Abstract Introduction: MYCN amplification is the genetic aberration most consistently associated with poor outcomes in alveolar rhabdomyosarcomas (aRMS) and neuroblastoma (NBL). It regulates key oncogenic processes including tumor cell survival, proliferation and metastasis. In aRMS, MYCN transcription is driven by the PAX3-FOXO1 gene fusion. MYCN and other short half-life mRNA transcripts depend on CDK9 to drive transcription of target genes to initiate and maintain the oncogenic program, making CDK9 blockade an effective therapeutic route against MYC-driven cancers. We have previously shown the activity and tolerability of enitociclib in tumor xenograft models. In this study, we investigated the activity of the selective small-molecule CDK9 inhibitor enitociclib (VIP152/BAY1521152) in preclinical models of pediatric MYCN-amplified aRMS and NBL. Methods: Cell-based viability assays were performed in aRMS (Rh30 and Rh41) and NBL (LAN1, SK-N-AS, SK-N-BE(2) and SK-N-MC) cell lines. Enitociclib antitumor activity was measured by Alamar Blue cell viability assay after 96 h exposure at 9 dose levels (8 nM-2 μM) with DMSO used as control. We performed combination screens of enitociclib with several FDA-approved drugs (n=215). Selected agents with IC50 values <1 μM were analyzed at constant dilution ratios of two inhibitors. Drug synergy was calculated by established methods using SynergyFinder 3.0. Target modulation was determined for RNA polymerase II (RNAPII), MYCN and apoptosis inducers caspase-3 and PARP, in addition to PAX3-FOXO1 and PLK1 protein levels in aRMS cells, by western blotting after continuous exposure with enitociclib for 24 h. Results: Significant cytotoxic activity, with IC50 values ranging from 48-182 nM and 39-123 nM was seen for aRMS cells and NBL cells, respectively. SynergyFinder found synergistic effects of enitociclib with irinotecan, carfilzomib, etoposide, bortezomib, selinexor or topotecan tested at clinically relevant concentrations in the aRMS cell line Rh41. Enitociclib induced apoptosis with cleavage of caspase-3 and PARP by western blotting in a dose- and time-dependent manner in addition to the depletion of phosphorylated RNAPII (Ser2) and MYCN protein levels. In aRMS cells, PAX3-FOXO1 fusion and PLK1 proteins were downregulated. Annexin V/propidium iodide staining confirmed a dose-dependent increase in apoptosis after enitociclib exposure. Conclusions: Our results suggest that CDK9 inhibition is potentially clinically relevant for MYCN-amplified solid tumors such as aRMS and NBL due to its significant antitumor activity and pharmacologic targetability. The data provide essential information on the distinct targets, biomarkers of activity and clinically feasible drug combinations for the development of enitociclib in clinical trials addressing an unmet need in pediatric oncology. Citation Format: Son Tran, Patrick Sipila, Melanie M. Frigault, Amy J. Johnson, Joseph Birkett, Raquel Izumi, Ahmed Hamdy, Beatrix Stelte-Ludwig, David P. Douglass, Anne-Marie Langevin, Norman J. Lacayo, Aru Narendran. Targeting CDK9 via the small-molecule inhibitor enitociclib as a therapeutic strategy to treat MYCN-amplified rhabdomyosarcoma and neuroblastoma in children [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1558.