Age-Related Cellular Factors Facilitate TET2 Mutant Clonal Hematopoiesis

Background and Significance:

Besides chemotherapy and inherited or acquired bone marrow failure, aging is one of the most important risk factors for the development of clonal hematopoiesis (CH), and both intrinsic and chronological factors may play a role. While initially asymptomatic and referred as to clonal hematopoiesis of indeterminate potential (CHIP), CH frequently leads to adverse clinical consequences with increased risk of cardiovascular disease, certain pulmonary disorders, and other diseases. CHIP carriers experience increased risk of progression to myeloid neoplasia (MN). The process of clonal expansion may be protracted, and its penetrance is difficult to assess due to competing mortalities.

While various genetic lesions have been described as common to CHIP, loss of function TET2 mutation (TET2^MT) is very common. The prevalence of TET2^MT CH increases with age; for instance, our earlier research demonstrates that most cases of myelodysplastic syndrome in patients over age 70 are likely from TET2^MT CHIP. TET2^MT are prototypic CH lesions, being both weakly leukemogenic in the context of healthy, young hematopoiesis and increasingly strong initiators of leukemogenic cascades in aging populations.

In the current study, we investigate whether the progression of CH is not only linked to cell intrinsic properties of TET2^MT clones, but also to other extrinsic age-related changes in bone marrow niche, including inflammation, loss of hematopoietic stem cell (HSC) fitness, and quantitative contraction of the HSC compartment. These factors are particularly important for TET2^MT CHIP because they may, perhaps in addition to TET2 deficient clones, be subjects of therapeutic or preventive interventions.

Methods and Results:

We developed novel murine models of Tet2 mutant (Tet2^+/- and Tet2^-/-) in B6CD45.2, B6TomatoRed^mTmG and CD45.1 JaxBoy, and utilized young (8-16 weeks) and old (>16 months) WT mice to dissect the role of cell intrinsic and extrinsic aging factors in CHIP evolution. We utilized various surface markers along with cell intrinsic fluorophores to monitor different hematopoietic compartments. Data analyses were performed in FlowJo™ and statistical analyses were performed using Dunnett's test (sample size n<8) or one-way ANOVA for multiple comparisons.

Our models enabled precise monitoring of CH using phenotypic markers. We performed an in vivo marrow competitive bone marrow repopulation assay, where grafts from either young or old WT donors from CD45.2 mice (90%) competed with Tet2^-/- B6TomatoRed^mTmG (10%) in congenic CD45.1 JaxBoy recipient mice irradiated with 9.2 gray ionizing radiation. The Tet2^-/- fractions traced by tdTomato fluorescence and the WT donor fraction traced as tdTomato negative and CD45.2 positive were monitored until sacrifice at 52 weeks.

5 months post-transplant, we observed that the expansion rate of Tet2^-/- clone was accelerated (>2-fold, n=5, p =0.006, t-test) when the competitor graft was from old donors compared to young donors. Marrow cells derived from young donors restricted the expansion of Tet2^-/- HSC due to better fitness, reducing the proportion of Tet2^-/- cells from 80.47% in the transplants from old WT donors to 33.80% in those from young donors (n=5, p=0.0002, t-test) 52 weeks post-transplant at the end of the experiment. In a further analysis, we observed that aging effects on hematopoiesis varied across compartments, notably contracting the T-cell compartment from 49.25% (young WT donors) to 25.08% (old WT donors) (n=5, p=0.0339) consistent with immune cell deficit with aging.

Using these models, we have identified that aging HSCs feature a lack of fitness that may contribute to the emergence and expansion of CH. Our data suggest that the age-associated attrition of normal HSCs allows the expansion of TET2^MT CHIP. Therefore, enhancing the fitness of a polyclonal, normal HSC compartment by targeting the factors that created such attrition, may restrict or delay clonal evolution and prevent associated morbidities.