The Oncogenic ΔNp73α Isoform Contributes to Aggressiveness in APL By Leading to ATRA Resistance through NANOG/BMP4 Axis Upregulation

Treatment approaches for Acute Promyelocytic Leukemia (APL) have undergone significant advancements with the incorporation of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO). Yet, the exact molecular mechanisms contributing to the variability in treatment response remain unclear. In this context, TP73 has been previously proposed as key protein related to prognostic in APL patients treated with ATRA+chemotherapy (Lucena-Araujo et al., Blood 2019 - doi.org/10.1182/blood.2019000239). The TP73 gene encodes several isoforms that can be divided in two main groups– TA, having the transactivation domain (TAD) and functions like TP53, and ΔNp73, which by lacking the TAD, can bind to DNA but cannot activate the same target genes. Here, we studied the role of ΔNp73α and TAp73 isoforms in modulating the therapeutic efficacy of ATRA and ATO in APL. First, we evaluated the gene expression of TP73 isoforms in bone marrow (BM) mononuclear cells from 98 APL patients (median age: 36.1, 26.7-47.3 years, 51% male) enrolled in the International Consortium of Acute Promyelocytic Leukemia (IC-APL), all treated with ATRA+chemotherapy. We found that the high ΔNp73 levels were associated with significantly lower overall survival rates (77.8%) compared to those with lower expression (96.6%). Next, using lentiviral based systems, we induced stable expression of ΔNp73α or TAp73 in NB4 (ATRA-sensitive) and NB4-R2 (ATRA-resistant) APL cell lines. We found that ΔNp73α overexpression (OE) increased the cell proliferation of both APL models. Next, we treated the APL cell lines with ATRA and ATRA+ATO to evaluate the role of ΔNp73α and TAp73 isoforms as modulators of drug response. In both APL models, neither TP73 isoform was associated with differential response to ATRA or ATO regarding drug induced apoptosis, compared to the empty vector (EV) control. Molecularly, we observed that ATO-treatment for 24 to 72h hours was associated with degradation of the ΔNp73α oncoprotein. Since ATO is associated with activation of TP53/TP73 signaling pathway, we tested whether TAp73-OE would restore ATO sensitivity in ATO resistant APL models. In ATO resistant APL cells, TAp73α-OE was able to revert the ATO resistance, suggesting the potential role of activation of TAp73 for ATO resistant cases. Also, ΔNp73α hindered the induction of ATRA-induced myeloid markers (CD11b, CD14, and CD15), suggesting an inhibitory role in differentiation processes. In this context, we evaluated the BMP4-ΔNp73-NANOG signaling cascade, identified as a positive inducer of stem cell-like phenotype in acute myeloid leukemia cells (Voeltzel et al., Cell Death Dis 2018 – doi.org/10.1038/s41419-018-1042-7). Our findings revealed increased expression of BMP4 and NANOG in ΔNp73α-OE cells following ATRA treatment, which was absent in non-treated cells, underscoring the significance of this signaling axis in APL response to ATRA. To explore new molecular targets associated with ATRA response in the context of TP73 isoforms in APL, we performed label-free quantification proteomic analysis in NB4 ΔNp73α-OE treated with ATRA. We found in ATRA-treated NB4 ΔNp73α-OE a negative regulation of proteins associated with mRNA splicing via spliceosome and a positive regulation of interleukin 8 production, compared to EV control. Finally, we performed in vivo experiments using a ΔNp73α- and TAp73α-OE model on murine APL blasts from the leukemic hCG-PM::RARA mice..Modified APL blasts were transplanted into NSG mice (8 weeks old, 50% males), and after two weeks of the transplant, mice were split in four groups of treatment (n=7/group), being treated for 21 days with ATRA (1.5 mg/Kg/day), ATO (5 mg/Kg/day), ATRA+ATO, and vehicles. Notably, ΔNp73α-OE in leukemic blasts led to a reduction of the ATRA-induced blast clearance in both BM and spleen. In conclusion, our study emphasizes the prognostic and therapeutic implications of ΔNp73α in APL. We observed that ΔNp73α-OE APL cells up-regulate NANOG and BMP4 in response to ATRA treatment, decreasing its efficacy, supporting the role of ΔNp73 expression in therapy response. This is particularly relevant in low and middle-income countries centers where ATO is not an available treatment option.