Primary resistance to ATP-competitive mTOR inhibitors for the treatment of solid tumors

The mammalian target of rapamycin (mTOR) functions to integrate nutrient and energy availability with extracellular growth factor signals to regulate macromolecular biosynthesis including protein translation and lipogenesis. This essential gene in conserved from yeast to humans and is a core metabolic regulatory element. Aberrant regulation of metabolism is a phenotype recognized as a hallmark of cancer, and it has been shown that increases in translation alone can be oncogenic. Additionally, many of the most common oncogenes in cancer alter the growth factor signaling network upstream of mTOR and these lesions likely activate mTOR in many cancers. Thus, repression of mTOR activity is an emerging therapeutic strategy for human cancer and there is both strong mechanistic and epidemiological evidence to suggest that attenuation of mTOR signaling may be broadly applicable. As a kinase with a defined small-molecule binding pocket, mTOR presents an attractive target for therapeutic intervention because of its conserved role in integrating many different oncogenic lesions and its central requirement for metabolic regulation./nThe successful application of mTOR inhibitors to clinical oncology will require the development of potent and selective inhibitors of this kinase and equally importantly, an understanding of which oncogenic lesions mark tumors as specifically sensitive to mTOR inhibition as well as independent biomarkers for in vivo efficacy. Fortuitously, mTOR is inhibited with near perfect selectivity by the natural product rapamycin, and analogs of this compound have been approved for the treatment of solid tumors. Rapamycin inhibits mTOR through a non-conserved non-competitive mechanism of action and only blocks the phosphorylation of certain substrates. ATP-competitive inhibitors of mTOR have recently been invented that occupy the kinase active site and block all substrate phosphorylation. In many preclinical models they are significantly more potent than rapamycin and as they enter clinical trials, questions about how to maximize their therapeutic index naturally arise. This may be challenging to ask for mTOR because it is not mutated in cancer and what genetic lesions mark cancers as susceptible or not to mTOR inhibitors has not been determined./nTo address the question of how best to apply ATP-competitive mTOR inhibitors to human cancer, I performed a large (~650) unbiased cell screen to identify markers for sensitivity and resistance to PP242, a tool compound that has been developed into the phase I clinical drug, MLN0128. In comparison to rapamycin, PP242 was a more effective compound and more cell lines were inhibited. Colon origin was significantly associated with resistance to both drugs. For PP242, mutations in the gene PIK3CA were a marker of sensitivity. I subsequently focused on colon cancer because it gave the strongest mTOR drug dependent signature, and one of resistance. A panel of mTOR and PI3K pathway inhibitors differentiated colon cancer cell lines based on RAS and PIK3CA genotypes. Ordering of colon cancer cell lines by sensitivity to PP242 revealed a striking resistance to mutations in KRAS within the already resistant colon cancer set. I identified that this KRAS specific resistance was due to a specific failure to inhibit phosphorylation of the translational repressor 4E-BP1 even when other mTOR substrates were inhibited. Resistance correlated with the amount of KRAS in the active GTP-bound form and was independent of canonical mitogen activated protein kinase signaling. Finally, introduction of mutant PIK3CA can sensitize even KRAS mutant colon cancer cells to PP242 and the mechanism is correlated with 4E-PB1 phosphorylation./nIn colon cancer cell lines I identified predictive markers of sensitivity and resistance and a biomarker that reported upon functional inhibition of mTOR in vivo. Cell lines have well documented shortcomings, and I worked to characterize a colon cancer patient-derived xenograft (PDX) model and apply it to early drug discovery to validate these findings. The PDX model uses metastatic colon cancer removed from patients that is then propagated in nude mice. Each patient tumor can be expanded into a cohort of identical tumors, and a drug trial can be conducted on an individual's tumor. In the colon cancer PDX model, PP242 was orally bioavailable and acutely inhibited mTOR signaling in many tumors. In a continuous dosing trial however, sensitivity to the drug paralleled what was observed in cell lines. Single KRAS mutant tumors were refractory to treatment. Overall, failure to durably inhibit 4E-BP1 phosphorylation correlated with failure to respond to treatment./nI discovered a set of cell lines that were resistant to the ATP-competitive mTOR inhibitor PP242 and identified a defect in inhibition of 4E-BP1 phosphorylation as giving rise to this phenotype. This mechanism of resistance appears to be common in KRAS mutant colon cancer cell lines as well as patients. The differential inhibition of distinct mTOR substrates I discovered reveals an additional layer of as yet uncharacterized biological control in this kinase signaling pathway. 4E-BP1 is a robust biomarker for ATP-competitive drugs and should be strongly considered for clinical use in trials of these agents.

Advisor: Shokat, Kevan M