Clonal Competition: Cisplatin Treatment Drives Dominance of TP53- over Tet2-Mutant Stem Cell Clones

Introduction: In the process of aging, somatic mutations accumulate in stem cells, and some mutations promote stem cell fitness leading to an increased proportion of variant cells in the blood, referred to as clonal hematopoiesis (CH). Some of the most commonly mutated genes that drive CH include DNMT3A, TET2, PPM1D, TP53, and SMC3. While it is known that most people acquire mutations in all of these drivers over time, only one clone will typically expand and become dominant in a given individual. This suggests external factors can drive clonal expansion in a genotype-specific manner. Here we sought to establish a mouse model to examine factors that drive specific clonal expansion and determine if there are clone-clone interactions in vivo that contribute to their dominance in certain contexts.

Methods: We established a mouse model in which stem cells of 5 different genotypes were competed head-to-head along with wild-type (WT) bone marrow. Mice were transplanted with Tet2^-/-, Dnmt3a^-/-, Smc3^+/-, Ppm1d^+/-,and TP53^-/- and WT whole bone marrow (WBM) cells for a 5-gene (5X) transplant. Clonal contribution was tracked using digital droplet PCR (ddPCR) to determine the variant allele frequency (VAF) of each genotype in peripheral blood (PB). Following up initial observations, 2X experiments were performed to test interactions between Tet2^-/- and TP53^-/- along with WT bone marrow, and 1X transplants examined the behavior of individual genotypes in a background of WT bone marrow. To test the impact of chemotherapy on clonal competition, one experimental arm of mice of each transplant setup (5X, 2X, and 1X) was treated with 3mg/kg/week cisplatin for at least 5 weeks. Before and after chemotherapy, VAF for each genotype in the PB was determined by ddPCR. Bone marrow (BM) samples were taken once mice started showing signs of disease at 24 weeks for the 5X mice and 14 weeks for the 1X and 2X transplants. BM was sorted for Lin-ScaI+ckit+ (LSK) hematopoietic stem and progenitor cells (HSPCS) and analyzed by flow cytometry and ddPCR.

Results: In the 5X transplant, Tet2-mutant cells dominated most frequently, followed by TP53 at 4 weeks after transplant in the PB (Table 1). After treatment with cisplatin for 7 weeks, TP53 cells expanded significantly faster compared to the control mice (ΔVAF of 21.6 vs 1.7) whereas Tet2 cell expansion was significantly attenuated (ΔVAF of 20.3 vs 29.4). At 24 weeks a similar trend was observed in the WBM and in the LSK fraction. To study this interaction in more depth, we focused on the competition between TP53- and Tet2-mutant clones in the presence of cisplatin treatment in the 2X transplant. Compared to the 5X transplant, Tet2 engraftment after 5 weeks was slightly higher in the 1X Tet2 and 2X transplanted mice as well as TP53 in the 1X TP53 mice (Table 1). Interestingly, TP53 engraftment was lowest in the 2X transplant. After 5 weeks of cisplatin treatment, there was a significant decrease of Tet2 cells (ΔVAF of -9.6 vs -4.8) when in competition with TP53 that was not present in the 1X Tet2 mice (Figure 1). In contrast, TP53 expanded after cisplatin treatment compared to control mice in the 2X transplant (ΔVAF of 15.4 vs 7.6) and 1X transplant (ΔVAF of 15.3 vs 9.5) (Figure 1), and TP53 became the dominant clone in the 2X setting. Overall we observed a negative correlation between Tet2- and TP53- mutant cells in the cisplatin treated 5X mice in the PB and BM that was even more striking in the 2X setting.

Conclusion: We were able to establish a model to study clonal competition in mice in vivo where clones with different genetic backgrounds showed different fitness. Our setup revealed a negative correlation between Tet2- and TP53-mutant clones that was enhanced by chemotherapeutic stress with cisplatin. The presence of both mutant clones resulted in a general suppression of expansion when compared to single transplants that might be due to direct interaction between the clones. Analysis of the inflammatory environment and RNA sequencing of the different populations within the BM is being performed to examine the mechanism underlying this phenotype.

Tseng:Navigate BioPharma Service Inc: Current Employment. Tovy:Novartis Research Institute: Current Employment.