Citations

Abstract Radio-resistant properties of melanomas undermine benefit of radiation therapy (RT). DNA-dependent protein kinase (DNA-PKcs) is essential for the non-homologous end joining (NHEJ) mediated repair DNA double-strand break (DSB). We evaluated radio-sensitizing effects of M3814, a selective oral inhibitor of DNA-PKcs, in patient-derived xenografts (PDXs) of melanoma brain metastases. M3814 (≥300 nM) inhibited RT-induced (5 Gy) auto-phosphorylation of serine-2056 of DNA-PKcs in primary cultures of M12, M15 and M27 PDX lines. Interestingly, inhibition of RT-induced DNA-PKcs by M3814 coincided with increased KAP1 phosphorylation, a DNA damage signaling regulated via ATM. As a measure of lasting DNA damage, persistent γH2AX foci were observed in 28% cells 24 hours after co-treatment with M3814 and RT as compared to RT controls, where only 12% cells had persistent γH2AX foci. In a clonogenic survival assay, M3841 augmented RT-induced killing of M12 cells in a dose dependent manner. However, a minimal 16 h exposure with ~300 nM M3814 was most effective treatment. Pharmacokinetics (PK) after single oral dose of 20 mg/kg M3814, showed considerably short half-life (~2.44 hours) and poor brain distribution in wildtype (WT) FBV mice (Kpuu, 0.027). Suggesting liability to efflux transporters, brain distribution of M3814 in TKO mice (triple knockouts for efflux transporters Mdr1a, Mdr1b and BCRP1) was ~11 fold higher than WT animals (kpuu, 0.215). Using this preliminary PK data, simulations were performed using simBiology software to define dosing regimen and schedule to maximize drug exposures in brain. Based on this in silico modeling, two regimens- (A: 125 mg/kg twice a day (at 0 and 7 hours), and B: 50 mg/kg dose followed by three additional doses of 35 mg/kg per day at 4 hour intervals), combined with a single fraction RT (3 Gy) delivered 10 min after the first M3814 dose in each regimen, were tested in athymic nude mice carrying M12 flank tumors. In this study, drug levels achieved in brain and plasma (121±95, 1914±1661 nM with regimens A and 79±51, 1205±664 nM with regimen B, respectively at 24 h), exceeded predictions. Consistent with a much higher accumulation of M3814 in flank tumor tissues (1.5 to 3.4 fold higher than plasma), therapy with M3814/RT had robust pharmacodynamics effects on DNA damage signaling both at 6 and 24 hours of treatment as compared to RT alone. In summary, M3814 is a promising radio-sensitizer in melanoma brain metastases. Further studies will determine efficacy and pharmacodynamics effects of M3814/RT regimens in relevant orthotopic models of brain metastases and address potential concerns of normal tissue toxicity. Citation Format: Jianxiong Ji, Emily J. Smith, Paige Sarkaria, Ann C. Mladek, Surabhi Talele, Katelyn Swanson, Afroz S. Mohammad, Lihong He, Zeng Hu, Katrina K. Bakken, Shiv K. Gupta, Danielle M. Burgenske, Gaspar J. Kitange, William F. Elmquist, Jann N. Sarkaria. Inhibition of DNA-PKcs by M3814 potentiates efficacy of ionizing radiation in patient derived xenografts of melanoma brain metastases [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 1381.

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.

2019 Background: The mammalian target of rapamycin (mTOR) functions within the PI3K/Akt signaling pathway as a critical modulator of cell survival. We previously demonstrated significant synergy of the mTOR inhibitor sirolimus with RT in glioma xenografts. Methods: The standard cohorts of 3 design was applied with dose escalation of weekly IV CCI-779 in combination with standard TMZ/RT. CCI-779 was given during both RT (60 Gy)/TMZ (75 mg/m2 daily) and adjuvant TMZ (200 mg/m2 daily x 5 every 28 days). Results: A total of 17 patients were enrolled. CCI-779 therapy during RT/TMZ was well tolerated at dose level 0 (25 mg CCI-779, n = 3) and dose level 1 (50 mg CCI-779, n = 6) with 1 of 9 patients experiencing a DLT (Gr 3 fatigue). Dose level 2 (75 mg CCI-779) exceeded the maximally tolerated dose with two of six patients with DLT (<75% CCI-779 delivered; Gr 4 dyspnea). Despite reasonable tolerance during RT/TMZ, the overall regimen was associated with a high rate of infection associated with lymphopenia. After a patient died from PCP on cycle 2 of adjuvant TMZ/CCI-779, antibiotic prophylaxis was mandated. Two other patients died of gram-negative sepsis despite prophylaxis: one after their first dose of CCI-779 and one during cycle 4 of adjuvant therapy. In contrast to our 18% grade 5 infection rate, only 4% grade 3 (no grade 4/5) infections were observed in 26 other CTEP-sponsored clinical trials involving 1,006 patients treated with CCI-779. Results from translational studies evaluating the effects of treatment on immune function will be presented. Further infections were avoided on this trial after CCI-779 therapy was limited to RT/TMZ. Conclusions: Although CCI-779 in combination with RT/TMZ was well-tolerated, adjuvant therapy with TMZ/CCI-779 was associated with an increased risk of opportunistic infections. [Table: see text]

Abstract New approaches are needed to overcome intrinsic therapy resistance in glioblastoma (GBM). Because GBMs exhibit sexual dimorphism and are reported to express steroid hormone receptors, we reasoned that signaling through the androgen receptor (AR) could mediate therapy resistance in GBM, much as it does in AR-positive prostate and breast cancers. We found that nearly half of GBM cell lines, patient-derived xenografts (PDX), and human tumors expressed AR at the transcript and protein level—with expression levels overlapping those of primary prostate cancer. Analysis of gene expression datasets also revealed that AR expression is higher in GBM patient samples than normal brain tissue. Multiple clinical-grade antiandrogens slowed the growth of and radiosensitized AR-positive GBM cell lines and PDXs in vitro and in vivo. Antiandrogens blocked the ability of AR-positive GBM PDXs to engage adaptive transcriptional programs following radiation and slowed the repair of radiation-induced DNA damage. These results suggest that combining blood–brain barrier permeable antiandrogens with radiation may have promise for patients with AR-positive GBMs.

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&lt;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&lt;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&lt;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

2072 Background: Human glioma stem cells and xenograft lines are common translational models in neuro-oncology but it has not been established if they are genetically and phenotypically comparable. This study aimed to determine if human glioma xenografts and stem cell-derived tumors had similar genotypes. Methods: Matched glioma stem cell cultures and subcutaneous xenograft lines were generated from four human glioblastoma specimens (BT114, BT116, BT120, BT132). Comparison was made between subcutaneous stem cell-derived tumors (flank) and xenografts established in nude mice. Copy number variation (CNV) and gene expression microarray studies were performed. Results: Various differences in copy number and gene expression were seen. Observed CNVs included regions within EGFR, myc, and p16 (INK). For example, EGFR copy number was two fold higher in xenografts vs. stem cell-derived tumor in one line (BT114). This difference was corroborated by western blot. Other differences included a heat shock protein homolog (DNAJA4), tetraspanin 13, and a p53 family target gene (ISG20L1). Two lines (BT114, BT116) had a greater than two fold increase in DNAJA4 expression in xenografts vs. stem cell-derived tumors (p = 0.04, 0.01). Two cell lines (BT116, BT120) had a two to eight fold increase in tetraspanin 13 expression in xenografts (p = 0.02, 0.05). However, neither copy number nor gene expression variations were consistent across all cell lines. Conclusions: Xenografts and glioma stem cell-derived tumors established from the same patient specimens have distinct genotypes. Further work is needed to establish if these differences are random or represent characteristic changes selected by different in vitro or in vivo pressures. However, these variations raise questions regarding which model is ideal for studying glioma biology, and which ones best replicate glioma characteristics in human patients.

e14507 Background: Serious gastrointestinal injury (SGI) is a rare but documented side effect that can occur independently with agents that affect the vascular endothelial growth factor (VEGF) receptor or with stereotactic body radiotherapy (SBRT). The risk of SGI in patients treated with the combination of SBRT sequentially followed by a VEGF inhibitor (VEGF-i) is not yet quantified. We explored the incidence of SGI in patients treated with SBRT with or without VEGF-i therapy at a single institution. Methods: From May 2008 to August 2011, 76 patients with 84 primary or metastatic intraabdominal lesions underwent SBRT (median dose, 50 Gy in 5 fractions). Twenty of these patients (26%) received a VEGF-i within two years of completing SBRT (bevacizumab, n=14; sorafenib, n=4; pazopanib, n=1; sunitinib, n=1). The most common site treated with SBRT was the liver (n=43, 57%), and the most common histology was colorectal adenocarcinoma (n=18, 24%). Other common histologies included melanoma of the skin (n=14, 18%) and non-small cell lung cancer (n=10, 13%). The incidence of SGI (CTCAE v4.0 grade 3 to 5 ulceration or perforation) after SBRT was obtained, and the relationship between SGI and VEGF-i was examined. Other factors that could potentially contribute to SGI, such as radiotherapy dose to bowel, were also analyzed. Results: Seven patients (9%) experienced SGI at a median 4.6 months (range, 3-17 months) after SBRT. All seven had received a VEGF-i prior to SGI and within 13 months of completing SBRT, and five received the VEGF-i within three months of SBRT. The six-month estimate of SGI in patients receiving a VEGF-i within three months of SBRT was 38%. No SGI was noted in the 63 patients not receiving VEGF-i therapy after SBRT. The Log-Rank test showed a significant correlation between SGI and VEGF-i therapy within three months of SBRT (p=0.0006) but not between SGI and maximum radiotherapy bowel dose (p=0.20). Conclusions: The combination of SBRT and VEGF-i therapy results in a much higher risk of SGI than would be expected with either treatment given independently. Local therapies other than SBRT should be considered if a patient is likely to be treated with a VEGF-i in the future.