Preclinical trial of a new dual mTOR inhibitor, MLN0128, using renal cell carcinoma tumorgrafts

Abstract

mTOR is a rational target in renal cell carcinoma (RCC) because of its role in disease progression. However, the effects of temsirolimus, the only first-generation mTOR inhibitor approved by the FDA for first-line treatment of metastatic RCC, on tumor reduction and progression-free survival are minimal. Second-generation mTOR inhibitors have not been evaluated on RCC. We compared the effects of temsirolimus and MLN0128, a potent second-generation mTOR inhibitor, on RCC growth and metastasis using a realistic patient-derived tissue slice graft (TSG) model. TSGs were derived from three fresh primary RCC specimens by subrenal implantation of precision-cut tissue slices into immunodeficient mice that were randomized and treated with MLN0128, temsirolimus, or placebo. MLN0128 consistently suppressed primary RCC growth, monitored by magnetic resonance imaging (MRI), in three TSG cohorts for up to 2 months. Temsirolimus, in contrast, only transiently inhibited the growth of TSGs in one of two cohorts before resistance developed. In addition, MLN0128 reduced liver metastases, determined by human-specific quantitative polymerase chain reaction, in two TSG cohorts, whereas temsirolimus failed to have any significant impact. Moreover, MLN0128 decreased levels of key components of the two mTOR subpathways including TORC1 targets 4EBP1, p-S6K1, HIF1α and MTA1 and the TORC2 target c-Myc, consistent with dual inhibition. Our results demonstrated that MLN0128 is superior to temsirolimus in inhibiting primary RCC growth as well as metastases, lending strong support for further clinical development of dual mTOR inhibitors for RCC treatment.

Keywords: mTOR inhibitor; renal cell carcinoma; tumorgrafts.

Figure 1

TSGs preserved histological features and biomarker expression of corresponding parental tumors. All three cases and their TSGs displayed intermediate to strong expression of CAIX (a–f) and patchy staining of CD10 (g–l). All three cases and their TSGs were negative for CD117 (m–r). Case 1 and its TSGs showed weak staining for CK7 (s and t). Cases 2 and 3 and their TSGs were negative for CK7 (u–x). Case 1 and its TSGs harbored the same mutation in VHL gene exon 3 (y and z). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

MLN0128 was superior to temsirolimus in tumor growth inhibition. (a) TSG volume was measured by MRI and 3D volumetric modeling. (b) Diagram of study timeline. (c) SGR of TSGs in mice treated with either MLN0128, temsirolimus or placebo in three cohorts. * indicates statistical significance (p < 0.05). The numbers on the columns are the numbers of mice in each arm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 3

MLN0128 was superior to temsirolimus in inhibiting metastases. mRCC cells were observed surrounding blood vessels (arrows in a–c) in livers of mice carrying TSG cohort 3. They expressed Ku70 (a), a human-specific nuclear antigen, CAIX (b) and were negative for CD10 (c). MLN0128 but not temsirolimus reduced the mean ratio of mRCC cells vs. total cells, measured as the ratio of human-specific GAPDH to total GAPDH by qRT-PCR (d). The effects were statistically significant as shown by p-values of Student’s t-test (e). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 4

MLN0128 downregulated key components of the two mTOR subpathways, TORC1 and TORC2. Quantitative immunoblot demonstrated downregulation of p-4EBP1, HIF1α, MTA1 and c-Myc in TSG cohort 1 by MLN0128 (a). In TSG cohort 3 (b), MLN0128 was superior to placebo and temsirolimus in downregulating p-4EBP1, p-S6K1 and HIF1α. In addition, temsirolimus significantly upregulated (S473)p-AKT compared to placebo. * indicates statistical significance (p < 0.05). (c) Expression of p-4EBP1 in parent tumors and derivative TSGs for cohorts 2 and 3, treated with placebo or MLN0128, as determined by immunohistochemistry. (d) Diagram of MLN0128 actions in RCC. The numbers on the columns are the numbers of mice in each arm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]