Abstract Introduction: Acute Myeloid Leukemia (AML) is a heterogeneous disease with cytogenetic abnormalities and gene mutations that are routinely used in clinical practice to stratify patients into three risk groups: low, intermediate, or high. Core binding factor (CBF) AML with inv(16) and t(8;21) involved in CBFP-MYH11 and RUNX1-RUNX1T1 gene fusions is associated with a better prognosis. Likewise, outcomes are inferior in patients with non-CBF (NCBF) AML. Despite efforts on genomics and transcriptomics to understand the AML heterogeneity, studies focused on proteomic profiles associated with AML cytogenetic features remain limited. Therefore, the objective of this study is to detect proteomic differences in CBF vs. NCBF pediatric AML as well as to identify proteomic markers associated with the therapeutic response after induction treatment. Methods: For this pilot study, we selected 16 patients (6 with CBF-AML and 10 with NCBF-AML) that were treated in the multi-center AML02 clinical trial (NCT00136084). Leukemic cells at diagnosis were used to generate an untargeted global proteomics profile using 16-plex TMT-LC/LC-MS/MS. Overall, 9800 unique proteins were identified and were evaluated for differences between CBF and non-CBF AML using non-parametric rank-sum statistical tests from the R Bioconductor RankProd package. An exploratory analysis was further performed to identify proteins associated with minimal residual disease after induction 1 (MRD1) in the 10 NCBF patients. Given 15 patients also had gene expression levels using Affymetrix U133A microarray available, Spearman's Rho correlation test between transcriptomics and proteomics was also performed. Results and Conclusion: As compared to CBF-AML, we identified 63 upregulated and 49 downregulated proteins (LogFC >1 and FDR <0.05) in NCBF-AML. The top 10 proteins included upregulated proteins CYP7B1, MYT1L, MMP8, MMP24, CTSG, MGLL, HOXD10, and downregulated proteins TPPP3, HLA-DRB3, MMP14. Within non-CBF AML patients, higher levels of MXRA7, DPYSL2, TNFSF13, PHGHDH proteins and lower levels of MMP8, KLKB1, VCAM, CEACAM1 proteins were predictive of MRD1 positivity after the first induction (LogFC>1, p<0.0001 and FDR <0.15). Though the sample size is small, we observed minimal statistically significant correlations (p<0.05) with 16.2% between leukemic cell transcriptome and proteome. This implies a potential for unleashing biological heterogeneity beyond cytogenetics using proteomics. Our ongoing work is focused on expanding the evaluation of proteomics with AML cytogenetic heterogeneity and clinical outcomes in a larger cohort of AML patients for identifying prognostic markers to refine patient classification and to discover novel therapeutic targets. Acknowledgments: This research was supported by NIH R01CA132946 and University of Florida Health Cancer Center Opportunity funds. Citation Format: Nam K. Nguyen, Huiyun Wu, Junmin Peng, Jeffrey E. Rubnitz, Stanley Pounds, Jatinder K. Lamba. Leukemic Cell Proteomic Profiling in Pediatric AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2535.