Clinical and Functional Studies Reveal That TP73 Isoforms Levels Are Associated with Prognosis and RA-Resistance in Acute Promyelocytic Leukemia
Authors: Ortiz Rojas César Alexander , Pereira-Martins Diego A , Weinhäuser Isabel , Almeida Luciana Yamamoto , Bianco Thiago Mantello , Figueiredo-Pontes Lorena Lobo , Koury Luisa C A , Melo Raul Antônio Morais , Bittencourt Rosane , Pagnano Katia B , Pasquini Ricardo , Nunes Elenaide C. , Fagundes Evandro Maranhão , Gloria Ana Beatriz F. , Kerbauy Fabio R. , Keating Armand , Tallman Martin S. , Ribeiro Raul , Dillon Richard , Ganser Arnold , Löwenberg Bob , Valk Peter , Sanz Miguel A. , Berliner Nancy , Rego Eduardo M 
Published: November 2019

Background: TP73 isoforms gained particular relevance in acute promyelocytic leukemia (APL) since Bernasola et al (JEM. 2004) demonstrated that TAp73 was directly regulated by the PML protein in the nuclear body. The isoforms differ in their transcriptional activity, with those lacking domains in the N-terminal part of the protein exerting a dominant negative effect on TP73 function. In a retrospective analysis of patients with APL treated in ICAPL study, Lucena-Araujo et al (Blood 2015) demonstrated the association between higher ΔNp73/TAp73 ratio values and poor clinical outcome. However,there is a diversity of TP73 isoforms and specially those lacking N-terminal domains (e.g.ΔEx2p73, ΔEx2-3p73 and ΔN'p73) may be relevant in APL genesis and therapy response. Aims: Here, we quantified transcript levels of TP73 N-terminal variants TAp73, ΔNp73, ΔEx2p73, ΔEx2-3p73 and ΔN'p73, as well as the C-terminal variants TP73αand TP73β, and determined whether there is a prognostic correlation. In addition, we evaluated the effect of ΔNp73overexpression on APL cell lines survival and differentiation in vitro and in vivo. Methods: Bone marrow (BM) samples from 98 patients (age, 18-74y) with newly diagnosed APL enrolled in the International Consortium on Acute Leukemia (ICAPL2006) were included. For comparison, BM mononuclear cells from 14 healthy donors (age, 18-60y) were also included. TP73 transcripts were determined by qPCR and using survival ROC curve analysis and the C-index we dichotomized patients into "low" and "high" expression. Proportional hazard model on overall survival (OS) and disease-free survival (DFS) was performed to evaluate prognosis. In addition, empty vector (EV) or ΔNp73α was transduced in NB4 and NB4R2 (RA-resistant) APL cell lines using a lentivirus system. The apoptosis rate was evaluated in both cell lines upon ATO (1 μM) treatment for 24, 48 and 72h. ΔNp73α, cleaved caspase 3 and Bcl-2 protein abundance was detected by western blotting. Granulocytic differentiation induced by RA-treatment (1 μM) alone or in combination with ATO (1 μM) for 72 h was assessed by CD11b surface levels. Finally, lethally irradiated 8-12 week old C57/BL6 Boy (CD45.1) were transplanted with hCG-PMLRARa Blasts (CD45.2) ΔNp73α or EV transduced (ΔNp73α group=16 and EV group=12). Results: Compared to normal BM, APL patients presented higher levels of ΔNp73 (p=.004), ΔEx2p73 (p=.008), and TP73β (p<.001) mRNA expression and lower of TAp73 (p<.001). ΔNp73 and ΔEx2p73 expression levels were directly correlated with TP73β (rho=.52 and .54, respectively), and TAp73 with TP73α (rho = .6). The 5y OS rate was significantly higher in patients with lower expression of ΔEx2p73 (78% vs. 97%, p<.05) and in patients with ΔEx2p73/TAp73 ratio values below the cut-off (77% vs. 100%, P<.04), and both variables were independent considering sex, age and leukocytes as confounders (HR=8, 95% CI:1.1-60.8 and HR=15.3, 95% CI: 2.1-1962,respectively). Furthermore, levels of ΔNp73 expression higher than the cut off were also associated with lower 5y OS rate (78% vs. 97%), establishing the fact that ΔNp73, ΔEx2p73 and TAp73 have similar patterns of expression and impact on APL treatment outcome. There was no association between TP73 isoform expression and DFS rate. Regarding the granulocytic differentiation, ΔNp73αoverexpression lead to RA-resistance in NB4 cells (p<.05), although the addition of ATO circumvented effect. As expected, ΔNp73αtransduced cells presented resistance to ATO induced apoptosis, which was detected at 48h (22.4 vs.49.8, p<.001) and 72h (47.3 vs.76.9, p=.032). We observed a partial clearance of ΔNp73α protein after 72h of ATO treatment, which may explain why ATO overcomes RA resistance induced by ΔNp73α, and why the apoptosis resistance to ATO was less evident after 72h compared to 48h. No significant difference in cleaved caspase 3 and Bcl-2 was detected during ATO treatment. No effect of ΔNp73αoverexpression was observed for NB4R2 cells. In vivo model showed no differences regarding peripheral blood counts, spleen weight (p>.05) or disease progression in ΔNp73α blasts compared to the control group (p=.095). Conclusions: Our results suggest that transcript levels of ΔNp73, ΔEx2p73 and TAp73 predict outcomes in APL patients treated with RA plus chemotherapy. Additionally, RA plus ATO combination therapy seemed to overcome the effect of ΔNp73 overexpression in vitro.

Disclosures
Figueiredo-Pontes: Novartis: Honoraria. Pagnano:Abbvie: Consultancy; Sandoz: Consultancy; Pint Pharma: Consultancy. Tallman:Danbury Hospital Tumor Board: Honoraria; 14th Annual Miami Cancer Meeting: Honoraria; International Conference in Leukemia: Honoraria; KAHR: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Nohla: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; New Orleans Summer Cancer Conference: Honoraria; Indy Hematology Review: Honoraria; Mayo Clinic: Honoraria; Orsenix: Membership on an entity's Board of Directors or advisory committees, Research Funding; UpToDate: Patents & Royalties; Salzberg Weill Cornall MSKCC Seminar in Hematologic Malignancies: Honoraria; Hematology Oncology of Indiana: Honoraria; ADC Therapeutics: Research Funding; Arog Pharmaceuticals: Research Funding; Cellerant Therapeutics: Research Funding; University of Oklahoma Medical Center: Honoraria; Bioline: Membership on an entity's Board of Directors or advisory committees, Research Funding; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees. Dillon:Abbvie: Consultancy, Honoraria; TEVA: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Löwenberg:Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands: Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Frame Pharmaceuticals: Equity Ownership; Hoffman-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees; Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherland: Membership on an entity's Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees; CELYAD: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Astex: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.