TP73 Isoforms (TAp73 and ΔNp73) Are Overexpressed in Acute Myeloid Leukemias and Potential Therapeutic Targets to Enhance Anti-Leukemia Activities of Bcl-2 and MDM2 Inhibitors

BackgroundTP73 is one of the TP53 family transcription factors and generates two isoforms, the transactivation p73 (TAp73) and the N-terminally truncated ΔNp73. TAp73 shares a homologous N-terminal activation domain with p53 and has similar pro-apoptotic function to p53. ΔNp73 lacks an activation domain and has activities distinct from TAp73. ΔNp73 has a dominant negative effect on the DNA binding of TAp73 and more importantly, of p53, since the DNA binding domain is homologous with that of TAp73 and highly similar to that of p53. In acute myeloid leukemias (AML), TP73 has been reported to be expressed except in acute promyelocytic leukemias. However, the association of TP73 isoforms with clinical and genetic characteristics and the regulation of the isoforms in AML have not been explored.

Results We determined copy numbers of ΔNp73 and TAp73 mRNA levels in 78 AML samples including 31 de novo AML using droplet digital PCR (ddPCR), which allows to determine the absolute quantity of the isoforms expressed, and investigated their clinical and biological relevance. ΔNp73 and TAp73 expression was detected in 93.6% and 98.7% of AML samples at variable levels (mean ± SEM, 10.6 ± 5.0, and 106.6 ± 33.7 copies/µL, for ΔNp73 and TAp73, respectively). ΔNp73 and TAp73 mRNA levels were highly correlated (R² = 0.72, P < 0.0001). Normal peripheral blood mononuclear cells and CD34+ hematopoietic cells showed little or no ΔNp73 and TAp73 expression (0.09 ± 0.09 and 0.42 ± 0.35 copies/µL, respectively), demonstrating that expression of ΔNp73 and TAp73 is 100 - 1,000 fold higher in AML as compared to normal hematopoietic cells. These data collectively suggests that transcriptional systems of both isoforms in AML cells are abnormally activated. Disease status (de novo or relapsed/refractory) and cytogenetic abnormalities did not correlate with ΔNp73 and TAp73 levels. However, AML with IDH1/2 mutations had 8.5-fold lower ΔNp73 expression than those with wild-type IDH1/2 (1.8 ± 0.8 vs 15.4 ± 7.7 copies/µL, P = 0.0069), with a similar trend for TAp73 (49.0 ± 20.3 vs 138.6 ± 51.4 copies/µL, P = 0.056). For de novo AML samples, those with DNMT3a and NRAS mutations had significantly higher ΔNp73, but not TAp73, than those without these mutations (21.6 ± 18.2 vs 2.5 ± 1.2 copies/µL, P = 0.017 and 5.6 ± 2.5 vs 9.7 ± 8.0 copies/µL, P = 0.047, respectively). These findings suggest that ΔNp73 and TAp73 can be differentially regulated in AML based on mutation status. To further explore the regulation of TP73 isoforms, we used MDM2 inhibitor Nutlin-3a to induce p53 which is a transcriptional inducer of ΔNp73. Indeed, MDM2 inhibition increased p73 protein levels, and knockdown of both TAp73 and ΔNp73 in AML cells enhanced apoptosis induction by Nutlin-3a (specific annexin V induction by 5 uM Nutlin-3a, 21.9 ± 1.3% vs 61.3 ± 5.2%, P = 0.0084 in OCI-AML3 cells with vector control vs Shp73, respectively), possibly due to the silencing of ΔNp73. AML cells with IDH1/2 mutations are more dependent on Bcl-2 than those without (Chan, Nat Med 2015). Intriguingly, (R)-2HG, the oncometabolite of mutant IDH1/2, reduced both TAp73 and ΔNp73 in AML cells and increased susceptibility to the Bcl-2 inhibitor ABT-199. These results imply a potential mechanism that regulates p73 isoforms by histone methylation or other epigenetic modifications in AML.

Conclusion Absolute quantitation of TP73 isoforms by ddPCR revealed high expression in AML cells compared to normal hematopoietic cells. The repressed expression of TP73 isoforms in AML cells with IDH1/2 mutations or by the oncometabolite (R)-2HG suggests that epigenetic modifications through (R)-2HG can regulate TP73 transcription and enhance the anti-leukemia effect by Bcl-2 inhibition. Finally, downregulation of TP73 isoforms enhances the efficacy of MDM2 inhibitor in AML, suggesting a potential therapeutic strategy to enhance MDM2 inhibitor-mediated p53 activation.