Figure 4. TETi selectively restricts the growth of TET2 mutant cells.

A, Schematic representation of the mixing experiment of Tet2^wt and Tet2^mt murine bone marrow in colony forming assay. B, Tet2^mt bone marrow (CD45.2) cells were mixed in the ratio of 1:2 with Tet2^+/+ (CD45.1) and grown in MethoCult for colony formation in the presence or absence of TETi76 (20 μM). On day 10 cells were harvested and the ratio of Tet2^mt/Tet2^+/+ was measured by flow cytometry using isoform specific antibodies. C, The ratio was plotted for two consecutive platings. D, Tet2^mt or TET2^wt mice were treated with TETi (50 mg/Kg, p.o., 5 days/week) for 8 weeks. The spleens were harvested and weights were plotted compared to vehicle control. E, Schematics of experimental design for in-vivo transplant experiment. F, C57BL6 PepBoy mice expressing CD45.1 surface marker on mononuclear hematopoietic cells were lethally irradiated and transplanted with a mixture donor mice bone marrows (5% Tet2^−/−, CD45.2; and 95% Tet2^+/+, CD45.1). Once mice fully recovered after transplant, the engraftement was accessed by isotype specific antibodies and TETi treatment (25mg/kg, s.c.) 5 days a week at 4 weeks were started. The engraftment of Tet2^mt cells in the PBMC was monitored and plotted. TETi76 prevented the clonal expansion of Tet2^mt cells in vivo. G, Tumor growth of SIGM5 cells in NSG mice (n=8/group) were monitored upon TETi76 treatment. Once controlled reached the maximum allowed limit of tumor burden, tumors were harvested and tumor weight is plotted. TETi76 significantly reduced the tumor size. Data are shown as mean±SEM; statistical significance (p values) from two tailed t-test are indicated; ** p<0.01; **** p<0.0001; ns: not significant.