Based on our previous testing for single agent activity, twelve solid tumor models and three leukemia models having intermediate sensitivity to each agent were selected for combination testing. For the leukemia models dexamethasone was substituted for cisplatin, as the latter agent has little activity against ALL (24
The intent of the in vitro
studies was to use rapamycin to establish whether mTORC1 inhibition altered the in vitro
response of the PPTP cell lines to standard cytotoxic agents. The method of analyzing for a shift in EC50
for a standard agent in the presence of a test agent is comparable to methods employed in evaluating the effect of RNA interference “knockdown” of specific gene products on response of cell lines to cytotoxic agents (27
). The methods have also been applied in combination testing studies analyzing chemical “knockdown” of activity of therapeutic targets by small molecule inhibitors (29
). Comparable results have been reported for the shift in EC50
method and for the combination indices method (29
). In vitro
there was a trend toward sub-additive effects when rapamycin was combined with melphalan, cisplatin or vincristine. By contrast, there was strong evidence for supra-additivity for dexamethasone against leukemia cell lines, consistent with previously published results (31
). The results obtained for the standard cytotoxic agents suggest that mTORC1 signaling results in enhanced cell death in the presence of these agents. This would be consistent with recent data showing that cells deficient in TSC function (hence with constitutively activated mTORC1 signaling) were more sensitive to the DNA damaging agent methylmethane sulfonate (MMS) (10
). However, in S. cerevisiae
, inhibition of TORC1 signaling reduces survival in cells challenged with MMS or camptothecin (11
), and rapamycin significantly decreases clonogenic survival of mammalian cells treated with topotecan in vitro
For in vivo testing, the addition of rapamycin to cyclophosphamide and to vincristine at their respective MTDs was relatively well tolerated, with little increase in toxicity observed. However, rapamycin significantly potentiated the toxicity of cisplatin administered to scid mice. Athymic nude mice, used for the propagation of glioblastoma models, tolerated cisplatin poorly at the higher dose, with exacerbation of toxicity by the addition of rapamycin.
Rapamycin or its analogs have previously been tested in combination with cytotoxic agents. Vincristine was reported to be synergistic against several mantle cell lymphoma cell lines in vitro
when combined with the rapalog RAD001 (everolimus) (33
), although there are no reports evaluating this combination in vivo.
However, at high concentrations (> 1 µM) rapamycin is a substrate for P-glycoprotein-mediated efflux and hence can reverse vincristine resistance (17
). In our study, combination of vincristine with rapamycin produced therapeutic enhancement in 4 of the 11 xenografts evaluable at one or both of the vincristine doses studied. For example, addition of rapamycin converted the response to vincristine from Progressive Disease 2 (PD2) to CR in D456 and Rh18 (0.5MTD), or MCR for SK-NEP-1 tumors. It seems unlikely that at the dose level used, rapamycin is enhancing the antitumor activity of vincristine through reversing P-glycoprotein-mediated drug efflux, as only slight enhanced toxicity was observed (as occurs with most multidrug resistance modulators). In the analysis of clinical trials for drug combinations in which a novel agent is added to a standard agent(s), the combination effect is compared to the effect of the standard agent(s) used alone in the same patient population. Our analysis of vincristine combined with rapamycin shows the combination to be effective in significantly extending EFS compared to single agent vincristine administered at its MTD in 6 of 9 solid tumor models. Thus, the vincristine/rapamycin combination demonstrates promising activity using this clinically relevant comparison.
Cyclophosphamide combined with rapamycin has been evaluated primarily for organ transplantation (34
), but not in the context of cancer chemotherapy. In the current study cyclophosphamide showed therapeutic enhancement for all 5 evaluable solid tumor xenografts, although the evaluation of therapeutic enhancement was not possible in some models because of the high level of activity of single agent cyclophosphamide used at its MTD. One important observation is that therapeutic enhancement was rarely observed when cyclophosphamide at 0.5MTD was combined with rapamycin. This highlights the importance of using agents with steep dose response curves like cyclophosphamide at or near their MTD in combination studies. These results suggest that the clinical success of combinations of alkylating agents with mTOR inhibitors will be dependent upon whether full doses of the alkylating agents can be administered.
Rapamycin, or the analog CCI-779 (temsirolimus) has been shown previously to potentiate apoptosis induced by cisplatin in several tumor cell models (13
), and to potentiate cisplatin antitumor activity in vivo
). Further, Beuvink et al (15
) have reported increased apoptosis activity when cisplatin was combined with the rapalog everolimus (RAD001) only in tumor cells with wild type p53. The purported mechanism of enhanced cell killing was through rapamycin blocking p53-mediated induction of p21CIP1
, leading to decreased G1 arrest, and enhanced apoptosis. In our analyzable studies, the combination of rapamycin with cisplatin did not show therapeutic enhancement using the lower, tolerable dose of cisplatin. The mechanism for increased systemic toxicity, when rapamycin is combined with cisplatin is unknown. Potentially, the combination could have similar effects on normal proliferating tissues in the intestine and bone marrow as reported by Beuvink et al (15
). The combination of rapamycin with cisplatin (0.63MTD) was significantly more effective than cisplatin at its MTD in 3 of 7 evaluable models in terms of prolonging EFS.
Rapamycin combined with vincristine, cyclophosphamide or dexamethasone was evaluated against three ALL models. The vincristine and rapamycin combination showed little evidence for superiority in comparison to the single agents, while interpretation of results for the cyclophosphamide and rapamycin combination were complicated by the high level of activity for single agent cyclophosphamide. Although as a single agent dexamethasone was tolerated at 30 mg/kg, in combination with rapamycin the maximum tolerated dose was 7.5 mg/kg. Even at this low dose, therapeutic enhancement was demonstrated for 1 of 3 xenografts. Rapamycin has been shown to reverse dexamethasone resistance in ALL cell lines through modulation of MCL1, but in this previous report, the therapeutic utility was not tested in vivo
). The value of combining dexamethasone with rapamycin will require examining a larger cohort of leukemia models as well as further evaluation of the increased toxicity.
The most appropriate methodology to apply for in vivo
combination testing remains an open question. While formal synergy testing can be performed using isobologram or response surface modeling methods, such methods require testing of several dose levels for each agent and require a very large number of animals for each combination tested against a single xenograft. As our goal is to develop a dataset to inform clinical prioritization for a wide range of histotypes and drug combinations, these formal synergy assessment methods are not suitable. For in vivo
combination testing, we favor a fixed-dose drug combination approach, as this minimizes the number of animals required per drug combination. Among fixed-dose approaches, testing for “therapeutic enhancement” appears to be the most clinically relevant strategy for evaluating combinations in vivo
, as its presence implies an enhanced therapeutic index (i.e. the combination has greater activity than either agent alone administered at the MTD) (25
Many published combination preclinical studies, particularly with agents lacking intrinsic antitumor activity and used to modulate the activity of cytotoxic agents (e.g., buthionine sulfoxamine given with melphalan, multidrug resistance modulators given with standard chemotherapy agents, etc.), failed to compare the combination effect to the effect of the cytotoxic agent administered at its MTD (35
). Clinically, it has been observed that many combinations required substantial reductions in the dose of the standard agent when the novel agent was used at biologically effective doses in humans. The failure of O6
-benzylguanine to enhance the clinical activity of nitrosoureas when tested in clinical trials, was primarily the result of the reductions in nitrosourea dosing required to safely administer nitrosoureas with O6
-benzylguanine in humans (36
). The “therapeutic enhancement” concept directly addresses this concern by comparing the activity of the combination to the activity of the single agents at their MTD.
The second method of evaluating combination treatments used allows for model based testing for supra- or sub-additivity. This approach is conceptually the same as the commonly applied method of summing log-cell kill values for single agents to estimate the log-cell kill expected for additivity. Application of linear regression modeling has the advantage of allowing the statistical evidence for claims of supra- or sub-additivity to be quantified. The interesting observation from these analyses is that some xenografts show consistent evidence for supra-additivity, suggesting that there is a subset of tumors for which combinations of cytotoxic agents and rapalogs may be particularly effective. Identifying the biological basis for supra-additivity for these xenografts could have direct clinical implications. One lesson learned from the PPTP evaluations of rapamycin combinations is that optimal application of the model based approach requires selecting durations of treatment for the single agents that are not so effective that treated animals without events are present at the end of the observation period.
Exactly how rapamycin enhances the in vivo
antitumor activity of cyclophosphamide and vincristine used at their respective MTDs remains to be explored. Rapamycin enhances some forms of DNA damage, and emerging evidence indicates that the TOR pathway regulates transit through mitosis in yeast. Rapamycin and related mTOR inhibitors also have antiangiogenic activity through inhibition of proliferation of endothelial cells and through impaired VEGF production (4
), and hence the interaction may be tumor cell-dependent or -independent. There is an apparent discrepancy between the in vitro
testing results, which primarily demonstrated sub-additivity, and the in vivo
testing results, for which therapeutic enhancement was commonly observed. It is important to note, however, that therapeutic enhancement can be present when the true interaction at the cellular level between the agents used in combination ranges from sub-additivity to supra-additivity. Particularly when the agents can be administered together at their single agent MTDs, there is the opportunity for a significant increase in efficacy for the combination (i.e., therapeutic enhancement) even when the model based assessment of the interaction shows sub-additivity.
In summary, we have evaluated both in vitro and in vivo efficacy for rapamycin combined with standard cytotoxic agents frequently used in treatment of childhood cancer. The predominant interaction in vitro was sub-additive activity, with the notable exception of supra-additivity for dexamethasone plus rapamycin for leukemia cell lines. In vivo, there were numerous models that showed therapeutic enhancement for combinations in which rapamycin was administered with either cyclophosphamide or vincristine at their respective MTDs. These results will be useful in planning combination clinical trials with rapalogs in pediatric cancer patients.
Statement of Translational Relevance
Rapamycin, a selective inhibitor of mTOR signaling, has shown significant antitumor activity against in vivo models of childhood cancer. Rapamycin analogs, everolimus and temsirolimus, are currently in phase I and phase II clinical development through the Children’s Oncology Group. Clinical development of rapamycin analogs will probably include combination of these agents with standard curative cytotoxic therapy. Consequently, we have evaluated the antitumor activity of standard cytotoxic agents that form the backbone for curative therapy, alone or in combination with rapamycin. These results will be of value in developing effective clinical protocols incorporating rapamycin analogs into standard of care for treatment of childhood malignancies.