The pre-clinical animal model studies presented demonstrate that not all GBM tumors will benefit equally from combined therapy with TMZ and the PARP inhibitor ABT-888. Specifically, ABT-888 combined with TMZ only enhanced survival in the two TMZ-naive xenograft lines (GBM12 and 22), while derivative tumor lines, which had been selected in vivo
for TMZ resistance (GBM12TMZ and GBM22TMZ), were unaffected by the addition of ABT-888 to TMZ therapy. Along with the lack of survival benefit with combined therapy for two other TMZ-resistant lines (GBM14TMZ, and GBM39TMZ), these results suggest that combined therapy with ABT-888 and TMZ may not be effective in GBM tumors that already have developed resistance to TMZ. These data are in contrast to several in vitro
and in vivo
studies that demonstrate improved efficacy of TMZ when combined with various PARP inhibitors including ABT-888 (8
). One of the key differences between these previous studies and the current study is the exclusive in vivo
evaluation of therapies using the unique Mayo GBM xenograft panel. In this model, primary patient tumor samples are implanted directly into mice, serially passaged as heterotopic xenografts, and used for therapy evaluations exclusively in the intracranial location. In contrast to typical cell culture models, propagation of tumors in the flank preserves key features of the primary patient tumor samples including MGMT promoter methylation status and inherent TMZ responsiveness (manuscript in preparation). In contrast, many of the previous studies were performed in non-GBM models, and all studies have been performed using tumor cell models which have been subjected to prolonged culture on plastic, which selects for characteristics that may be far removed from primary tumors. From these observations, we believe that the Mayo xenograft panel provides a robust platform for testing novel TMZ-sensitizing strategies for GBM therapy.
The data presented demonstrate that the lack of a TMZ-sensitizing effect of ABT-888 in certain tumor lines is not due to a failure to effectively inhibit PARP activity. PAR formation was effectively suppressed in flank tumor from both GBM12 and GBM12TMZ with the ABT-888 dosing regimen used for the majority of the studies (15 mg/kg/day; ). While the blood-brain barrier potentially could limit access of the drug to the intracranial tumors, the parental GBM12 line lacks an intact blood-brain barrier (J. Poduslo and J. Sarkaria, unpublished data), and ABT-888 effectively penetrates an intact blood-brain barrier and shows demonstrable accumulation in the CNS (20
). Consistent with effective inhibition of PARP activity in intracranial tumors, ABT-888 effectively sensitized the GBM12 and GBM22 xenograft lines ( and ). Moreover, a higher dose ABT-888 regimen (40 mg/kg/day), which would provide dose levels in mice that would be supra-therapeutic in humans (21
), was equally ineffective in the GBM14TMZ resistant tumor line. Thus, ABT-888 was ineffective in a subset of tumor lines despite effective suppression of PARP activity in the resistant tumors.
Resistance to TMZ therapy requires integrity of both short-patch BER pathway and the MGMT repair protein in order to repair cytotoxic N3-methyladenine and O6-methylguanine lesions, respectively, and abrogation of either pathway leads to significant increased cell killing after TMZ treatment (reviewed in (5
)). TMZ resistance in the GBM12TMZ and GBM14TMZ lines can be reversed with the MGMT inhibitor O6-benzylguanine and both lines demonstrate a marked up-regulation of MGMT protein and mRNA levels (unpublished data). In conjunction with the lack of TMZ sensitization by ABT-888, these data would be consistent with incomplete disruption of BER in these tumor lines by PARP inhibition. In support of this possibility, several cell culture models of PARP deficiency demonstrate slowed kinetics of BER without complete abrogation of BER activity (22
). The key cytotoxic lesion induced by TMZ and processed by BER is N3-methyladenine, which can lead to cytotoxicity only when encountered by a replication fork during S-phase (11
). Since cell cultures grown in vitro
typically have a much higher S-phase fraction than tumors grown in vivo
, we speculate that any delayed kinetics of BER following ABT-888 may not be manifest as increased cell killing in our TMZ-resistant models because of the much longer average time available to a cell prior to replication. Differential effects of PARP inhibition on BER between the TMZ sensitive and resistant tumor lines also could explain the results observed. Future studies will address the mechanisms of PARP-mediated sensitization in our xenograft model and will specifically measure rates of various DNA repair processes involved in processing TMZ-induced damage.
The current set of studies was designed to guide clinical development of ABT-888 in GBM. While these results need to be validated with other clinically used PARP inhibitors, there are several important observations that may guide the general development of PARP inhibitor based TMZ-sensitizing strategies in GBM. First, of the 6 xenograft lines tested, only the two that were inherently sensitive to TMZ were effectively sensitized by ABT-888, while ABT-888 combined with TMZ was ineffective in TMZ-resistant lines. These data suggest that combined therapy with TMZ and a PARP inhibitor likely will be more effective in newly diagnosed GBM patients, and that PARP inhibition combined with TMZ in patients who have progressed on TMZ is less likely to provide significant benefit. Second, for the two tumor lines in which robust sensitization to TMZ were observed, there were no observed radiosensitizing effects of ABT-888. Although this is a limited data set, these observations reduce our enthusiasm for studies integrating PARP inhibitors with radiation monotherapy in patients who are not suitable candidates for combined TMZ/RT therapy. Third, the efficacy of TMZ was reduced with latter cycles of therapy in TMZ-naïve tumors. This observation is similar to clinical experiences in which over 30% of newly diagnosed patients progress while receiving TMZ therapy (2
), and this may reflect relatively early development of TMZ resistance in these tumors. Given the lack of efficacy of combined therapy in TMZ-resistant tumors, these data suggest that PARP inhibitors may be most effective when integrated early during therapy before resistance develops. While these observations remain to be confirmed in clinical trials, we believe the studies performed in the Mayo GBM xenograft model have helped delineate a potential strategy for optimizing the integration of PARP inhibitors with TMZ for therapy of GBM patients.