Patient-derived Orthotopic Xenograft Research Articles

BIOL-24. USING NEEDLE WASH SPECIMEN AFTER STEREOTACTIC BIOPSY TO DEVELOP PDOX MODELS OF DIPG AND DIFFUSE MIDLINE GLIOMAS

Abstract Obtaining viable tissue remains a major barrier for lab-based studies, especially in brain tumors deemed surgically unresectable such as diffuse midline gliomas. Surgical biopsy is performed for those tumors invested in deep and vital brain structures however, biopsied specimens are usually of small volume without additional tissue for research after clinical testing. To overcome the barrier of limited surgical specimen volume, we partnered with neurosurgeons to develop a protocol which procures viable cells from discarded biopsy needle after stereotactic biopsies to develop patient-derived orthotopic xenograft (PDOX) models. After procurement of surgical specimen and prior to disposal of biopsy needle, each stereotactic biopsy needle was washed with FBS media twice. Wash solution was collected and immediately transported on ice to the research lab. Cell number was counted for in vitro culture and for orthotropic in vivo tumor implantation. To date, 29 samples of biopsy needle washes have been obtained including 12 diffuse midline gliomas of the brain stem, 8 supratentorial diffuse midline gliomas, and 3 pineal region tumors with harvested cell number from no visible cells to 5.3 million cells (median 1 million). 14 tumors have been orthotopically implanted into matched locations of immunodeficient mouse brains. Tumor formation was confirmed in 4 out of 15 (28.5%), including 2 brain stem tumors, 1 infiltrating astrocytoma, and 1 midline glioma, after implantation of cell numbers as low as 12,500. 3 models were successfully sub-transplanted to passages II and III. Histological examination confirmed the replication of key pathological features of the originating patient tumors. In conclusion, we have developed a novel protocol to procure clinical specimen by using wash solution of stereotactic brain tumor biopsy needles for surgically unresectable tumors. Our work demonstrates a new possibility for clinical sample acquisition and tumor modeling, opening new frontiers in the field of pediatric brain tumor research.

OTHR-02. CHIMERIC AURORA A KINASE (AURKA) DEGRADERS EFFICIENTLY TARGET N-MYC

Abstract MYC family proteins are primary drivers of oncogenic processes in a variety of cancer histologies. N-myc overexpression and amplification induce aggressive pediatric cancers, the most common of which are solid extracranial tumors in children (neuroblastoma, NB) and malignant pediatric CNS tumors (medulloblastoma, MB). Due to the oncogenic addiction observed in these tumor types, N-myc is considered an attractive therapeutic target. However, direct small molecule targeting of N-myc remains technically challenging. Alternative approaches to target this pathway include inhibiting proteins thought to stabilize N-myc, such as Aurora A kinase (AURKA). While inhibition of AURKA was found to be effective in preclinical tumor studies, no AURKA inhibitors have been approved for clinical use due to a lack of efficacy. Here, we aim to develop chimeric degrader molecules suitable for preclinical and clinical use that degrade AURKA and concomitantly reduce N-myc levels. We used automated solid-phase synthesis to generate a library of >1000 chimeric degraders using derivatives of six known AURKA ligands (Series 1-6). Due to the limitations of using N-myc-driven MB cells in vitro, we employed NB cells as the proof-of-concept model. While the degraders based on five of the ligands showed efficient AURKA degradation, only degraders based on series 3 and 6 could also diminish N-myc. Selected series were further characterized in pharmacokinetic and pharmacodynamic experiments using NB tumor xenografts implanted in the flank in CD1-nude mice. In summary, chimeric degraders based on two individual AURKA ligands efficiently degraded AURKA and N-myc in vitro and in vivo, providing the foundation for further development of novel therapies for patients with N-myc-driven cancers. Further studies will focus on assessing antitumor properties of lead candidates in NB and patient-derived orthotopic MB xenograft models.

DIPG-16. ACUTE VERSUS PROLONGED CONVECTION-ENHANCED DELIVERY OF ALISERTIB INTO H3 K27M-MUTANT MODELS OF DIFFUSE MIDLINE GLIOMA

Abstract Convection-enhanced delivery (CED) is a potentially promising strategy to administer therapeutics directly into the brainstem of children harboring H3 K27-altered diffuse midline glioma (DMG). While early-phase clinical trials have demonstrated the safety of this technique, efficacy has yet to be achieved. Here, we describe how different variations of CED may be exploited to extend survival in a DMG murine patient-derived orthotopic xenograft model. Either 1-day (8 µL/hr) or 7-day (1 µL/hr) continuous CED infusions were performed to deliver an equivalent volume of the aurora kinase inhibitor alisertib to the mouse brainstem. Bioluminescence and magnetic resonance images were acquired to monitor tumor progression, validate CED catheter positioning, and analyze infusion-related imaging changes. Both infusion regimens significantly prolonged survival versus control (median survival benefit of 6.5 and 8 days, p=0.03 and p=0.01, for 1-day and 7-day CED, respectively). Postmortem examination of brains revealed no signs of tissue necrosis, cavitary lesions, or cellular (inflammatory) infiltrate at the site of infusion. However, the tumor parenchyma surrounding the cannula tract was markedly hypocellular in treated animals. Alisertib induced a robust increase in H3 K27 trimethylation along with decreased H3 K27M and Ki-67. Our findings indicate that CED of alisertib is well tolerated and effective, in both the acute and prolonged setting, underscoring the potential of this approach in the management of DMG.

EPEN-04. DEVELOPMENT OF B7-H3-TARGETED CAR T CELL THERAPY FOR EPENDYMOMA

Abstract Targeted treatment options are desperately needed for ependymomas (EPN). Chimeric antigen receptor (CAR) T cells have immense potential to positively transform treatment outcomes; however, little is known regarding antitumor efficacy of CAR T cells against EPNs. We found that B7-H3 is highly expressed on pediatric brain tumor patient-derived orthotopic xenografts from EPN patients, making it a promising target for CAR-T cells. The project’s goal is to develop a safe, effective B7-H3 targeted CAR T cell immunotherapy for EPN. We generated second generation human B7-H3-CAR T cells and examined their anti-EPN cytolytic activity, expansion, and cytokine production. We found that B7-H3-CAR T cells had potent in vitro antitumor activity against all EPN cell lines tested (1425, ST1, CPITT, ST2, EPI, L6SJ). In repeated-stimulation assays, CAR T cells expanded 7-10 times, and up to one million-fold when co-cultured with EPN cells. Interestingly, while the EPN cells were found to secrete high levels of C-C motif chemokine ligand 2 (CCL2) which is consistent with published data, our Multiplex analysis of co-culture supernatants shows that CCL2 production increased up to 10-fold when EPN cells were co-cultured with CAR T cells. Finally, we demonstrate that B7-H3-CAR T cells had antitumor activity in a supratentorial-EPN xenograft mouse model (1425 cell line) without apparent toxicities, although 4 out of 12 tumors re-lapsed. Recurrent tumor analysis is underway. Together, our data shows establishment of a clinically relevant model to study resistance to CAR T cell therapy in EPN, and preliminary mechanistic insight into CAR T cell persistence and modulation through CCL2 signaling. Given that little is known about the function of CCL2 and its receptor, C-C chemokine receptor type 2 (CCR2) in EPNs or CAR T cells, future work will focus on defining these biological mechanisms, enabling us to develop improvements to B7-H3-CAR T cells for EPN.

DIPG-15. CONCURRENT GB-13 (IL13.E13K-PE4E) IMPROVES THE EFFICACY OF RADIATION THERAPY IN DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Diffuse midline glioma with H3 K27 alterations (DMG) is the most aggressive primary malignant brain tumor in the pediatric population. Radiation therapy (RT) is the standard-of-care; however, it has not demonstrated a significant improvement in overall survival or stalled disease progression, and children usually succumb to disease within 12-15 months of diagnosis. Consequently, finding efficient treatments for DMG remains one of the most critical unmet needs in modern neuro-oncology. Interleukin 13 receptor subunit alpha 2 (IL-13Rα2) is a cell-surface receptor upregulated in ~80% of DMG versus normal brain, posing a potentially promising therapeutic target. Immunotherapies harnessing IL-13Rα2 to selectively delivery cytotoxic payloads, such as pseudomonas exotoxin A (PE), to tumor cells have been previously demonstrated to be safe in DMG patients and efficacious in preclinical models of DMG with high IL-13Rα2 expression using convection-enhanced delivery (CED). Furthermore, there is evidence that PE-based therapies can sensitize cells to RT. In this study, we utilized in vitro and in vivo models of DMG to evaluate clinically-relevant combination treatments of RT and the IL-13/PE immunotoxin GB-13 (IL13.E13K-PE4E). GB-13 improved the effectiveness of low-dose RT in multiple DMG cell lines by promoting caspase activation, which boosted apoptotic cell death. Intratumoral administration of GB-13 via chronic CED decreased tumor burden and prolonged survival in both DMG patient-derived orthotopic xenograft and genetically engineered mouse models. When GB-13 and RT were administered concurrently, this impact became even more prominent, informing future preclinical and clinical investigation of a potentially efficacious therapeutic approach in a subset of DMG.

Relationship between efficacy of phase I/II drug ispinesib in medulloblastoma, molecular subtypes, and p53-mutant status.

e14001 Background: KIF11, a mitotic kinesin, is responsible for assembly and maintenance of mitotic spindle during mitosis. Tumor cells can upregulate KIF11 to enhance tumor proliferation. Many cancers overexpress KIF11, including medulloblastoma (MB), the most common malignant brain tumor in children. MB is molecularly classified into 4 molecular subtypes: sonic-hedgehog (SHH)-activated MB, Wingless (WNT)-activated, and less characterized Group 3 and 4 subtypes. Group 3 and 4 MBs are the most aggressive subtypes and Group 3 MBs have the worst survival outcome. p53 mutation defines a high-risk group in MB patients. We evaluated the efficacy of a Phase I/II KIF11 inhibitor, ispinesib on MB. Methods: We employed in-vitro proliferation assays (CCK), flow cytometry (Annexin V, Propidium Iodide), western blot, Hoechst staining, and two patient-derived orthotopic xenograft (PDOX) mouse models for in-vivo drug testing. Results: Ispinesib effectively inhibited proliferation at nanomolar concentrations for all 5 MB cell lines. A comparison of IC50 (day 4) indicated that p53-mutational-status did not influence sensitivity to ispinesib. In all five cell lines, regardless of p53 mutational status, ispinesib led to G2/M arrest. However, different p53-mutational-status led to different modes of cell death. High levels of necrotic cell death were observed in p53-mutant, while apoptosis was the predominant mode of cell death in p53-wildtype, on flow cytometry. Apoptotic nuclei were more significantly observed in p53-wildtype (p = 0.002) while p53-mutant cell line demonstrated more micronuclei (p = 0.0302) and multi-nucleation (p= 0.0161), compared to respective DMSO-control cells on Hoechst staining. Ispinesib has demonstrated effective blood brain barrier penetration in brain xenografts. We compared 2 PDOX models of MB. Ispinesib significantly improved survival outcome of Group 3 MB (Control=6, Treated=7, Log-rank p=0.0003), but worsened the survival outcome of p53-mutant SHH MB (Control=14, Treated=11, Log-rank p=0.169). This differential efficacy of ispinesib was recapitulated in the matched 3D-neurosphere cultures of both PDOX models. Cancer-stem-cell (CSC)-enriched neurospheres derived from aggressive Group 3 subtype demonstrated susceptibility to 7-day in-vitro treatment with ispinesib (AUC=0.7743). Monolayer tumor cells demonstrated similar susceptibility (AUC=0.7757). Area under curve (AUC)>0.7 was the significant cut-off. In contrast, both CSC-enriched neurospheres (AUC=0.379) and monolayer tumor cells (AUC=0.491) derived from p53-mutant SHH MB did not demonstrate susceptibility to ispinesib, consistent with matching PDOX models. Conclusions: Preclinically, our results indicate feasibility of ispinesib treatment in MB, specifically aggressive Group 3 MB. Importantly, p53-mutant SHH MB represents a poor molecular-subtype candidate for ispinesib therapy.

A phase 0/Ia study of BI 907828 concentrations in brain tissue and a nonrandomized, open-label, dose escalation study of BI 907828 in combination with radiotherapy in patients with newly diagnosed glioblastoma (GBM).

TPS2081 Background: Murine double minute 2 (MDM2) directly regulates the stability of the tumor suppressor p53, and dysregulation of this pathway can critically disrupt cellular response to genomic stress and lead to cell death. BI 907828 is a highly potent inhibitor of the MDM2-p53 interface that triggers accumulation of p53 and subsequent apoptosis in multiple GBM patient-derived xenografts (PDXs) with wild-type p53. Moreover, the combination of BI 907828 and radiotherapy provides significant survival extension in multiple orthotopic GBM PDXs. Methods: An integrated Phase 0/1 trial design is being used to evaluate the potential for BI 907828 to achieve a therapeutic concentration in brain tumor tissue and to test the safety of the combination with radiation. The co-primary endpoints for the Phase 0 study are i) measured total concentration of BI 907828 and ii) calculated unbound concentration of BI 907828 in contrast and non-contrast enhancing regions of tumor. Key inclusion criteria for the Phase 0 study are patients at least 18 years of age, ECOG performance status of 0 or 1, histologic or radiologic new diagnosis of GBM, and planned surgical resection. The first approximately 6 patients enrolled in the Phase 0 will be treated at the starting dose of 30 mg, and the future patients enrolled will be dosed at higher levels (45 mg or 60 mg) provided there are no safety concerns. If dose-limiting toxicities (DLTs) are encountered, then a Bayesian Logistic Regression Model (BLRM) with overdose control will be used to guide the dose level decisions for the additional patients. Multiple, image-registered tumor samples and intra-operative plasma samples will be analyzed for BI 907828 by LC-MS/MS. Intra-tumoral variation in BI 907828 will be correlated with pharmacodynamic effects of MDM2 inhibition (elevation of p53, p21, MDM2, GDF15, PUMA). Those patients completing the Phase 0 study and who meet additional eligibility criteria can enroll on the Phase 1a portion of the study. The pathologic inclusion criteria are TP53 wild-type, IDH1/2 wild-type, MGMT unmethylated GBM. Co-primary endpoints for the Phase 1a trial are i) occurrence of DLTs during and within 21 days after completion of radiation therapy, and ii) occurrence of adverse events throughout the treatment period. Tolerability of inter-patient dose-escalation of BI 907828 (30, 45, 60 mg) combined with radiation therapy (60 Gy in 30 fractions) will be evaluated with dose-escalation guided by a two-parameter BLRM with overdose control. Patients will remain on BI 907828 monotherapy following completion of radiotherapy until progression. To date, two patients have enrolled on the Phase 0 study, and one patient went on to enroll on the Phase 1a portion of the study. Clinical trial information: NCT05376800 .

Highly Selective Targeting of Pancreatic Cancer in the Liver with a Near-Infrared Anti-MUC5AC Probe in a PDOX Mouse Model: A Proof-of-Concept Study.

Accurately identifying metastatic disease is critical to directing the appropriate treatment in pancreatic cancer. Mucin 5AC is overexpressed in pancreatic cancer but absent in normal pancreas tissue. The present proof-of-concept study demonstrates the efficacy of an anti-mucin 5AC antibody conjugated to an IR800 dye (MUC5AC-IR800) to preferentially label a liver metastasis of pancreatic cancer (Panc Met) in a unique patient-derived orthotopic xenograft (PDOX) model. In orthotopic models, the mean tumor to background ratio was 1.787 (SD ± 0.336), and immunohistochemistry confirmed the expression of MUC5AC within tumor cells. MUC5AC-IR800 provides distinct visualization of pancreatic cancer liver metastasis in a PDOX mouse model, demonstrating its potential utility in staging laparoscopy and fluorescence-guided surgery.

A novel preclinical model of craniospinal irradiation in pediatric diffuse midline glioma demonstrates decreased metastatic disease.

Diffuse midline glioma (DMG) is an aggressive pediatric central nervous system tumor with strong metastatic potential. As localized treatment of the primary tumor improves, metastatic disease is becoming a more important factor in treatment. We hypothesized that we could model craniospinal irradiation (CSI) through a DMG patient-derived xenograft (PDX) model and that CSI would limit metastatic tumor. We used a BT245 murine orthotopic DMG PDX model for this work. We developed a protocol and specialized platform to deliver craniospinal irradiation (CSI) (4 Gy x2 days) with a pontine boost (4 Gy x2 days) and compared metastatic disease by pathology, bioluminescence, and MRI to mice treated with focal radiation only (4 Gy x4 days) or no radiation. Mice receiving CSI plus boost showed minimal spinal and brain leptomeningeal metastatic disease by bioluminescence, MRI, and pathology compared to mice receiving radiation to the pons only or no radiation. In a DMG PDX model, CSI+boost minimizes tumor dissemination compared to focal radiation. By expanding effective DMG treatment to the entire neuraxis, CSI has potential as a key component to combination, multimodality treatment for DMG designed to achieve long-term survival once novel therapies definitively demonstrate improved local control.

Abstract 3570: Preservation of cellular heterogeneity in 3D and 2D cultures of pediatric solid tumors

Abstract Over the past 10 years, we have developed and characterized over 280 orthotopic patient derived xenografts (O-PDXs) representing 23 different types of pediatric solid tumors. The O-PDXs have undergone comprehensive characterization including genomics, transcriptomics, epigenomics, histologic review and analysis of cellular heterogeneity. All data, tissue samples and cryopreserved O-PDXs are freely available with no obligation to collaborate through the Childhood Solid Tumor Network (CSTN) at www.stjude.org/CSTN/. While these in vivo models are useful for preclinical testing of drugs and immunotherapy, researchers are limited by throughput, expense and expertise. High throughput ex vivo culture systems that more faithfully recapitulate the cellular heterogeneity and arrested differentiation in the patient tumors are urgently needed to test new drug combinations. Solid tumor cell lines have undergone extensive selection and adaptation in culture, and most have lost the cellular heterogeneity found in patient tumors. In order to overcome this barrier in the field, we have developed a 3D-to-2D workflow. Previous attempts to produce 2D ex vivo cultures from patient tumors or O-PDXs have had limited success because most cells die upon initial plating, they are difficult to cryopreserve for future studies and they lose key cell populations found in the patient tumors. Our new workflow was designed to overcome two sources of stress when adapting patient tumors or O-PDXs to culture. The first source of stress is transitioning from the in vivo environment to ex vivo in culture medium. The second source of stress is the transition from extensive 3D cell-cell contacts in vivo to a more limited cell-cell contact ex vivo. To overcome these two sources of stress, we adapted cultures to 2D in a stepwise manner. First, we dissociate O-PDX tumors into single cell suspension and then rapidly re-aggregate them in 3D spheres for ex vivo culture. After the spheres have grown for 7 days, they are transitioned to 2D by seeding cells in culture dishes coated with extracellular matrix components. Importantly, the 3D and 2D cultures preserve the cellular heterogeneity of the O-PDX and patient tumor and they can be cryopreserved. When injected back into immunocompromised mice, they recapitulate the molecular and cellular features of the original tumors. We have shown that the 3D and 2D cultures can be genetically manipulated using lentiviral vectors and can be used for high-throughput drug screening. Matched 3D and 2D cryovials are now available to the international biomedical research community for rhabdomyosarcoma through the CSTN. Neuroblastoma, osteosarcoma, Ewing sarcoma and retinoblastoma are expected to be available in late 2023. We anticipate this will accelerate the testing of oncology drugs for pediatric solid tumors in response to the RACE act through the PIVOT program. Citation Format: Justina D. McEvoy, Melody Allensworth, Mariajose Franco, Natalie McDonald, Brittney Gordon, Anand Patel, Elizabeth Stewart, Michael Dyer. Preservation of cellular heterogeneity in 3D and 2D cultures of pediatric solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3570.

Abstract 1718: Development of potent estrogen receptor beta agonists for treating glioblastoma

Abstract Background: Glioblastoma (GBM) is the most prevalent and aggressive type of adult brain cancers with low 5-year overall survival rates. Epidemiologic data suggest estrogen may decrease brain tumor growth. The lack of powerful, selective brain permeable estrogen receptor beta (ERβ) ligands to promote its anticancer action is limiting the therapeutic promise of ERβ. The goal of this project is to create a new class of brain permeable ERβ agonists for the treatment of GBM. Method: We have used four different assays to identify potent leads. (1) ERβ and estrogen receptor alpha (ERα) reporter assays, (2) Polar Screen Estrogen Receptor (ER) Competitive Binding Assays, (3) Cell viability assays using GBM cells, and (4) Activity in WT and ERβ-KO GBM cells. CellTiter-Glo Cell Viability and colony formation assays were used to test the efficacy of ERβ agonists. Invasion was measured by matrigel invasion chamber assays and apoptosis was measured by Caspase-Glo® 3/7 and Annexin V assays. Flow cytometry was used to evaluate the cell cycle. Western blotting, RNA-Seq, RT-qPCR, and ERβ KO cells were used in the mechanistic studies. Both orthotopic patient-derived xenografts and GBM cell line-derived (CDX) xenografts were used to test the ERβ agonist's activity in vivo. Results: Indanone and tetralone-oximes or keto-oximes have been hypothesized, produced, and evaluated as new ER agonists. Due to its high action in ERβ reporter assays, specific binding to ERβ in polar screen assays (EC50 91 nM), and potent growth inhibitory activity (IC50 7.4) in GBM cells, CIDD-97 was discovered as a possible hit by screening a library of ~60 compounds. CIDD-97 is significantly more selective for ERβ than ERα (40-fold). Treatment with CIDD-97 markedly reduced U251 cell viability while having little to no effect on U251-ER-KO cells and normal human astrocytes. Further, CIDD-97 treatment decreased the expression of stemness markers in patient derived GSC lines and promoted apoptosis. Additionally, CIDD-ERβ agonist improved TMZ's ability to reduce GBM cell survival. The activation of ERβ target genes in cells treated with CIDD-97 was verified by using RNA-Seq and RTqPCR. PK studies confirmed that CIDD-0149897 has systemic exposure, was found in the brain, and its maximal detectable levels suggested that it has good BBB permeability. Mice tolerated daily intraperitoneal treatment of CIDD-0149897 (50 mg/kg) with a 7-day repeat dosage with no evidence of toxicity. Additionally, when compared to vehicle CIDD-97 (50 mg/kg/i.p.) treatment significantly decreased tumor growth in xenograft models and extended the survival of tumor-bearing mice in orthotopic GSC models. Conclusion: Collectively, these findings pointed to CIDD-97 as a possible ERβ agonist and is easily adaptable to clinical use alongside current chemo- and radiation-therapy regimens, offering GBM patients an additional means of improving survival. Citation Format: Uday P. Pratap, Michael Tidwell, Henriette U. Balinda, Suryavathi Viswanadhapalli, Gangadhara Reddy Sareddy, Stanton McHardy, Andrew Brenner, Ratna K. Vadlamudi. Development of potent estrogen receptor beta agonists for treating glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1718.

Abstract 2796: Tumor pharmacokinetics, pharmacodynamics and efficacy analysis of WEE1 inhibitor, zn-c3 in patient-derived xenograft models of glioblastoma

Abstract WEE1 kinase regulates G2/M checkpoint transition via phosphorylation of CDK1 and prevents entry into mitosis. WEE1 inhibition increases genomic instability due to G2/M checkpoint inactivation and can result in mitotic catastrophe as monotherapy or when combined with DNA damaging agents. In this study, we evaluated the efficacy as well as pharmacokinetic and pharmacodynamic properties of ZN-c3, a best-in-class WEE1 inhibitor, in orthotopic patient-derived xenograft (PDX) models of GBM. Mice with intracranial tumors were randomized to 80 mg/kg ZN-c3 PO QD x 2 weeks vs. placebo. ZN-c3 levels in plasma and contrast-enhancing tumor tissue were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Unbound fractions were determined by equilibrium dialysis. Immunohistochemistry was performed to assess levels of pCDK1, phospho-Histone H3 (pHH3), and cleaved caspase 3 (CC3). The median unbound concentrations of ZN-c3 were 315 nmol/L and 15 nmol/kg in plasma and tumor tissue, respectively - higher than the biochemical IC50 of ZN-c3 for inhibition of WEE1 (3.8nM). An increase in pHH3(+) cells (0.40% vs. 6.57%, p=0.03) indicated drug-induced mitotic entry upon ZN-c3 treatment. ZN-c3 was potently cytotoxic across multiple patient-derived xenograft (PDX)-derived cell lines (IC50- 150-10000nM). Time and dose-escalation experiments exhibited target engagement at concentrations lower than IC50 values within one hour of treatment. Basal levels of WEE1 and pCDK1 were correlated with cell viability in response to ZN-c3 across multiple PDX cell lines. Overall, ZN-c3 is well tolerated, achieves pharmacologically-relevant unbound concentrations in GBM PDX models, and is associated with significant checkpoint modulation. Preclinical evaluation of ZN-c3 in combination with radiation therapy and DNA damage response pathway inhibitors is currently underway, which will support future combinatorial strategy for glioblastoma treatment. Citation Format: Shwetal Mehta, An-Chi Tien, Sonam Patel, Tigran Margaryan, Anilkumar Thaghalli Shivanna, Leonel Elena, Jennifer Molloy, William Knight, Ruiheng Hon, Mackenna Elliott, Mariya Stavnichuk, Zorana Opachich, Yu-Wei Chang, Chelsea Montgomery, Sarah Himes, Barbara Hopkins, Artak Tovmasyan, Nader Sanai. Tumor pharmacokinetics, pharmacodynamics and efficacy analysis of WEE1 inhibitor, zn-c3 in patient-derived xenograft models of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2796.

Abstract 3118: Predicting response to PARP inhibitors in pediatric cancer via synthetic lethal networks

Abstract Introduction: PARP inhibitors (PARPi) are known to have a synthetic lethal (SL) relationship with BRCA1/2 mutated cancers (ie: BRCA loss confers sensitivity to PARPi), but heterogeneity in response exists. PARPi have also found utility in BRCA-non mutated cancers, suggesting other factors may contribute to response. Recently, PARPi trials in pediatric solid tumors have had mixed results. SL interactions have been shown to exist in a network relationship, as such the aim of this study was to identify a clinically relevant SL signature (including synthetic dosage lethality and synthetic rescue) that would predict response to PARPi in pediatric cancers. Methods: Pre-treatment genomic copy number alteration, RNA sequencing, and survival data from 519 pediatric cancer patients (INFORM Consortium, 91% solid tumor) were obtained. SL partner genes for PARPi were selected if co-alteration of the drug target (PARP1/2/3) and candidate gene was 1) found to be under-represented in the patient transcriptome, 2) associated with improved survival, and 3) the genes were phylogenetically similar to PARP1/2/3. The 100 gene signature was validated on two independent pediatric cancer drug screens: in-vivo orthotopic patient-derived xenograft (OPDX) mouse models (n=24) and in-vitro PRISM cell lines (n=54). It was also validated on a cisplatin (PARP surrogate) treated pediatric osteosarcoma clinical trial (n=37). Comparisons were made to 1) a signature generated with the same pipeline using adult TCGA data, 2) a validated adult synthetic lethality pipeline (SELECT), and 3) a published 60-gene PARPi response signature (BRCAness score). Results: The pediatric signature predicted response in all drug screens evaluated. Four cell line screens: Talazoparib (AUC 0.72, p = 0.003), Rucaparib (AUC 0.69, p = 0.01), Niraparib (AUC 0.67, p = 0.03), Olaparib (AUC 0.64, p = 0.06), and two OPDX screens: Talazoparib (AUC 0.74, p = 0.03), Olaparib (AUC 0.74, p = 0.03). The pediatric signature outperformed both adult signatures (AUCs 0.34-0.61, all p’s >0.05), and the BRCAness score (AUCs 0.41-0.53, p’s >0.05) except in the Olaparib cell line (BRCAness: AUC 0.68, p = 0.01). In the pediatric osteosarcoma dataset, the pediatric signature predicted death (AUC 0.68) and two-year recurrence (AUC 0.72), outperforming the other measures (death: AUC 0.49-0.66; two-year recurrence: AUCs 0.36-0.44). Discussion: We developed a pediatric derived, clinically relevant, synthetic lethal signature that predicts response to PARPi in pan-pediatric cancer drug screens and in a cisplatin treated osteosarcoma clinical trial. Our findings support the goal of identifying precision oncology frameworks to improve the care of children with cancer. Citation Format: Matthew Nagy, Fiorella Schischlik, Kun Wang, Nishanth Ulhas Nair, E. Michael Gertz, Lipika R. Pal, Natalie Jaeger, Christopher Previti, Dina ElHarouni, Stefan M. Pfister, Eytan Ruppin. Predicting response to PARP inhibitors in pediatric cancer via synthetic lethal networks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3118.

Abstract 4665: Enhancing preclinical testing of agents in patient-derived pediatric solid tumor orthotopic xenografts with pharmacokinetics

Abstract Understanding a potential therapeutic agent’s in vivo pharmacokinetics (PK) is important in translating findings from preclinical murine efficacy models to patients. In consideration of this, PK evaluations play a prominent up-front role in our pediatric solid tumor testing pipeline. The aim of such PK studies is to define an agent’s PK under our experimental conditions – i.e. our mouse strain, husbandry, chow, source and lot of agent, and formulation. We ultimately relate our mouse plasma exposures to the known or anticipated exposures in humans or children, thereby deriving a clinically relevant dose (CRD). Preclinical testing of novel agents and combinations are performed simultaneously with the testing of standard of care agents using the CRD and schedule to allow for more unbiased assessment of efficacy. To date, our group has studied the PK of over 35 agents, predominantly investigational new drugs, in both normal and patient-derived orthotopically xenografted (PDOX) immuno-deficient mice. When possible, rhabdomyosarcoma and neuroblastoma PDOXs were destructively sampled over time to evaluate an agent’s intra-tumor exposure. All samples were quantified using sensitive, specific, and research- or qualified-tier LC-MS/MS assays. Roughly 70% of the agents had adequate tumor penetration, approaching or exceeding concentrations in the plasma. This was unsurprising, given that many test agents had reportedly high volume of distribution (Vd) values. While most of our mouse PK exposure parameters (e.g. Cmax, AUC) were in line with available previous reports, several agents varied appreciably, generally trending lower. We speculate that this may be due to differing study conditions, test agent physical forms, or formulations. CRD estimates were based upon unbound (when available) steady-state plasma AUC equivalencies between mice and humans for a defined clinical dosage regime. Some latitude was permitted in the recommended CRD, and being within two-fold of clinical target was generally considered acceptable. The resultant mouse CRDs and updated PK results for studied agents will be presented, with this information also being openly available upon request and/or at http://cstn.stjude.cloud. In conclusion, we have found that PK-driven, clinically relevant dosing enhances the efficiency and rigor in our in vivo efficacy testing program. Such an approach minimizes false positive signals arising from supra-clinically relevant doses and exposures in mouse models, allowing us to focus on efficacious agents and regimens for rapid clinical translation. Citation Format: Burgess B. Freeman, Kaley Blankenship, Michael A. Dyer, Elizabeth Stewart. Enhancing preclinical testing of agents in patient-derived pediatric solid tumor orthotopic xenografts with pharmacokinetics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4665.

Abstract 5056: ABN401 demonstrates robust anti-tumor efficacy in orthotopic PDX models of MET-driven NSCLC brain metastases in a pre-clinical study

Abstract Background Brain metastases (BM) are manifested in approximately 30-50% of patients with non-small cell lung cancer (NSCLC) and are associated with catastrophic implications on prognosis and survival. The development of blood-brain barrier-penetrant therapies for brain metastases of NSCLC is imperative and is a major clinical hurdle. ABN401, a highly potent and selective c-MET inhibitor, shows promising anti-tumor activity without significant safety concerns in patients with advanced NSCLC. Here, we report that ABN401 exhibits potent anti-tumor efficacy in pre-clinical orthotopic patient-derived xenografts (PDXs) derived from MET-driven NSCLC brain metastases. Methods Anti-tumor efficacy of ABN401 was assessed in two lung cancer PDXs (LU5349 and LU5406) from BM models harboring high-level MET amplification grown in NOD/SCID mice. Subcutaneous PDXs (n=14/group) were treated with ABN401 (60 mg/kg QD, 125 mg/kg QD, and 165 mg/kg QD) or control groups. Established PDXs were orthotopically implanted into the brain (n=14/group) and were treated with ABN401 (125mg/kg QD), or control groups. The tumor volume of orthotopic model was monitored using magnetic resonance imaging (MRI). Brain, plasma, and tumor samples were collected for pharmacokinetics (PK) and pharmacodynamics (PD) correlation analysis. Results The efficacy of ABN401(125 mg/kg QD) was confirmed in the subcutaneous PDX models (LU5349 and LU5406). Treatment with ABN401 showed up to 95% and 99% tumor growth inhibition in the LU5349 and LU5406 models, respectively. ABN401 (125 mg/kg QD) treatment induced complete or near-complete tumor growth regressions in the orthotopic tumor model. ABN401 also showed a desirable PK and safety profile with no significant changes in body weight. Conclusions ABN401 treatment showed robust anti-tumor efficacy in PDX models of MET-driven NSCLC brain metastases. Our findings suggest that ABN401 has excellent potential for treating NSCLC patients with BM and supports its ongoing clinical development. Citation Format: Nirmal Rajasekaran, Kyung Eui Park, Saehyung Lee, Na Young Kim, Suk-Jae Chung, Young Kee Shin, Jun Young Choi. ABN401 demonstrates robust anti-tumor efficacy in orthotopic PDX models of MET-driven NSCLC brain metastases in a pre-clinical study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5056.

Abstract 1083: MEKi and SOS1i as a combination treatment option for KRAS mutant endometrial cancer

Abstract Endometrial cancer (EC) is the most common cancer of the female genital tract, resulting in over 75,000 deaths per year worldwide. Despite recent advances in medical oncology research, chemo- and radiotherapy are still the standard of care treatments for recurrent or metastatic EC and prognosis for these patients remains poor, with median survival rates of less than one year. Approximately 15-30% of the more common type I EC harbors oncogenic KRAS mutations, which have been linked to disease progression. Preclinical studies have shown that MEK inhibitors exhibit strong anti-tumor activity in KRAS mutant EC models. In the clinical setting however, MEKi monotherapy has proven unsuccessful in KRAS mutant EC patients, suggesting that combinations with other targeted agents may be needed to achieve increased signaling modulation and enhanced therapeutic activity. As suggested by our previous work in KRAS mutant lung, colorectal and pancreatic cancer, the combined inhibition of MEK and SOS1 may overcome adaptive resistance to MEKi by blocking SOS1-mediated reactivation of MAPK signaling. We evaluated whether this hypothesis can also be applied in the context of EC. Here we treated preclinical models of EC with a combination of clinically relevant doses of a MEKi (trametinib) and BI-3406, a potent and selective orally bioavailable SOS1 inhibitor. Treatment of KRAS mutant EC cells resulted in decreased activity of the KRAS-MAPK pathway, as measured by ERK phosphorylation. This effect was further enhanced by co-treatment with BI-3406. In vivo, the combination of MEKi with SOS1i synergistically decreased tumor growth in two KRAS G12D mutant orthotopic patient-derived xenograft models of EC. The anti-tumor effect of the combination was comparable to the effect of paclitaxel, the standard of care treatment in EC. This suggests that the combination of MEKi with SOS1i may be a potential therapeutic approach for the treatment of EC patients with tumors harboring KRAS mutations. Citation Format: Kaja Kostyrko, Melanie Hinkel, Marco H. Hofmann, Francesca Trapani. MEKi and SOS1i as a combination treatment option for KRAS mutant endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1083.

Abstract 4672: In-vivo tumor implantation site exhibits differential immune response in solid tumors

Abstract Successful discovery and development of cancer targeted therapeutics depends on testing these agents in the clinically most relevant translational models. Emerging evidence highlights the importance of the local tumor microenvironment in evaluating the efficacy of new therapeutics, especially for immunotherapies. Historically, patient-derived xenograft (PDX) modeling involves subcutaneous implantation which minimally represents the actual human tumor site. Here, we use PBMC humanized PDX models to demonstrate differences in the tumor microenvironment, overall immune response, and the pharmacological outcome, between subcutaneous and orthotopic PDX models. These findings highlight the importance of testing cancer therapeutics in the most physiologically relevant setting for greater translation into clinical success. Citation Format: Rajeev Shrimali, Jasmine Borroel, Carmen Sunico, Bridget Corcoran, Bianca Carapia, Rowan Prendergast, Jantzen Sperry, Jonathan Nakashima. In-vivo tumor implantation site exhibits differential immune response in solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4672.

Establishment of an orthotopic model of lung cancer by transthoracic lung puncture using tumor fragments.

Orthotopic models of lung cancer have been widely utilized, and the purpose of this study was to demonstrate the viability of our proposed modified modeling approach. A total of 50 female BALB/c mice were implanted with 1×1×1 mm fragments of a tumor sample into the left lung lobe. After 2 months of observation, the mice were humanely euthanized through CO2 inhalation. The macroscopic specimens were photographed, and the most representative neoplastic lesions were collected for histological analysis. Small-animal positron emission tomography/computed tomography (PET/CT) scans were conducted on 6 randomly selected mice. Local tumor formation, ipsilateral thoracic tissue infiltration, the contralateral chest wall, right lung metastases, and distant kidney metastases were observed in these models. Overall, the tumor development and metastasis rates were 60.86% (28/46) and 57.14% (16/28), respectively. The 3 mice that had a small-animal PET/CT scan developed a local tumor, but no distant metastases were observed. This modified method was deemed reliable, reproducible, minimally invasive, straightforward, and comprehensible; it might serve as the foundation for developing patient-derived orthotopic xenografts of lung cancer.

Development and characterization of a patient‑derived orthotopic xenograft of therapy‑resistant breast cancer.

Numerous years of cell line‑based studies have enhanced the current understanding of cancer and its treatment. However, limited success has been achieved in treating hormone receptor‑positive, HER2‑negative metastatic breast cancers that are refractory to treatment. The majority of cancer cell lines are unsuitable for use as pre‑clinical models that mimic this critical and often fatal clinical type, since they are derived from treatment‑naive or non‑metastatic breast cancer cases. The aim of the present study was to develop and characterize patient‑derived orthotopic xenografts (PDOXs) from patients with endocrine hormone receptor‑positive, HER2‑negative metastatic breast cancer who had relapsed on therapy. A patient who progressed on endocrine hormone therapy provided her tumor via a biobank. This tumor was implanted in mice. It was then serially passaged by implanting PDOX tumor fragments into another set of mice to develop further generations of PDOXs. These tissues were characterized using various histological and biochemical techniques. Histological, immunofluorescence and western blot analyses indicated that the PDOX tumors retained a similar morphology, histology and subtype‑specific molecular features to that of the patient's tumor. The present study successfully established PDOXs of hormone‑resistant breast cancer and characterized them in comparison with those derived from the original breast cancer tissue of the patient. The data highlight the reliability and usefulness of PDOX models for studies of biomarker discovery and preclinical drug screening. The present study was registered with the clinical trial registry of India (CTRI; registration no.CTRI/2017/11/010553; registered on 17/11/2017).

Rabies virus glycoprotein 29 (RVG29) promotes CAR-T immunotherapy for glioma

Chimeric antigen receptor T cell (CAR-T) therapy has limited efficacy for treating glioma because of the infiltrative nature of the blood–brain barrier (BBB) and T cell exhaustion. Conjugation with rabies virus glycoprotein (RVG) 29 enhances the brain-related efficacy of various agents. Here we assess whether RVG enhances the ability of CAR-T cells to cross the BBB and improves their immunotherapy. We generated 70R CAR-T cells (anti-CD70 CAR-T modified with RVG29) and validated their tumor-killing efficacy in vitro and in vivo. We validated their effects on tumor regression in a human glioma mouse orthotopic xenograft model as well as in patient-derived orthotopic xenograft (PDOX) models. The signaling pathways activated in 70R CAR-T cells were revealed by RNA sequencing. The 70R CAR-T cells we generated showed effective antitumor function against CD70+ glioma cells both in vitro and in vivo. 70R CAR-T cells were better able to cross the BBB into the brain than CD70 CAR-T cells under the same treatment conditions. Moreover, 70R CAR-T cells significantly promote the regression of glioma xenografts and improve the physical characteristics of mice without causing overt adverse effects. RVG modification enables CAR-T cells to cross the BBB, and stimulation with glioma cells induces 70R CAR-T cells to expand in a resting state. The modification of RVG29 has a positive impact on CAR-T therapy for brain tumors and may have potential in CAR-T therapy for glioma.