Mechanisms of acquired resistance to rapalogs in metastatic renal cell carcinoma

Abstract

The mechanistic target of rapamycin (mTOR) is an established therapeutic target in renal cell carcinoma (RCC). Mechanisms of secondary resistance to rapalog therapy in RCC have not been studied previously. We identified six patients with metastatic RCC who initially responded to mTOR inhibitor therapy and then progressed, and had pre-treatment and post-treatment tumor samples available for analysis. We performed deep whole exome sequencing on the paired tumor samples and a blood sample. Sequence data was analyzed using Mutect, CapSeg, Absolute, and Phylogic to identify mutations, copy number changes, and their changes over time. We also performed in vitro functional assays on PBRM1 in RCC cell lines. Five patients had clear cell and one had chromophobe RCC. 434 somatic mutations in 416 genes were identified in the 12 tumor samples. 201 (46%) of mutations were clonal in both samples while 129 (30%) were acquired in the post-treatment samples. Tumor heterogeneity or sampling issues are likely to account for some mutations that were acquired in the post-treatment samples. Three samples had mutations in TSC1; one in PTEN; and none in MTOR. PBRM1 was the only gene in which mutations were acquired in more than one post-treatment sample. We examined the effect of PBRM1 loss in multiple RCC cell lines, and could not identify any effect on rapalog sensitivity in in vitro culture assays. We conclude that mTOR pathway gene mutations did not contribute to rapalog resistance development in these six patients with advanced RCC. Furthermore, mechanisms of resistance to rapalogs in RCC remain unclear and our results suggest that PBRM1 loss may contribute to sensitivity through complex transcriptional effects.

Fig 1. PBRM1 downregulation has no effect on growth inhibition by rapamycin in the ccRCC line 786-O.

(A) PBRM1 expression in 786-O cells stably expressing different PBRM1 shRNAs was assessed by immunoblotting. (B) 786-O cells stably expressing control shRNAs or PBRM1 shRNAs 889, 890 and 994 were treated with DMSO or 20nM rapamycin (RAPA) for 24, 48 and 72hr. Cell number was quantified using Crystal Violet, and normalized to day 1 (24 hr).

Fig 2. Rapamycin has similar effects in growth inhibition of RCC 704 cells independent of PBRM1 addback.

(A) Immunoblot analysis of the PBRM1 null A704 cell line stably transfected with doxycycline inducible constructs expressing empty vector (EV), wild-type PBRM1 (WT) or mutant Q1298* PBRM1, treated with or without doxycycline (Dox, 1ug/ml) for 5 days. (B) Growth of these same A704 derivative cell lines treated with or without doxycycline and with DMSO (control) or rapamycin (RAPA, 20nM) for 2, 4, or 6 days. Cell growth was assessed by Crystal Violet staining. (C) Proportional growth of A704 derivative cell lines derived from panel B. Rapamycin inhibited the growth of all 3 cell lines to a similar extent, and doxycycline treatment had little or no effect on growth. (D, E) These same A704 derivative cell lines were plated at 200 cells per 10cm dish and treated with or without Doxycycline and DMSO control or Rapamycin (20nM) for 30 days, then formalin fixed and stained with Crystal Violet. D, Image of colonies after crystal violet staining. E, Quantitation of colonies > 2mm in diameter per 10 cm dish. The bar is placed at the median value.