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Abstract: Myristoylation is the post-translational modification of proteins with a 14-carbon fatty- acid by N-myristoyltransferase 1 (NMT1) or NMT2. Myristoylation is key to protein membrane binding and cell survival. Bioinformatics data suggests NMT mRNA expression impacts overall survival (OS) in AML. We characterized NMT protein levels in AML patients. We performed a retrospective and prospective cohort study, including adult patients newly diagnosed with AML in Edmonton from April 2014 until September 2016, excluding acute promyelocytic leukemia. In addition, a small group of control marrow aspirates were obtained. We assayed marrow aspirate or peripheral blood for NMT1/2 protein levels by fluorescence activated cell sorting with intracytoplasmic staining by custom mouse anti-NMT1- Alexa-Fluor-647 or anti-NMT2-FITC, expressed as Mean Fluorescent Intensity (MFI) relative to IgG1k isotype control. NMT1/2 MFI was determined for the bulk blast, CD34+, and CD34+38- blast subpopulations. Clinical data was analysed by t-test or ANOVA. Relapse-free survival (RFS) and OS were dichotomized with a receiver operator curve, followed by a Kaplan-Meier analysis with a log-rank test for significance or univariate and multivariate Cox regression, with p < 0.05 as significant. Censoring, survival, and clinical parameters were defined by European LeukemiaNet (ELN) guidelines and REMARK criteria. Recruitment reached 105 patients. Median age was 67.1 years, with 57 ELN intermediate-risk patients. Median follow-up and OS are 1.54 and 0.95 years, respectively. NMT1 MFI was consistent through all samples. NMT2 MFI was higher in lymphocytes (mean MFI = 3.01 ± 0.08) vs. monocytes (mean MFI = 0.98 ± 0.03, p < 0.001) NMT2 MFI did not vary between AML bulk blast (mean MFI = 1.13 ± 0.03), CD34+ blast (mean MFI = 1.21 ± 0.03), and CD34+38- blast populations (mean MFI 1.22 ± 0.04) (p = 0.145). NMT2 MFI was significantly higher in control CD34+ cells (mean MFI = 1.65 ± 0.11, p < 0.001) and CD34+38- cells (mean MFI = 1.91 ± 0.17, p < 0.001) versus their AML counterparts. NMT2 MFI was significantly higher in AML with the cytogenetic abnormality inv(16) (mean MFI = 1.67 ± 0.05, p < 0.001), and slightly lower with mutated NPM1 (mean MFI = 1.09 ± 0.04, p = 0.004). NMT2 MFI was not associated with ELN risk groups or remission status. (p=0.166, 0.303). NMT2 MFI receiver operator curve analysis for OS in ELN intermediate-risk AML in CD34+38- cells generated a cut-off 1.28 (AUC=0.697, p=0.021) for sensitivity=50% and specificity=80%. Kaplan-Meier analysis was significant with respect to RFS in the CD34+38- population (p = 0.038) and OS in the bulk blast population (p = 0.005). Kaplan-Meier analysis was significant with respect to OS for bulk blast (p = 0.048) and CD34+38- population (p = 0.014) when analyzing the intermediate-risk, age < 65 years patient subgroup. NMT2 was not significant for RFS or OS versus the ELN scheme by univariate or multivariate Cox regression. NMT2 MFI for all intermediate-risk patients was not significant for RFS on univariate Cox regression, but was significant on multivariate regression (p = 0.019). NMT2 MFI for OS in all intermediate-risk patients was significant on univariate Cox regression (p = 0.007) but not significant on multivariate analysis. Age and WBC at diagnosis were used as covariates. We analyzed how NMT1 and NMT2 MFI relate to clinical factors, cytogenetics, molecular abnormalities, ELN risk group, and clinical outcomes in AML. NMT1 was not associated with any of these factors. NMT2 MFI was intermediate in normal hematopoietic stem cells, higher in lymphocytes, and lower in monocytes. This suggests that regulation of NMT2 protein levels may influence early lymphoid/myeloid lineage commitment, possibly by modulation of the T-cell receptor pathway. The cytogenetic abnormality inv(16) showed significantly higher NMT2 MFI, possibly secondary to control of NMT2 expression by the transcriptional regulators RUNX2 and RUNX3. NMT2 MFI was moderately lower in patients with NPM1. NMT2 MFI was not associated with ELN risk group, or achievement of CR with first induction chemotherapy. Higher NMT2 MFI generally predicted worsened outcomes in the ELN intermediate-risk population, with this effect being driven by the CD34+38- blast subsets. This may represent an indirect measure of the stemness of the blast population, and the LSC burden in patients with AML. NMT2 MFI may be a novel biomarker for prognosis in intermediate-risk AML, and further investigation is warranted.