S193: GENOMIC AND FUNCTIONAL IMPACT OF TP53 INACTIVATION IN JAK2V617F MYELOPROLIFERATIVE NEOPLASMS: A TRANSGENIC MOUSE MODEL APPROACH.

Abstract

Background: MPN are characterized by increased proliferation of myeloid cells and a risk of transformation to AML. MPN are due to the acquisition of mutations (JAK2, CALR, MPL) leading to JAK2 pathway activation but cooperating mutations involved in MPN evolution have been described. Mutations of TP53 in JAK2V617F patients are linked to AML transformation, illustrated by development of leukemias after retroviral overexpression of JAK2V617F in TP53 inactivated (KO) cells transplanted in irradiated animals, thus a possible cooperation between TP53 inactivation and JAK2 signaling in transformation process has been postulated. However, JAK2V617F signaling has also been reported as able to reduce p53 expression per sesuggesting a redundant role of p53 inactivation in JAK2V617F cells. Aims: Determine the role of TP53 in JAK2V617F induced genetic and phenotypic modifications. Methods: To better understand the role of TP53 in MPN phenotype and evolution in steady state hematopoiesis, we developed a transgenic model of vav-cre induced JAK2V617F expression in TP53 knock-out (KO) animals and analyzed phenotype, survival, genetic modifications using stem cell compartments RNA-Seq and response to therapy of this triple transgenic mice. Results: JAK2V617F vav-inductible-Tg/TP53 KO mice were generated to recombine JAK2V617F at an endogenous level in TP53 inactivated hematopoietic cells. During follow-up, JAK2V617F/TP53KO mice developed the same phenotype than JAK2V617F transgenic mice without leukemia. In competitive repopulations with grafts with different ratio of JAK2V617F, JAK2V617F/TP53KO and wild type cells, we observed that JAK2V617F/TP53KO cells outcompete JAK2V617F cells. In order to better define TP53-dependent and independent pathways linked to these phenotypes, LT-HSC, ST-HSC, MPP, CMP, MEP and GMP from normal, JAK2V617F and JAK2V617F/TP53 KO mice were cell sorted and RNA-Seq analysis was performed. Principal component analysis demonstrated that JAK2V617F/TP53KO cells were closer to normal cells than JAK2V617F cells whatever the (stem) cell compartment, illustrating that JAK2V617F-induced modifications are largely p53 dependent. Further analysis demonstrated that approximatively half of JAK2V617F deregulated genes in the different compartments are TP53 dependent including the IFN pathways. In order to validate this finding, mice repopulated with a mix of WT and JAK2V617F (either p53 KO or wild-type) cells were treated for 8 weeks with recombinant murine pegylated IFN-a. JAK2V617F reconstituted animals entered in complete hematological remission while JAK2VF/TP53KO reconstituted animals did not, illustrating that loss of TP53 in this context induced resistance to IFN-a. On the other hand, since MPN develop in the same way in JAK2V617F-only and in JAK2V617F/TP53KO mouse, this suggests that JAK2V617F-specific pathways also found differentially expressed in JAK2V617F/TP53KO are linked to the MPN phenotype. KEGG and GO analysis demonstrated that these genes were mainly implicated in cytokine response, cell proliferation, differentiation, and leukemia evolution illustrating that the development of MPN and its possible risk of transformation in this mouse model is largely TP53 independent. Summary/Conclusion: Taken together, our results show that a large part of genetic modifications induced by JAK2V617F mutation are p53 dependent but MPN phenotype is not, TP53 loss is insufficient to induce quick leukemic transformation in steady-state hematopoiesis in Jak2 V617F MPN despite it increases LT-HSC cell proliferation and finally that TP53 loss could be involved in IFN resistance in MPN.