Evaluation of the TP53/TP73 pathway in the engraftment of primary acute promyelocytic leukemia cells using a xenotransplantation model

Author(s)

Diego Antonio Pereira Martins

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-05-02

Examining board members

Eduardo Magalhães Rego; José Barreto Campello Carvalheira; Emmanuel Griessinger; Fabíola Traina

• Advisor: Eduardo Magalhães Rego

Abstract

Current murine xenotransplantation models used as in vivo study models of human leukemic processes, particularly in acute myeloid leukemia (AML) have failed to reconstruct the pathophysiological processes related to the disease due to their non-similarity to the human bone marrow (BM) niche. Thus, the evaluation of the processes of self-renewal, differentiation, and transformation of human hematopoietic (HSCs) and leukemic (LSCs) stem cells, as well as their evaluation of the effectiveness of new treatment modalities, must be carried out in species-specific microenvironments. This is supported by the fact that some leukemic subtypes (categorized with favorable prognosis regarding the disease course), such as the acute promyelocytic leukemia (APL), present recurrent graft failure in xenotransplantation models. Despite their favorable prognosis against other subtypes of AML, this subtype still has a high risk of relapse often associated with drug-resistance. In this sense, it is believed that genetic variations that confer greater resistance to the leukemic clone may influence the grafting capacity of these cells, favoring in vivo evaluation. Previous studies from our group have shown that a higher ΔNp73/TAp73 expression ratio is associated with a worse prognosis in APL (lower overall survival [OS] and disease-free survival [DFS]) and resistance to cytarabine-induced apoptosis. ΔNp73, a truncated form of the TP73 gene, acts as a potent inhibitor of the transcriptional activity of TP53 and TAp73 proteins, thus playing an important role in cell proliferation and death. Thus, the oncogenic activity of the TP73 gene is determined by the balance between its transcriptionally active (TAp73) and inactive (ΔNp73) isoforms, and this relationship is correlated with the clinical prognosis and treatment failure in several human neoplasms. Thus, the present work aims to determine whether alterations in the TP53/TP73 pathway are associated with an engraftment capacity of cells isolated from APL patients in humanized xenotransplantation models. Moreover, we investigated the mechanisms by which the TP53/TP73 pathway can confer grafting advantages to PML-RARA+ cells, such as changes in cell proliferation, viability, and differentiation. Furthermore, we evaluated the transcriptome of patients with APL and AML with different expression levels of ΔNp73/TAp73, to identify possible targets of TP53/TP73 regulation. In CD34+ cells isolated from healthy donors, overexpression of ΔNp73α/β isoforms resulted in increased cell proliferation and colony formation in long-term cultures (35 days) compared to the empty vector control (pMEG). In APL cell lines (NB4 and NB4-R2), overexpression of ΔNp73α/β promoted an increase in cell proliferation, viability in response to cytotoxic agents, and decreased granulocytic differentiation induced by all-trans retinoic acid. Conversely, overexpression of TAp73α promoted the reversal of all phenotypes observed with ΔNp73, resulting in sensitization to arsenic trioxide (ATO) in an ATO-resistant APL model. Furthermore, in vivo patient derived xenotransplant models using NSGS mice demonstrated that primary APL blasts transduced with ΔNp73 had a superior engraftment in the BM, peripheral blood, and spleen of the mice. In the context of AML, patients with high expression of ΔNp73 isoforms had higher leukocyte counts, with the ΔNp73/TAp73 ratio being associated with lower OS and DFS. Furthermore, at the transcriptional level, patients with high ΔNp73 were associated with the terms \"HSC UP\" and \"LSC UP\", and negatively correlated with the terms \"GMP UP\" and \"TP53 regulates cell cycle arrest\". In cell lines, the overexpression of the ΔNp73α/β isoforms resulted in proliferative and cell survival advantages in cell models that did not contain mutations in the TP53 gene (MOLM13, MV4-11, OCI-AML3 and HL60), although no effect was observed in the cell lines containing mutations in TP53 (TF-1 and KG1). The silencing of TP53 gene expression in wild-type TP53 cells reduced the effects of overexpression of ΔNp73α/β isoforms. Mechanistically, high ΔNp73 expression resulted in reduced TP53 expression and increased DHODH expression. In conclusion, overexpression of ΔNp73 isoforms conferred cell survival advantages on AML cells, resulting in engraftment of primary cells from patients diagnosed with APL in a xenotransplantation model. (AU)

• FAPESP's process: 17/23117-1 - Evaluation of TP53/TP73 pathway in engraftment of acute promyelocytic leukemia cells in xenotransplantation model
• Grantee: Diego Antonio Pereira Martins
• Support Opportunities: Scholarships in Brazil - Doctorate