Sarkaria, Jann N.

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<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

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.

2031 Background: The mammalian target of rapamycin (mTOR) functions within the PI3K/Akt pathway as a critical modulator of cell survival. Preclinical studies in GBM indicate that the combination of mTOR inhibitors, such as everolimus (RAD001), with either radiation therapy (RT) or temozolomide (TMZ) provide increased tumor cell killing. Methods: Newly diagnosed GBM pts were eligible for the study. RAD001 was dosed orally at 70 mg/week weekly, starting 1 week prior to RT/TMZ, and continued throughout RT/TMZ, adjuvant TMZ and then until progression. This was a single arm phase II design powered to detect a true overall survival at 12 months (OS12) of 73% (vs 58% in historical controls). Secondary endpoints were toxicity, response rate, and time to progression (TTP). A subgroup of patients with measurable residual disease were eligible for the PET imaging component of the study, consisting of an 18FLT-PET/CT scan performed before and after the initial two doses of RAD001 but before the first dose of RT or TMZ. Results: 103 patients were accrued to phase II of which 100 were evaluable. The median age was 60.5 years (23-81), median ECOG PS was 1, 46 patients had GTR, 33 STR, and the remainder had biopsy at diagnosis. Treatment tolerance was acceptable: 17% patient had at least one grade 3 hematologic toxicity; 14% had at least one grade 4 hematologic toxicity, 42% had at least one grade 3 non-heme toxicity, while 12% had at least one grade 4 non-heme toxicity. No increased incidence of infectious complications was observed and there were no treatment related deaths. Median PFS was 5.3 months (1.3-21.4), with 22 patients progression free. Mature OS data will be available at the meeting. MGMT methylation status analysis is ongoing. Of the pts who had evaluable FLT-PET data, three of eight (37.5%) had a drop in SUVmax >25% after two treatments of RAD001. Conclusions: RAD001 with standard of care chemoradiation had moderate toxicity. Serial 18FLT-PET was feasible for evaluating drug-induced changes in tumor proliferation following RAD001. Final outcome data and association of MGMT status with outcome will be reported at the meeting.

2046 Background: Vorinostat (VOR) is a histone deacetylase inhibitor that represents a rational targeted agent in GBM treatment. Given its single-agent activity in recurrent disease (Galanis,et al, 2009) and radiosensitizing properties, this phase I/II trial was designed to test the addition of VOR to standard chemoradiation in newly diagnosed GBM patients (pts): the phase I portion of the trial is the focus of this report. Methods: A standard cohorts of three design was used to assess the safety of VOR in combination with RT and concomitant TMZ and establish the phase II dose of the combination. VOR was given orally days 1 - 5 every wk beginning with the first dose of RT (total dose 60 Gy) and (75mg/m²/day). Following a 4 - 6 week rest, pts received up to 12 cycles of standard adjuvant TMZ in combination with VOR on days 1-7 and 15 – 21 of each cycle; dose was based on NABTT trial 04-03 (Lee, et al, 2012). Results: The phase I component is complete with 15 pts, 12 pts at dose level 0 (VOR 300 mg/day days 1 - 5, weekly x 6 wks), and 3 pts at dose level 1 (VOR 400 mg/day, days 1 – 5 weekly x 6 wks) in combination with RT/TMZ. Dose limiting toxicity (DLT) in dose level 1 included grade 3 fatigue in 2 pts, grade 3 wound dehiscence in 1 pt, and grade 4 neutropenia and thrombocytopenia in 1 pt. In dose level 0, 1/6 pts had DLT (gr 3 dyspnea). An MTD expansion cohort of 6 additional patients was added to dose level 0; one patient experienced grade 4 thrombocytopenia and grade 3 fatigue, and 1 patient experienced grade 3 febrile neutropenia. In the 12 pts treated in the phase II dose, most common toxicities were hematologic, including lymphopenia (gr 3/4 in 66.7%), thrombocytopenia (gr 3 in 16.7%, gr 4 in 16.7%) and neutropenia (gr 3 in 16.7%, gr 4 in 8.3%). Grade 3 fatigue was observed in 8.3% of the pts. Conclusions: MTD for VOR in combination with TMZ/RT in newly diagnosed GBM patients is 300 mg/d, days 1 - 5 weekly during RT. Toxicity was primarily hematologic. This dose was used in the recently completed phase II trial of the combination (110 pts). RNA expression profiling in patient samples is in process to assess vorinostat responsive signatures observed in preclinical models. Clinical trial information: NCT00731731.

2014 Background: PFS6 is low (2.3%) and med OS brief (4 mos) for recurrent GBM pts who progress after prior RT/TMZ and bevacizumab (Bev), and then receive non-Bev containing regimens (Reardon et al, Br J Canc 2012). Following VEGFi exposure, tumor cell resistance potentially may result from activation of Ras/Raf/MEK/ERK (MAPK) and PI3K/Akt/mTOR signaling pathways. Temsirolimus (mTOR inhibitor) and sorafenib (Raf, PDGFR, VEGFR inhibitor) have previously shown limited single agent activity in GBM. Methods: Recurrent GBM pts who showed radiographic progression despite prior surgery, RT + temozolomide, and VEGFi (initially or at recurrence) received sorafenib 200 mg PO bid and temsirolimus 20 mg IV weekly (the NCCTG N0572 phase I MTD) until progression. All pts had received < 2 prior chemo regimens. A two-stage design was used, with 90% power to detect an increase in PFS6 from 8% to 23%, requiring 41 evaluable pts. The primary endpoint was PFS6; secondary endpoints included OS, TTP, and ORR. Toxicity (TOX) was assessed using CTC ver 3.0. Results: 44 evaluable pts were accrued. Four of the first 40 (10%) reached PFS6, but this did not meet the pre-study threshold for success. Median TTP was 1.8 mos. (40 events, 95% CI: 1.5 – 2.3); median OS was 5 mos. (36 events, 95% CI: 6.6 – 3.1), and no objective responses were reported. Eight pts are alive; 4 have not progressed (median F/U 3.1 mos.; max, 12.2 mos.). Grade 3+ non-heme AE were observed in 51% (23/45; fatigue-5, hypophosphatemia-7, hypercholesterolemia-5, hypertriglyceridemia-5). One grade 4 hematologic AE (thrombocytopenia) occurred. 13.3% (6/45 pts) went off due to TOX, and 71.1% (32/45) due to disease progression. Conclusions: Sorafenib plus temsirolimus was tolerable, but the primary endpoint threshold for success (PFS6) was not met in post-VEGFi recurrent GBM patients. Nevertheless, PFS6 (10%) and OS (5 mos.) compared favorably to contemporary series of Bev-refractory, recurrent GBM pts who are subsequently treated with a non-Bev containing regimen. Supported by NCI CA-25224. Clinical trial information: NCT00329719.