p53 has been an unwitting target of traditional cancer therapeutics since the development of radiotherapy and genotoxic chemotherapy. These treatments rely on the cellular DNA damage response for success, which is why they are often less effective in the ~50% of cancers with mutant p53. A recent flurry of drug discovery efforts has identified cleaner and safer strategies to harness the tumor suppressive power of p53 for selective elimination of cancer cells. This growing list includes targeted therapeutics such as Nutlin-3, which binds to and blocks MDM2; PRIMA-142
, which reactivates mutant p53; and RITA43
, which binds directly to p53 and stabilizes it. However, the clinical worth of these drugs is limited a priori
by the pleiotropic character of the p53 network. Depending on the context, p53 can promote survival or death. The key question then becomes: what determines the cellular response to p53 activation? We have employed Nutlin-3 as a paradigm to answer this question. Nutlin-3 elicits cell cycle arrest or apoptosis in a cell-type specific manner6,8
. Our functional genomics approach has successfully identified gene modules that enforce survival in Nutlin-3-resistant cells. This has led us not only to a greater understanding of the p53 network, but also to the identification of new combinatorial strategies to augment the therapeutic efficacy of Nutlin-3.
Our identification of ATM as an antagonist of p53 dependent-apoptosis was totally unexpected. Current dogma maintains that ATM activates p53 in response to DNA damage by phosphorylating specific serines in both p53 and MDM2, thus preventing their interaction and leading to p53 stabilization44
. According to this view, ATM functions as a p53 agonist during the apoptotic response to DNA damage37
. However, a uniform view of ATM in the response to genotoxic therapies is prevented by the observation that ATM has also been shown to activate pro-survival pathways upon DNA damage, such as the NF-κB pathway41,45
. In fact, the role of ATM can be switched from pro-survival to pro-apoptotic depending on the extent of DNA damage39
. As mentioned above, radiation and genotoxic drugs have been widely used in the clinic for years. One strategy to increase the efficacy of these treatments has been to target components of the DNA damage repair machinery. As such, inhibitors of upstream signaling kinases in these pathways (e.g. ATM, ATR, DNA-PK) have been employed to confer radio- and/or chemo-sensitivity upon cancer cells46
. Unfortunately, many of the known inhibitors of these kinases, including wortmannin and caffeine, lack specificity and inhibit a wide range of PI3K-like kinases46
. The development of KU-55933 imparts specificity on this system, targeting ATM with a much lower IC50 than previous inhibitors26
. However, even specific inhibitors would still rely on the introduction of high levels of DNA damage and its associated pleiotropic effects. Our finding that a combination of non-genotoxic p53 activation and ATM inhibition is capable of eliciting an apoptotic response in cancer cells is a conceptual breakthrough, as it does not induce a DNA damage response above stochastic levels, which is already high in tumors. This is the first report showing that ATM is required for survival upon pharmacological activation of p53 in the absence of exogenously introduced DNA damage. Future studies will investigate how exactly ATM promotes its anti-apoptotic effects in this scenario.
Our 53BP1 studies provide additional support for the notion that ATM activity can be decoupled from other components of the DNA damage response pathway. Previous work showed that 53BP1 knockdown allows cells to escape Nutlin-3-induced cell cycle arrest and impairs Nutlin-3-induced upregulation of p2138
. We confirm a role for 53BP1 in the anti-proliferative effects of Nutlin-3, and, conversely, show that ATM inhibition converts cell cycle arrest into apoptosis without affecting expression of p21. In this report the authors did not test the role of ATM directly, but used caffeine, a broad inhibitor of PI3Ks, which suggested that one or more PI3Ks were required for full induction of p21 and cell cycle arrest in their system. However, they used a cell line (MCF7) displaying constitutive DNA damage signaling as seen by H2A.X foci, and a dose of Nutlin-3 that did not produce maximum induction of p21. In contrast, our screen was performed in cells with no apparent constitutive DNA damage signaling and with a dose of Nutlin-3 that leads to maximum p21 induction. Thus, while their screen was prone to identify a role for DNA damage signaling in complementing pharmacological inhibition of MDM2 for cell cycle arrest, ours was not.
The MET network plays an important role in many malignancies. Upon activation by HGF, MET activates various intracellular pathways promoting proliferation, survival, and invasion47
. MET is hyperactivated or overexpressed in many cancers, making it an attractive target for therapeutic intervention22,29,47
. Crizotinib was originally developed as a MET-specific inhibitor30
, however, it also inhibits ALK (anaplastic lymphoma kinase)31
. ALK is a receptor tyrosine kinase within the insulin-receptor superfamily with roles in development and function of the nervous system31
. However, ALK is not essential, as knockout mice are viable and mostly normal31
. ALK is frequently translocated in lung cancer48
. Crizotinib underwent clinical trials for NSCLC patients with MET amplification or ALK translocations, which ultimately determined that in this case cancers with ALK translocations were better targets for Crizotinib49
. We show here that Crizotinib sensitizes cells to p53-induced cell death. This strongly implicates the MET pathway in survival signaling upon p53 activation, as none of the cell lines used in this study has ALK translocations or detectable ALK expression (Cancer Cell Line Project, Welcome Trust Sanger Institute). Furthermore, we found that a second MET inhibitor, SU1127432
, also sensitizes to Nutlin-3-induced apoptosis. Interestingly, although MET has been depicted as a target of p53 repression33
, we did not observe changes in MET expression upon non-genotoxic activation of p53 in Nutlin-3-resistant cells. However, we found that MET is strongly silenced in BV173 cells, which could partially explain why they undergo apoptosis upon Nutlin-3 treatment. These results not only reveal a new functional module affecting outcome to p53 activation, but demonstrate the ability of synthetic lethal screens to resuscitate drugs that may not have been effective as single agents, despite the great time and cost spent on their development.
Much of the research on mechanisms of cell fate choice to p53 activation has focused on p53-centric events, such as p53 post-translational modifications, p53-binding factors and regulation of p53 DNA binding activity. The overarching view generated by these studies is that cell fate choice is determined by differential transactivation of p53 target genes involved in cell cycle arrest (e.g. p21, 14-3-3σ) versus apoptosis (e.g. PUMA)1,17
. In contrast, our functional genomics approach revealed that the p53 response can be converted from cell cycle arrest into apoptosis without affecting expression of these key genes. Therefore, our results reinforce an alternative model where cell fate choice to p53 activation is governed by p53 autonomous mechanisms8,18,36
. This alternative view is more consistent with our increasing appreciation of cancer as disease of gene networks, rather than individual gene modules or linear pathways.
Although we focused our efforts on two key pathways, ATM and MET, we are fully aware that many other relevant SLN pathways identified in our screen remain unexplored. We hope that making the results of our screen available to the community will inspire other teams to identify additional strategies to modulate the p53 response for therapeutic purposes and to generate a greater understanding of the p53 network.