Philip Earwaker

The outlook for patients with advanced renal cell cancer (RCC) has been improved by targeted agents including inhibitors of the PI3 kinase (PI3K)-AKT-mTOR axis, although treatment resistance is a major problem. Here, we aimed to understand how RCC cells acquire resistance to PI3K-mTOR inhibition. We used the RCC4 cell line to generate a model of in vitro resistance by continuous culture in PI3K-mTOR kinase inhibitor NVP-BEZ235 (BEZ235, Dactolisib). Resistant cells were cross-resistant to mTOR inhibitor AZD2014. Sensitivity was regained after 4 months drug withdrawal, and resistance was partially suppressed by HDAC inhibition, supporting an epigenetic mechanism. BEZ235-resistant cells up-regulated and/or activated numerous proteins including MET, ABL, Notch, IGF-1R, INSR and MEK/ERK. However, resistance was not reversed by inhibiting or depleting these pathways, suggesting that many induced changes were passengers not drivers of resistance. BEZ235 blocked phosphorylation of mTOR targets S6 and 4E-BP1 in parental cells, but 4E-BP1 remained phosphorylated in resistant cells, suggesting BEZ235-refractory mTORC1 activity. Consistent with this, resistant cells over-expressed mTORC1 component RAPTOR at the mRNA and protein level. Furthermore, BEZ235 resistance was suppressed by RAPTOR depletion, or allosteric mTORC1 inhibitor rapamycin. These data reveal that RAPTOR up-regulation contributes to PI3K-mTOR inhibitor resistance, and suggest that RAPTOR expression should be included in the pharmacodynamic assessment of mTOR kinase inhibitor trials.

BACKGROUND: In patients with advanced hepatocellular carcinoma (HCC), the multikinase inhibitor sorafenib is the only systemic treatment that has been shown to increase overall survival. However, similar to other tyrosine kinase inhibitors, most patients achieve disease stabilisation radiologically, and only 2-3% of patients achieve a partial response. Recent exploratory subgroup analyses of the large phase 3 trials have demonstrated that patients with chronic hepatitis C virus (HCV) infection associated HCC survive longer than those who are negative for HCV. The mechanism underlying this currently remains unknown. A small number of cases of complete response to sorafenib treatment have now been reported worldwide, however a prolonged response has only been reported in 2 cases, both of whom had HCV-related HCC. CASE PRESENTATION: A 55 year old gentleman was diagnosed with hepatocellular carcinoma and concomitant chronic hepatitis C viral infection. He progressed following transarterial chemoemoblisation treatment and was commenced on sorafenib treatment. His serum alphafetoprotein level normalised within 2 months of treatment and he achieved an almost complete radiological response. This response was maintained for 20 months before the patient progressed. A 75 year old lady was diagnosed with advanced hepatocellular carcinoma and concomitant chronic hepatitis C viral infection. She was commenced on sorafenib treatment but required early dose reductions due to palmar plantar erythrodysesthesia, and liver decompensation. Despite this she achieved an excellent serological and radiological response that was maintained for 24 months. CONCLUSIONS: Our two cases show that patients with HCV-associated HCC can attain excellent responses to sorafenib treatment that is durable. Furthermore, such exceptional responses can be achieved even with dose reductions and treatment breaks.

<b>Introduction</b>: Advanced clear cell renal cell cancer (CCRCC) is incurable, but molecularly targeted treatments have improved the prognosis. One such molecular target is the mammalian target of rapamycin (mTOR) and two rapalogues (everolimus and temsirolimus) have been licensed for the treatment of advanced CCRCC, but resistance to treatment is a significant problem. Novel mTOR kinase inhibitors show more complete blockade of mTOR downstream targets, and greater <em>in vitro</em> anti proliferative activity, although resistance remains a problem. <b>Hypotheses</b>: 1) CCRCC cell lines, resistant to mTOR kinase inhibition could be generated <em>in vitro</em> through continuous culture in drug, and 2) mediators of resistance could be identified through screening cells at the mRNA and protein/phosphoprotein level, or by examining the signalling in resistant cells to identify the re-activation of key mTOR targets. <b>Methods</b>: Resistant cells were created by continuous culture in the PI3K-mTOR kinase inhibitor BEZ235. Resistance was assessed by CellTiter-Glo viability assays and clonogenic assays. mRNA microarray and antibody arrays were conducted. Intra-cellular signalling was assessed by western blotting. The functional relevance of identified markers of resistance were assessed with small molecule inhibitors and siRNA protein depletion. <b>Results</b>: BEZ235 resistant cells were created and had a 14 fold higher growth inhibitory 50 (GI₅₀) concentration compared to sensitive cells (99 nM vs. 7 nM). Array screening of the cells identified markers, but not mediators of resistance (MET, Abl, MEK/ERK and the Notch pathway). In BEZ235 resistant cells, the downstream mTOR target 4E-BP1 was rephosphorylated, despite evidence of on-going blockade of another mTOR target, S6. Recovery of 4E-BP1 phosphorylation was associated with increased protein expression of the mTOR binding partner RAPTOR, and could be reduced by depletion of RAPTOR, or by the addition of rapamycin. Both of these interventions partially reversed BEZ235 resistance. <b>Conclusions</b>: RAPTOR protein up-regulation, represents a novel mechanism of resistance to mTOR kinase inhibition and can be partially overcome by rapamycin. The combination of BEZ235 and rapamycin warrants further investigation to evaluate its potential to overcome resistance to mTOR kinase inhibition in RCC.