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.

Purpose Effective sensitizing strategies potentially can extend the benefit of TMZ therapy in GBM patients. We previously demonstrated that robust TMZ-sensitizing effects of the 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, U251TMZ), inherent TMZ-resistance (T98G), and TMZ-sensitive (U251, 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 3-10 μM veliparib. In vivo, combined TMZ/veliparib, compared to 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 μM) 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.

6-Acetyl-8-cyclopentyl-5-methyl-2-([5-(piperazin-1-yl)pyridin-2-yl]amino)pyrido(2,3-d)pyrimidin-7(8H)-one [palbociclib (PD-0332991)] is a cyclin-dependent kinase 4/6 inhibitor approved for the treatment of metastatic breast cancer and is currently undergoing clinical trials for many solid tumors. Glioblastoma (GBM) is the most common primary brain tumor in adults and has limited treatment options. The cyclin-dependent kinase 4/6 pathway is commonly dysregulated in GBM and is a promising target in treating this devastating disease. The blood-brain barrier (BBB) limits the delivery of drugs to invasive regions of GBM, where the efflux transporters P-glycoprotein and breast cancer resistance protein can prevent treatments from reaching the tumor. The purpose of this study was to examine the mechanisms limiting the effectiveness of palbociclib therapy in an orthotopic xenograft model. The in vitro intracellular accumulation results demonstrated that palbociclib is a substrate for both P-glycoprotein and breast cancer resistance protein. In vivo studies in transgenic mice confirmed that efflux transport is responsible for the limited brain distribution of palbociclib. There was an ∼115-fold increase in brain exposure at steady state in the transporter deficient mice when compared with wild-type mice, and the efflux inhibitor elacridar significantly increased palbociclib brain distribution. Efficacy studies demonstrated that palbociclib is an effective therapy when GBM22 tumor cells are implanted in the flank, but ineffective in an orthotopic (intracranial) model. Moreover, doses designed to mimic brain exposure were ineffective in treating flank tumors. These results demonstrate that efflux transport in the BBB is involved in limiting the brain distribution of palbociclib and this has critical implications in determining effective dosing regimens of palbociclib therapy in the treatment of brain tumors.

Purpose Wee1 regulates key DNA damage checkpoints, and in this study, the efficacy of the Wee1 inhibitor MK-1775 was evaluated in GBM xenograft models alone and in combination with radiation and/or temozolomide (TMZ). Experimental design In vitro MK-1775 efficacy alone and in combination with TMZ, and the impact on DNA damage was analyzed by western blotting and γH2AX foci formation. In vivo efficacy was evaluated in orthotopic and heterotopic xenografts. Drug distribution was assessed by conventional mass spectrometry (MS) and matrix-assisted laser desorption/ionization (MALDI) -MS imaging. Results GBM22 (IC50 = 68 nM) was significantly more sensitive to MK-1775 compared to 5 other GBM xenograft lines including GBM6 (IC50 >300 nM), and this was associated with a significant difference in pan-nuclear γH2AX staining between treated GBM22 (81% cells positive) and GBM6 (20% cells positive) cells. However, there was no sensitizing effect of MK-1775 when combined with TMZ in vitro. In an orthotopic GBM22 model, MK-1775 was ineffective when combined with TMZ, while in a flank model of GBM22, MK-1775 exhibited both single agent and combinatorial activity with TMZ. Consistent with limited drug delivery into orthotopic tumors, the normal brain to whole blood ratio following a single MK-1775 dose was 5%, and MALDI-MS imaging demonstrated heterogeneous and markedly lower MK-1775 distribution in orthotopic as compared to heterotopic GBM22 tumors. Conclusions Limited distribution to brain tumors may limit the efficacy of MK-1775 in GBM.

Poly (ADP-ribose) polymerase PARP inhibitors, including talazoparib (TAL), potentiate temozolomide (TMZ) efficacy in multiple tumor types, however TAL-mediated sensitization has not been evaluated in orthotopic glioblastoma (GBM) models. This study evaluates TAL ± TMZ in clinically relevant GBM models. TAL at 1–3 nmol/L sensitized T98G, U251 and GBM12 cells to TMZ, and enhanced DNA damage signaling and G2/M arrest in vitro. In vivo cyclical therapy with TAL (0.15 mg/kg twice daily) combined with low dose TMZ (5 mg/kg daily) was well tolerated. This TAL/TMZ regimen prolonged tumor stasis more than TMZ alone in heterotopic GBM12 xenografts [median time to endpoint: 76 days vs. 50 days TMZ (p=0.005), 11 days placebo (p <0.001)]. However, TAL/TMZ did not accentuate survival beyond that of TMZ alone in corresponding orthotopic xenografts (median survival 37 vs. 30 days with TMZ (p=0.93), 14 days with placebo, p<0.001). Average brain and plasma TAL 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 TAL in Bcrp−/− versus WT mice did not differ, while 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 TAL accumulation in MDCKII cells. These results indicate that TAL has significant MDR1 efflux liability that may restrict delivery across the blood-brain barrier, and this may explain the loss of TAL-mediated TMZ sensitization in orthotopic versus heterotopic GBM xenografts.