Background: Mutations in TP53 (TP53mut) are present in approximately 10% of acute myeloid leukemia (AML) patients and represent a unique subtype with a very poor prognosis. Although the exact mechanisms of TP53mut driven leukemogenesis remain elusive, transcriptional changes associated with suppression of the TP53 signaling pathway are considered to confer a dismal prognosis on TP53mut patients.
Aims: Using molecular profiling of a large cohort of AML patients (n=654) we identify a new poor prognostic subgroup in AML that is TP53-wt but shares strong similarities with TP53mut patients. Patients included in this new group are characterized by increased expression of the oncogenic isoform of the TP73 gene – ΔNp73, which lacks the transactivation domain but still binds to chromatin, negatively regulating the expression TP53 itself and its target genes as well as TAp73 (the tumor-suppressor form of the TP73 gene).
Methods: We find that the transcriptional program of ΔNp73high AMLs strongly resembles that of TP53mut AML and is enriched for stemness signatures like “17LSC”, while ΔNp73low AML is correlated with more mature GMP signatures. In line with TP53mut AML, ΔNp73high/TP53-wt frequently co-occurs with mutations in spliceosome-related genes.
Results: Lentiviral overexpression (OE) of ΔNp73 in healthy CD34+ cells results in increased cell proliferation and stemness. ΔNp73-OE in several TP53-wt AML models imposes drug resistance against several used cytotoxic therapies in AML (e.g., Cytarabine, Venetoclax±Azacytidine), which is not seen in TP53mut AML cells, suggesting that high ΔNp73 is associated with downregulation of the TP53 signaling pathway. These observations were validated using ex vivo treated primary AML samples (n=60), where increased ΔNp73high samples were more resistant to several cytotoxic therapies, including VEN+AZA schemes. Lentiviral ΔNp73-OE in primary APL patient samples (n=10) allowed efficient engraftment in vivo using xenograft mouse models with colonization of secondary organs and splenomegaly, which was not observed in when ΔNp73-OE was not overexpressed.
Chromatin accessibility data on AML patients revealed a differentially accessible region in intron 2 (+24 Kb after the transcription start site, referred to as peak B) of the TP73 gene, with unknown regulatory functions on TP73 expression. To evaluate whether this region would act as a potential enhancer region to drive ΔNp73 expression we generated CRISPR/Cas9 knockout MOLM13 cells lacking the peak B region (MOLM13-KO). MOLM13-KO cells exhibited reduced ΔNp73 expression, and loss of this region significantly impaired the ability to up-regulate ΔNp73 expression upon high dose Cytarabine-induced stress. Chromatin immunoprecipitation analysis demonstrated complete loss of ΔNp73 but not TAp73 binding at TP53 regulatory regions, as well as at TP53 downstream targets, such as CDKN1A, allowing the re-expression of TP53 targets. Consequently, MOLM13-KO cells displayed decreased cell survival, in comparison to control.
Summary/Conclusion: We demonstrate for the first time the importance of ΔNp73 isoforms as oncogenic hits that mimic poor-risk TP53mut AML, which will aid in diagnostic patient stratification. Functionally, high ΔNp73 levels resulted in increased resistance to cytotoxic therapy and allowed engraftment of otherwise notoriously difficult to engraft primary APL patient samples. We identify a novel enhancer driving the expression of the TP73 oncogenic isoforms. Our study provides important insight into the molecular mechanisms underlying the poor risk characteristics of a TP53-wt patient subgroup expressing ΔNp73.
Keywords: Acute myeloid leukemia, Chemoresistance, TP53, Venetoclax