Mladek, Ann C.

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.

Abstract Glioblastoma (GBM) represents one of the most aggressive cancer types with the vast majority of patients succumbing to disease within the first five years. This dire prognosis reflects the limited efficacy of our frontline therapies which include radiation therapy and temozolomide (TMZ) chemotherapy. The cellular response to these therapies is critically mediated by DNA damage response signaling networks that are regulated by Ataxia Telangiectasia Mutated (ATM) and Ataxia Telangiectasia And Rad3-Related Protein (ATR). Preliminary studies from our laboratory suggest the ATR inhibitor VX-970 has single agent efficacy in both established and primary GBM cell lines. Moreover, combined treatment with TMZ demonstrated therapeutic responses that, following regression analysis, were deemed synergistic (p<0.001). Validation studies using siRNA confirmed ATR as a central mediator of TMZ sensitivity as knockdown of ATM elicited no added benefit when tested in combination. VX-970 also enhanced radiation effects in neurosphere formation assays to suggest it may also be used as a radiosensitizer. While the specific mechanism responsible for these observations has yet to be determined, VX-970 treatment was found to exert anti-proliferative effects in a dose-dependent manner. This collection of work suggests that ATR inhibition may represent a worthwhile treatment strategy in GBM. Citation Format: Danielle Burgenske, Ann Mladek, Jann Sarkaria. The selective ATR inhibitor VX-970 enhances the therapeutic effects of standards of care in glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B21.

Abstract Clinical data indicate that certain regions within glioblastoma (GBM) have a relatively intact blood-brain barrier (BBB), but controversy surrounds whether associated heterogeneous delivery may limit efficacy for otherwise highly active drugs with poor brain distribution. In this study, the efficacy of molecularly targeted therapies was compared in relevant GBM patient-derived xenograft (PDX) models grown either as heterotopic or orthotopic tumors. The impact of further disrupting BBB integrity in non-responsive orthotopic tumors was evaluated by over-expressing vascular endothelial growth factor (VEGF) and measuring drug distribution by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). The brain:plasma ratio for five targeted agents was determined by LC-MSMS and treatment efficacy was evaluated in target-relevant GBM PDX lines: erlotinib (brain:plasma = 0.02 ± 0.02; efficacy tested in GBM6), palbociclib (brain:plasma = 0.06; efficacy tested in GBM22), AZD1775 (brain:plasma = 0.05; efficacy tested in GBM22), SAR405838 (brain:plasma = 0.01 ± 0.003; efficacy tested in GBM108), and AZD4547 (brain:plasma = 0.045 ± 0.02; efficacy tested in GBM150). Each of these drugs was effective in relevant GBM PDX flank tumor models with a 27% to 218% prolongation in the median time to exceed their pre-specified endpoint compared to placebo treatment (p≤0.02 for each drug). In contrast, none of these drugs were effective in prolonging survival in the same target-relevant orthotopic tumor models (-6% to 8% extension in median survival; p>0.05 for each drug). Consistent with partial and heterogeneous disruption of the BBB, MALDI-MSI of erlotinib, AZD1775 or SAR405838 distribution within orthotopic tumors demonstrated highly heterogeneous drug levels with large regions within each tumor approaching the low drug levels observed within surrounding normal brain. To further evaluate whether heterogenous drug distribution in orthotopic tumors could account for poor treatment efficacy, GBM108 was transduced with lentiviral constructs encoding for VEGF or empty vector (EV). While both GBM108-sublines were readily detectable with gadolinium-enhanced magnetic resonance imaging (MRI), the GBM108-VEGF tumors had a significantly more disrupted BBB, as evidenced by a more uniform and intense distribution of a brain-impenetrant TexasRed-dextran vascular marker. Similarly, the distribution of SAR405838, measured by MALDI-MSI, was markedly higher and more uniform in the GBM108-VEGF tumors when compared to the GBM108-EV tumors. Enhanced delivery of SAR405838 into orthotopic GBM108-VEGF models translated into a marked enhancement in treatment efficacy in comparison to GBM108-EV with a median survival prolongation of 37 vs. 4 days, respectively (p=0.0055). Collectively, these data highlight the importance of testing novel therapeutic agents in orthotopic tumor models and suggest that limited brain penetration for many molecules may significantly limit their efficacy in brain tumors that have a partially intact BBB. In the face of continued failure to develop effective targeted agents for GBM, these in vivo results highlight the importance of re-evaluating the dogma in neuro-oncology that the BBB is fully disrupted in GBM and, therefore, drug delivery across the BBB is not a major factor limiting treatment efficacy. Citation Format: Jann N. Sarkaria, David Calligaris, Daniel Ma, Karen Parrish, Ann C. Mladek, Janice Laramy, Minjee Kim, Shuangling Zhang, Mark Schroeder, Brett L. Carlson, Katrina Bakken, Aaron Johnson, Nathalie Agar, William Elmquist. The critical importance of the blood-brain barrier in modulating the response to otherwise highly effective targeted therapies in patient-derived orthotopic glioblastoma xenografts. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B25.

Abstract Microtubule-targeting agents (MTA) have been employed in the treatment of many cancers for decades. BAL101553 is a highly soluble prodrug of BAL27862, a novel, small molecule, microtubule-depolymerizing agent that induces tumor cell death by activating the ‘spindle assembly checkpoint’. Given intravenously or orally, the drug penetrates the brain and has anti-cancer activity in diverse tumor models refractory to standard MTA or radiotherapy (RT). In this study, BAL101553 was evaluated in orthotopic xenografts from 16 GBM PDX models; 7 of 16 lines demonstrated significant (p<0.01) increases in median survival with BAL101553 versus placebo (range in median survival extension 24-87%). The combination of BAL101553 with conventional therapies for GBM (RT and temozolomide (TMZ) was then evaluated in select lines. In the MGMT methylated GBM12 line, combination of RT with TMZ increased survival compared to placebo (median survival 80 days vs. 23 days, respectively; p<0.001). Extended BAL101553 monotherapy provided a short but significant extension in survival (median survival 31 days, p<0.001), while extended BAL101553 dosing during and after RT/TMZ (median survival 85 days) did not extend survival relative to RT/TMZ alone (p = 0.56). In contrast, in the MGMT unmethylated GBM6 line, combination of RT and extended BAL101553 increased survival (median 90 days, p<0.001) relative to either treatment alone (median survival BAL101553 63 days; RT 69 days) or placebo (46 days). Additionally, the combination of BAL101553 with TMZ (median survival 70 days) was more effective than TMZ alone (median survival 60 days; p = 0.009). Consistent with the unmethylated MGMT status, the TMZ/RT combination (median survival 66 days) was similar to RT alone (p = 0.62), but the combination of extended BAL101553 with RT/TMZ (median survival 101 days; p<0.001 compared to other combination groups) was significantly more effective. To further evaluate whether BAL101553 is a true radiosensitizer, a second GBM6 study was performed. Also here, combination of RT (20Gy, 2wks) with extended BAL101553 dosing (median survival 66 days) significantly extended survival compared to RT alone (median survival 54 days; p = 002). Interestingly, when BAL101553 dosing was limited to 2 weeks with RT, there was no increase in median survival (58 days; p = 0.16). To evaluate effects on tumor repopulation during RT, the efficacy of an extended RT schedule (36 Gy, 6 wks) with or without 6 weeks of BAL101553 was evaluated. In this case, BAL101553 given during the RT schedule (median survival 78 days) extended median survival as compared to RT alone (61 days; p<0.001). Collectively, these data demonstrate that BAL101553 has broad single agent activity across a panel of GBM PDX models and suggests that combination with RT/TMZ therapy may provide additional benefits for survival extension. Citation Format: Ann C. Mladek, Jenny L. Pokorny, Heidi Lane, Felix Bachmann, Mark A. Schroeder, Katrina K. Bakken, Brett L. Carlson, Paul A. Decker, Jeanette E. Eckel-Passow, Jann N. Sarkaria. The novel tubulin-binding ‘tumor checkpoint controller’ BAL101553 has anti-cancer activity alone and in combination treatments across a panel of GBM patient-derived xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4781.