Abstract
Certain somatic mutations result in clonal expansion of mutated hematopoietic cells — the phenomenon known as clonal hematopoiesis (CH). CH accelerates with age and is associated with an ~11-fold increased risk of hematologic malignancies and all-cause mortality. Recent evidence has also associated CH with lung cancer and emphysema, which is the most severe form of chronic obstructive pulmonary disease (COPD). A recent clinical study suggested that one of the most common genetic mutations in CH, ASXL1 mutation (mt), was strongly associated with CH in individuals with a history of cigarette smoking, which is strongly linked to emphysema. However, the mechanisms by which ASXL1 mutations are linked to smoking and contribute to emphysema are not fully understood.
To examine the association between ASXL1 mutationsand smoking, we established a mouse model using treatment with nanoparticulate carbon black (nCB), which mimics heavy smoke exposure-induced emphysema. Mice were administered nCB intranasally three days a week over four weeks to promote chronic lung inflammation, alveolar destruction, and emphysema. One year after nCB exposure, we performed deep duplex sequencing on the peripheral blood of mice with or without emphysema to examine the development of natural CH clones. Strikingly, expanded Asxl1-mutant hematopoietic clones were more prevalent in mice with emphysema compared to other common CH-associated mutations such as Dnmt3a and Tet2, mirroring observations in humans.
Next, since COPD and CH are correlated, we investigated whether the Asxl1 mutation in hematopoietic cells contributes to emphysema in the context of nCB exposure. We used Asxl1Y588X transgenic mice (Asxl1-mt), which express a C-terminal–truncated ASXL1 protein in the hematopoietic system (Vav1 promoter). After administering nCB to 8-week-old Asxl1-mt or WT mice, the animals were maintained for an additional 4 weeks. Significantly more innate immune cells were detected in lungs of Asxl1-mt compared to WT mice. Histological analysis of the lungs revealed that Asxl1-mt mice exhibit a higher prevalence of emphysema in the context of nCB. Next, we examined the levels of cytokine expression in the lungs to determine why the Asxl1-mt hematopoietic cells expand abnormally. Inflammatory cytokines, G-CSF and GM-CSF, which promote cell proliferation and inflammation, were more abundant in lungs of Asxl1-mt compared to WT mice.
We further examined the molecular mechanisms underlying the inflammatory response. A prior study revealed that nCB treatment increased HIF-1α protein expression in macrophages, which regulates inflammatory cytokine release and emphysema development. Importantly, Asxl1-mt macrophages exposed to nCB showed higher levels of HIF-1α protein expression than WT. Previous studies indicated that HIF-1α transcription depends on the activation of NF-kB pathway. Given that the C-terminus of ASXL1-WT is known to be essential for suppressing the NF-κB pathway, reduced ASXL1 function may lead to increased HIF-1α expression, as well as G-CSF and GM-CSF, and their inflammatory response. This may explain the abnormal inflammatory response and profound induction of emphysema in the context of the ASXL1-mutation relative to WT.
Chronic Myelomonocytic Leukemia (CMML) has been associated with COPD, and the Asxl1-mt mouse develops hematologic malignancies, including CMML with age. Thus, we investigated whether experimental emphysema would affect the development of hematologic malignancies in Asxl1-mt mice. Indeed, Asxl1-mt mice with emphysema induced by nCB showed accelerated hematologic malignancies compared to the untreated group. CMML is associated with an increase in inflammatory monocytes and co-mutations of ASXL1 and TET2. We found that inflammatory monocytes were increased in Asxl1-mt mice with nCB treatment and observed positive selection (expansion) of naturally arising Tet2 mutant clones in these mice compared to the untreated group. Overall, Asxl1-mt mice with nCB developed hematologic malignancies more rapidly due to increased inflammatory monocytes and a potentially higher frequency of Tet2 mutations.
In summary, these studies demonstrate a causal relationship between cigarette smoke, CH with ASXL1 mutation, emphysema, and malignancy development, and implicate the HIF-1α – NF-kB axis as an underlying mechanism. This work underscores the distinct mechanisms through which CH affects human health and may lead to novel therapeutic interventions.
