Bakken, Katrina K.

Abstract There is an unmet need to identify novel targets that can sensitize temozolomide (TMZ) or prevent resistance in GBM. We have demonstrated that retinoblastoma binding protein 4 (RBBP4) interacts with p300 to modulate expression of genes involved in homologous recombination (HR), including RAD51. In vitro, RBBP4- or p300-shRNA significantly sensitized TMZ in patient derived xenograft (PDX) GBM43 cells (relative fluorescence for 100µM TMZ treated control shNT cells was 0.89 ± 0.1 vs 0.47± 0.09 and 0.39 ± 0.01 for shRBBP4 and shp300, respectively (p< 0.01)). TMZ sensitization increased DNA damage signaling through phosphorylation of KAP1 (p-KAP1) and p-CHK1. Moreover, RBBP4- or p300-shRNA delayed the repair of TMZ-induced DSBs evidenced by persistent gH2AX. Silencing RBBP4 or p300 reduced acetylation of lysine 27 of histone H3 (H3K27Ac) within promoters of HR genes regulated by RBBP4/p300 complex. Thus, RBBP4/p300 complex controls gene expression through p300-mediated histone acetyltransferase (HAT) activity, suggesting that p300 inhibitors could sensitize GBM to TMZ. Accordingly, CBP/p300 inhibitor CPI1612 significantly suppressed H3K27Ac and HR repair genes, including RAD51. Moreover, CPI1612 sensitized TMZ in GBM43 (synergy score = 258), and TMZ/CPI1612 significantly suppressed growth of GBM39 PDX cells compared with either drug alone (confluence (%) was 92 ± 1.0 (DMSO), 76.5 ± 4.6 (10 µM TMZ), 62 ± 3.4 (10 nM CPI1612) and 21.9 ± 3.2 (TMZ 10 µM/CPI-CPI1612 10 nM). CPI1612 enhanced TMZ-induced DSBs with increased damage signaling through p- KAP1 and persistent gH2AX. Pharmacodynamics studies in GBM39 orthotopic mice models revealed that oral CPI1612 penetrates the brain and accumulate in tumor regions and suppresses H3K27Ac without significant weight loss in mice that received placebo, TMZ, CPI1612 alone or combined TMZ/CPI-1612, demonstrating good animal tolerability. Collectively, these findings are encouraging that CBP/p300 inhibition by the brain penetrant CPI-1612 is a potential strategy for enhancing the efficacy of TMZ in GBM.

Abstract The efficacy of standard radiation therapy (RT) in glioblastoma (GBM) is limited, and there is a strong rationale to develop effective radiosensitizing agents. ATM is a key regulator of DNA damage induced by RT, and small molecule ATM inhibitors have shown radio-sensitizing effects in cancer cells, and may improve radio-sensitivity in GBM. Here, we evaluated the brain penetrant ATM inhibitor AZD1390 in combination with RT in GBM cell lines and patient derived xenografts (PDXs). AZD1390 (30 nM and higher) suppressed ionizing radiation-(5 Gy, IR) induced phosphorylation of ATM-Serine1981 and downstream signaling proteins Kap1, Chk2 and H2AX in GBM established cell lines (U251 and U87) and PDX lines (GBM12, GBM39 and GBM43). In cell cycle analysis, 30nM AZD1390 enhanced the IR induced G2/M arrest in U251 (80.6% with AZD1390/IR vs. 64.6% with IR alone, p= 0.01) and PDX lines GBM43 (61.9% vs. 25.7%, p= 0.01) and GBM39 (40.9% vs. 25.4%, p= 0.01). Moreover, in a U251 clonogenic survival assay, AZD1390 sensitized cells to IR-(5 Gy) induced killing (0.24% survival with AZD1390/RT vs. 2.3% with IR alone, P=0.01). For in vivo radiation combination studies, mice with established orthotopic tumors were irradiated to the cerebrum through opposed laterals with a 225 kVp stereotactic animal irradiator. While the hard palate and upper tongue were in the radiation target, the oropharynx and hypopharynx were spared with this radiation set up. A pilot study of RT combined with AZD1390 was evaluated in mice with established U251 orthotopic tumors using cohorts of 5 mice per arm. AZD1390 was given concurrently with conventionally fractionated radiation (2 Gy x 5 fractions) at a dose of 20 mg/kg PO delivered either daily or twice daily, with or without the efflux inhibitor elacridar. RT alone was well tolerated with minimal weight loss but was ineffective (median survival 30 days vs. 33 days in control group, p=0.5). In contrast, all of the RT/AZD1390 treatment arms exhibited enhanced but tolerable weight loss and had significant extension in symptom-free survival that currently extends beyond 65 days (median survival no reached). In conclusion, AZD1390 is an effective radio-sensitizer in vitro, and survival benefit from AZD1390 combined with conventionally fractionated RT in U251 models is encouraging. Further evaluation of efficacy across additional PDX models and careful analysis of the pharmacodynamic effects in tumor vs. normal tissues will be important to understand the therapeutic window for this agent in combination with RT. Citation Format: Jiajia Chen, Shiv K. Gupta, Gaspar J. Kitange, Ann C. Mladek, Brett L. Carlson, Lihong He, Zeng Hu, Katrina K. Bakken, Danielle M. Burgenske, Margaret A. Connors, Jann N. Sarkaria. Targeting ATM with AZD1390 for radio-sensitization of glioblastoma patient derived xenografts [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 6506.

Abstract Despite aggressive treatment that involves surgery, radiation and temozolomide therapy, a significant morbidity from glioblastoma (GBM) recurrence highlights the pressing unmet medical need to develop effective novel therapies for GBM. Murine Double Minute 2 (MDM2) is an important regulator of the p53 tumor suppressor, which promotes cell cycle arrest and apoptosis in response to DNA damage. Here we have assessed the efficacy of RG7388, a purported brain penetrant MDM2-inhibitor, alone or combined with radiation therapy (RT) in patient-derived xenograft (PDX) models of GBM. In vitro, RG7388 suppressed viability of GBM PDX short-term stem cell cultures of p53 wildtype lines with MDM2-amplification (GBM46 and 108, IC50 25 and 11 nM, respectively) or without MDM2-amplification (GBM10 and 14, IC50 43.5 and 9.1 nM, respectively). Serum proteins appear to reduce efficacy of RG7388, which is supported by the notion that adding 2.5% bovine serum albumin to a serum free stem cell cultures increased the IC50 by 3 to 5 fold. At the molecular level, both RG7388 and RT could induce p53 signaling in GBM10, but a more robust induction was observed with the combination. In athymic nude mice, RG7388 readily distributes into normal brain as measured by LCMS-MS at 1 hour after five daily oral doses (70 mg/kg) the average RG7388 concentrations in brain and plasma were 2167 ng/g and 4423 ng/ml, respectively. In an initial efficacy study using flank tumors established from GBM10, one week of dosing at 70 mg/kg/day RG7388 alone was ineffective, while combining RG7388 with focal radiation (4 daily doses of 5 Gy each) had a moderate 15 day delay in tumor progression. However, in an MDM2-amplified GBM108 line with a relatively intact blood-brain barrier, one week of daily RG7388 dosing, either alone or in combination with RT was remarkably effective (median survival 71 days with RG7388 alone vs. 29 days with vehicle, p<0.0001; and 262 days for RG7388/RT vs. 74 days with RT, p=0.017). Taken together, RG7388 alone or in combination with RT is highly effective in an MDM2-amplified pre-clinical model of GBM. Determining impact of serum protein binding on drug pharmacokinetics and further evaluation of pharmacodynamic effects and orthotopic therapy in additional p53 wildtype PDX lines will help to increase our understanding if RG7388/RT therapy can be used in a larger population of GBM tumors. Citation Format: Shiv K. Gupta, Ann C. Mladek, Surabhi Telele, Afroz S. Mohammad, Lihong He, Zeng Hu, Katrina K. Bakken, Danielle M. Burgenske, Brett L. Carlson, William F. Elmquist, Jann N. Sarkaria. Brain penetrant MDM2 inhibitor RG7388 extends survival benefit of radiation treatment in select glioblastoma patient-derived xenograft models [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 6276.

Abstract Robust function of the p53 tumor suppressor pathway is critical when treating with DNA-damage inducing agents such as radiation therapy (RT), which is a key component of standard care for GBM. MDM2 is an important negative regulator of p53 stability and MDM2 is amplified in approximately 14% of GBM. Based on the concept that suppression of MDM2 can reactivate p53 function and potentially have single agent or combinatorial effects, multiple MDM2 inhibitors have been developed. Here we report in vitro and in vivo efficacy and pharmacodynamic (PD) effects of a BBB-penetrant MDM2-p53 antagonist, BI-MDM2, in GBM patient-derived xenograft (PDX) models. In vitro studies in p53 wild-type (WT) lines with or without MDM2 amplification demonstrate IC50 values in cell viability assays of 2-12 nM in serum-free culture and 5-35 nM in serum-containing culture after seven days of treatment. In vivo studies were performed in p53 WT lines: the MDM2-amplified GBM108, and the non-amplified GBM14, grown as orthotopic tumors in nude mice. Weekly oral treatment at 2 mg/kg of BI-MDM2 doubled median survival (placebo, 28 days (d) vs 2 mg/kg BI-MDM2, 57 d. p<0.0001) while weekly dosing at 10 mg/kg extended the median over 5 fold (176 d. p<0.0001) with half of the mice still living at 223 days post-inoculation. To assess the PD properties of this compound, mice bearing orthotopic GBM108 were treated three weeks after inoculation with a single dose of 2 or 10 mg/kg BI-MDM2. Normal brain, tumor, and plasma were collected at 24 and 48 hours (h) after dosing and downstream p53 transcriptional targets, p21 and PUMA were evaluated by qRT-PCR. p21 mRNA relative quantification detection at 24h increased by 1.5-10.5-fold in the 2 mg/kg group of animals compared to vehicle while the 10 mg/kg dose led to an increase by 2-25 fold compared to vehicle. PUMA was minimally affected by the 2 mg/kg dose but increased 1.2-17-fold over vehicle in the 10 mg/kg group at 24h. The observed increases in p21 and PUMA varied little between the 24 and 48h timepoints. In an orthotopic GBM14 PDX efficacy study, BI-MDM2 was combined with 20 Gy RT delivered in 10 fractions over 2 weeks with dosing limited to 2 weeks of therapy or until mice reached a moribund state. While two doses of 10 mg/kg BI-MDM2 alone had a modest effect on survival (41 d vs 31 d with placebo; p=0.002), continued dosing until moribund further extended median survival (82 d with BI-MDM2; p=0.001). The combination of two doses of BI-MDM2 with 2 weeks of RT extended survival as compared to RT alone (107 d vs. 69 d, respectively; p=0.019), while with extended drug dosing combined with 2 weeks of RT, median survival has not yet been reached (>125 d). Taken together, these results suggest that BI-MDM2 is a promising therapeutic agent that may provide significant anti-tumor efficacy either alone or in combination with RT in both MDM2 amplified and non-amplified p53 WT patients. Citation Format: Ann C. Mladek Tuma, Shiv Gupta, Surabhi Talele, Afroz Shareef Mohammad, Katrina K. Bakken, Helen He, Zeng Hu, Margaret A. Connors, Danielle M. Burgenske, Brett L. Carlson, William F. Elmquist, Ulrike Weyer-Czernilofsky, Jann N. Sarkaria. A promising blood-brain-barrier penetrant MDM2-p53 antagonist, BI-MDM2, increases survival in orthotopic, glioblastoma patient-derived xenograft models [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 4190.

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 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

Abstract INTRODUCTION Neuroimaging dogma for glioblastoma asserts that hyperintensity on T1Gd MRI reveals the bulk of the tumor, while T2/FLAIR signal indicates edema. However, it is unclear whether this edema results from immune response or increased tumor cells. Further, one significant driver of the known sex differences in glioblastoma may be differences in immune response, due to the X-linkage of many immune genes. Based on this, we hypothesized that assumptions regarding tumor cellularity in T2/FLAIR images should be tailored to the biological sex of the patient. METHODS Using a retrospective cohort of 18 primary glioblastoma patients receiving multiple image-localized biopsies (82 total) and standard MRI, we assessed: distance of biopsy from T1Gd and T2 areas; a pathologist’s score of percent tumor cell density; and an imaging-based invasion metric, D/ρ. This metric is derived from the biomathematical Proliferation-Invasion model of glioma growth, which features two parameters, net growth rate (ρ) and net invasion rate (D). Their ratio D/ρ is related to degree of invasion, and is estimated from volumetric measurements of MRI abnormalities. Additionally, 25 patient-derived xenograft models implanted in females were grown until moribund, at which point brains were excised and stained for DAPI (to show all cells) and Lamin (to highlight tumor cells). Image processing of lamin-stained sections defines contours of intensity correlating with cell density. RESULTS Outside both the T1Gd and T2 region, male patient biopsies had higher tumor cell densities than females. Males also tended to have higher invasion metrics. Although each set derived from different patients, preclinical metrics of invasion were positively correlated with clinical invasion in females but negatively correlated in males. CONCLUSION Our preliminary finding that cell distribution patterns correlate with imaging metrics differently between the sexes supports the hypothesis that the degree of tumor cell density represented on certain MRI sequences may be sex-specific.