Patient-derived Orthotopic Xenograft Research Articles

Capmatinib is an effective treatment for MET-fusion driven pediatric high-grade glioma and synergizes with radiotherapy

BackgroundPediatric-type diffuse high-grade glioma (pHGG) is the most frequent malignant brain tumor in children and can be subclassified into multiple entities. Fusion genes activating the MET receptor tyrosine kinase often occur in infant-type hemispheric glioma (IHG) but also in other pHGG and are associated with devastating morbidity and mortality.MethodsTo identify new treatment options, we established and characterized two novel orthotopic mouse models harboring distinct MET fusions. These included an immunocompetent, murine allograft model and patient-derived orthotopic xenografts (PDOX) from a MET-fusion IHG patient who failed conventional therapy and targeted therapy with cabozantinib. With these models, we analyzed the efficacy and pharmacokinetic properties of three MET inhibitors, capmatinib, crizotinib and cabozantinib, alone or combined with radiotherapy.ResultsCapmatinib showed superior brain pharmacokinetic properties and greater in vitro and in vivo efficacy than cabozantinib or crizotinib in both models. The PDOX models recapitulated the poor efficacy of cabozantinib experienced by the patient. In contrast, capmatinib extended survival and induced long-term progression-free survival when combined with radiotherapy in two complementary mouse models. Capmatinib treatment increased radiation-induced DNA double-strand breaks and delayed their repair.ConclusionsWe comprehensively investigated the combination of MET inhibition and radiotherapy as a novel treatment option for MET-driven pHGG. Our seminal preclinical data package includes pharmacokinetic characterization, recapitulation of clinical outcomes, coinciding results from multiple complementing in vivo studies, and insights into molecular mechanism underlying increased efficacy. Taken together, we demonstrate the groundbreaking efficacy of capmatinib and radiation as a highly promising concept for future clinical trials.

CD57 defines a novel cancer stem cell that drive invasion of diffuse pediatric-type high grade gliomas.

Diffuse invasion remains a primary cause of treatment failure in pediatric high-grade glioma (pHGG). Identifying cellular driver(s) of pHGG invasion is needed for anti-invasion therapies. Ten highly invasive patient-derived orthotopic xenograft (PDOX) models of pHGG were subjected to isolation of matching pairs of invasive (HGGINV) and tumor core (HGGTC) cells. pHGGINV cells were intrinsically more invasive than their matching pHGGTC cells. CSC profiling revealed co-positivity of CD133 and CD57 and identified CD57+CD133- cells as the most abundant CSCs in the invasive front. In addition to discovering a new order of self-renewal capacities, i.e., CD57+CD133- > CD57+CD133+ > CD57-CD133+ > CD57-CD133- cells, we showed that CSC hierarchy was impacted by their spatial locations, and the highest self-renewal capacities were found in CD57+CD133- cells in the HGGINV front (HGGINV/CD57+CD133- cells) mediated by NANOG and SHH over-expression. Direct implantation of CD57+ (CD57+/CD133- and CD57+/CD133+) cells into mouse brains reconstituted diffusely invasion, while depleting CD57+ cells (i.e., CD57-CD133+) abrogated pHGG invasion. We revealed significantly increased invasive capacities in HGGINV cells, confirmed CD57 as a novel glioma stem cell marker, identified CD57+CD133- and CD57+CD133+ cells as a new cellular driver of pHGG invasion and suggested a new dual-mode hierarchy of HGG stem cells.

PK-PD-efficacy modeling of brigimadlin in MDM2-amplified glioblastoma.

2071 Background: Brigimadlin (BI 907828) is a potent small molecule inhibitor of the p53-MDM2 pathway currently in a phase 0/1 clinical trial in combination with radiotherapy in newly diagnosed MGMTunmethylated glioblastoma (GBM). To inform development of brigimadlin for GBM, we developed a pharmacokinetic (PK)/ pharmacodynamic (PD)/ efficacy model using GBM patient derived xenografts (PDXs). Methods: Brigimadlin PK parameters were evaluated in FVB wild-type and knockout (Mdr1a/b-/-Bcrp1-/-) mice and in athymic nude mice. Drug binding was measured by rapid equilibrium dialysis, and drug levels quantified by LC/MS-MS. Dose-response relationships were evaluated in subcutaneous and orthotopic PDXs across a range of doses. Response to brigimadlin (2 mg/kg weekly) +/- RT (2 Gy x 10 fractions) was evaluated in orthotopic PDXs. Results: Brigimadlin significantly delayed median time to regrowth of GBM108 ( MDM2amplified) subcutaneous tumors (vs. placebo; p<0.05) at dose levels of 0.25 mg/kg (1.3-fold), 0.5 mg/kg (1.5-fold), 1 mg/kg (2.6-fold), and 2 mg/kg (5.3-fold). A single dose of brigimadlin resulted in corresponding intra-tumoral drug levels 24 hours post-dose: 84 nM (0.25 mg/kg), 175 nM (0.5 mg/kg), 398 nM (1 mg/kg) and 924 nM (2 mg/kg) and dose-dependent upregulation of p53 target genes (p21 and PUMA). In contrast, GBM14 ( MDM2non-amplified) showed regrowth delay (1.6-fold) only at 2 mg/kg (p=0.008). Brigimadlin was highly bound (fraction unbound = 0.0006 in brain; 0.0017 in GBM tumor tissue), and CNS distribution was limited by efflux; in wild-type mice Kp,uu_brain = 0.002 ± 0.003; in Mdr1a/b-/-Bcrp1-/- mice Kp,uu = 0.043 ± 0.025. Efficacy of brigimadlin was compared in orthotopic GBM108 PDXs grown in athymic nude mice vs. immuno/efflux-deficient (Rag2-/-Mdr1a-/-Bcrp1-/-) mice. In nude mice, median survival was extended at 2 mg/kg brigimadlin (1.4-fold, p=0.009), with corresponding intra-tumoral drug levels of 29 nM. In efflux deficient mice, median survival was extended (p<0.05) at 0.25 mg/kg (1.5-fold), 0.5 mg/kg (1.5-fold), 1 mg/kg (1.8-fold), and 2 mg/kg (>4-fold). Measurement of intra-tumoral drug levels is in progress. In combination with RT in GBM108, brigimadlin (2 mg/kg x 2 weeks) extended median survival vs. vehicle (66 vs. 50 days; p=0.012) and enhanced median survival following RT (128.5 vs. 82 days; p=0.009). In GBM14, 2 mg/kg brigimadlin (2 mg/kg x 12 weeks) extended median survival (39.5 vs. 32.5 days; p=0.0004) and enhanced survival following RT (97 vs. 81 days; p<0.0001). Conclusions: Efficacy of brigimadlin is dependent on adequate CNS delivery. Studies in subcutaneous PDX demonstrated intra-tumoral total drug levels in the mid-nanomolar range provide meaningful tumor effects in a highly sensitive, MDM2 amplified PDX. In orthotopic PDXs with and without MDM2 amplification, brigimadlin provided further benefit in combination with RT. These data support ongoing clinical evaluation of brigimadlin in GBM.

Pre-clinical modeling of navtemadlin pharmacokinetics (PK), pharmacodynamics (PD), and efficacy in glioblastoma, IDH-wildtype.

2012 Background: Navtemadlin (nvtm) is a potent, selective, orally available small molecule inhibitor of murine double minute 2 homologue (MDM2), which has completed a Ph 0 surgical window of opportunity study in glioblastoma, IDH-wildtype (GBM; Lee SNO 2022). To optimally interpret the clinical data, an extensive evaluation of nvtm PK, PD, and efficacy was performed in GBM patient derived xenografts (PDXs). Methods: Response to nvtm +/- radiotherapy (RT) was characterized in vitro and in vivo across a panel of GBM PDXs with various TP53 and MDM2 alterations. Efficacy, PK, and PD were evaluated in flank or orthotopic PDX models. p53 pathway activation was evaluated by qPCR, Western blot, and IHC. Nvtm brain penetration was evaluated in immunodeficient abcb1a/b-/- (P-glycoprotein; P-gp), abcg2-/- (BCRP), or double-knockout mice. Binding was assessed by rapid equilibrium dialysis, and nvtm concentrations by LC/MS-MS and MALDI-MSI. Results: Nvtm decreased in vitro viability of TP53WT GBM PDX explant cultures with IC50s <100 nM, but was ineffective in TP53-mutant GBM. In flank PDX models, nvtm (100 mg/kg daily for 4 weeks) delayed tumor regrowth by 1.9 to 4.8-fold in TP53 WT PDXs without MDM2amplification (n=9 models), and more markedly by 20.1 to 29.4-fold in MDM2amplified models (n=4; 2 PDXs did not recur). In a flank PDX dose-response study, nvtm delayed the growth of MDM2amplified GBM108 at 25 mg/kg vs. placebo (61 vs. 11 days; p=0.014; mean intra-tumoral nvtm = 590 nM). In contrast, growth of MDM2 non-amplified GBM14 was delayed only at 100 mg/kg (37 vs. 15 days; p=0.082; mean intra-tumoral nvtm = 2990 nM). In both flank models, p53 target gene upregulation was similar between 25 and 100 mg/kg and did not correlate with regrowth. Nvtm was highly bound (fraction unbound = 0.090 in culture media and 0.014 in GBM tissue homogenate). In mice after oral dosing, distribution to CNS was limited by P-gp, with a brain to plasma ratio of 0.009 and unbound ratio of 0.005, whereas BCRP deficiency did not affect CNS distribution. With orthotopic GBM108, nvtm efficacy was evaluated in nude vs. efflux-deficient mice. In nude mice, nvtm was ineffective at doses up to 100 mg/kg; however, in efflux deficient mice, the mean intra-tumoral nvtm level at 25 mg/kg was similar to flank tumors (690 nM) and survival was significantly extended (134 vs. 62 days, p=0.0002) with additional benefit at 100 mg/kg (>300 vs. 62 days, p=0.002). Conclusions: Nvtm showed significant efficacy in TP53 WT GBM flank PDX models but efficacy in orthotopic PDX was highly dependent on CNS penetration. Targeting nvtm levels based on the in vitroIC50 may significantly underestimate the in vivo effective concentration . In vivo studies showed lower intra-tumoral nvtm levels are required to significantly inhibit growth of MDM2amplified compared with non-amplified tumors.

DsRNAi-mediated silencing of PIAS2beta specifically kills anaplastic carcinomas by mitotic catastrophe

The E3 SUMO ligase PIAS2 is expressed at high levels in differentiated papillary thyroid carcinomas but at low levels in anaplastic thyroid carcinomas (ATC), an undifferentiated cancer with high mortality. We show here that depletion of the PIAS2 beta isoform with a transcribed double-stranded RNA–directed RNA interference (PIAS2b-dsRNAi) specifically inhibits growth of ATC cell lines and patient primary cultures in vitro and of orthotopic patient-derived xenografts (oPDX) in vivo. Critically, PIAS2b-dsRNAi does not affect growth of normal or non-anaplastic thyroid tumor cultures (differentiated carcinoma, benign lesions) or cell lines. PIAS2b-dsRNAi also has an anti-cancer effect on other anaplastic human cancers (pancreas, lung, and gastric). Mechanistically, PIAS2b is required for proper mitotic spindle and centrosome assembly, and it is a dosage-sensitive protein in ATC. PIAS2b depletion promotes mitotic catastrophe at prophase. High-throughput proteomics reveals the proteasome (PSMC5) and spindle cytoskeleton (TUBB3) to be direct targets of PIAS2b SUMOylation at mitotic initiation. These results identify PIAS2b-dsRNAi as a promising therapy for ATC and other aggressive anaplastic carcinomas.

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.

Patient tissue-derived FGFR4-variant and wild-type colorectal cancer organoid development and anticancer drug sensitivity testing

ObjectivesFGFR4-variant and wild-type colorectal cancer (CRC) organoids were developed to investigate the effects of FGFR4-targeted drugs, including FGFR4-IN and erdafitinib, on CRC and their possible molecular mechanism. MethodsClinical CRC tissues were collected, seven CRC organoids were developed, and whole exome sequencing (WES) was performed. CRC organoids were cultured and organoid drug sensitivity studies were conducted. Finally, an FGFR4-variant (no wild-type) CRC patient-derived orthotopic xenograft mouse model was developed. Western blot measured ERK/AKT/STAT3 pathway-related protein levels. ResultsWES results revealed the presence of FGFR4-variants in 5 of the 7 CRC organoids. The structural organization and integrity of organoids were significantly altered under the influence of targeted drugs (FGFR4-IN-1 and erdafitinib). The effects of FGFR4 targeted drugs were not selective for FGFR4 genotypes. FGFR4-IN-1 and erdafitinib significantly reduced the growth, diameter, and Adenosine Triphosphate (ATP) activity of organoids. Furthermore, chemotherapeutic drugs, including 5-fluorouracil and cisplatin, inhibited FGFR4-variant and wild-type CRC organoid activity. Moreover, the tumor volume of mice was significantly reduced at week 6, and p-ERK1/2, p-AKT, and p-STAT3 levels were down-regulated following FGFR4-IN-1 and erdafitinib treatment. ConclusionsFGFR4-targeted and chemotherapeutic drugs inhibited the activity of FGFR4-variant and wild-type CRC organoids, and targeted drugs were more effective than chemotherapeutic drugs at the same concentration. Additionally, FGFR4 inhibitors hindered tumorigenesis in FGFR4-variant CRC organoids through ERK1/2, AKT, and STAT3 pathways. However, no wild-type control was tested in this experiment, which need further confirmation in the next study.

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.

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

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...

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.