Patient-derived Orthotopic Xenograft Model Research Articles

Synergistic anti-tumor efficacy of mutant isocitrate dehydrogenase 1 inhibitor SYC-435 with standard therapy in patient-derived xenograft mouse models of glioma

Clinical outcomes in patients with WHO grade II/III astrocytoma, oligodendroglioma or secondary glioblastoma remain poor. Isocitrate dehydrogenase 1 (IDH1) is mutated in > 70% of these tumors, making it an attractive therapeutic target. To determine the efficacy of our newly developed mutant IDH1 inhibitor, SYC-435 (1-hydroxypyridin-2-one), we treated orthotopic glioma xenograft model (IC-BT142AOA) carrying R132H mutation and our newly established orthotopic patient-derived xenograft (PDX) model of recurrent anaplastic oligoastrocytoma (IC-V0914AOA) bearing R132C mutation. In addition to suppressing IDH1 mutant cell proliferation in vitro, SYC-435 (15 mg/kg, daily x 28 days) synergistically prolonged animal survival times with standard therapies (Temozolomide + fractionated radiation) mediated by reduction of H3K4/H3K9 methylation and expression of mitochondrial DNA (mtDNA)-encoded molecules. Furthermore, RNA-seq of the remnant tumors identified genes (MYO1F, CTC1 and BCL9) and pathways (base excision repair, TCA cycle II, sirtuin signaling, protein kinase A, eukaryotic initiation factor 2 and α-adrenergic signaling) as mediators of therapy resistance. Our data demonstrated the efficacy SYC-435 in targeting IDH1 mutant gliomas when combined with standard therapy and identified a novel set of genes that should be prioritized for future studies to overcome SYC-435 resistance.

Evaluation of an EZH2 inhibitor in patient-derived orthotopic xenograft models of pediatric brain tumors alone and in combination with chemo- and radiation therapies

AbstractBrain tumors are the leading cause of cancer-related death in children. Tazemetostat is an FDA-approved enhancer of zeste homolog (EZH2) inhibitor. To determine its role in difficult-to-treat pediatric brain tumors, we examined EZH2 levels in a panel of 22 PDOX models and confirmed EZH2 mRNA over-expression in 9 GBM (34.6 ± 12.7-fold) and 11 medulloblastoma models (6.2 ± 1.7 in group 3, 6.0 ± 2.4 in group 4) accompanied by elevated H3K27me3 expression. Therapeutic efficacy was evaluated in 4 models (1 GBM, 2 medulloblastomas and 1 ATRT) via systematically administered tazemetostat (250 and 400 mg/kg, gavaged, twice daily) alone and in combination with cisplatin (5 mg/kg, i.p., twice) and/or radiation (2 Gy/day × 5 days). Compared with the untreated controls, tazemetostat significantly (Pcorrected < 0.05) prolonged survival times in IC-L1115ATRT (101% at 400 mg/kg) and IC-2305GBM (32% at 250 mg/kg, 45% at 400 mg/kg) in a dose-dependent manner. The addition of tazemetostat with radiation was evaluated in 3 models, with only one [IC-1078MB (group 4)] showing a substantial, though not statistically significant, prolongation in survival compared to radiation treatment alone. Combining tazemetostat (250 mg/kg) with cisplatin was not superior to cisplatin alone in any model. Analysis of in vivo drug resistance detected predominance of EZH2-negative cells in the remnant PDOX tumors accompanied by decreased H3K27me2 and H3K27me3 expressions. These data supported the use of tazemetostat in a subset of pediatric brain tumors and suggests that EZH2-negative tumor cells may have caused therapy resistance and should be prioritized for the search of new therapeutic targets.This study confirms the preservation of EZH2 overexpression in 22 patient-derived orthotopic xenograft models of pediatric brain tumors. The authors demonstrate the activity of an FDA-approved EZH2 inhibitor, tazemetostat, alone and in combination with radiation in a subset of the models, and identifies EZH2-negative cells as potential cause of therapy resistance.

High Incidence of Lymph-node Metastasis in a Pancreatic-cancer Patient-derived Orthotopic Xenograft (PDOX) NOG-Mouse Model.

Our laboratory pioneered the patient-derived orthotopic xenograft (PDOX) model. An important goal of PDOX-model development is facile visualization of metastasis in live mice. In the present report we evaluated tumor growth and metastasis in pancreatic cancer PDOX NOG [Non-obese diabetes (NOD)/Scid/IL2Rγnull]-and nude-mouse models using red fluorescent protein (RFP)-expressing tumor stroma to visualize the primary tumor and metastasis. A patient-derived pancreatic cancer was initially implanted in transgenic RFP-expressing nude mice. Then, tumor fragments, which acquired RFP expressing stroma while growing in RFP-expressing nude mice were orthotopically implanted in nude and NOG mice. The primary pancreatic tumor and metastasis were observed 8 weeks after implantation. Lymph-node metastases expressing red fluorescence were detected only in NOG mice. Significantly faster growth of primary pancreatic tumors and a higher incidence of lymph-node metastasis occurred in NOG mice compared to nude mice. RFP-expressing tumor stroma, which traffics together with cancer cells to lymph nodes, is useful to observe tumor behavior, such as lymph-node metastasis in a PDOX NOG-mouse model which can be used for evaluation of novel anti-metastatic agents, as well as personalized therapy to identify effective drugs.

Oral-recombinant Methioninase Converts an Osteosarcoma from Methotrexate-resistant to -sensitive in a Patient-derived Orthotopic-xenograft (PDOX) Mouse Model.

Osteosarcoma is the most common bone sarcoma. Although surgery and chemotherapy are initially effective, the 5-year survival is approximately 60% to 80%, and has not improved over three decades. We have previously shown that methionine restriction (MR) induced by oral recombinant methioninase (o-rMETase), is effective against osteosarcoma in patient-derived orthotopic xenograft (PDOX) nude-mouse models. In the present report, the efficacy of the combination of oral o-rMETase and methotrexate (MTX) was examined in an osteosarcoma PDOX mouse model. An osteosarcoma-PDOX model was previously established by implanting tumor fragments into the proximal tibia of nude mice. The osteosarcoma PDOX models were randomized into four groups: control; o-rMETase alone; MTX alone; combination of o-rMETase and MTX. The mice were sacrificed after 4 weeks of treatment. The combination of o-rMETase and MTX showed significantly higher efficacy compared to the control group (p=0.04). The combination also showed significantly higher efficacy compared to MTX alone (p=0.04). No significant efficacy of o-rMETase alone or MTX alone compared to control was shown (p=0.21, 1.00, respectively). Only the combination of o-rMETase and MTX reduced the cancer-cell density in the osteosarcoma tumor. rMETase converted an osteosarcoma PDOX from MTX-resistant to MTX-sensitive and thereby shows future clinical potential.

The Molecular Subtype of Adult Acute Lymphoblastic Leukemia Samples Determines the Engraftment Site and Proliferation Kinetics in Patient-Derived Xenograft Models.

In acute lymphoblastic leukemia (ALL), conventional cell lines do not recapitulate the clonal diversity and microenvironment. Orthotopic patient-derived xenograft models (PDX) overcome these limitations and mimic the clinical situation, but molecular stability and engraftment patterns have not yet been thoroughly assessed. We herein describe and characterize the PDX generation in NSG mice. In vivo tumor cell proliferation, engraftment and location were monitored by flow cytometry and bioluminescence imaging. Leukemic cells were retransplanted for up to four passages, and comparative analyses of engraftment pattern, cellular morphology and genomic hotspot mutations were conducted. Ninety-four percent of all samples were successfully engrafted, and the xenograft velocity was dependent on the molecular subtype, outcome of the patient and transplantation passage. While BCR::ABL1 blasts were located in the spleen, KMT2A-positive cases had higher frequencies in the bone marrow. Molecular changes appeared in most model systems, with low allele frequency variants lost during primary engraftment. After the initial xenografting, however, the PDX models demonstrated high molecular stability. This protocol for reliable ALL engraftment demonstrates variability in the location and molecular signatures during serial transplantation. Thorough characterization of experimentally used PDX systems is indispensable for the correct analysis and valid data interpretation of preclinical PDX studies.

The Application of [68Ga]-Labeled FAPI-04 PET/CT for Targeting and Early Detection of Pancreatic Carcinoma in Patient-Derived Orthotopic Xenograft Models.

[18F]FDG as a probe of PET/CT is a radiolabeled glucose analogue taken up by most cells, but its batch activity is limited. [68Ga]FAPI-04 is a promising alternative based on a fibroblast activation protein-specific inhibitor (FAPI) labeled with radiotracer FAP. Here, a series of databases suggested that FAP expression was significantly different in pancreatic cancer compared to normal tissue. The FAP-positive fibroblasts were evaluated around the tumor cells and the stroma. A patient-derived orthotopic xenograft (PDOX) model of pancreatic adenocarcinoma (PDAC) exhibits significantly higher quantitative uptake of [68Ga]FAPI-04 (P < 0.05) than [18F]FDG PET/CT in various organs. Because of relatively high (T/M) ratios, the [68Ga]FAPI-04 is excellent for B-mode ultrasound, NIRF, and PET/CT. Thus, [68Ga]FAPI-04 PET displayed a better tumor specificity and can be a potential application for the early detection of pancreatic cancer.

Chloroquine Combined With Rapamycin Arrests Tumor Growth in a Patient-derived Orthotopic Xenograft (PDOX) Mouse Model of Dedifferentiated Liposarcoma.

Dedifferentiated liposarcoma (DDLS) is a type of soft-tissue sarcoma with a poor prognosis due to distant metastasis and resistance to chemotherapy. The antimalarial drug chloroquine (CQ) can induce apoptosis in cancer cells. CQ in combination with rapamycin (RAPA), an mTOR inhibitor, has shown efficacy on osteosarcoma and other types of cancer. In the present study the efficacy of RAPA combined with CQ on the treatment of a DDLS patient-derived orthotopic xenograft (PDOX) model was investigated. A patient-derived DDLS was transplanted into the left retroperitoneum of nude mice to establish a DDLS PDOX nude-mouse model. The mice were randomly divided as follows: untreated control group; CQ group; RAPA group; combined CQ and RAPA group (n=7 for all groups). During the treatment period, tumor volume was measured every 3-4 days with calipers. After 2 weeks treatment, the mice were sacrificed, and H&E staining was performed for histological evaluation. The TUNEL assay was performed to detect apoptosis. The combination of CQ and RAPA arrested tumor growth in the DDLS PDOX compared to the untreated control (p=0.009) and was significantly more effective than RAPA alone (p=0.009). RAPA alone slowed tumor growth, but the difference was not statistically significant (p>0.05). CQ was not active alone (p>0.05). The number of apoptotic TUNEL-positive cells was significantly higher in the CQ plus RAPA group than in the other groups (p=0.02). Combination therapy with CQ and RAPA arrested tumor growth in a DDLS PDOX model by inducing apoptosis.

Convection enhanced delivery of EGFR targeting antibody-drug conjugates Serclutamab talirine and Depatux-M in glioblastoma patient-derived xenografts.

BackgroundEGFR targeting antibody-drug conjugates (ADCs) are highly effective against EGFR-amplified tumors, but poor distribution across the blood–brain barrier (BBB) limits their efficacy in glioblastoma (GBM) when administered systemically. We studied whether convection-enhanced delivery (CED) can be used to safely infuse ADCs into orthotopic patient-derived xenograft (PDX) models of EGFRvIII mutant GBM.MethodsThe efficacy of the EGFR-targeted ADCs depatuxizumab mafodotin (Depatux-M) and Serclutamab talirine (Ser-T) was evaluated in vitro and in vivo. CED was performed in nontumor and tumor-bearing mice. Immunostaining was used to evaluate ADC distribution, pharmacodynamic effects, and normal cell toxicity.ResultsDose-finding studies in orthotopic GBM6 identified single infusion of 2 μg Ser-T and 60 μg Depatux-M as safe and effective associated with extended survival prolongation (>300 days and 95 days, respectively). However, with serial infusions every 21 days, four Ser-T doses controlled tumor growth but was associated with lethal toxicity approximately 7 days after the final infusion. Limiting dosing to two infusions in GBM108 provided profound median survival extension of over 200 days. In contrast, four Depatux-M CED doses were well tolerated and significantly extended survival in both GBM6 (158 days) and GBM108 (310 days). In a toxicity analysis, Ser-T resulted in a profound loss in NeuN+ cells and markedly elevated GFAP staining, while Depatux-M was associated only with modest elevation in GFAP staining.ConclusionCED of Depatux-M is well tolerated and results in extended survival in orthotopic GBM PDXs. In contrast, CED of Ser-T was associated with a much narrower therapeutic window.

Oral-recombinant Methioninase Lowers the Effective Dose and Eliminates Toxicity of Cisplatinum for Primary Osteosarcoma of the Mammary Gland in a Patient-derived Orthotopic Xenograft Mouse Model.

Primary osteosarcoma of the mammary gland is a very rare disease, accounting for under 1% of all mammary gland malignancies. There is no established first-line treatment, and prognosis is poor compared to conventional breast cancer. We previously demonstrated the efficacy of cisplatinum and eribulin in a patient-derived orthotopic xenograft (PDOX) mouse model of primary breast osteosarcoma. However, these drugs show significant clinical toxicity. All cancers are addicted to methionine (Hoffman effect). In the present study, we determined whether methionine restriction with oral recombinant methioninase (o-rMETase) would lower the effective dose of cisplatinum in a PDOX model of primary osteosarcoma of the mammary gland, thereby reducing its toxicity. Mouse PDOX models of primary osteosarcoma of the breast were randomized into the following groups: control; cisplatinum (weekly at 3 or 6 mg/kg); twice-daily o-rMETase; or o-rMETase combined with 3 mg/kg cisplatinum, with treatment for 2 weeks. Cisplatinum at 6 mg/kg significantly inhibited breast osteosarcoma growth compared with the untreated control and mice treated with 3 mg/kg cisplatinum (p=0.01 and 0.009, respectively). There was no significant difference in tumor growth between mice treated with cisplatinum at 3 mg/kg and the control (p=0.16). Combination therapy with cisplatinum at 3 mg/kg and twice daily o-rMETase regressed the osteosarcoma of the mammary gland (p=0.009), similar to the inhibition by cisplatinum at 6 mg/kg alone. Cisplatinum at 6 mg/kg caused a significant loss of mouse body weight, compared to the control (p=0.02). There was no significant body-weight loss with the combination therapy of o-rMETase and cisplatinum at 3 mg/kg, compared to the untreated control. o-rMETase halved the effective dose of cisplatinum, thereby eliminating cisplatinum toxicity, demonstrating a future clinical strategy for therapy of osteosarcoma of the breast.

Establishment of Patient-derived Orthotopic Xenografts (PDX) as Models for Pancreatic Ductal Adenocarcinoma.

Pancreatic cancer (PC) is one of the leading causes of cancer-related death. The purpose of the present study was to establish a patient-derived orthotopic xenograft model (PDOX) for pancreatic ductal adenocarcinoma (PDAC), thus providing a tumor microenvironment resembling that of the human pancreas to identify novel potential biomarkers and treatment regimens. PDAC tissue samples were received from 35 patients, following informed consent, and three mouse strains were implemented. Successful PDOX engraftment was performed in nonobese diabetic/severe combined immunodeficient (NOD/SCID) and NOD/SCID gamma (NSG) mice. Nonetheless, we found a higher rate of successful engraftment and tumor growth in NSG compared to NOD/SCID mice, possibly owning to the different level of immunosuppression and more specifically of the natural killer cells presence. Our suggested PDOX model represents a preclinical cancer research model with a high affinity for the patient's tumor microenvironment, thus enabling the acceleration of PDAC research.

Predictive modeling of resistance to SMO inhibition in a patient-derived orthotopic xenograft model of SHH medulloblastoma.

BackgroundInhibition of the sonic hedgehog (SHH) pathway with Smoothened (SMO) inhibitors is a promising treatment strategy in SHH-activated medulloblastoma, especially in adult patients. However, the problem is that tumors frequently acquire resistance to the treatment. To understand the underlying resistance mechanisms and to find ways to overcome the resistance, preclinical models that became resistant to SMO inhibition are needed.MethodsTo induce SMO inhibitor resistant tumors, we have treated a patient-derived xenograft (PDX) model of SHH medulloblastoma, sensitive to SMO inhibition, with 20 mg/kg Sonidegib using an intermitted treatment schedule. Vehicle-treated and resistant models were subjected to whole-genome and RNA sequencing for molecular characterization and target engagement. In vitro drug screens (76 drugs) were performed using Sonidegib-sensitive and -resistant lines to find other drugs to target the resistant lines. One of the top hits was then validated in vivo.ResultsNine independent Sonidegib-resistant PDX lines were generated. Molecular characterization of the resistant models showed that eight models developed missense mutations in SMO and one gained an inactivating point mutation in MEGF8, which acts downstream of SMO as a repressor in the SHH pathway. The in vitro drug screen with Sonidegib-sensitive and -resistant lines identified good efficacy for Selinexor in the resistant line. Indeed, in vivo treatment with Selinexor revealed that it is more effective in resistant than in sensitive models.ConclusionsWe report the first human SMO inhibitor resistant medulloblastoma PDX models, which can be used for further preclinical experiments to develop the best strategies to overcome the resistance to SMO inhibitors in patients.

Fluorescent Anti-MUC5AC Brightly Targets Pancreatic Cancer in a Patient-derived Orthotopic Xenograft.

Overexpression of mucin-5AC (MUC5AC) makes it a targetable biomarker in pancreatic cancer. The present study evaluated tumor targeting with a MUC5AC antibody conjugated to a near-infrared dye in a patient-derived orthotopic xenograft (PDOX) mouse model. MUC5AC monoclonal antibody was conjugated to the near-infrared dye IRDye800CW to synthesize MUC5AC-IR800. PDOX models were established by implanting a high-MUC5AC-expressing patient-derived pancreatic tumor on the pancreas of nude mice. After 4 weeks of PDOX tumor growth, mice were imaged after receiving MUC5AC-IR800 (75 μg) intravenously. In the PDOX models, MUC5AC-IR800 selectively and brightly targeted the pancreatic tumor (tumor to background ratio: 2.46±0.465). MUC5AC-IR800 provides distinct visualization of pancreatic tumors. MUC5AC-IR800 may be used clinically in the future to improve pancreatic cancer resection. This novel fluorescent probe is also promising for targeting of pre-malignant pancreatic lesions with subsequent resection under fluorescence guidance.

Brain penetration and efficacy of tepotinib in orthotopic patient-derived xenograft models of MET-driven non-small cell lung cancer brain metastases

Central nervous system-penetrant therapies with intracranial efficacy against non-small cell lung cancer (NSCLC) brain metastases are urgently needed. We report preclinical studies investigating brain penetration and intracranial activity of the MET inhibitor tepotinib. After intravenous infusion of tepotinib in Wistar rats (n = 3), mean (±standard deviation) total tepotinib concentration was 2.87-fold higher in brain (505 ± 22 ng/g) than plasma (177 ± 20 ng/mL). In equilibrium dialysis experiments performed in triplicate, mean tepotinib unbound fraction was 0.35% at 0.3 and 3.0 µM tepotinib in rat brain tissue, and 4.0% at 0.3 and 1.0 µM tepotinib in rat plasma. The calculated unbound brain-to-plasma ratio was 0.25, indicating brain penetration sufficient for intracranial target inhibition. Of 20 screened subcutaneous patient-derived xenograft (PDX) models from lung cancer brain metastases (n = 1), two NSCLC brain metastases models (LU5349 and LU5406) were sensitive to the suboptimal dose of tepotinib of 30 mg/kg/qd (tumor volume change [%TV]: –12% and –88%, respectively). Molecular profiling (nCounter®; NanoString) revealed high-level MET amplification in both tumors (mean MET gene copy number: 11.2 and 24.2, respectively). Tepotinib sensitivity was confirmed for both subcutaneous models at a clinically relevant dose (125 mg/kg/qd; n = 5). LU5349 and LU5406 were orthotopically implanted into brains of mice and monitored by magnetic resonance imaging (MRI). Tepotinib 125 mg/kg/qd induced pronounced tumor regression, including complete or near-complete regressions, compared with vehicle in both orthotopic models (n = 10; median %TV: LU5349, –84%; LU5406, –63%). Intracranial antitumor activity of tepotinib did not appear to correlate with blood–brain barrier leakiness assessed in T1-weighted gadolinium contrast-enhanced MRI.

Osteosarcoma Patient-derived Orthotopic Xenograft (PDOX) Models Used to Identify Novel and Effective Therapeutics: A Review.

Recurrent osteosarcoma is recalcitrant with poor response rates to first-line chemotherapy due to heterogeneity and metastatic potential. This disease requires novel drug discovery and precision treatment. The osteosarcoma patient-derived orthotopic xenograft (PDOX) mouse model mimics the clinical disease and has identified effective clinically-approved drugs and experimental agents, especially drug combinations, that hold much clinical promise. Effective treatment for drug-resistant osteosarcoma includes regorafenib, as monotherapy, and temozolomide-irinotecan, trabectedin-irinotecan, sorafenib-everolimus, sorafenib-palbociclib, and olaratumab-doxorubicin-cisplatinum, as combinations. The PDOX model can be used to improve the outcome of osteosarcoma patients, including individualized, precision therapy.

Inhibitors of Bcl-2 and Bruton\u2019s tyrosine kinase synergize to abrogate diffuse large B-cell lymphoma growth in vitro and in orthotopic xenotransplantation models

Numerous targeted therapies have been developed for diffuse large B-cell lymphoma, but the results of late-stage clinical trials were mostly disappointing and have led to very few new regulatory approvals. Here, we use single and combinatorial drug response profiling to show that the combined inhibition of the anti-apoptotic protein Bcl-2 and of the tyrosine kinase BTK with the small molecules venetoclax and ibrutinib efficiently kills DLBCL cells in vitro. High Bcl-2 expression due to either BCL2 amplifications or translocations, in conjunction with chronic active BCR signaling accurately predict responses to dual Bcl-2/BTK inhibition. Orthotopic xenotransplantation and patient-derived xenograft models confirm that the combinatorial is superior to single-agent treatment in reducing the lymphoma burden. Combinatorial treatment further efficiently overcomes both primary and acquired resistance to venetoclax, which we could link to reduced expression of the Bcl-2 family members Bcl-XL and Bcl-2A1 under ibrutinib. We found in a Swiss DLBCL cohort that ~15% of patients are projected to respond to the venetoclax/ibrutinib combination based on their high Bcl-2 expression and nuclear NF-κB localization. Our data show that drug sensitivities exposed by drug response profiling can be attributed to specific mutational signatures and immunohistochemical biomarkers, and point to combined Bcl-2/BTK inhibition as a promising therapeutic strategy in DLBCL.

EXTH-09. TUMOR PHARMACOKINETICS, PHARMACODYNAMICS AND RADIATION SENSITIZATION IN PATIENT-DERIVED XENOGRAFT MODELS OF GLIOBLASTOMA TREATED WITH THE AURORA KINASE A INHIBITOR LY3295668

Abstract Cell-cycle deregulation is at the crux of all malignancies, including glioblastoma (GBM). Aurora Kinase A (AURKA) plays a central role in G2/M transition and faithful chromosome segregation. In this study, we evaluated the pharmacokinetics, pharmacodynamics, and radiation sensitization properties of LY3295668, a highly specific AURKA inhibitor, in orthotopic patient-derived xenograft (PDX) models of GBM. Mice with intracranial tumors were randomized to 50 mg/kg LY3295668 PO BID x 4 days vs. placebo. LY3295668 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 pAURKA (T288), phospho-Histone H3 (pHH3), and cleaved caspase 3 (CC3). For survival studies, mice with intracranial tumors were randomized to four cohorts – vehicle, radiotherapy, LY3295668 monotherapy, and LY3295668 plus radiotherapy. The median unbound concentration of LY3295668 was 270.88 nmol/L and 22.33 nmol/kg in plasma and tumor tissue, respectively – significantly higher than the biochemical IC50 of LY3295668 for AURKA inhibition (0.8 nM). A decrease in pHH3(+) cells (0.8% vs. 6.4%, p=0.036) indicated drug-induced mitotic arrest and was accompanied by an increase in CC3(+) cells (6.4% vs. 8.0%, p=0.67). Combination of LY3295668 with radiotherapy prolonged survival compared to either therapy alone in orthotopic GBM PDX models. LY3295668 is well tolerated, achieves pharmacologically-relevant unbound concentrations in GBM PDX models, and is associated with significant target modulation. Preclinical combination of LY3295668 with radiation therapy leads to synergistic effects and supports future clinical study of this multimodal strategy in glioblastoma patients.

EXTH-38. TARGETING KIF11 TO RADIOSENSITIZE GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most lethal primary brain tumor, with a 5 year survival rate of only 5%. The standard of care for GBM is maximal surgical resection of the tumor, followed by irradiation and chemotherapy. Despite treatment, tumors recur in almost 100% of patients. There are subpopulations of cells in GBM that are radioresistant and chemoresistant, and new treatments will need to inclusively target these cells. KIF11 is a mitotic protein that drives bipolar spindle formation and is crucial for successful completion of mitosis. We previously reported that KIF11 is overexpressed in GBM over normal brain tissue, and that inhibiting KIF11 in a patient-derived xenograft (PDX) GBM mouse model increased survival. However, in this model, tumors recurred after treatment was stopped, indicating that treatment may have had a cytostatic effect, rather than cytotoxic. Importantly, it has been reported that cells are most vulnerable to irradiation when they are in mitosis. Because using a KIF11 inhibitor arrests cells in mitosis, we hypothesized that combining irradiation and a KIF11 inhibitor would radiosensitize GBM cells, and lead to greater tumor cell death. In this study, we found that combination therapy increased cell death over vehicle or either treatment used alone in patient-derived GBM cells in vitro. Additionally, we found that inhibiting KIF11 combined with radiotherapy increased survival over vehicle or monotherapy in orthotopic PDX models. Our results demonstrate that combining KIF11 inhibitors with radiotherapy is a promising potential therapy for GBM.

STEM-02. THERAPEUTIC INTERVENTION OF LUNG-, BREAST-, AND MELANOMA-BRAIN METASTASIS

Abstract BACKGROUND The incidence of brain metastases (BM) is tenfold higher than that of primary brain tumours. BM predominantly originate from primary lung, breast, and melanoma tumours with a 90% mortality rate within one year of diagnosis, posing a large unmet clinical need to identify novel therapies against BM.This unmet clinical need is largely attributed to a small population of cancer stem cells (CSCs), termed BM-initiating cells (BMICs), that are able to escape a primary tumour, drive metastasis and facilitate the formation of a secondary tumour in the brain. METHODS Using a large in-house biobank of patient-derived BMIC lines, the Singh Lab has generated murine orthotopic patient-derived xenograft models of BM and captured a “premetastatic” population of BMICs that have just seeded the brains of mice before forming clinically detectable tumours: a cell population that is impossible to detect in human patients but represents a therapeutic window wherein metastasizing cells can be targeted and eradicated before establishing clinically detectable tumours. RESULTS RNA sequencing of pre-metastatic BMICs from all three primary tumour models with subsequent Connectivity Map analysis identified a lead compound that exhibits selective anti-BM activity in vitro. Preliminary in vivo work has shown that this lead compound reduces the tumor burden of treated mice compared to vehicle control while providing a significant survival advantage. Ongoing mechanistic investigations aim to delineate the protein target of this compound in the context of the observed selective anti-BMIC phenotype. CONCLUSION Identification of novel small molecules that target premetastatic BM cells could prevent the formation of BM and dramatically improve the prognosis of at-risk cancer patients.

NIMG-75. ANALYZING THE INTERFACE BETWEEN MRI AND DRUG DISTRIBUTION USING ORTHOTOPIC GBM-DERIVED XENOGRAFT (PDX) MODELS

Abstract INTRODUCTION Glioblastoma (GBM) is a diffusely invasive primary brain tumor with significant spread of tumor cells to the periphery of visible image abnormality. Enhancement of Gadolinium (Gd) contrast agent on magnetic resonance imaging (MRI) has historically been considered a confirmation of local breakdown of the blood brain barrier (BBB) and sufficient drug delivery to the bulk of tumors. In this work, we used GBM-derived xenograft (PDX) models to compare drug delivery in GBM brain for high and low BBB-permeable drugs. MATERIALS AND METHODS Five patient-derived orthotopic xenograft models from two GBM cell lines (GBM39 and GBM12) were co-dosed with erlotinib and osimertinib, two drugs with low and high BBB-permeability, respectively. T1Gd and T2-weighted MRIs were acquired from all animals prior to model sacrifice. Tumors were manually segmented on denoised and standardized MRIs and intensity patterns were captured using first and second order statistical features in the moving 3x3 kernel. We compared drug levels found in Matrix Assisted Laser Desorption Ionization (MALDI) in T1Gd enhancement, T2 enhancement, and normal brain. We also performed linear regression modeling to predict drug levels using MRI features. Model performance was measured using root mean squared error (RMSE). RESULTS Our analysis showed correlations between imaging features and MALDI drug levels. Osimertinib had a uniform distribution across the brain for all animals and all cell lines, consistent with our expectation for a high BBB-penetrant drug. Erlotinib showed the highest drug levels in T2 for GBM39 and in T1Gd for GBM12. Regression models showed promising results for predicting Erlotinib with a low RMSE of 0.037. CONCLUSION Our preliminary results suggest MRI can be predictive of drug levels for low-BBB penetrant drugs. Understanding the relationship between MRIs and drug distribution in diffuse tumors can be beneficial to developing effective treatment.

Anti-invasive efficacy and survival benefit of the YAP-TEAD inhibitor verteporfin in preclinical glioblastoma models.

Glioblastoma (GBM) remains a largely incurable disease as current therapy fails to target the invasive nature of glioma growth in disease progression and recurrence. Here, we use the FDA-approved drug and small molecule Hippo inhibitor Verteporfin (VP) to target YAP-TEAD activity, known to mediate convergent aspects of tumor invasion/metastasis, and assess the drug's efficacy and survival benefit in GBM models. Up to 8 low-passage patient-derived GBM cell lines with distinct genomic drivers, including 3 primary/recurrent pairs, were treated with VP or vehicle (VEH) to assess in vitro effects on proliferation, migration, invasion, YAP-TEAD activity, and transcriptomics. Patient-derived orthotopic xenograft (PDX) models were used to assess VP's brain penetrance and effects on tumor burden and survival. VP treatment disturbed YAP/TAZ-TEAD activity; disrupted transcriptome signatures related to invasion, epithelial-to-mesenchymal, and proneural-to-mesenchymal transition, phenocopying TEAD1-knockout effects; and impaired tumor migration/invasion dynamics across primary and recurrent GBM lines. In an aggressive orthotopic PDX GBM model, short-term VP treatment consistently diminished core and infiltrative tumor burden, which was associated with decreased tumor expression of Ki67, nuclear YAP, TEAD1, and TEAD-associated targets EGFR, CDH2, and ITGB1. Finally, long-term VP treatment appeared nontoxic and conferred survival benefit compared to VEH in 2 PDX models: as monotherapy in primary (de novo) GBM and in combination with Temozolomide chemoradiation in recurrent GBM, where VP treatment associated with increased MGMT methylation. We demonstrate combined anti-invasive and anti-proliferative efficacy for VP with survival benefit in preclinical GBM models, indicating potential therapeutic value of this already FDA-approved drug if repurposed for GBM patients.