Childhood cancers are the main cause of death for disease in children between 0 and 15 years of age. Although many advances have been done in their treatment, patients with high-risk and aggressive tumors still have a very poor prognosis. In this context, neuroblastoma and medulloblastoma are certainly the more common high-risk cancers, especially in the cases in which MYCN amplification occurs. Hence, searching a new effective therapy is extremely needful.
MYCN, like others oncogene, induces replication stress, a condition in which replication fork progression is prematurely interrupted leading to fork stalling or collapse. This requires activation of several cellular response to restrain it and to avoid DNA damage and finally to preserve cell survival. Therefore we believe that interfering these protective mechanisms could exacerbate MYCN-induced replication stress leading cancer cells to death.
It is well documented the role of PARP and CHK1 proteins in both replication stress response and DNA damage repair. In addition, we have recently shown that PARP and CHK1 inhibition enhances replication stress and causes mitotic catastrophe in MYCN amplified and overexpressing neuroblastoma cells, in agreement with their firmly established role in controlling RS and DNA repair.
Based on these findings obtained in cellular models, we propose to translate them into in vivo models using two tumor xenograft: human MYCN amplified neuroblastoma cells and mouse tumor-derived medulloblastoma spheres, in order to validate the anti-tumor activity of PARP and CHK1 inhibition.
The drugs we have planned to use are already approved for human administration. Therefore, by eventually showing that targeting the replication stress response by their means is possible and effective, our work will be immediately translatable into clinical trials for a number of MYC(N)-dependent deadly tumors including, but not limited to, neuroblastoma and medulloblastoma.
Understanding the molecular basis of aggressive and therapy-resistant tumors is a challenge in cancer research. Many efforts have been made to counteract this disease with successful results, but high-risk and rare tumors as well as some childhood cancers, still do not have an effective therapy. Therefore, attempting to gain this purpose is an absolute priority. Our project propose a novel route to target MYC(N)-dependent tumors.
As widely reported, MYCN is one of the main oncogenes that, if amplified or overexpressed, induces RS and cancer transformation. Being an ¿undruggable¿ target so far, many emerging studies are focused on targeting MYCN indirectly: one of the most promising theory is to target MYCN-dependent RS.
PARP and CHK1 proteins have a pivotal role in RS control and in DNA repair. PARP proteins are a family of nuclear proteins that catalyze mono and poly ADP-ribosilation (PARylation) of target substrates involved in many cellular processes. PARP1 and PARP2 have long been known to function as DNA damage sensors and, through their PARylation activity, as recruiters of DNA repair factor. A role for PARP molecules in replication stress had been previously established (Bryant HE et al., 2009). More recently, PARP activity was shown to act in concert with RECQ1 helicase and RAD51, to stabilize reversed forks induced by multiple genotoxic treatments and/or by oncogene-induced RS, thus preventing their collapse and the consequent accumulation of DNA damage (Berti M et al., 2013; Neelsen KJ et al., 2013; Ray-Chaudhuri A et al., 2012).
ATR-CHK1 axis is the most important pathway activated in RS-response to ensure the stabilization and the repair of the replication forks, and to prevent mitosis entry with unreplicated or damaged genome. Indeed, CHK1 the first kinase activated by ATR, activates in turn a number of targets that halt cell cycle in S and G2 phases under RS and is frequently upregulated in cancer cells with high proliferative index. Moreover, the expression of Chk1 is under the control of oncogenes such as Myc or E2F, which might be responsible for its enhanced levels in certain tumors (Verlinden L et al., 2007; Houglund A et al., 2011). Thus, targeting RS via PARP and CHK1 inhibition may be a relevant new strategy to counteract cancer progression.
In this scenario, PARP and CHK1 simultaneous inhibition increases the level of RS in MYCN amplified and overexpressing neuroblastoma cells, leading cancer cells in mitosis with unrepaired DNA, eventually culminating in mitotic catastrophe (Colicchia V et al., 2017). Although other studies proved the efficacy of this combination scheme in tumors such as mammary, ovarian and gastric cancer, little is known about its outcome in pediatric tumors and in particular in RS-dependent tumors (Booth L et al., 2013; Kim H et al., 2016; Yuping Y et al., 2017). Hence, based on our recent work and our solid preliminary data, we suppose that targeting RS via PARP and CHK1 inhibition may be a new therapeutic strategy also in pediatric MYCN-dependent tumors. Addressing this hypothesis in animal models will be extremely needful to test the anti-tumor activity of PARP inhibitors in combination with CHK1 inhibitors in vivo and to pave the way to its translation in clinical trials for human neuroblastoma and medulloblastoma treatment.
Based on the extensive literature also supporting our findings, our project appears particularly feasible. Indeed, even if we prove the efficacy of different PARP (olaparib and talazoparib) and CHK1 (PF-477736 and MK-8776) inhibitors in vitro, we choose for our in vivo experiments the most clinically validated olaparib and MK-8776. We also propose to use doses translated from previously human studies to mice administration according the rule of body surface area (Reagan-Shaw S et al., 2007) in order to accomplish safe and effective doses in animal models. In addition, the introduction of CHK1 inhibitors in cancer therapy has been strongly limited by their toxicity, so far. In contrast, our observations in vitro suggest the possibility to reduce the effective doses by combination of CHK1 and PARP inhibitors, probably extending their therapeutic index for the treatment of MYCN-dependent tumors. Finally, the protocol for animal experimentation has already been approved by the Italian Ministry of Health to Prof. Giuseppe Giannini.
For all these reasons, we believe that our project may produce reasonable progresses in MYCN-dependent tumors treatment, such as neuroblastoma and medulloblastoma.