Elmquist, William F.

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

9048 Background: Brain metastases continue to be the major cause of morbidity and mortality in patients with metastatic melanoma. The impact of BRAF mutations and effectiveness of BRAF inhibitors on the brain metastases in these patients is lacking. Methods: Preclinical studies were conducted to assessthe steady-state brain and plasma distribution of vemurafenib, a BRAF inhibitor in FVB wild-type and Mdr1a/b-/-Bcrp1-/- mice deficient in the drug efflux transporters, p-glycoprotein (P-gp) and breast cancer resistant protein (BCRP). A retrospective analysis of patients with metastatic melanoma treated at University of Minnesota from August 2011 to December 2012 was conducted. A similar analysis of cases treated at Mayo Clinic is underway. Results: The preclinical studies in mice show that both P-gp and BCRP play a significant role in limiting the brain distribution of vemurafenib. Retrospective analysis was performed on 57 patients with Stage IIIc /IV cutaneous and mucosal melanoma. Patients with BRAF mutation had a higher incidence rate of brain metastases compared to patients without BRAF mutation, although it was not statistically significant (Incidence ratio=1.56; 95% CI=0.70-3.48; P=0.27). Vemurafenib neither reduced the incidence of brain metastases (Incidence ratio = 0.89; 95% CI: 0.30-2.60; P=0.83) nor made significant difference in overall survival. It was observed that treatment with BRAF inhibitor led to improvement in extracranial disease but did not affect progression of intracranial disease. Conclusions: In concordance with preclinical data which indicates that P-gp and BCRP play a significant role in limiting the brain distribution of vemurafenib, the retrospective analysis shows that there is improvement in extracranial disease but progression in intracranial disease with treatment with BRAF inhibitor in patients with metastatic malignant melanoma with BRAF mutation. Development of BRAF inhibitors that are not substrates for P-gp and BCRP or concomitant use of P-gp and BCRP inhibitors with vemurafenib, may be needed in order to control or prevent intracranial disease in these patients. Further analysis to improve statistical power of our observation is underway.

Abstract Glioblastoma (GBM) is increasingly recognized as a disease involving dysfunctional cellular metabolism. GBMs are known to be complex heterogeneous systems containing multiple distinct cell populations and are supported by an aberrant network of blood vessels. A better understanding of GBM metabolism, its variation with respect to the tumor microenvironment, and resulting regional changes in chemical composition is required. This may shed light on the observed heterogeneous drug distribution, which cannot be fully described by limited or uneven disruption of the blood–brain barrier. In this work, we used mass spectrometry imaging (MSI) to map metabolites and lipids in patient-derived xenograft models of GBM. A data analysis workflow revealed that distinctive spectral signatures were detected from different regions of the intracranial tumor model. A series of long-chain acylcarnitines were identified and detected with increased intensity at the tumor edge. A 3D MSI dataset demonstrated that these molecules were observed throughout the entire tumor/normal interface and were not confined to a single plane. mRNA sequencing demonstrated that hallmark genes related to fatty acid metabolism were highly expressed in samples with higher acylcarnitine content. These data suggest that cells in the core and the edge of the tumor undergo different fatty acid metabolism, resulting in different chemical environments within the tumor. This may influence drug distribution through changes in tissue drug affinity or transport and constitute an important consideration for therapeutic strategies in the treatment of GBM. Significance: GBM tumors exhibit a metabolic gradient that should be taken into consideration when designing therapeutic strategies for treatment. See related commentary by Tan and Weljie, p. 1231

Abstract Glioblastoma, the most common and lethal of brain tumors, is both highly invasive and proliferative. This allows tumor cells to infiltrate into regions of the brain with an intact blood brain barrier and be protected from effective therapeutics. Thus, an ideal glioblastoma therapy needs to target cellular components that drive both invasion and proliferation, with inhibitors that penetrate the blood brain barrier. The mitotic kinesin KIF11 meets these criteria and it can be targeted with ispinesib, a highly specific small molecule inhibitor. However, to be effective, ispinesib needs to cross the blood brain barrier and be retained within brain long enough to target glioblastoma cells when they are vulnerable, during mitosis.. We have examined the factors that affect distribution of ispinesib to both brain and glioblastoma. We find that delivery of ispinesib is limited by P-gp and Bcrp-mediated drug efflux at the blood brain barrier. Consequently, ispinesib levels are significantly lower in the infiltrative tumor margin relative to the tumor core, where the blood brain barrier is defective. We also show that elacridar—an inhibitor of the P-gp and Brcp efflux transporters—enhances delivery of ispinesib, and that co-administration of ispinesib with elacridar markedly slows tumor proliferation and prolongs survival in a mouse model of this disease. These results demonstrate the feasibility and efficacy of combining a potentially ideal therapeutic with a compound that enhances brain retention of this therapeutic, and provides support for utilizing this approach in clinical investigations of KIF11 inhibitors in GBM.

Abstract OBJECTIVES Dogma suggests that for brain tumors, regions of enhancement on T1-weighted gadolinium contrast enhanced (T1Gd) magnetic resonance imaging (MRI) correlate with intravenously delivered drug distribution as enhancement indicates a compromised blood-brain barrier (BBB). However, poor response to intravenous therapies highlights the importance of the diffuse disease beyond enhancing regions. This study investigated whether imaging features can provide an accurate prediction of drug distribution. METHODS Eight brain tumor patients (7 gliomas and 1 metastatic adenocarcinoma) were included in this Phase 0 trial. Presurgery T1-weighted, T1Gd, T2-weighted gadolinium contrast enhanced (T2Gd), and T2-weighted Fluid Attenuated Inversion Recovery (T2FLAIR) MRIs were acquired. All images underwent bias correction using the N4 algorithm, standardization of intensities, and registration. Prior to incision, patients received both an antibiotic cefazolin (6% BBB penetrance) and levetiracetam (80% BBB penetrance), an anti-seizure drug. Subsequently, multiple blood samples and image-guided biopsies were taken and analyzed for drug concentration using liquid chromatography mass spectrometry. Biopsy drug levels are reported as Brain-Plasma Ratio (BPR), the ratio of biopsy concentration relative to plasma concentration. Mean image intensity was extracted from an 8x8 mm window surrounding each biopsy location. Regression analysis was performed to determine which combination of image types were linearly predictive of BPR for both drugs. Correlations were also analyzed according to the biopsy location radiographic appearance. RESULTS Regression analysis revealed that T2Gd intensity was linearly predictive of cefazolin BPR and FLAIR intensity was linearly predictive of levetiracetam BPR (p=0.009 and 0.041, respectively). Grouping samples according the the radiographic appearance revealed that levetiracetam BPR had a similar pattern of values to that of FLAIR intensities and cefazolin BPR had a similar pattern to T2, further supporting the regression analysis results. CONCLUSIONS Local concentrations of drug may be related to T2-weighted signals (T2Gd and T2FLAIR) rather than the gadolinium distribution on T1Gd images.

Abstract Glioblastoma (GBM) is an aggressive and infiltrative primary brain tumor with a median survival of 14.6 months following the current treatment strategy of radiation and chemotherapy. Therefore, there is a need to develop strategies to enhance the efficacy of chemo-radiation treatments for GBM. DNA damage response signaling pathways play a critical role in DNA repair and cell survival following radiation therapy and the inhibition of these pathways could augment the cytotoxicity associated with radiation providing a radiosensitizing effect. Ataxia Telangiectasia and Rad3-Related Protein (ATR) is a key regulator of the DNA damage response network and VX-970 is the first potent and selective inhibitor of ATR to enter clinical trials. Preliminary in vitro studies from our lab to determine a dose dependent effect of VX-970 in combination with a radiation dose of 5 Gy on the cell survival indicated that administration of radiation led to an enhancement in the cell death with an increasing dose of VX-970 in the U251 human GBM cell line. We evaluated the BBB penetration of VX-970 and studied the role of efflux transporters on the brain exposure of VX-970 in preparation for efficacy studies in PDX models of GBM. Brain distribution studies were performed in wild-type and Mdr1a/b-/- Bcrp1-/- (triple knockout) FVB mice (n=4) following intravenous administration of 20 mg/kg VX-970. Plasma and brain samples were collected at 7 time points post dosing and were analyzed using LC-MS. The brain-to-plasma (B/P) ratio in transporter (Pgp and Bcrp) knockout mice was 17.5 as opposed to 0.8 in transporter intact wild-type mice. This 22-fold increase in the B/P ratio in the triple knockout mice indicates that Pgp and/or Bcrp play a significant role in the efflux of VX-970 from the brain thereby limiting its brain penetration. Oral administration of VX-970 at a dose of 20mg/kg in transporter intact wild-type mice indicated an oral bioavailability of 38%. Steady state brain distribution studies were also performed in wild-type and triple knockout FVB mice following intraperitoneal implantation of the Alzet osmotic pumps to release VX-970 at a rate of 10ul/hr for 48 hours. We observed that the B/P ratio in transporter (Pgp and Bcrp) knockout mice was 12.6 as opposed to 0.12 in transporter intact wild-type mice We are in the process of conducting additional studies in Pgp and Bcrp single knockout mice to determine a more precise status of the role of efflux transporters in the brain delivery of VX-970. The free fraction of VX-970 was found to be 6.1% in plasma indicating that it has a relatively high unbound fraction as compared to other chemotherapeutics for brain delivery. We are in the process of determining free fraction in the brain and relating the concentration of free drug to a dose range associated with effective radiosensitization through efficacy studies in the PDX models of GBM. This PK-PD relationship will help guide future clinical trials. Citation Format: Surabhi Talele, Afroz Mohammad, Minjee Kim, Danielle Burgenske, Ann C. Mladek Tuma, Jann N. Sarkaria, William F. Elmquist. CNS delivery of VX-970: A selective ATR inhibitor for radiosensitization in GBM [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 4858.

Abstract Background: Bioluminescence imaging (BLI) of firefly luciferase expressing cells is widely used for analysis of growth, metastasis and drug response in tumor xenografts. D-luciferin, a commonly used luciferase substrate, has limited brain distribution that may undermine BLI for intracranial xenografts. The synthetic luciferin cyclic alkylamino-luciferin (CycLuc1) is a purported brain penetrant luciferase substrate not yet tested in intracranial xenografts. We compared BLI with CycLuc1 and D-luciferin in a glioblastoma (GBM) xenograft model. Methods: GBM6 cells were lentivirally transduced to express LUC2=tdTomato fusion protein. In vivo BLI were performed in orthotopic and heterotopic xenografts established in athymic nude mice. Concentrations of D-luciferin and CycLuc1 were measured by LC-MS/MS. Results: In a dose-response analysis, CycLuc1 at 5 mg/kg provided robust and reproducible imaging; however, an increased photon flux was observed with 10 mg/kg, while escalation to 15 or 20 mg/kg provided marginal additional gains in photon flux. In comparison of CycLuc1 (5 or 25 mg/kg) and D-luciferin (30 or 150 mg/kg, respectively) with serial, crossover BLI at both dose levels, CycLuc1 resulted in less variability and significantly higher photon flux as compared to D-luciferin in GBM6 intracranial xenografts (15 days after implantation). On average BLI with 25 mg/kg CycLuc1 resulted in ~8 fold greater photon flux from these early-stage xenografts as compared to 150 mg/kg D-luciferin (2.9±0.6x106 vs. 3.3±2.8x105 p/sec/cm2, p<0.001). Interestingly, bioluminescence emission with CycLuc1 and D-luciferin was comparable in xenografts imaged 28 days after implantation. In samples harvested after last imaging session, there was no significant difference in substrate distribution in intracranial tumors (tumor to plasma ratios for CycLuc1 and D-luciferin were 0.012 ± 0.020 and 0.012 ± 0.015, respectively; p=0.89). These results suggest that intracranial tumors at early stage may comprise relatively intact blood-brain barrier, which is disrupted at later stage. Consistent with notion that suboptimal delivery may interfere with D-luciferin mediated BLI in intracranial xenografts, photon flux in heterotopic tumors with D-luciferin or CycLuc1 was comparable (4.9±4.6x108 vs. 1.3±0.8x108 p/sec/cm2, p=0.10). Conclusions: These findings demonstrate that CycLuc1 may be a superior BLI substrate as compared to D-luciferin for monitoring tumor growth of intracranial GBM xenografts. Citation Format: Shiv K. Gupta, Minjee Kim, Bianca-Maria Marin, Lihong He, Katelyn Swanson, Tuma Tuma, Brett L. Carlson, Mark A. Schroeder, William F. Elmquist, Jann N. Sarkaria. Synthetic luciferin, CycLuc1, improves bioluminescence imaging for intracranial glioblastoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4112.

Abstract Controversy exists surrounding whether heterogeneous disruption of the blood–brain barrier (BBB), as seen in glioblastoma (GBM), leads to adequate drug delivery sufficient for efficacy in GBM. This question is especially important when using potent, targeted agents that have a poor penetration across an intact BBB. Efficacy of the murine double minute-2 (MDM2) inhibitor SAR405838 was tested in patient-derived xenograft (PDX) models of GBM. In vitro efficacy of SAR405838 was evaluated in PDX models with varying MDM2 expression and those with high (GBM108) and low (GBM102) expression were evaluated for flank and orthotopic efficacy. BBB permeability, evaluated using TexasRed-3 kDa dextran, was significantly increased in GBM108 through VEGFA overexpression. Drug delivery, MRI, and orthotopic survival were compared between BBB-intact (GBM108-vector) and BBB-disrupted (GBM108-VEGFA) models. MDM2-amplified PDX lines with high MDM2 expression were sensitive to SAR405838 in comparison with MDM2 control lines in both in vitro and heterotopic models. In contrast with profound efficacy observed in flank xenografts, SAR405838 was ineffective in orthotopic tumors. Although both GBM108-vector and GBM108-VEGFA readily imaged on MRI following gadolinium contrast administration, GBM108-VEGFA tumors had a significantly enhanced drug and gadolinium accumulation, as determined by MALDI-MSI. Enhanced drug delivery in GBM108-VEGFA translated into a marked improvement in orthotopic efficacy. This study clearly shows that limited drug distribution across a partially intact BBB may limit the efficacy of targeted agents in GBM. Brain penetration of targeted agents is a critical consideration in any precision medicine strategy for GBM. Mol Cancer Ther; 17(9); 1893–901. ©2018 AACR.

Abstract Poly ADP-ribose polymerase (PARP) inhibitors, including talazoparib, potentiate temozolomide efficacy in multiple tumor types; however, talazoparib-mediated sensitization has not been evaluated in orthotopic glioblastoma (GBM) models. This study evaluates talazoparib ± temozolomide in clinically relevant GBM models. Talazoparib at 1–3 nmol/L sensitized T98G, U251, and GBM12 cells to temozolomide, and enhanced DNA damage signaling and G2–M arrest in vitro. In vivo cyclical therapy with talazoparib (0.15 mg/kg twice daily) combined with low-dose temozolomide (5 mg/kg daily) was well tolerated. This talazoparib/temozolomide regimen prolonged tumor stasis more than temozolomide alone in heterotopic GBM12 xenografts [median time to endpoint: 76 days versus 50 days temozolomide (P = 0.005), 11 days placebo (P < 0.001)]. However, talazoparib/temozolomide did not accentuate survival beyond that of temozolomide alone in corresponding orthotopic xenografts [median survival 37 vs. 30 days with temozolomide (P = 0.93), 14 days with placebo, P < 0.001]. Average brain and plasma talazoparib concentrations at 2 hours after a single dose (0.15 mg/kg) were 0.49 ± 0.07 ng/g and 25.5±4.1 ng/mL, respectively. The brain/plasma distribution of talazoparib in Bcrp−/− versus wild-type (WT) mice did not differ, whereas the brain/plasma ratio in Mdr1a/b−/− mice was higher than WT mice (0.23 vs. 0.02, P < 0.001). Consistent with the in vivo brain distribution, overexpression of MDR1 decreased talazoparib accumulation in MDCKII cells. These results indicate that talazoparib has significant MDR1 efflux liability that may restrict delivery across the blood–brain barrier, and this may explain the loss of talazoparib-mediated temozolomide sensitization in orthotopic versus heterotopic GBM xenografts. Mol Cancer Ther; 16(12); 2735–46. ©2017 AACR.