Dissecting the Interactions between Leukemic Cells and the Aging Bone Marrow Niche

AML is a hematological disorder with a median age at onset of 70 years. TP53 mutations are observed in 5-10% of young adult AML cases, while TP53 mutation frequency increases significantly to more than 30% in older patients. Mutations result in inhibition of p53 function and are a strong predictor of inferior response to induction chemotherapy. Leukemic Stem Cells (LSCs) drive AML escape to chemotherapy and present with similar properties to normal adult hematopoietic stem cells (HSCs) depending on the bone marrow (BM) niche for proliferation and treatment survival.

However, how TP53 LSCs interact with the BM niche and if there are changes in the leukemic niche upon aging that increase TP53 leukemogenesis is not known.

This project focuses on characterizing the changes in the TP53 leukemic BM niche upon aging. We aim at establishing a mouse model of Tp53 leukemia to investigate whether the old leukemic BM niche increases leukemogenesis compared with the young and whether it can be targeted to improve disease outcome.

For characterization of leukemia comparing young vs old BM niche and for the survival study, we induced Tp53-driven leukemia in old and young CreERT2-Rosa26 knock-in mice crossed with Tp53flox mice. Mice were sacrificed according to the following end-point criteria: >20% weight reduction, >20M/mL of white blood cells, and an accused decrease in the mobility. At sacrifice, mice undergo necropsy and BM is stained for HSC and progenitor characterization by flow cytometry analysis.

Further, we isolated HSCs from Tp53 knock-out and wild-type littermate mice and performed transplantations in lethal-irradiated (9Gy) twelve-week-old Ly5.1 female recipient mice. 200 p53KO and p53WT Ly5.2 HSCs were injected into the retroorbital eye of irradiated female mice together with 300,000 total BM cells from wild-type Ly5.1 mice to support reconstitution of the hematopoietic system. After 6 weeks, mice were bled to check the engraftment of transplanted cells. The engraftment was checked every month after this time point. Mice were sacrificed according to the previously explained criteria. At sacrifice, mice undergo necropsy and BM as previously explained.

To investigate the Tp53 leukemic niche, we crossed Tp53flox mice with acRFP X CreERT2-Rosa26 knock-in mice. P53 depletion was induced by systemic injection of Tamoxifen (TAM) in young (12 weeks) and old (80 weeks) mice. Next, we transplanted p53KO and p53WT RFP+ HSCs into young and aged irradiated recipients and young and aged non-irradiated immunocompromised NSGBW mice. Recipient mice were analyzed according to our previously established FASTi3D whole mount histology protocol to image LSCs and their surrounding BM niche.

Survival analysis shows that old p53KO mice die faster compared to young p53KO mice. In agreement with previous findings, BM immunophenotype of HSCs and hematopoietic progenitors supports that p53KO mice died due to a hematological disorder (lymphoma).

Upon transplantation of young p53KO LSCs into irradiated young and old mice, survival analysis shows that old p53KO recipients dye significantly faster compared to both old p53WT recipients and young p53KO recipients.

FASTDi3D histological analysis reveals that donor cells in young recipients are located near the endosteal region, while, in the old ones, RFP+ cells are located further from the endosteum. Interestingly, while in general donor-derived cells in the old recipients are located closer to the vessels than in young recipients, p53KO LSC transplanted into old recipients are found further to the vessels compared to p53KO cells transplanted into young recipients.

Our data show that old Tp53KO mice die faster compared to young Tp53KO mice and that this outcome is largely driven by a hematological malignancy that develops faster in the old microenvironment. By whole-mount histological analysis, our results show that old Tp53 LSCs are located in different BM niches compared to wild-type HSCs and to young Tp53 LSCs. Molecular characterization by scRNA-seq of niche cells is currently ongoing to gain insight into the different cell types that interact with Tp53 LSCs in young and aged mice. Improving our understanding of the role of microenvironment in the progression of TP53 leukemia might be critical for the rational integration of novel treatment strategies that seek to increase the response rate in elderly patients.

Florian:MoglingBio: Consultancy, Current holder of stock options in a privately-held company.