Cigarette Smoking Is Associated with Worse Prognosis in MDS and Causes Inflammation and Hematopoietic Alterations in Murine Models

Introduction:

Cigarette Smoking and associated carcinogens have been associated strongly with solid tumors but the causative links with myeloid malignancies have not been well elucidated. Smoking has also been linked with clonal hematopoiesis and blood mutations, but it is not clear whether this is due to direct genotoxic effects of carcinogens or other more indirect mechanisms. We explored the effects of smoking on hematopoiesis in vivo by developing novel mouse models of exposure with a smoke machine and also with intra-tracheal administration of smoking condensates.

Methods:

A retrospective cohort of Myelodysplastic syndrome (MDS) patients was developed, using the Montefiore Einstein Cancer Center Electronic Data Warehouse and clinical outcomes were correlated with smoking histories. Mice were exposed to cigarette smoking in a smoke machine 3 times a week, with 6 cigarettes per day exposure. Cigarette smoke condensate was used for chemical analysis and used for intra-tracheal treatment of an independent cohort of mice for systemic measures of inflammation and hematopoietic function.

Results:

Our cohort of MDS patients (N=128) included 37% Hispanic and 29% African American cases, with median age of 75yrs. Overall survival was found to be significantly reduced in subjects with previous smoking histories (N=78) when compared to never smokers (N= 58), (log rank P value = 0.04; Median Survival for smokers was 2130 days vs 4620 days in never smokers). This difference was not due to significant differences in usual prognostic variables (age, sex, IPSS, blast counts, mutations) between the groups.

Next, to determine the effects of smoking in vivo, we optimized a cigarette smoke machine (Busco Tobacco Smoke Generator) that allowed us to expose mice (C57B/6) to 6 cigarettes a day, 3 times a week for one month. Mice exposed to cigarette smoking exhibited signs of reduced activity and showed raised levels of inflammatory mediators, Interleukin-1b (IL1b) and histamine, while the anti-inflammatory protein Interleukin -10 decreased when compared to controls. Mice exposed to cigarette smoking showed reduced white cell counts. Increased hemoglobin was also seen reflecting exposure to smoke. FACs analysis of bone marrow stem and progenitor populations did not reveal any quantitative differences. To assess for qualitative alterations we performed competitive transplantation experiments using CD45.2 donors exposed to smoking using CD45.1 recipients. Smoking exposure led to reduced donor chimerism at 4 months after transplants, when compared to controls. This difference was reduced at 8 and 12 months, demonstrating that smoking exposure led to reduced progenitor functionality, reflected in early chimerism defects.

Cigarette smoke condensate (CSC) collected from the smoke machine's artificial lung was chemically analyzed and showed high levels of nicotine and known carcinogens Acetaldehyde, Formaldehyde, Benzo(a)pyrene, Toluene, Benzene, and Nitrosamines (NNN, NAT, NAB, NNK).

Next to determine the exact functional effects of chemicals in the CSC, without the effects of the smoke itself, we administered CSC via intratracheal injections chronically for 4 months (3 times a week) to an independent cohort of mice. Mice again demonstrated an increase in inflammatory IL-1b and histamine and murine bronchial macrophages treated with CSC exhibited to an inflammatory transcriptomic signature and proteomic validation of increased IL-1b. Blood counts showed a significant decrease in white blood counts demonstrating bone marrow suppression. Lastly, treatment of primary human CD34+ stem and progenitors with CSC resulted in a decreased production of red and white blood cell colonies, validating the in vivo findings of bone marrow suppression.

Conclusion: We demonstrate that history of cigarette smoking is associated worse overall survival in MDS. We also demonstrate that exposure to cigarette smoking as well as to the chemicals in smoke condensate lead to inflammation and hematopoietic alterations in murine models. These novel data suggest that systemic inflammation and associated hematopoietic suppression are potential causes of adverse outcomes seen in myeloid malignancies in smokers.

Verma:Curis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Halia: Research Funding; Calico: Membership on an entity's Board of Directors or advisory committees; Bioconvergent health: Current equity holder in private company; Clinstreet: Current equity holder in private company; Bristol Myers Squib: Research Funding; Prelude: Research Funding; Stelexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Shastri:NACE & PeerView: Honoraria; Kymera: Research Funding; Jassen: Consultancy; Gilead, Rigel, Kymera: Consultancy; Ryvu therapeutics: Research Funding; Geron: Speakers Bureau.