Sani H. Kizilbash

Delineation of MGMT Hypermethylation as a Biomarker for Veliparib-Mediated Temozolomide-Sensitizing Therapy of Glioblastoma

BACKGROUND Sensitizing effects of poly-ADP-ribose polymerase inhibitors have been studied in several preclinical models, but a clear understanding of predictive biomarkers is lacking. In this study, in vivo efficacy of veliparib combined with temozolomide (TMZ) was evaluated in a large panel of glioblastoma multiforme (GBM) patient-derived xenografts (PDX) and potential biomarkers were analyzed. METHODS The efficacy of TMZ alone vs TMZ/veliparib was compared in a panel of 28 GBM PDX lines grown as orthotopic xenografts (8-10 mice per group); all tests of statistical significance were two-sided. DNA damage was analyzed by γH2AX immunostaining and promoter methylation of DNA repair gene O6-methylguanine-DNA-methyltransferase (MGMT) by Clinical Laboratory Improvement Amendments-approved methylation-specific polymerase chain reaction. RESULTS The combination of TMZ/veliparib statistically significantly extended survival of GBM models (P < .05 by log-rank) compared with TMZ alone in five of 20 MGMT-hypermethylated lines (average extension in median survival = 87 days, range = 20-150 days), while the combination was ineffective in six MGMT-unmethylated lines. In the MGMT promoter-hypermethylated GBM12 line (median survival with TMZ+veliparib = 189 days, 95% confidence interval [CI] = 59 to 289 days, vs TMZ alone = 98 days, 95% CI = 49 to 210 days, P = .04), the profound TMZ-sensitizing effect of veliparib was lost when MGMT was overexpressed (median survival with TMZ+veliparib = 36 days, 95% CI = 28 to 38 days, vs TMZ alone = 35 days, 95% CI = 32 to 37 days, P = .87), and a similar association was observed in two nearly isogenic GBM28 sublines with an intact vs deleted MGMT locus. In comparing DNA damage signaling after dosing with veliparib/TMZ or TMZ alone, increased phosphorylation of damage-responsive proteins (KAP1, Chk1, Chk2, and H2AX) was observed only in MGMT promoter-hypermethylated lines. CONCLUSION Veliparib statistically significantly enhances (P < .001) the efficacy of TMZ in tumors with MGMT promoter hypermethylation. Based on these data, MGMT promoter hypermethylation is being used as an eligibility criterion for A071102 (NCT02152982), the phase II/III clinical trial evaluating TMZ/veliparib combination in patients with GBM.

Discordant In Vitro and In Vivo Chemopotentiating Effects of the PARP Inhibitor Veliparib in Temozolomide-Sensitive versus -Resistant Glioblastoma Multiforme Xenografts

Purpose: Effective sensitizing strategies potentially can extend the benefit of temozolomide (TMZ) therapy in patients with glioblastoma (GBM). We previously demonstrated that robust TMZ-sensitizing effects of the [poly (ADP-ribose) polymerase] (PARP) inhibitor veliparib (ABT-888) are restricted to TMZ-sensitive GBM xenografts. The focus of this study is to provide an understanding for the differential sensitization in paired TMZ-sensitive and -resistant GBM models. Experimental Design: The impact of veliparib on TMZ-induced cytotoxicity and DNA damage was evaluated in vitro and in vivo in models of acquired TMZ resistance (GBM12TMZ-mgmtHigh, GBM12TMZ-mgmtLow, and U251TMZ), inherent TMZ resistance (T98G), and TMZ-sensitive (U251 and GBM12). In vivo drug efficacy, pharmacokinetics, and pharmacodynamics were analyzed using clinically relevant dosing regimens. Results: Veliparib enhanced TMZ cytotoxicity and DNA-damage signaling in all GBM models in vitro with more pronounced effects in TMZ-resistant lines at 3 to 10 μmol/L veliparib. In vivo, combined TMZ/veliparib, compared with TMZ alone, significantly delayed tumor growth and enhanced DNA-damage signaling and γH2AX levels in the sensitive GBM12 xenograft line but not in the resistant GBM12TMZ lines. The pharmacokinetic profile of veliparib was similar for GBM12 and GBM12TMZ tumors with Cmax (∼1.5 μmol/L) in tissue significantly lower than concentrations associated with optimal in vitro sensitizing effects for resistant tumors. In contrast, robust suppression of PARP-1 expression by shRNA significantly increased TMZ sensitivity of U251TMZ in vitro and in vivo. Conclusions: In vitro cytotoxicity assays do not adequately model the therapeutic index of PARP inhibitors, as concentrations of veliparib and TMZ required to sensitize TMZ-resistant cancer cells in vivo cannot be achieved using a tolerable dosing regimen. Clin Cancer Res; 20(14); 3730–41. ©2014 AACR.

Efficacy of PARP Inhibitor Rucaparib in Orthotopic Glioblastoma Xenografts Is Limited by Ineffective Drug Penetration into the Central Nervous System

PARP inhibition can enhance the efficacy of temozolomide and prolong survival in orthotopic glioblastoma (GBM) xenografts. The aim of this study was to evaluate the combination of the PARP inhibitor rucaparib with temozolomide and to correlate pharmacokinetic and pharmacodynamic studies with efficacy in patient-derived GBM xenograft models. The combination of rucaparib with temozolomide was highly effective in vitro in short-term explant cultures derived from GBM12, and, similarly, the combination of rucaparib and temozolomide (dosed for 5 days every 28 days for 3 cycles) significantly prolonged the time to tumor regrowth by 40% in heterotopic xenografts. In contrast, the addition of rucaparib had no impact on the efficacy of temozolomide in GBM12 or GBM39 orthotopic models. Using Madin-Darby canine kidney (MDCK) II cells stably expressing murine BCRP1 or human MDR1, cell accumulation studies demonstrated that rucaparib is transported by both transporters. Consistent with the influence of these efflux pumps on central nervous system drug distribution, Mdr1a/b−/−Bcrp1−/− knockout mice had a significantly higher brain to plasma ratio for rucaparib (1.61 ± 0.25) than wild-type mice (0.11 ± 0.08). A pharmacokinetic and pharmacodynamic evaluation after a single dose confirmed limited accumulation of rucaparib in the brain is associated with substantial residual PARP enzymatic activity. Similarly, matrix-assisted laser desorption/ionization mass spectrometric imaging demonstrated significantly enhanced accumulation of drug in flank tumor compared with normal brain or orthotopic tumors. Collectively, these results suggest that limited drug delivery into brain tumors may significantly limit the efficacy of rucaparib combined with temozolomide in GBM. Mol Cancer Ther; 14(12); 2735–43. ©2015 AACR.

Is the blood–brain barrier really disrupted in all glioblastomas? A critical assessment of existing clinical data

The blood-brain barrier (BBB) excludes the vast majority of cancer therapeutics from normal brain. However, the importance of the BBB in limiting drug delivery and efficacy is controversial in high-grade brain tumors, such as glioblastoma (GBM). The accumulation of normally brain impenetrant radiographic contrast material in essentially all GBM has popularized a belief that the BBB is uniformly disrupted in all GBM patients so that consideration of drug distribution across the BBB is not relevant in designing therapies for GBM. However, contrary to this view, overwhelming clinical evidence demonstrates that there is also a clinically significant tumor burden with an intact BBB in all GBM, and there is little doubt that drugs with poor BBB permeability do not provide therapeutically effective drug exposures to this fraction of tumor cells. This review provides an overview of the clinical literature to support a central hypothesis: that all GBM patients have tumor regions with an intact BBB, and cure for GBM will only be possible if these regions of tumor are adequately treated.

Central nervous system prophylaxis in diffuse large B-cell lymphoma.

Central nervous system (CNS) involvement with diffuse large B‐cell lymphoma (DLBCL) is a relatively uncommon manifestation; with most cases of CNS involvement occuring during relapse after primary therapy. CNS dissemination typically occurs early in the disease course and is most likely present subclinically at the time of diagnosis in many patients who later relapse in the CNS. CNS relapse in these patients is associated with poor outcomes. Based on a CNS relapse rate of 5% in DLBCL and weighing the benefits against the toxicities, universal application of CNS prophylaxis is not justified. The introduction of rituximab has significantly reduced the incidence of CNS relapse in DLBCL. Different studies have employed other agents for CNS prophylaxis, such as intrathecal chemotherapy and high‐dose systemic agents with sufficient CNS penetration. If CNS prophylaxis is to be given, it should be preferably administered during primary chemotherapy. However, there is no strong evidence that supports any single approach for CNS prophylaxis. In this review, we outline different strategies of administering CNS prophylaxis in DLBCL patients reported in literature and discuss their advantages and drawbacks.

Incidence, Characteristics, and Implications of Seizures in Patients With Glioblastoma:

Background: Seizures in patients with glioblastoma are associated with worse quality of life. However, their incidence, clinical characteristics, and prognostic implications are less well characterized. Objective: This study was undertaken to provide a contemporary experience along with benchmark data relevant to the above in patients with glioblastoma. It also sought to reexplore improved survival with seizures, as observed by others. Methods: In this single-institution study, patients with glioblastoma from 2010 through 2014 had their medical records reviewed in detail. Results: Among 122 patients, 58 (48%) had a seizure history. Of these, 67% had more than 1, 41% had generalized seizures, and most received antiseizure medication (most commonly levetiracetam). The median survival for patients with seizures was 1.66 years and 0.87 years for those without (hazard ratio for risk of death with seizures: 0.72; 95% confidence interval: 0.43, 1.21; P = .22 by the log-rank test). Conclusion: Seizures are common in patients with glioblastoma and, in contrast to earlier reports, are not associated with a statistically significant improvement in survival.

Restricted Delivery of Talazoparib Across the Blood–Brain Barrier Limits the Sensitizing Effects of PARP Inhibition on Temozolomide Therapy in Glioblastoma

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.

Barriers to Effective Drug Treatment for Brain Metastases: A Multifactorial Problem in the Delivery of Precision Medicine

The treatment of metastatic lesions in the brain represents a serious unmet medical need in the field of neuro-oncology. Even though many effective compounds have demonstrated success in treating peripheral (non-CNS) tumors with targeted agents, one aspect of this lack of success in the brain may be related to poor delivery of otherwise effective compounds. Many factors can influence the brain delivery of these agents, but one key barrier is a heterogeneously “leaky” BBB that expresses efflux transporters that limit the BBB permeability for many targeted agents. Future success in therapeutics for brain metastases must take into account the adequate delivery of “active, free drug” to the target, and may include combinations of targeted drugs that are appropriate to address each individual patient’s tumor type. This review discusses some issues that are pertinent to precision medicine for brain metastases, using specific examples of tumor types that have a high incidence of brain metastases.

Abstract CT074: Phase I/II study of VAL-083 in patients with recurrent glioblastoma

Glioblastoma (GBM) is the most common brain cancer. Front-line systemic therapy with temozolomide (TMZ) is often ineffective due to O6-methylguanine-DNA-methyltransferase (MGMT)-mediated resistance and patients with recurrent glioma have limited treatment options and very poor prognosis. Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent that readily crosses the blood-brain barrier and has demonstrated activity against GBM in prior NCI-sponsored clinical trials. VAL-083 induces cross-links at N7 of guanine causing double-strand DNA breaks and apoptosis independent of MGMT expression against multiple GBM cell lines and cancer stem cells in vitro. VAL-083 cytotoxic activity also appears to be less dependent on wild type p53 compared to other alkylating agents. The main goal of this clinical trial was to determine an appropriate dose for VAL-083 for advancement to Phase II/III trials as a potential new treatment for refractory GBM. METHOD: Open-label, single-arm Phase I dose-escalation study (3+3 design) of IV treatment with VAL-083 on days 1, 2, 3 of a 21-day cycle, until MTD was reached. In Phase II, additional patients with GBM are treated at the MTD to gather further safety and outcomes data. Patients must have histologically confirmed GBM, previously treated with surgery and radiation and must have failed both TMZ and bevacizumab, unless contraindicated. RESULTS: Phase I has been completed and 40 mg/m2/d confirmed as the MTD. 29 GBM patients were enrolled in Phase I across 9 dose cohorts (1.5 - 50 mg/m2/d). Myelosuppression was mild; no drug-related serious adverse events were reported at doses ?40 mg/m2/d. Dose limiting toxicities (DLT), consisting of thrombocytopenia, were observed at 50 mg/m2/d and at an interim 45 mg/m2/d cohort. Platelet nadir occurred around day 20 and resolved rapidly and spontaneously. Pharmacokinetic analyses show dose-dependent linear systemic exposure with a short 1-2h plasma terminal half-life; average Cmax 781 ng/mL (5.3μM) at 40 mg/m2/d resulting in estimated CNS concentrations within the IC50 range observed for GBM cell-lines in vitro. A 14 patient Phase II expansion cohort was enrolled at 40 mg/m2/d. Safety observations in the Phase II expansion cohort to date are consistent with Phase I: Observed myelosuppression is mild, with the exception of 1 patient previously treated with CCNU who developed grade 4 thrombocytopenia. To date, 20 GBM patients (6 patients in Phase I and 14 patients in Phase II) have been treated with VAL-083 at therapeutic doses of 30 or 40 mg/m2/d. CONCLUSIONS: VAL-083 at 40 mg/m2/d on days 1, 2, 3 of a 21-day cycle exhibits a favorable safety profile and the Phase I part of the study showed a trend toward clinically meaningful improved survival in refractory GBM patients. Updated safety and outcomes data from the Phase II expansion cohort will be presented. ClinicalTrials.gov Identifier NCT01478178. Citation Format: Kent C. Shih, Manish R. Patel, Nicholas Butowski, Gerald S. Falchook, Sani H. Kizilbash, Jeffrey A. Bacha, Dennis M. Brown, Anne Steino, Richard Schwartz, Sarath Kanekal, Lorena M. Lopez, Howard A. Burris. Phase I/II study of VAL-083 in patients with recurrent glioblastoma. [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 CT074.

Abstract 340: Talazoparib enhances low-dose temozolomide efficacy in flank glioblastoma models, but intracranial efficacy is constrained by limited brain distribution

BACKGROUND: Talazoparib (TAL) is a potent poly (ADP-ribose) polymerase (PARP) inhibitor that robustly enhances in vitro sensitivity to temozolomide (TMZ) in several tumor models. We assessed the in vitro and in vivo efficacy of TAL combined with TMZ in glioblastoma (GBM) models. METHODS: Established glioma cell lines (T98G, U251) and the GBM12 patient derived xenograft (PDX) line were treated in vitro with TAL (1-10 nM) ± TMZ (2-300 μM). Antitumor efficacy was assessed with CyQUANT, clonogenic and primary neurosphere assays; cell cycle analysis with flow cytometry; DNA damage signaling with phospho-specific western blots for pKap1, pChk1, pChk2 and fluorescent immunocytochemistry for γH2AX and replication protein A foci. Brain and plasma concentrations of TAL in wild-type (WT), Mdr1a/b(-/-), Bcrp(-/-) and Mdr1a/b(-/-)Bcrp(-/-) knockout (KO) mice were assessed with LC-MS/MS. The tolerability of TAL (0.05 - 0.30 mg/kg/day orally, divided twice daily (div. bid)) and TMZ (5-50 mg/kg/day orally) was tested in athymic nude mice. Subsequently, in vivo combination TAL/TMZ efficacy was assessed with survival analyses in intracranial and flank GBM12 PDX models. RESULTS: TAL 1-3 nM sensitizes the TMZ resistant T98G glioma cell line to TMZ at high TMZ concentrations (30-300 μM) and is associated with G2 cell cycle arrest and enhanced DNA damage signaling. TAL 1-3 nM also enhances the cytotoxic efficacy of lower concentrations of TMZ (10-30 μM) in the TMZ sensitive U251 cell line. At least 3 nM TAL is required to enhance the in vitro efficacy of low TMZ concentrations (2-10 μM) in the TMZ sensitive GBM12 PDX line. PK studies in non-tumor bearing WT FVB mice treated with a single dose of TAL 0.15 mg/kg revealed mean brain and plasma concentrations of 0.49 ng/g (1.3 nM) and 25.5 ng/ml (67.1 nM) respectively at 2 hours. The mean brain/plasma ratio at 2 hours was unchanged in Bcrp(-/-) KO mice compared to WT mice (1.2% vs. 2.0%), but was significantly increased in Mdr1a/b(-/-) KO mice (22.8%) and Mdr1a/b(-/-)Bcrp(-/-) KO mice (23.9%). Standard doses of TMZ (50 mg/kg/day, days 1-5) combined with low doses of TAL (0.05 mg/kg/day, div. bid) were toxic in nude mice. Higher doses of TAL (0.30 mg/kg/day, div. bid) required substantial TMZ dose reductions (5 mg/kg/day, days 1-5) for tolerability. The addition of TAL (0.30 mg/kg/day, div. bid) to low-dose TMZ (5 mg/kg/day) prolonged tumor stasis in flank GBM12 xenografts (median time to endpoint 76 vs. 50 days, p = 0.005). However this regimen was ineffective in intracranial GBM12 xenografts (median survival 37 vs. 30 days, p = 0.93). CONCLUSIONS: TAL is a potent PARP inhibitor that enhances the efficacy of TMZ in both in vitro GBM models and flank GBM12 PDX models, but not in the corresponding GBM12 intracranial xenografts. This lack of intracranial efficacy is associated with limited brain distribution due to active efflux mediated by Mdr1 at the blood-brain barrier. Citation Format: Sani H. Kizilbash, Kenneth Chang, Shiv K. Gupta, Ryo Kawashima, Karen Parrish, Ann C. Mladek, Brett L. Carlson, Katrina K. Bakken, Mark A. Schroeder, Gaspar J. Kitange, Paul A. Decker, Yuqiao Shen, William F. Elmquist, Jann N. Sarkaria. Talazoparib enhances low-dose temozolomide efficacy in flank glioblastoma models, but intracranial efficacy is constrained by limited brain distribution. [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 340.