Impact of Traditional and E-Cigarette Smoking Metabolites on Acute Myeloid Leukemia
Authors: Alqahtani Shaikha , Ma Huaxian , Figueroa Mary , Poche Alaina , Nam Deokhwa , Chandra Joya 
Published: December 2024

Introduction:
Acute myeloid leukemia (AML) is a highly aggressive hematological malignancy with a notably poor prognosis. FMS-like tyrosine kinase 3 (FLT3) mutant AML is linked to a worse prognosis, which is found in 25% of AML cases among adolescents and young adults (AYA). Cigarette smoking leads to reduced survival rates in patients with AML. Tobacco products, including cigarettes, smokeless tobacco, and e-cigarettes contain nitrosamines like N′-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Many of these tobacco metabolites have been associated with oxidative stress and adaptive responses which may confer resistance to chemotherapy but have not been examined in the context of AML and smoking. Since AYA cancer patients are prone to unhealthy habits such as smoking and vaping, understanding the effects of nicotine metabolites on FLT3-mutant AML has potential to lead to better clinical management of patients who use or have used tobacco products.
This study investigates the effects of cigarette smoke condensate (CSC) and tobacco-specific nitrosamines (TSNAs) on FLT3-mutated AML cell lines and in vivo models to elucidate how smoking compounds influence AML progression and oxidative stress responses.
Methods:
FLT3-ITD-mutant AML cell lines (MOLM-13, MOLM-14) and non-FLT3 OCI-AML3 cells were treated with cigarette smoke condensate (CSC), NNN, and NNK, or vehicle controls for 2 weeks. Cell viability and proliferation were assessed using the trypan blue exclusion method. Oxidative stress was evaluated by measuring intracellular glutathione (GSH), peroxide, and superoxide levels at days 2, 5, 9, and 14 post treatment. In vivo, NOD-SCID mice aged 6-8 weeks, were xenografted Luciferase-tagged MOLM-13 cells we treated with CSC, NNN, or NNK or vehicle for 2 weeks prior to injection. Tumor growth was monitored bi-weekly using bioluminescence imaging for 22 days until sacrifice. Mice tissues were analyzed for heme oxygenase-1(HO-1) expression using immunohistochemistry assay (IHC) in spleen and liver tissues. Statistical analysis was performed using GraphPad Prism version 10.
Results:
In vitro, exposure of MOLM 13 cells to CSC (10 μg/mL), NNN (1 μM), and NNK (1 μM) every 48-72 hours for a total duration of 2 weeks did not significantly affect cell proliferation and viability.There was an adaptive oxidative stress response, marked by initial GSH decline followed by compensatory increase by day 9 after MOLM 13 treatment specifically in NNK and CSC group. In vivo, CSC exposure had the highest leukemia burden by imaging compared to NNN, NNK and vehicle.
Interestingly, females had a higher leukemia burden by imaging, which was most pronounced on day 18 after injection in the NNK-treated group. In contrast, the leukemia burden in males did not show any significant difference. IHC of HO-1 which is an indicative of oxidative stress response, was significantly upregulated in female liver tissues exposed to NNK and to lesser degree in NNN group. As for spleen tissues, HO-1 expression was significantly upregulated in NNK group in both males and females.
Conclusion
This study highlights the significant impact of smoking metabolites on AML progression and emphasizes the need for further research into underlying mechanisms. Gender differences in response to nitrosamines but not CSC suggest sex-specific factors in evaluating smoking-related cancer risks. Contrasting in vitro and in vivo findings stress the importance of considering the tumor microenvironment impact. Future research will explore tobacco products' impact on AML outcomes and whether oxidative stress mediates accelerated leukemia growth. Understanding these mechanisms could inform targeted therapies and improve survivorship in AML patients with a smoking history.