Rapamycin weekly maintenance dosing and the potential efficacy of combination sorafenib plus rapamycin but not atorvastatin or doxycycline in tuberous sclerosis preclinical models

Abstract

Background: Tuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor syndrome, characterized by hamartomatous growths in the brain, skin, kidneys, lungs, and heart, which lead to significant morbidity. TSC is caused by mutations in the TSC1 or TSC2 genes, whose products, hamartin and tuberin, form a tumor suppressor complex that regulates the PI3K/Akt/mTOR pathway. Early clinical trials show that TSC-related kidney tumors (angiomyolipomas) regress when treated with the mammalian target of rapamycin (mTOR) inhibitor, rapamycin (also known as sirolimus). Although side effects are tolerable, responses are incomplete, and tumor regrowth is common when rapamycin is stopped. Strategies for future clinical trials may include the investigation of longer treatment duration and combination therapy of other effective drug classes.

Results: Here, we examine the efficacy of a prolonged maintenance dose of rapamycin in Tsc2+/- mice with TSC-related kidney tumors. Cohorts were treated with rapamycin alone or in combination with interferon-gamma (IFN-g). The schedule of rapamycin included one month of daily doses before and after five months of weekly doses. We observed a 94.5% reduction in kidney tumor burden in Tsc2+/- mice treated (part one) daily with rapamycin (8 mg/kg) at 6 months <or= age < 7 months, (part 2) weekly with rapamycin (16 mg/kg) at 7 months <or= age < 12 months, and (part 3) daily with rapamycin (8 mg/kg) at 12 months <or= age < 13 months; but we did not observe any improvement with combination IFN-g plus rapamycin in this study. We also used a Tsc2-/- subcutaneous tumor model to evaluate other classes of drugs including sorafenib, atorvastatin, and doxycycline. These drugs were tested as single agents and in combination with rapamycin. Our results demonstrate that the combination of rapamycin and sorafenib increased survival and may decrease tumor volume as compared to rapamycin treatment alone while sorafenib as a single agent was no different than control. Atorvastatin and doxycycline, either as single agents or in combination with rapamycin, did not improve outcomes as compared with controls.

Conclusion: Our results indicate that prolonged treatment with low doses of mTOR inhibitors may result in more complete and durable TSC-related tumor responses, and it would be reasonable to evaluate this strategy in a clinical trial. Targeting the Raf/Mek/Erk and/or VEGF pathways in combination with inhibiting the mTOR pathway may be another useful strategy for the treatment of TSC-related tumors.

Figure 1

Comparison of single agent rapamycin treatment to rapamycin plus IFN-g treatment in Tsc2^+/- mice using a dosing schedule that includes weekly maintenance rapamycin treatment. Severity of kidney disease in Tsc2^+/- mice was quantified by scoring (a) total kidney cystadenomas and (b) kidney lesion subtype for indicated treatment groups (see treatment details in methods and Table 1). Lesion subtypes include cystic, pre-papillary, papillary, and solid; for a definition of each subtype and scoring details, see Tables 2 and 3. The red error bars in (a) indicate a statistically significant difference (p < 0.05) relative to the untreated cohort. Statistical analysis was not done for the lesion subtype date. These data show a significant reduction of kidney disease in both treatment groups but no difference between single agent rapamycin and combination rapamycin plus IFN-g. Error bars shown indicate standard error.

Figure 2

Improved survival and decreased tumor growth in nude mice bearing Tsc2^-/- tumors with combination rapamycin plus sorafenib treatment. (a) Average tumor volume over time. Error bars shown indicate standard error. (b) Survival curve for indicated treatment cohorts. Based on survival analysis, rapamycin plus sorafenib is more effective than single agent rapamycin. Based on tumor volume analyses, the rapamycin plus sorafenib treated group is not statistically different from the rapamycin treated group on day 44. However, on the last day that tumor volume data was available for all mice (day 43), there was a significant difference between tumor volumes from the rapamycin plus sorafenib group compared with the single agent rapamycin group. Although rapamycin is an effective single agent, sorafenib is not. This data is summarized in Table 4.

Figure 3

Atorvastatin does not decrease tumor growth or increase survival in nude mice bearing Tsc2^-/- tumors. (a) Average tumor growth over time for atorvastatin and atorvastatin plus rapamycin treated animals. Error bars shown indicate standard error. (b) Survival over time for atorvastatin and atorvastatin plus rapamycin cohorts. Based on survival analysis and comparison of tumor volumes on days 26 and 42, atorvastatin was not effective as a single agent or in combination with rapamycin in this preclinical study. This data is summarized in Table 5.

Figure 4

Doxycycline does not decrease tumor growth or increase survival in nude mice bearing Tsc2^-/- tumors. (a) Average tumor growth over time for doxycycline and doxycycline plus rapamycin treated animals. Error bars shown indicate standard error. (b) Survival over time for doxycycline and doxycycline plus rapamycin cohorts. Based on survival analysis and comparison of tumor volumes on days 26 and 42, doxycycline was not effective as a single agent or in combination with rapamycin in this preclinical study. This data is summarized in Table 5.

Figure 5

Sorafenib, atorvastatin and doxycycline do not affect whole blood rapamycin levels in nude mice bearing Tsc2^-/- tumors. (a) Tumor rapamycin levels from rapamycin treated group (n = 5) and sorafenib plus rapamycin combination treated group (n = 4). (b) Whole blood rapamycin levels from rapamycin treated group (n = 8), atorvastatin plus rapamycin combination treated group (n = 8) and doxycycline plus rapamycin combination treated group (n = 8). Rapamycin levels were measured 24 hours after the last dose of rapamycin for all groups.