Low Regimen Therapeutic Strategies to Mitigate TET2 Mutant Clonal Hematopoiesis

Circulating myeloid cells carrying somatic mutations characteristic of myeloid neoplasms (MN) are also found in a significant proportion of otherwise healthy, mostly elderly individuals in a prodromal condition called clonal hematopoiesis of indeterminate potential (CHIP). CHIP carriers have an increased risk (10-25x) of MN, including treatment-related MN in cancer survivors who received chemotherapy and/or ionizing radiation. These individuals also face an increased risk of cardiovascular disease and pulmonary atrial hypotrophy (PAH), among other diseases. However, there are currently no known therapeutic options to address CHIP. Recent reports suggest that the age-related inflammatory microenvironment profoundly affect the evolution of CHIP (Guarnera L & Jha BK. Semin Hematol. 2024). TET2 mutation (TET2^MT) is the second most frequent somatic lesion in CHIP. The loss of TET2 results in an aberrant pattern of 5-methyl cytosine (mC), creating a volatile pre-neoplastic state in hematopoietic stem and progenitor cells (HSPCs). Clonal evolution of TET2^MT HSPCs is strongly age dependent. TET2^MT often occurs as a founding lesion, implying that eliminating the TET2^MT clone would impact CHIP at an early ontogenetic stage. We assessed two distinct strategies to target TET2-deficient clones: (i) a synthetic lethal approach, where the TET2^MT clone can be targeted by blocking residual activity (Guan et al., Blood Cancer Discov. 2021) while simultaneously increasing the fitness of TET2 proficient normal HSPCs and (ii) an anti-inflammatory regimen, where given that TET2^MT clone creates and persists in pro-inflammatory milieu, targeting inflammatory pathways may prevent clonal evolution. In this study, we screened three nontoxic and well tolerated anti-inflammatory molecules (Nicotinamide riboside (NR), Danazol, and Ibuprofen) along with TETi76, a known TET inhibitor that restricts the evolution of TET2^MT by inhibiting residual TET-dioxygenase activity. Here, we report two complimentary low regimen strategies for restricting TET2^MT CHIP in vivo.

We developed Tet2^-/- B6 CD45.2 TomatoRed ^mTmG murine models to facilitate easy monitoring of the Tet2^mt graft. Competitive bone marrow transplant coupled with spectral flow cytometry was used to evaluate clonal burden and its impact on polyclonal normal hematopoiesis. Each graft (2x10⁶ cells; 10% Tet2^-/-TomatoRed⁺ 90% WT Bl6CD45.2) were transplanted into JaxBoy CD45.1 recipients. Three weeks post-transplant, mice were randomized into five treatment groups (n=8/group): Vehicle, TETi76 (25mg/kg), NR (300mg/kg), Ibuprofen (40mg/kg), or Danazol (35mg/kg). Peripheral blood was analyzed by flow at 3, 6, 24, and 52 weeks using CD45.1, CD45.2, and Tomato to assess CHIP expansion. Mice were sacrificed at 52 weeks and analysis of blood, bone marrow, and spleen were performed by spectral flow cytometry.

At the time of sacrifice, the Tet2^-/- fractions traced by the TomatoRed were as follows: 75% (vehicle), 43% (TETi76, p= 0.003), 61% (NR, p=0.054), 62% (ibuprofen p=0.21) and 48% (Danazol p=0.0187). The mean of Tet2^-/- fraction in the control group was slightly higher than in the groups treated with anti-inflammatory drugs. Our data indicate that synthetic lethality induced by TETi76 restricted the evolution of Tet2^-/- HSPCs and simultaneously restored normal hematopoiesis. The anti-inflammatory treatments moderately slowed Tet2^-/- CHIP expansion. Among the other drugs used, Danazol showed a significant reduction in overall Tet2^-/- fraction, while ibuprofen had no significant effect. HPSC differentiation analysis showed that CD11b⁺ populations were decreased in mice treated with TETi76 (3-fold; p =0.054), with moderate effects observed with (<1.5-fold) compared to vehicle control. Interestingly, treatments mitigated the myeloid skewing of Tet2^-/- fraction reflected in an increased CD3⁺ population (5-fold; p=0.003) with milder effects observed for Danazol (<2-fold, p=0.18), NR (<2 fold; p= 0.41), and Ibuprofen (3-fold, p=0.0469) compared to the vehicle control.

In summary, treatment with TETi76 slowed the expansion of Tet2^mt CHIP in vivo. The effects of anti-inflammatory drugs were moderate. A complete to partial restoration of normal hematopoiesis was observed by TETi76. Our results indicated this low regimen, non-toxic treatment could be a potential option for preventing TET2^MT CHIP.