Abstract A06: Murine models of mutant p53 clonal hematopoiesis elucidate mechanisms of expansion of p53 mutant subclones by MDM2 inhibition
Authors: Pourebrahim Russell , Montoya Rafel Heinz , Ostermann Lauren , Andreeff Michael 
Published: May 2023

Abstract
Unleashing the pro-apoptotic effects of p53 by pharmacologic inhibition of MDM2 has been an attractive therapeutic strategy in hematologic malignancies with intact p53. However, concerns were expressed regarding the selection of TP53-mutant subclones with potential GOF activities. It is not clear whether pre-existing sub-clones with TP53 mutation(s) undergo malignant transformation or clonal expansion after exposure to MDM2i therapy. We generated traceable conditional somatic TP53R172H mutant mouse models 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-CreERT2, or Mx1-cre with a conditional knock-in mutant Trp53 allele that constitutively expresses WT Trp53 and converts to mutant (Mut) Trp53R172H upon Cre-mediated recombination. To trace the recombined hematopoietic cells, we used the Rosa26mTmG reporter allele as previously described and performed a pulse-chase experiment. LysM-CreERT2;mTmG;Trp53R172H/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 the Mdm2i therapy may cooperate with Trp53 mutation and transform the myeloid cells to AML, we induced the p53 mutation by tamoxifen injection and treated LysM-CreERT2;mTmG;Trp53fx/fx and LysM-CreERT2;mTmG;Trp53R172H/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. We transplanted LysM-CreERT2;mTmG;Trp53R172H/fx and control 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 p53 mutant mice transplanted with AML after MDM2i treatment, suggesting that MDM2 inhibition promotes the proliferation of p53-mutant myeloid subclones in the context of AML. We sorted the GFP+ cells and transplanted them into syngeneic mice. No leukemia arose after transplantation. We here established the first in vivo model of p53-mutant clonal hematopoiesis using a new p53 mutant conditional allele. The expanded population of p53 mutant subclone did not transform into AML. However, in the presence of fully transformed AML cells, the p53 mutant clonal hematopoiesis expanded. This finding is consistent with reports of transitory expansion of p53 mutant cells in patients with myelofibrosis treated with MDM2 inhibitor.
Citation Format: Russell Pourebrahim, Rafel Heinz Montoya, Lauren Ostermann, Michael Andreeff. Murine models of mutant p53 clonal hematopoiesis elucidate mechanisms of expansion of p53 mutant subclones by MDM2 inhibition [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A06.