Traceable Mouse Models for Tracking p53-Mutant Subclones in Acute Myeloid Leukemia

Background: Unleashing the pro-apoptotic effects of p53 by pharmacologic inhibition of MDM2 is an attractive therapeutic strategy in hematologic malignancies with intact p53 ^1-3. However, concerns were expressed regarding the selection of TP53-mutant subclones with potential GOF activities after treatment with MDM2 inhibitor (MDM2i) ^4-8. The emergence of newly detectable TP53 mutations over the course of therapy was also observed in patients receiving intensive chemotherapy ⁹. Although resistance to treatment leads to selection of TP53-mutant subclones, it is not known whether pre-existing clonal hematopoiesis (CH) sub-clones with TP53 mutation(s) undergo malignant transformation or clonal expansion after exposure to MDM2i therapy.

Methods: We generated a traceable Tamoxifen-inducible conditional somatic TP53^R172H mutant mouse model to monitor and track the expansion of p53 mutant myeloid cells over the course of therapy with MDM2i. We combined a myeloid-specific Cre line, LysM-CreER^T2, as the Cre driver with a conditional knock-in mutant Trp53 allele that constitutively expresses WT Trp53 and converts to mutant (Mut) Trp53^R172H upon Cre-mediated recombination (WT to mutant, wm), named Trp53^wmR172H13,14. Tamoxifen mediated activation of Cre driven by the myeloid-specific LysM promoter, switches the WTp53 to Mutp53^R172H, which corresponds to TP53^R175H, a recurrent TP53 mutation in human AML. To trace the recombined hematopoietic cells, we used the Rosa26^mTmG reporter allele and performed a pulse-chase experiment in LysM-CreER^T2;mTmG;Trp53^fx/fx and LysM-CreER^T2;mTmG;Trp53^R172H/fx mice.

Results: Analysis of peripheral blood and the bone marrow (BM) after tamoxifen injection revealed a population of GFP⁺ myeloid cells, confirming efficient recombination. LysM-CreER^T2;mTmG;Trp53^R172H/fx mice did not develop leukemia after 1 year upon multiple tamoxifen inductions, suggesting that Trp53 mutation in mature myeloid cells is not leukemogenic. Having a GFP marker in myeloid cells allowed us to monitor the population of GFP+ myeloid cells in peripheral blood after each tamoxifen pulse. The GFP+ cells slowly disappeared from peripheral blood over time and were completely undetectable after 1 month following induction, suggesting that p53 mutation in myeloid cells does not extend the half-life of myeloid cells. Next, to assess if Mdm2 inhibition may cooperate with Trp53 mutation and transform the myeloid cells to AML, we induced the p53 mutation by tamoxifen injection and treated LysM-CreER^T2;mTmG;Trp53^fx/fx and LysM-CreER^T2;mTmG;Trp53^R172H/fx mice with the MDM2i, idasanutlin, for 10 days. There were no significant differences in the number of GFP+ myeloid cells between the two groups at baseline, however, in the p53-mutant group, the population of GFP⁺ cells was significantly decreased and in some mice was fully eradicated after MDM2i treatment. This suggests that p53-mutant protein may have some residual apoptotic activity that is activated after treatment with an MDM2i. In contrast to p53-mutant group, the GFP+ population in the p53-null group did not significantly change in the treated group. Next, we sought to examine if the same response can be achieved in the context of leukemia. Therefore, we transplanted LysM-CreER^T2;mTmG;Trp53^R172H/fx and control LysM-CreER^T2;mTmG;Trp53^fx/+ mice with syngeneic MLL-AF9 transformed leukemia cells and treated them with idasanutlin 1 week after transplant. Strikingly, the population of GFP⁺ cells was highly expanded in LysM-CreER^T2;mTmG;Trp53^R172H/fx mice transplanted with AML after MDM2i treatment, suggesting that MDM2 inhibition promotes the proliferation of p53-mutant myeloid subclones in the context of AML. To examine if the expanded population was due to transformation of p53-mutant myeloid cells, we sorted the GFP⁺ cells and transplanted them into syngeneic mice. No leukemia arose after transplantation, suggesting that the expansion of p53-mutant myeloid cells in the context of leukemia was not due to leukemic transformation of p53-mutant cells but rather due to unrestrained proliferation.

Conclusion: Overall, these data indicate that the prior existing TP53-mutant subclones are expanded upon idasanutlin treatment without being transformed. Therefore, monitoring of TP53-mutant subclones during the course of treatment is warranted.

Andreeff:Breast Cancer Research Foundation: Research Funding; Kintor Pharmaceutical: Research Funding; Chimerix: Current holder of stock options in a privately-held company; Pinot Bio: Research Funding; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI: Membership on an entity's Board of Directors or advisory committees; Oxford Biomedical UK: Research Funding; AstraZeneca: Research Funding; Syndax: Consultancy, Research Funding; Senti Bio: Consultancy, Research Funding; Daiichi-Sankyo Inc.: Consultancy, Research Funding; Reata: Current holder of stock options in a privately-held company; Medicxi: Consultancy; Glycomimetics: Consultancy; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Aptose: Consultancy, Membership on an entity's Board of Directors or advisory committees; Brooklyn ITX: Research Funding; Oncolyze: Current holder of stock options in a privately-held company.