Carlson, Brett L.

Abstract The Mayo Clinic collection of patient-derived xenograft (PDX) models has been developed from the direct implantation of fresh glioblastoma samples into immunodeficient mice. Over 95 models have been extensively characterized with regards to histology, invasion, flank and orthotopic growth rates, and molecular profiling. Evaluation of orthotopic tumors demonstrated clear infiltration of tumor cells into the brain parenchymal in 90% of PDXs with contralateral hemisphere involvement in 64%. Extensive molecular profiling confirmed that our collection of PDXs captures the genetic heterogeneity of GBM, with the majority of known alterations represented at a frequency similar to the TCGA including gain of chromosome 7 and loss of chromosome 10. TERT promoter mutations were the most frequent overall (86%) followed by homozygous deletion of CDKN2A (70%). Multiple alterations were identified in the p53 pathway, including TP53 mutations (36%) and MDM2/4 (10%/2%) amplification. PI3-kinase pathway alterations mainly involved PTEN (48%) while MAP kinase pathway alterations were present in a subset of PDXs (NF1 alterations 17%, BRAF 4%). IDH mutations were found in only two models. Gene expression analysis revealed that our PDX collection includes all GBM subtypes with classical being the most represented (46% vs mesenchymal 31% and proneural 23%). MGMT promoter methylation was also observed in 45% of PDXs. The high fidelity of PDX models to their human counterpart makes these models ideal for testing standard of care and novel targeted therapies. To assess if PDXs are predictive of patient treatment response, 37 orthotopic PDX lines were treated with radiation (RT), temozolomide (TMZ), or concurrent RT/TMZ. Across all PDXs, RT and TMZ demonstrated median survival extension of 90 and 46 days relative to vehicle. Concurrent RT/TMZ further extended survival by 146 days. MGMT promoter methylation was strongly associated with response to TMZ-based strategies but not RT alone.This observation suggests that our PDX collection possesses clinically relevant predictive value. To date, we have characterized treatment responses to over 135 regimens including both GBM standards of care and targeted strategies in our PDX models. Recent lab efforts strive to define the effect of blood brain barrier disruption on drug delivery. With over 20 years of experience, our group has extensive expertise in the development of clinically relevant study designs focused on examining efficacy, tolerability, and pharmacokinetic/pharmacodynamic assessments. Since 2016, we have shared over 5200 individual samples (tumor tissue, cells, DNA/RNA, protein extracts, tissue microarray slides) in both internal and external collaborators. Our models have been utilized in over 41 NIH funded grants, including 33 funded RO1s, as well as by the pharmaceutical community with 50 working relationships to date (20 contracts, 30 collaborations). Finally, our PDX collection has been used in over 120 peer-reviewed published manuscripts. Citation Format: Danielle Burgenske, Ann C. Mladek, Rachael A. Vaubel, Shulan Tian, Mark A. Schroeder, Zeng Hu, Brett L. Carlson, Paul A. Decker, Jeanette E. Eckel-Passow, Jann N. Sarkaria. Evaluation of novel therapeutics using models from the Mayo Clinic GBM patient-derived xenograft (PDX) collection [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1113.

Abstract Lisavanbulin (LIS; BAL101553) is the prodrug of BAL27862, a microtubule-binding, tumor checkpoint controller and potential radiosensitizer. These studies evaluated optimal integration of LIS with standard of care radiation therapy (RT) and/or temozolomide (TMZ) using GBM PDX models. Distribution across the blood brain barrier was evaluated after a single 30 mg/kg oral LIS dose, and concentrations of the active metabolite BAL27862 were measured by liquid chromatography-tandem mass spectrometry. Similar BAL27862 concentrations were detected in the brain (B) and plasma (P) at both two (B:P ratio 1.29) and six hours (B:P ratio 1.64) post-dose. An in vivo screen of LIS monotherapy across 14 orthotopic GBM PDX models showed significant survival benefit (p<0.01) in seven models (median survival extension 24-87%). Extending from these results, LIS was evaluated in several of the sensitive models in combination with RT +/- TMZ. Two MGMT unmethylated PDXs, GBM6 and GBM150, were treated with vehicle or two weeks of RT +/- LIS. LIS dosing during the RT dosing period did not significantly improve median survival in either line (GBM6 survival with RT 54 days vs RT/LIS 58 days, p=0.16; GBM150 RT 86 days vs RT/LIS 101 days, p=0.21). However, prolonged LIS dosing from the start of RT until mice reached a moribund state demonstrated added benefit (GBM6 median 90 days vs RT 69 days, p=0.0001; GBM150 median 143 days vs RT 73 days, p=0.06). In GBM6, prolonged LIS dosing also significantly extended survival when combined with 2 weeks of RT/TMZ (median 101 days vs 66 days, p<0.0001), while LIS alone or RT/TMZ resulted in similar median survivals (63 days vs 66 days, respectively; p=0.68). This same RT/TMZ/LIS benefit was not seen in the MGMT methylated GBM12. Subsequent experiments were performed to evaluate integration of prolonged LIS dosing with concurrent RT/TMZ followed by 3 cycles of adjuvant TMZ (‘Stupp’ regimen). In MGMT methylated GBM39, LIS alone did not significantly extend survival, but LIS addition to the Stupp regimen doubled median survival (Stupp 249 days vs Stupp/LIS 502 days, p=0.0001). GBM150 demonstrated equal benefit from LIS alone or Stupp regimen (median 118 days vs 123 days, p=0.49). Stupp/LIS showed no additional survival benefit (median 98 days, p=0.97). In a second MGMT unmethylated, TMZ-resistant GBM26 PDX, LIS alone or combined with the Stupp regimen provided significant survival benefit: median survival 53 days for vehicle vs. 80 days for LIS (p=0.0001), 114 days for RT only (p<0.0001), 147 days for RT/LIS (p=0.30 relative to RT), 121 days for ‘Stupp’ regimen alone (p=0.57 relative to RT), and 172 days for Stupp/LIS (p=0.04 relative to Stupp). A follow-up GBM39 study revealed a significant increase in the mitotic marker phospho-histone H3 with LIS treatment relative to vehicle-treated controls (p=0.01) while Ki67 levels were similar (p=0.15). This suggests that LIS induces a mitotic arrest associated with microtubule deregulation. Collectively, these data provide a strong rationale to evaluate lisavanbulin (BAL101553) with RT +/- TMZ in GBM and provided the basis for an ongoing Phase I clinical trial. Citation Format: Danielle M Burgenske, Ann C Mladek, Jenny L Pokorny, Heidi A Lane, Felix Bachmann, Rachael A Vaubel, Mark A Schroeder, Katrina K Bakken, Lihong He, Zeng Hu, Brett L Carlson, Surabhi Talele, Gautham Gampa, Matthew L Kosel, Paul A Decker, Jeanette E Eckel-Passow, William F Elmquist, Jann Sarkaria. Modeling the clinical paradigm of lisavanbulin (BAL101553) deployment in patient-derived xenografts (PDX) of glioblastoma (GBM) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C096. doi:10.1158/1535-7163.TARG-19-C096

Abstract Cell survival following radiation therapy (RT) is critically modulated by multiple DNA damage response pathways including the p53 tumor suppressor, which promotes cell cycle arrest and/or apoptosis in the face of DNA damage. Murine Double Minute 2 (MDM2) targets p53 for degradation and suppresses its functions. The MDM2 locus is amplified in approximately 14% of glioblastoma (GBM) tumors and is a promising target for individualized therapy. In these studies, we tested the MDM2 small molecule inhibitor KRT-232, alone and in combination with RT, in MDM2-amplified and/or p53 wildtype patient-derived xenograft (PDX) models of GBM in vitro and in vivo. KRT-232 suppressed GBM PDX cell explant culture viability in three MDM2-amplified lines (GBM46, 108, and 148) and two non-amplified but p53 wildtype lines (GBM10 and 39) with similar IC50s ranging from 300-800nM in FBS culture conditions. As expected, little suppression was observed in a p53 mutant (GBM43) line. Many drugs have limited distribution in the brain, and to understand limitations surrounding KRT-232, LCMS-MS was used to compare brain and plasma levels of drug in mice. Preliminary pharmacokinetic analysis using FVB wildtype mice compared to mice which maintain triple knockout of the efflux transporters Mdr1a, Mdr1b and Bcrp1–/– (TKO) treated with a single 10 mg/kg oral dose of KRT-232 and harvested 2 hours later demonstrate an 8% brain/plasma ratio of KRT-232 in the TKO mice, while levels were undetectable in WT mice. In light of these data, the efficacy of KRT-232 was initially tested in vivo in an MDM2-amplified GBM108 PDX with an artificially disrupted BBB afforded by VEGFA lentiviral expression. In this model, daily dosing with KRT-232 alone produced a 100 day prolongation in survival as compared to vehicle. In a subsequent experiment, just one week of daily KRT-232 dosing, either alone or in combination with RT, in the control GBM108 PDX with a relatively intact BBB, was remarkably effective; median survival for placebo 22 days, KRT-232 alone 46 days, RT alone 31 days, and the KRT-232/RT combination 77 days. Despite low BBB penetration as determined by pharmacokinetic studies, KRT-232 alone and in combination with RT is an effective treatment in a pre-clinical model of glioblastoma. Future studies will evaluate KRT-232 pharmacodynamic responses as well as PDX orthotopic combination therapy in additional MDM2 amplified and non-amplified/p53 wildtype lines to understand if this therapy can be used in a larger population of GBM tumors. Citation Format: Ann C Mladek, Shiv Gupta, Minjee Kim, Afroz Shareef Mohammad, Katrina K Bakken, Helen He, Zeng Hu, Danielle M Burgenske, Brett L Carlson, William F Elmquist, Jann Sarkaria. MDM2 inhibitor KRT-232 extends survival in glioblastoma patient-derived xenograft models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C051. doi:10.1158/1535-7163.TARG-19-C051

1509 Background: Lack of MGMT methylation in GBM tumors is associated with a worse outcome with combined radiation (RT) and temozolomide (TMZ) therapy, and it is unclear whether non-methylated tumors benefit from TMZ treatment. Methods: The potential benefits of concomitant or adjuvant TMZ relative to methylation status were evaluated in 10 GBM tumors established from patient samples and maintained by serial passage in mice. For each tumor line, 32 mice with established orthotopic xenografts were randomized to A) placebo, B) TMZ PO daily × 5 (120 mg/kg or 66 mg/kg, as indicated) C) RT, 2 Gy bid × 10 fx., D) combined TMZ and RT, and mice were followed until moribund. Efficacy was evaluated by regression modeling and Kaplan-Meier statistical analyses. Results: Six lines with non-methylated MGMT (GBM10, 14, 26, 28, 36, 43) and 4 with methylated MGMT (GBM8, 12, 16, 34) were evaluated, and complete results are available in 6 lines. In all tumor lines and at both dose levels, there was a significant benefit for TMZ relative to placebo (p<0.003). Both methylated tumors (GBM8 and 12) and one non-methylated tumor (GBM14) were markedly sensitive to TMZ with greater than a 50 day extension in survival with monotherapy. Individually and in a pooled analysis, there was no evidence for a synergistic interaction between TMZ and RT in the non-methylated tumors (p=0.91 for pooled analysis). In contrast, there was a striking degree of synergy in GBM12 (p<0.001) and at least additive effects of combined treatment in GBM8 (5 of 8 mice still surviving in the RT/TMZ arm). In a pooled analysis of these 2 MGMT methylated tumors, there was a trend towards synergy (p=0.11). Conclusions: TMZ treatment did not sensitize MGMT non-methylated tumors to radiation, and with one exception, these non-methylated tumors were only modestly sensitive to TMZ alone. In contrast, treatment with TMZ appears to sensitize MGMT-methylated tumors to radiation and monotherapy is associated with marked and significant benefit. [Table: see text] No significant financial relationships to disclose.

Abstract Purpose: Glioblastoma is the most frequent and lethal primary brain tumor. Development of novel therapies relies on the availability of relevant preclinical models. We have established a panel of 96 glioblastoma patient-derived xenografts (PDX) and undertaken its genomic and phenotypic characterization. Experimental Design: PDXs were established from glioblastoma, IDH-wildtype (n = 93), glioblastoma, IDH-mutant (n = 2), diffuse midline glioma, H3 K27M-mutant (n = 1), and both primary (n = 60) and recurrent (n = 34) tumors. Tumor growth rates, histopathology, and treatment response were characterized. Integrated molecular profiling was performed by whole-exome sequencing (WES, n = 83), RNA-sequencing (n = 68), and genome-wide methylation profiling (n = 76). WES data from 24 patient tumors was compared with derivative models. Results: PDXs recapitulate many key phenotypic and molecular features of patient tumors. Orthotopic PDXs show characteristic tumor morphology and invasion patterns, but largely lack microvascular proliferation and necrosis. PDXs capture common and rare molecular drivers, including alterations of TERT, EGFR, PTEN, TP53, BRAF, and IDH1, most at frequencies comparable with human glioblastoma. However, PDGFRA amplification was absent. RNA-sequencing and genome-wide methylation profiling demonstrated broad representation of glioblastoma molecular subtypes. MGMT promoter methylation correlated with increased survival in response to temozolomide. WES of 24 matched patient tumors showed preservation of most genetic driver alterations, including EGFR amplification. However, in four patient–PDX pairs, driver alterations were gained or lost on engraftment, consistent with clonal selection. Conclusions: Our PDX panel captures the molecular heterogeneity of glioblastoma and recapitulates many salient genetic and phenotypic features. All models and genomic data are openly available to investigators.

Abstract Background: IDH mutated gliomas are critically dependent on glutaminase for glutamate biosynthesis. CB-839 is a novel glutaminase-1 inhibitor which has demonstrated efficacy in both genetically engineered and patient derived IDH mutated glioma cells, especially in combination with radiation. These preclinical studies evaluate the pharmacodynamic and pharmacokinetic impact of combining CB-839 with TMZ in patient derived xenograft (PDX) glioma models. Methods: GBM164 is a PDX model derived from an IDH1 mutant, 1p/19q non-codeleted glioma. D- and L-2HG levels in untreated GBM164 tumors were compared to GBM6 tumors (IDH wildtype glioma PDX) by GC/MS. Athymic nude mice bearing GBM164 flank tumors were treated with CB-839 (200 mg/kg PO BID x 9 doses) and/or temozolomide (50 mg/kg PO daily x 5 doses) and sacrificed four hours after the last dose to harvest plasma, normal brain and flank tumor. Tumor metabolomics were assessed by GC/MS. DNA damage signaling in tumors was assessed by Western blotting for KAP1 / Chk1 / Chk2 phosphorylation and H2AX / Rad51. Tumor, plasma and brain pharmacokinetics were assessed by LC/MS/MS. Results: Total 2HG levels in GBM164 were 18-fold higher than GBM6. CB-839 monotherapy reduced tumor glutamate (18% reduction, p = 0.15) and aspartate (30% reduction, p = 0.06) when compared to vehicle, however these changes did not reach statistical significance. The combination of CB-839 and TMZ more significantly reduced both tumor glutamate (30% reduction, p = 0.03) and aspartate (34% reduction, p &lt; 0.001) when compared to TMZ monotherapy. CB-839 did not significantly increase DNA damage compared to vehicle, and the combination of CB-839 and TMZ did not significantly increase DNA damage compared to TMZ monotherapy. The mean tumor: plasma ratios of CB-839 concentrations were 0.68 and 0.58 in mice treated with CB-839 monotherapy and CB-839/TMZ, respectively. The mean brain: plasma ratios of CB-839 concentrations were 0.07 and 0.12 in mice treated with CB-839 monotherapy and CB-839/TMZ, respectively. Conclusion: The addition of CB-839 to TMZ significantly reduces glioma aspartate and glutamate in an IDH1 mutant PDX glioma model, without any impact on DNA damage. Survival studies are in progress to assess the efficacy of CB-839 when used in combination with RT and/or TMZ. Citation Format: Sani Haider Kizilbash, Danielle M. Burgenske, Samuel McBrayer, Sandhya Devarajan, Shiv K. Gupta, Taro Hitosugi, Lihong He, Mark A. Schroeder, Brett L. Carlson, Marina Gelman, Charles A. Kunos, Joel M. Reid, Alex A. Adjei, Jann N. Sarkaria. The addition of CB-839 to temozolomide significantly reduces glioma aspartate and glutamate in an IDH mutated patient derived glioma xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3870.

Abstract Background Predictive molecular biomarkers to select optimal treatment for patients with glioblastoma and other cancers are lacking. New strategies are needed when large randomized trials with correlative molecular data are not feasible. Methods Gene signatures (GS) were developed from 31 orthotopic glioblastoma patient-derived xenografts (PDXs), treated with standard therapies, to predict benefit from radiotherapy (RT-GS), temozolomide (Chemo-GS), or the combination (ChemoRT-GS). Independent validation was performed in a heterogeneously treated clinical cohort of 502 glioblastoma patients with overall survival as the primary endpoint. Multivariate Cox analysis was used to adjust for confounding variables and evaluate interactions between signatures and treatment. Results PDX models recapitulated the clinical heterogeneity of glioblastoma patients. RT-GS, Chemo-GS, and ChemoRT-GS were correlated with benefit from treatment in the PDX models. In independent clinical validation, higher RT-GS scores were associated with increased survival only in patients receiving RT (P = 0.0031, hazard ratio [HR] = 0.78 [0.66–0.92]), higher Chemo-GS scores were associated with increased survival only in patients receiving chemotherapy (P < 0.0001, HR = 0.66 [0.55–0.8]), and higher ChemoRT-GS scores were associated with increased survival only in patients receiving ChemoRT (P = 0.0001, HR = 0.54 [0.4–0.74]). RT-GS and ChemoRT-GS had significant interactions with treatment on multivariate analysis (P = 0.0009 and 0.02, respectively), indicating that they are bona fide predictive biomarkers. Conclusions Using a novel PDX-driven methodology, we developed and validated 3 platform-independent molecular signatures that predict benefit from standard of care therapies for glioblastoma. These signatures may be useful to personalize glioblastoma treatment in the clinic and this approach may be a generalizable method to identify predictive biomarkers without resource-intensive randomized trials.