated, achieves pharmacologically relevant unbound concentrations in GBM PDX models, and is associated with significant target modulation. Ongoing studies are evaluating in vivo survival benefits from combining VT03989 inhibition with radiation therapy Citation Format: Tigran Margaryan, Yu-Wei Chang, Jennifer Molloy, Barbara Hopkins, Mackenna Elliott, Ernesto Luna Melendez, Connor White, Nader Sanai, An-Chi Tien, Artak Tovmasyan, Shwetal Mehta. Tumor pharmacokinetics and pharmacodynamics assessment of TEAD inhibitor VT03989 in patient-derived xenograft models of glioblastoma [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 1660.

Abstract 4971: Integrating artificial intelligence and functional precision oncology for individualized cancer therapies

Abstract Precision oncology seeks to tailor cancer treatments to individual patients. Patient-derived xenografts (PDXs) have emerged as a promising platform for selecting efficacious, personalized therapies and developing new oncology drugs. Here, we present a workflow for selecting and validating treatments for patients with colorectal cancer (CRC) that integrates orthotopic PDX (O-PDX) models, molecular profiling, machine learning, and in vivo pharmacological validation. Of the nine tumor biopsies collected, seven were successfully developed into O-PDX models (78%) with a median time to establishment of 119 days. Models were molecularly profiled for gene expression and were serially passaged to perform orthotopic pharmacology studies to validate test agents selected by oncologists as well those suggested by CertisAITM, a novel ensemble of machine learning models for the prediction of drug response in human cancers. The correlation between CertisAI's predicted therapy responses and the actual observed tumor growth inhibition (TGI) was r = 0.7, encompassing 37 distinct treatments across studies from six patients. This research collectively illustrates that the integration of artificial intelligence with functional in vivo assays offers a powerful platform for precision oncology, enabling the identification and validation of tailored treatments for individual cancer patients. Citation Format: Fernando Eguiarte-Solomon, Rowan Prendergast, Bianca Carapia, Javier Rodriguez, Kristen Buck, Derrick Gorospe, Elizabeth Valencia, Jose Lopez-Ramos, Itzel Gutierrez, Rafaella Pippa, Yuan-Hung Chien, Warren Andrews, Long Do, Jantzen Sperry, Jonathan K. Nakashima. Integrating artificial intelligence and functional precision oncology for individualized cancer therapies [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 4971.

Abstract 2271: Conservation and faithful representation of circular extrachromosomal DNA in orthotopic patient-derived medulloblastoma xenografts

Abstract Background: Extrachromosomal DNA (ecDNA) is a driver of tumor heterogeneity and is associated with poor survival in many different adult and childhood cancer types. Patient-derived xenograft (PDX) models facilitate preclinical drug testing in an in vivo environment that enables analysis of tumor growth and survival and molecular tumor profiling. These models, combined with single-cell profiling, can be used to study intratumoral heterogeneity under therapeutic pressure and the emergence of drug resistance. Here we investigate the abundance and conservation of ecDNA in PDX models with the aim of assessing the faithful representation of ecDNA compared to the tumors from which they are derived. Methods: Whole-genome sequencing (WGS) was applied to 27 medulloblastoma (MB) PDX models and their tumors of origin using Amplicon Architect, a tool used to reconstruct focally amplified regions of DNA to provide insights into the landscape of cancer genomes. The frequency of ecDNA in these models was also compared to that of ecDNA in the MB patient population. Optical genome mapping enabled accurate and reliable ecDNA sequence reconstructions. Multiome single-cell RNA and ATAC-sequencing of a PDX tumor enabled analysis of ecDNA conservation and intratumoral heterogeneity compared to similar data previously obtained from the primary tumor. Results: Comparative analyses revealed that ecDNA is largely conserved in PDX models, emphasizing their relevance as faithful models of the primary tumors. In addition, we find that ecDNA is significantly more frequent in MB PDX models (16 out of 27 analyzed patient-derived tumors) as compared to their frequency in the overall patient population (~18% of all medulloblastoma tumors contain ecDNA), suggesting ecDNA as a driver for successful establishment of MB PDX models. Single-cell analysis revealed that ecDNA is omnipresent in almost all PDX tumor cells, while its presence was limited to only ~9% of tumor cells in the primary tumor. Conclusion: Analysis of ecDNA in medulloblastoma primary tumors and in PDX models revealed that ecDNA is preserved in PDX models. This is an important characteristic that renders patient-derived mouse xenografts as faithful models of their primary tumors. These results emphasize the relevance of PDX models for preclinical in vivo studies aimed at analyzing the role of ecDNA under therapeutic pressure and for analyzing clonal selection along with molecular evolution of ecDNA during the development of therapeutic resistance. Citation Format: Rishaan Kenkre, Owen Chapman, Jens Luebeck, Vineet Bafna, Robert Wechsler-Reya, Jill Mesirov, Lukas Chavez. Conservation and faithful representation of circular extrachromosomal DNA in orthotopic patient-derived medulloblastoma xenografts [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 2271.

Abstract 3070: CDK12 is a novel therapeutic target in glioblastoma

Abstract Glioblastoma (GBM), the most common primary brain tumor in adults carries a highly unfavorable prognosis of only one to two years despite treatment with the standard of care, involving surgery, temozolomide and radiation. Patient-derived xenograft (PDX) GBM cultures were assessed for their susceptibility to loss of CDK12 function, which was either mediated genetically (shRNA) or by the novel inhibitor, SR-4835. GBM metabolism was measured by utilizing extracellular flux analysis as well as LC/MS based carbon tracing analyses. In vivo efficacy was determined by orthotopic GBM models in mice. Based on Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and shRNA library screens in multiple GBM cell cultures we identified CDK12 as essential for the growth of GBM. Consistently, loss of function of CDK12 (genetically and pharmacologically) led to a reduction of growth of GBM patient-derived xenograft (PDX) lines in vitro. Notably, SR-4835 reduced GBM growth in the low nanomolar range and synergistically enhanced the potency of temozolomide. Extracellular flux analysis and metabolite screening analyses, including carbon tracing (U13C-Glucose, U13C-Glutamine) suggested that loss of function of CDK12 elicited a substantial inhibition of cellular respiration, causing energy deprivation with subsequent cell death with apoptotic features. On the molecular level, loss of function of CDK12 increased ATF4 along with pro-apoptotic Noxa, whereas anti-apoptotic Mcl-1 decreased. In turn, adenoviral mediated ectopic expression of Mcl-1 inhibited cell death mediated by loss of function of CDK12 in GBM cells. In an orthotopic murine PDX model of GBM animals receiving treatment with SR-4835 had a significantly longer overall survival without induction of toxicity as compared to mice treated with vehicle. In summary, these findings suggest that CDK12 is a novel treatment target for GBM and that CDK12 is an important driver of energy metabolism in GBM to partially mediate apoptotic resistance. Moreover, SR-4835 enhanced the potency of temozolomide in GBM models. Therefore, the novel CDK12 inhibitor, SR-4835 should be tested in a clinical trial in patients suffering from GBM. Citation Format: Jeong-Yeon Mun, Chang Shu, Qiuqiang Gao, Mike-Andrew Westhoff, Georg Karpel-Massler, Markus D. Siegelin. CDK12 is a novel therapeutic target in glioblastoma [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 3070.

Abstract 2864: Transcriptional repressor REST controls autophagy in Sonic Hedgehog medulloblastoma through the VHL-HIF1α axis

Abstract Introduction: Medulloblastoma (MB) is the most common malignant brain tumor in children. It is categorized into four molecular subgroups, each with unique genetic and epigenetic alterations, molecular signatures, and differences in clinical features and treatment responses. Targeted therapy for MB has failed in the clinic, prompting new studies of mechanisms involved in MB tumorigenesis and progression. Experimental Procedure: The transcriptomic information of the MB patients was obtained from the publicly available RNAseq datasets and was analyzed for differential expression and clustering analysis. Patient-derived orthotopic xenografts of medulloblastoma tumors with high- and low-REST expression were histologically and biochemically studied for autophagy markers. Furthermore, the SHH-MB cell lines were studied to get a mechanistic understanding of the REST-driven autophagy in this MB subgroup. Summary: Our data showed that autophagy, a process responsible for cellular recycling, and genes associated with this pathway exhibit distinctive expression patterns in human MB subgroups. Specifically, we demonstrate that autophagy is induced in one of the MB subgroups (Sonic Hedgehog-SHH) in response to elevated expression of the RE1-silencing transcription factor (REST), a transcriptional repressor and a canonical regulator of neuronal differentiation genes. REST is also a driver of SHH-MB metastasis. Pharmacological inhibition of autophagy decreases MB cell growth, suggesting it is a pro-survival pathway in REST-driven SHH MBs. In mechanistic studies, we found REST-dependent autophagy induction to involve upregulation of the hypoxia-inducible factor 1-alpha (HIF1α) due to REST-mediated silencing of the von Hippel-Lindau (VHL) gene. The VHL gene product promotes HIF1α ubiquitination and proteasomal degradation, and its loss in SHH-MBs with elevated REST expression drives HIF1α stabilization and nuclear localization to activate target autophagy-related genes. Conclusion: Our work is the first to link VHL to MB pathology. VHL loss of function in cancers has been mainly attributed to mutational events. Here, we report a novel mechanism of VHL loss, namely its epigenetic silencing by REST. Our findings provide the foundation for future pre-clinical investigations to examine the feasibility of autophagy blockade as a therapeutic strategy for REST-driven SHH MBs. Citation Format: Ashutosh Singh, Donghang Cheng, Jyothishmathi Swaminathan, Yan Zheng, Nancy Gordon, Vidya Gopalakrishnan. Transcriptional repressor REST controls autophagy in Sonic Hedgehog medulloblastoma through the VHL-HIF1α axis [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 2864.

Abstract 6620: PDX models of TNBC established from pre- and post-therapy tumors identify vulnerabilities of resistant disease

Abstract Chemotherapy, along with the PD1 inhibitor pembrolizumab, is the recommended standard of care for patients with primary triple negative breast cancer (TNBC). Nearly half of TNBC patients treated with standard neoadjuvant chemotherapy (NACT) have excellent responses. However, those patients with significant residual cancer burden (RCB) after NACT are at high risk of recurrence or metastatic relapse within two years. Due to the challenges posed by the inter- and intratumoral heterogeneity of TNBC, it is critical to develop appropriate models for predicting and overcoming therapy resistance. We established a collection of 92 orthotopic patient-derived xenograft (PDX) models of TNBC from 84 patients before, during, and after NACT while they were enrolled in the ARTEMIS trial (NCT02276443) at MD Anderson Cancer Center. Serial biopsies were obtained from patients prior to treatment (pre-NACT), from poorly responsive disease after four cycles of Adriamycin and cyclophosphamide (AC, mid-NACT), after a 3-month course of additional chemotherapy and/or different experimental therapies in cases of AC resistance (post-NACT), and from the metastatic lesions of two patients. The collection includes 12 longitudinal sets. Models were established from chemo-sensitive and -resistant tumors, but engraftment success was higher from those cancers that proved to be treatment resistant (RCB II/III). The PDX collection includes models encompassing a broad heterogeneity of chemotherapy responses, histologic features, and molecular TNBC subtypes. In addition, the majority of models develop spontaneous lung metastases. Whole exome sequencing demonstrated conservation of mutations between patient tumors and corresponding PDX models, with TP53 being the most commonly mutated gene. A similar comparison of patient and PDX transcriptomes revealed conservation of signaling pathway signatures, with those related to immune and stromal interactions being the most variable, as anticipated when adapting human tumors to growth in mice. The subclonal architecture of the tumors exhibited little overall change throughout NACT, suggesting that selection for resistant subclones is not likely to be a significant contributor to resistance. Tumor cells from the longitudinal PDX collections were subjected to high throughput drug susceptibility profiling to identify targetable vulnerabilities associated with resistance to NACT. We found that post-NACT tumors exhibited enhanced sensitivity to drugs targeting protein homeostasis pathways. Preclinical studies with pevonedistat validated the neddylation pathway as an effective target in chemoresistant TNBC. Citation Format: Amanda L. Rinkenbaugh, Yuan Qi, Reid T. Powell, Shirong Cai, Jiansu Shao, Faiza Baameur Hancock, Lei Guo, Xiaomei Zhang, Sabrina Jeter-Jones, Chunxiao Fu, Rebekah Gould, Jason B. White, Clifford Stephan, Gloria V. Echeverria, Peter J. Davies, Stacy Moulder, W. Fraser Symmans, Jeffrey T. Chang, Helen Piwnica-Worms. PDX models of TNBC established from pre- and post-therapy tumors identify vulnerabilities of resistant disease [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 6620.

Abstract 525: A comprehensive approach: Copper chelation therapy modulates epigenetic, kinase signaling and metabolic pathways in diffuse midline gliomas (DMG)

Abstract Diffuse Midline Gliomas (DMG) are incurable pediatric brain tumors driven by histone mutation H3K27M, globally reducing H3K27 trimethylation altering the epigenome and deregulating gene expression. These mutations often partner with and are strongly integrated into kinase signaling and metabolic alterations, driving aggressive tumor growth with many mechanisms which can compensate for one another and therefore evade traditional single agent therapies. Copper over-accumulation has been linked to epigenetic, kinase and metabolic dysregulation in various cancers and neurological disorders. Copper chelators are clinically approved for use in pediatric Wilson’s Disease patients, improve neurological symptoms and are under clinical investigation for several cancers. We propose copper chelators may simultaneously target these mechanisms in DMG. We performed transcriptomic interrogation of patient datasets, in vitro investigations in a panel of DMG cell lines and an in vivo investigation using an orthotopic xenograft model of DIPG (SU-DIPGVI-Luc). Copper chelator tetraethylenepentamine-pentachloride (TEPA) was used to treat DMG cells. Transcriptomic (RNA-Seq), proteomic, phospho-proteomic and metabolomic (Mass Spectrometry) investigations, western blots and immunofluorescent microscopy determined efficacy and mechanism. In vivo investigation consisted of survival study with concurrent luminescence imaging, histology, and copper assay. We demonstrate copper chaperone transcript expression is increased in DMG patient samples compared to non-DMG samples. In vitro, apoptosis and growth assays demonstrated H3K27M DMG were more sensitive to TEPA than H3-WT DMG. In H3K27M DMG, TEPA downregulated ATP, GTP and SAM metabolism which corresponded with downregulated kinase signaling and altered H3K27 trimethylation and DNA methylation patterns around cell cycle genes. Affected genes included those regulating centrosome segregation and ATP synthesis. Further transcriptomic and proteomic investigations highlighted TEPA downregulated G2-M phase kinase pathways, with immunofluorescence demonstrating TEPA reduced expression of mitotic marker p-H3S10 and epigenetic methylators EZH2 and DNMT1. In vivo, TEPA reduced brain copper accumulation, eliminated tumors and increased survival in an orthotopic patient-derived xenograft DMG model. Collectively, our data indicates TEPA induced multi-modal targeting of M-phase cell cycle progression through epigenetic, kinase signaling and metabolic mechanisms. Efficacy was observed both in vitro and in vivo, providing a promising basis for future investigation of copper chelators in DMG. We propose copper-chelation is a viable therapeutic strategy for DMG patients warranting clinical investigation. Citation Format: Filip Michniewicz, Riccardo Cazzoli, Jessica Bell, Federica Saletta, Ensieh Poursani, Jourdin R. Rouaen, Tyler Shai-Hee, Vu Pham, Toni Rose Jue, Matthew Biery, Liesl Bramberger, Terry Lim, Aaminah Khan, Federico M. Giorgi, Daniele Mercatelli, Chelsea Mayoh, Giuseppe Cirillo, Alexander Macmillan, Renee Whan, Christopher T. Barlow, Maria Tsoli, Nicholas Vitanza, Scott Berry, Pouya Faridi, David Ziegler, Orazio Vittorio. A comprehensive approach: Copper chelation therapy modulates epigenetic, kinase signaling and metabolic pathways in diffuse midline gliomas (DMG) [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 525.

Abstract 6867: TWEAK-Fn14 signaling in glioblastoma-associated myeloid cells promotes tumor invasion via CCL5-CCR5 axis

Abstract Glioblastoma remains difficult to treat with immunotherapy due to the immunosuppressive tumor microenvironment and the ability of glioma cells to invade diffusely into normal brain parenchyma. While it is known that glioblastoma-associated myeloid cells (GAMs) comprise a large proportion of the tumor microenvironment and can suppress adaptive and innate immune responses through various mechanisms, detailed mapping of myeloid lineages in glioblastoma is still missing. Further characterization of pro-tumoral myeloid-lineages in glioblastoma is important for identifying myeloid-based drug targets that may synergize with T cell-based therapies and produce a more meaningful anti-tumor immune response. Towards this end, we examined numerous scRNAseq datasets derived from glioblastoma patients and identified expression of TNFRSF12A, the gene encoding the TNF- family receptor, Fn14, in a subset of GAMs enriched for expression of cytokines such as CCL3, CCL5, IL-6, and IL-8. This result is intriguing since TWEAK (TNFSF12), the ligand for Fn14, is abundantly expressed within numerous patient-derived glioblastoma cell lines, namely GBM6, GBM8, GBM22, and GBM39. To validate Fn14 protein expression in GAMs, we isolated murine bone-marrow derived macrophages and treated them with glioma-conditioned media, which induced Fn14 gene expression. We then conducted in vitro experiments utilizing the human microglia line HMC3, the murine microglia line SIM-A9, and primary murine bone-marrow-derived macrophages to assess the effects of Fn14 activation in myeloid cells. We found that treatment of Fn14+ myeloid cells with TWEAK ligand upregulates CCL3, CCL5, IL-6, and IL-8 expression. We tested the conditioned media generated from TWEAK-treated Fn14+ myeloid cell lines and found that it promotes glioblastoma invasion in a CCL5-dependent manner. This aligns with previous reports demonstrating the CCL5-CCR5 signaling in glioma cells promotes invasion through calcium-dependent matrix metalloproteinase 2. To assess the spatial distribution of this myeloid subpopulation, we performed RNAscope hybridization against AIF1, TMEM119, CCR5, CCL5, and Fn14 transcripts on sections of murine gliomas generated using the RCAS/tv-a model of human glioblastoma. We found an abundance of Fn14+ plus AIF1+ as well as Fn14+plus TMEM119+ myeloid cells throughout the tumor core and rim of murine tumors and identified CCL5 expression in pseudo-palisades bordering necrotic regions. Our results to date suggest that Fn14 expression in GAMs can promote the invasion of tumor cells into normal brain parenchyma by release of CCL5. Currently, we are utilizing orthotopic patient-derived xenograft models co-implanted with Fn14+/- myeloid cells to test the hypothesis that TWEAK-Fn14 signaling in GAMs provides strong pro-tumoral signals in the glioblastoma microenvironment. Citation Format: Angad Beniwal, Matthew Dufault, Pranjali Kanvinde, David Nascari, Ryan Eghlimi, Adarsha Malla, Dolores Hambardzumyan, Henrique Borges da Silva, Graeme Woodworth, Jeffrey Winkles, Nhan Tran. TWEAK-Fn14 signaling in glioblastoma-associated myeloid cells promotes tumor invasion via CCL5-CCR5 axis [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 6867.

Abstract 6908: Integrating PDX GBM in vivo models with patient history and whole exome sequencing: Advancing relevance and precision in preclinical studies

Abstract Glioblastoma poses a significant challenge in oncology, necessitating improved translational models that faithfully replicate the intricate tumor microenvironment. Patient-derived xenograft (PDX) models, are key in glioblastoma multiforme (GBM) preclinical research. PDX models (orthotopic and subcutaneous) provide a more clinically relevant representation compared to CDX xenografts. Analyzing PDX tumors in their native environment allows for a nuanced understanding of GBM biology, tumor-stroma interactions, and treatment responses. This abstract explores the rationale and advantages of utilizing PDX GBM in vivo models coupled with patient diagnosis and treatment history, empowered by tumor whole exome sequencing (WES), to enhance the relevance of preclinical studies in evaluating GBM-specific treatments. TD2 has access to over 100 GBM PDX tumors models with fully annotated patient details and treatment history. Moreover, 68 of these PDXs also have WES data, that allows most appropriate model selection to ensure alignment with the therapeutic development strategy. Below is a detailed analysis of two GBM PDX models, GBM6 and GBM46, using patient history and WES data to define criteria for success in assessing novel preclinical therapeutic agents. GBM6, was obtained prior to treatment from a 65-year-old man with glioblastoma multiforme, that contains 642 mutations, including EGFR VIII amplification. GBM46, was obtained from a 55-year-old man with prior treatment, contained 646 mutations, including RAS pathway alterations and EGFR (MET and FLT3) amplification. When grown subcutaneously the anti-tumor activity of cetuximab against GBM6 and GBM46 resulted in 41% and 48% growth inhibition, respectively. Traditional treatments have also been evaluated in the orthotopic setting, with temozolomide (TMZ) showing a nominal improvement in lifespan (11%) as expected for the unmethylated MGMT found in the GBM6 model. By contrast, GBM46 from a patient with prior treatment with TMZ, OSI-774, radiation, and BCNU, exhibited a 58% increase in overall lifespan when treated with TMZ alone. Orthotopic PDX GBM models preserve many molecular characteristics, enabling the study of intrinsic GBM mutations for developing novel therapeutic strategies. While orthotopic models better capture GBM's dynamic nature, traditional xenograft systems remain valuable as rapid screening tools. Models with defined clinical treatment history and WES data allows for testing new agents in models that more closely resemble the clinical development path for a given new agent and aids in deciphering factors influencing therapeutic resistance. These models have the potential to reshape preclinical research paradigms, accelerating the translation of promising therapeutic agents increasing the likelihood of clinical benefit for patients with glioblastoma multiforme. Citation Format: Erin E. Trachet, Paul M. Gonzales, Stephen Gately. Integrating PDX GBM in vivo models with patient history and whole exome sequencing: Advancing relevance and precision in preclinical studies [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 6908.

Targeted dual degradation of HER2 and EGFR obliterates oncogenic signaling, overcomes therapy resistance, and inhibits metastatic lesions in HER2-positive breast cancer models

AimsHuman epidermal growth factor receptor 2 (HER2) is an oncogenic receptor tyrosine kinase amplified in approximately 20% of breast cancer (BC). HER2-targeted therapies are the linchpin of treating HER2-positive BC. However, drug resistance is common, and the main resistance mechanism is unknown. We tested the hypothesis that drug resistance results mainly from inadequate or lack of inhibition of HER2 and its family member epidermal growth factor receptor (EGFR). MethodsWe used clinically relevant cell and tumor models to assess the impact of targeted degradation of HER2 and EGFR on trastuzumab resistance. Trastuzumab is the most common clinically used HER2 inhibitor. Targeted degradation of HER2 and EGFR was achieved using recombinant human protein PEPDG278D, which binds to the extracellular domains of the receptors. siRNA knockdown was used to assess the relative importance of EGFR and HER2 in trastuzumab resistance. ResultsBoth HER2 and EGFR are overexpressed in all trastuzumab-resistant HER2-positive BC cell and tumor models and that all trastuzumab-resistant models are highly vulnerable to targeted degradation of HER2 and EGFR. Degradation of HER2 and EGFR induced by PEPDG278D causes extensive inhibition of oncogenic signaling in trastuzumab-resistant HER2-positive BC cells. This is accompanied by strong growth inhibition of cultured cells, orthotopic patient-derived xenografts, and metastatic lesions in the brain and lung of trastuzumab-resistant HER2-positive BC. siRNA knockdown indicates that eliminating both HER2 and EGFR is necessary to maximize therapeutic outcome. ConclusionsThis study unravels the therapeutic vulnerability of trastuzumab-resistant HER2-positive BC and shows that an agent that targets the degradation of both HER2 and EGFR is highly effective in overcoming drug resistance in this disease. The findings provide new insights and innovations for advancing treatment of drug-resistant HER2-positive breast cancer that remains an unmet problem.