Acute promyelocytic leukemia (APL) is driven by the oncogene PML-RARA which is generated by fusion of the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARA) genes, and which strongly interferes with downstream signalling and the architecture of multiprotein structures known as PML nuclear bodies (NBs). NB disruption is a diagnostic hallmark of APL, yet the significance of this phenomenon to disease pathogenesis and treatment response remains poorly understood. The majority of APL patients can now be cured with combination therapy with arsenic trioxide (ATO) and ATRA (All Trans-Retinoic Acid), which synergize promoting re-formation of disrupted Pml NBs. To date, the importance of NB disruption has only been studied in vitro. To address this, we generated a knock-in mouse model with targeted NB disruption achieved through mutation of key zinc-binding cysteine residues (C62A/C65A) in the RING domain of Pml. Homozygous PmlC62A/C65A mice are viable, and developmentally normal. At a cellular level, Pml NB disruption was confirmed and treatment with ATO was associated with defective Pml SUMOylation and degradation. A key feature of APL fusion proteins is the capacity to homodimerise (mediated by the fusion partner e.g. PML), which is not a feature of wild-type RARα. This forced homodimerisation of RARα has been shown to be critical for APL pathogenesis. We investigated whether Pml NB disruption could cooperate in vivo with forced RARα homodimerisation (mediated artificially by linking RARα to the dimerisation domain of the NFκB p50 subunit). While no leukemias arose in PmlC62A/C65A mice, p50-RARα mice expressing PmlC62A/C65A presented a doubling in the rate of leukemia development (p<0.0001) compared to PmlWT-p50-RARα, leading to a penetrance comparable to that observed in previously published PML-RARα transgenic models. Moreover, the latency period to onset of leukemia was significantly reduced in the context of NB disruption (p=0.008). ATRA treatment significantly improved the survival of mice transplanted with PmlWT-p50-RARα or Pml-RARα leukemic blasts, but not with PmlC62A/C65A-p50-RARα. These data reveal not only the key role of PML-RARα expression-induced NB disruption in APL development, but also the importance of re-formation of NBs for an effective response to differentiating drug.
While formation of the PML-RARA fusion is considered an initiating event in APL pathogenesis, it is insufficient for the full leukemic phenotype. Exome sequencing studies have consistently identified presence of cooperating mutations. Since Pml and Pml NB have established roles in DNA repair and in the maintenance of genomic stability, we speculated that loss of NB integrity could affect these functions. Whole exome sequencing revealed a pattern of higher genomic instability in PmlC62A/C65A-p50-RARα leukemia as compared to PmlWT-p50-RARα, with detection of mutations found in human APL, including Kras, Ptpn11 and Usp9y. Using DNA repair reporter assays, we demonstrated that DNA repair via both non-homologous end joining (NHEJ; p=0.01) and homologous recombination (HR; p=0.006) pathways was less efficient in PmlC62A/C65A primary cells than in PmlWT cells. Importantly, using a PML-RARα-inducible cell line, comparable defects in the NHEJ and HR pathways, which were PML-RARα dependent, were identified. These data were also supported by an increase in sister-chromatid exchange (p<0.0001) and chromosome abnormality (p=0.0002) in the context of PmlC62A/C65A versus PmlWT. Interestingly, the kinetic of repair of ionising radiation (IR)-induced DNA double-strand breaks, assessed by analysis of γH2AX foci formation and clearance, was not affected. None of the DNA repair players analysed (e.g. Blm, Rad51 and 53BP1) failed to form foci in response to IR. However, their basal levels of foci were significantly greater in the presence of PmlC62A/C65A (p<0.04; quantified using Amnis ImageStreamX Mk II imaging flow cytometer). Additionally, we found that Rad51 foci showed a defect in localisation post-IR when PmlC62A/C65A was expressed, with impairment of Rad51 co-localisation and interaction with γH2AX. Altogether, our data therefore highlight the significant contribution of Pml NB to the effectiveness of DNA damage repair processes, and the manner in which their disruption mediated by the PML-RARα oncoprotein can assist APL pathogenesis.
Hills:TEVA: Honoraria. Grimwade:TEVA: Research Funding.
Asterisk with author names denotes non-ASH members.