Purine Nucleotide Availability Regulates mTORC1 Activity through the Rheb GTPase

Abstract

Pharmacologic agents that interfere with nucleotide metabolism constitute an important class of anticancer agents. Recent studies have demonstrated that mTOR complex 1 (mTORC1) inhibitors suppress de novo biosynthesis of pyrimidine and purine nucleotides. Here, we demonstrate that mTORC1 itself is suppressed by drugs that reduce intracellular purine nucleotide pools. Cellular treatment with AG2037, an inhibitor of the purine biosynthetic enzyme GARFT, profoundly inhibits mTORC1 activity via a reduction in the level of GTP-bound Rheb, an obligate upstream activator of mTORC1, because of a reduction in intracellular guanine nucleotides. AG2037 treatment provokes both mTORC1 inhibition and robust tumor growth suppression in mice bearing non-small-cell lung cancer (NSCLC) xenografts. These results indicate that alterations in purine nucleotide availability affect mTORC1 activity and suggest that inhibition of mTORC1 contributes to the therapeutic effects of purine biosynthesis inhibitors.

The mTORC1 Signaling Network Senses Changes in Cellular Purine Nucleotide Levels

“…Interestingly, 16-hour treatment with AVN-944 depleted guanylates but not adenylates and lead to decreased Rheb protein levels in A549 cells, which were rescued with excess exogenous guanine, partially at 1 hour and fully when co-treated for the 16-hours with AVN-944 (Figure 7A, 7B, and S7A), the paradigm used by Emmanuel et al (Emmanuel et al, 2017). However, our laboratory stock of A549 cells exhibited complete resistance to this compound for effects on mTORC1 signaling, even at doses 10-fold higher than that which leads to Rheb depletion, further demonstrating a separation of the Rheb effects from those on mTORC1 (Figure 7C).…”

“…The study by Emmanuel et al (Emmanuel et al, 2017) also demonstrated that mTORC1 signaling is sensitive to purine nucleotides. However, the authors conclude that mTORC1 activity is inhibited by guanylate depletion through effects on Rheb-GTP binding and farnesylation leading to loss of Rheb lysosomal localization.…”

“…Just prior to submission of this manuscript, a study by Emmanuel et al (Emmanuel et al, 2017) showed that inhibition of the de novo purine synthesis pathway, with a compound that like LTX inhibits GART, also inhibits mTORC1 signaling. The authors conclude that mTORC1 activity is regulated by guanylate availability through a mechanism leading to decreased farnesylation of Rheb, whereas we find a dominant and acute role for adenylates.…”

“…As shown in Figure 6A, we also do not detect changes in endogenous Rheb that are consistent with defects in farnesylation, rather the protein is degraded over time with purine depletion using any one of multiple different purine synthesis inhibitors. However, the findings of Emmanuel and colleagues (Emmanuel et al, 2017) prompted us to determine whether the effects of long-term purine depletion on Rheb degradation could be through changes in guanylates, even if they were unrelated to the acute sensing of adenylates by mTORC1.…”

“…To more closely compare the differences between the two studies, we used the two lung cancer cell lines (A549 and A427) and IMPDH inhibitor (AVN-944) primarily used in the Emmanuel et al study (Emmanuel et al, 2017), and examined time-dependent effects on mTORC1 signaling and Rheb. Interestingly, 16-hour treatment with AVN-944 depleted guanylates but not adenylates and lead to decreased Rheb protein levels in A549 cells, which were rescued with excess exogenous guanine, partially at 1 hour and fully when co-treated for the 16-hours with AVN-944 (Figure 7A, 7B, and S7A), the paradigm used by Emmanuel et al (Emmanuel et al, 2017).…”