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Neuroblastomas account for approximately 15% of all childhood cancer deaths. Clinical complete remission is achieved in many stage 4 neuroblastoma patients, but the high risk of relapse and the accompanying treatment-resistant nature of these tumors is still a challenge. We have previously identified higher frequencies of mutations that affect both the p53 and the pRb pathway, such as the homozygous loss of CDKN2A or co-amplification of MDM2 and CDK4. In addition, both cyclin D1 and MDM2 overexpression is a common characteristic of primary neuroblastoma. These genes act upstream of pRb and p53 and hence play a major role in cell cycle regulation. To study whether these G1 checkpoint aberrations render cells more sensitive to CDK4 or MDM2 inhibition, we exposed a series of 11 cell lines to the most promising CDK4 (ribociclib, palbociclib, and abemaciclib) and MDM2 (SAR405838, HDM-201, and idasanutlin) inhibitors. These cell lines have well-defined mutational properties: homozygous CDKN2A deletion, co-amplification of CDK4 and MDM2, TP53 mutation, and MYCN amplification. Whereas sensitivity for the MDM2 inhibitors inversely correlated with TP53 mutation status, it did not correlate to other G1 checkpoint aberrations. Similarly, there was no clear correlation seen for the CDK4 inhibitors. To further explore this, we generated cell lines with inducible overexpression of CDK4, MDM2, or both. The overexpression did indeed not increase sensitivity to CDK4 or MDM2 inhibitors. Next, we were interested whether combined treatment with the most potent CDK4 and MDM2 inhibitors, abemaciclib and idasanutlin respectively, would be of added value. Cells were exposed to 10 different concentrations of both compounds and Bliss Independence values were calculated as a measure of synergy. Interestingly, combined treatment resulted in slight antagonism in the range of clinically relevant doses in most cell lines. This adverse effect was supported by cell cycle analyses, which showed a lower apoptotic fraction, as well as PARP and caspase 3 cleavage, after combined treatment, as opposed to MDM2 inhibition alone. As was previously suggested in the first clinical trials with CDK4 inhibitors, neither CDK4 nor CDKN2A status is a clear biomarker for CDK4 inhibitor sensitivity. Our results suggest that MDM2 and CDKN2A status also fail as biomarkers for MDM2 inhibitor sensitivity. Further testing is necessary to identify (other) biomarkers for these inhibitors. Moreover, the logical rationale to combine CDK4 and MDM2 inhibitors in neuroblastoma patients with both pRb and p53 pathway disturbances should be taken with precaution, as first results do not show a beneficial response. In vivo experiments to confirm this finding are currently ongoing. Citation Format: Nil Schubert, Linda Schild, Stijn van Oirschot, Kaylee Keller, Lindy Alles, Lindy Vernooij, Marloes Nulle, Emmy Dolman, Marlinde van den Boogaard, Jan Molenaar. Combined targeting of the p53 and pRb pathway in neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A49.