KO of 5-InsP7 kinase activity transforms the HCT116 colon cancer cell line into a hypermetabolic, growth-inhibited phenotype

The inositol pyrophosphates 5-InsP(7) (diphosphoinositol pentakisphosphate) and 1,5-InsP(8) (bis-diphosphoinositol tetrakisphosphate) are highly energetic cellular signals interconverted by the diphosphoinositol pentakisphosphate kinases (PPIP5Ks). Here, we used CRISPR to KO PPIP5Ks in the HCT116 colon cancer cell line. This procedure eliminates 1,5-InsP(8) and raises 5-InsP(7) levels threefold. Expression of p53 and p21 was up-regulated; proliferation and G1/S cell-cycle transition slowed. Thus, PPIP5Ks are potential targets for tumor therapy. Deletion of the PPIP5Ks elevated [ATP] by 35%; both [ATP] and [5-InsP(7)] were restored to WT levels by overexpression of PPIP5K1, and a kinase-compromised PPIP5K1 mutant had no effect. This covariance of [ATP] with [5-InsP(7)] provides direct support for an energy-sensing attribute (i.e., 1 mM K-m for ATP) of the 5-InsP(7)-generating inositol hexakisphosphate kinases (IP6Ks). We consolidate this conclusion by showing that 5-InsP(7) levels are elevated on direct delivery of ATP into HCT116 cells using liposomes. Elevated [ATP] in PPIP5K(-/-) HCT116 cells is underpinned by increased mitochondrial oxidative phosphorylation and enhanced glycolysis. To distinguish between 1,5-InsP(8) and 5-InsP(7) as drivers of the hypermetabolic and p53-elevated phenotypes, we used IP6K2 RNAi and the pan-IP6K inhibitor, N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine (TNP), to return 5-InsP(7) levels in PPIP5K(-/-) cells to those of WT cells without rescuing 1,5-InsP8 levels. Attenuation of IP6K restored p53 expression but did not affect the hypermetabolic phenotype. Thus, we conclude that 5-InsP(7) regulates p53 expression, whereas 1,5-InsP(8) regulates ATP levels. These findings attribute hitherto unsuspected functionality for 1,5-InsP(8) to bioenergetic homeostasis.