The RAS oncogene hits the brake pedal on P53.
We show that mutations in the RAS oncogene sensitizes cells to drugs that target replication stress. We found that overactive RAS blocked the gene transcription of P53. These findings suggest that cancer patients with intact P53 but mutant RAS may still benefit from ATR or CKH1 inhibitors.
Understanding how cancer cells could deal with replication stress.
Cancer cells override cell cycle checkpoint mechanisms to force themselves to proliferate. A notorious example are mutations of the RAS oncogene. However, this forced cell cycle progression comes at the cost of DNA replication stress. Replication stress can be defined as the stalling and collapsing of the replication machinery. This can cause serious DNA damage, such as double-stranded breaks. However, cells can mitigate replication stress by activating the intra-S-phase checkpoint. This checkpoint involves a cascade of enzymes including ATR, CHK1, and WEE1. Thus cancer cells experience replication stress and rely heavily on the intra-S-phase. Hence drugs against these enzymes have been developed and are currently being evaluated in cancer patients. However we still have an incomplete picture of which specific gene mutations render cancer cells sensitive to these drugs.
Consequences of mutant RAS on drug responses.
We set out to perform mechanistic studies in a very well-controlled model system of cultured cells. We introduced an inducible gene construct encoding HRAS with a well-known mutation (G12V) in an otherwise normal cell line (RPE-hTert), as well as in a cancer cell line (U2OS). We also engineered those cells to express fluorescent markers that reveal the cell cycle phase as well as replication stress-induced DNA damage (FUCCI4 system and truncated 53BP1 respectively). Thus we were able to carefully monitor responses to replication stress-inducing drugs, and monitor the fates of individual cells.
Gene transcription events underlie cell fate decisions
We found that the activation of mutant RAS made cells much more sensitive to treatment with an ATR or CHK1 inhibitor in combination with a low dose of gemcitabine - a widely used therapeutic drug. Our careful genome-wide gene expression analysis in replicating cells showed that the only acute effect of these drugs was an increase in P53 target genes. Initially, we were somewhat disappointed by this, because P53 is an extremely well-described transcription factor, and it is known to activate a gene expression program that can arrest or repair cells after DNA damage.
P53 response with a twist
However, our enthusiasm increased a lot when we realized that this P53-dependent response was strongly reduced in the cells expressing mutant RAS. And even more surprisingly, we saw that the mRNA levels of TP53, which encode the P53 protein, were also reduced. This was surprising because the current paradigm is that P53 activity is almost exclusively controlled by the speed of its degradation via a protein called MDM2. However, our data now clearly demonstrate that RAS-signaling can strongly affect the P53 program simply by repressing the gene transcription of P53. We propose that transcription of P53 should never be overlooked when studying DNA damage responses.
We were not able to identify which transcription factor downstream of RAS was responsible for the repression of P53, and unbiased approaches to screen for such factor will need to be done in the future. Furthermore, it remains to be investigated how our findings can eventually be translated to cancer patients. Our data suggest that P53 does not have to be mutated in order for cancer cells to be sensitive to ATR or CHK1 inhibitors: RAS mutations can indirectly inactivate P53 too. Previous work demonstrated that P53 mutations sensitizes cells to these inhibitors, but phase I and phase II clinical trials failed to show a clear difference between drug responses in P53-mutant versus P53-wild type cancers. Our novel RAS-P53 connections indicate that cancers can deploy multiple routes to P53 inactivation. We hope that cancer patients carrying RAS mutations can also benefit from this class of drugs in the future. Interestingly, we also knocked down P53 in RAS-mutant cells and we observed that this combination sensitized cells even further to CHK1 inhibitors. A combination of RAS mutations and P53 mutations is seen in a substantial number of patients, and perhaps these patients could benefit most from inhibitors of the intra-S-phase checkpoint. The search to identify the optimal mutation spectrum to predict which individual patient can benefit most from ATR- or CHK1-inhibitors is far from over.