The Cause and Consequence of Replication Stress in Adult T-Cell Leukemia

EP300 and its homolog CBP are transcriptional co-activators of genes involved in a broad range of cellular processes, including proliferation, differentiation, apoptosis and DNA repair. Inactivating mutations in both EP300 and CBP have been reported in developmental disorders, such as Rubinstein-Taybi Syndrome, and several hematological malignancies. Most recently, somatic hypermutations in EP300 (but not CBP) were identified in Adult T-cell leukemia/lymphoma (ATLL) patients, diagnosed in Northern America. The mechanistic contribution of EP300 dysregulation to NA-ATLL etiology is currently unknown.

ATLL is caused by infection by the Human T-cell Lymphotrophic Virus Type-1 (HTLV-1), that is endemic to Japan, the Caribbean basin, South America, southern parts of North America, Eastern Europe and certain areas of Africa. Unlike patients diagnosed in Japan (J-ATLL), NA-ATLL patient display a severe case of chemo-refractory disease with very poor prognosis. While prominent differences in the transcriptional and epigenetic landscape, particularly the p300 mutational status, has been identified between J-ATLL and NA-ATLL patients, the mechanistic contribution of p300 dysregulation to disease severity is yet to be established. Studies have revealed that the independent inhibition of EP300 in human cells results in a unique transcriptional landscape, involving the differential expression of genes involved in regulating the cell cycle, DNA replication and DNA damage response.

Here, we show that EP300-mutated ATLL cells display a protracted S-phase, extensive genomic instability, dysregulations in DNA replication and repair dynamics. Using a powerful locus specific single molecule assay and genome-wide DNA fiber analysis, we show that perturbed DNA replication in EP300-mutated ATLL cells is characterized by elevated origin firing, increased replisome pausing and prominent nucleolytic degradation of nascent DNA at collapsed forks. The elevated origin firing in the absence of EP300 is likely driven by the inherent endogenous increase in c-MYC expression, which has been previously seen in EP300-mutated MCF7 cells. The striking replication pausing, fork collapse and nascent strand degradation observed in EP300-deficient NA-ATLL cells results in the persistence of single stranded DNA (ssDNA) genome-wide, resulting in the hyperphosphorylation of RPA at Ser4/8. Inhibition of Mre11 nuclease partially rescued the ssDNA accumulation indicating a dysregulation in downstream mechanisms that restrain nuclease activity at stalled forks. Elevated endogenous genomic instability, observed in ATLL cells during the S-phase is exacerbated by EP300 deficiency, even in the absence of exogenous stress/damage.

Analysis of the consequences of S-phase abnormalities and genomic instability revealed elevated micronuclei and cytosolic DNA in ATLL cells exposed to DNA damage. Preliminary studies evaluating the innate immune system activation (cGAS-STING-pIRF3) by immunoblotting, in response to stress, has led to some intriguing results. There is a clear separation in the ability of J-ATLL versus NA-ATLL cells in activating the innate immune system, a response that appears to be EP300 independent. These results raise the possibility of the contribution of a suppressed innate immune response, a phenomenon seen in many fast-progressing cancers, to the etiology of NA-ATLL. In addition to increasing our understanding of the mechanisms contributing to ATLL in general, the results from this study have helped identify new mechanism-based treatment regimens that could target the replicative and innate immune system vulnerabilities observed in North American ATLL patients.

No relevant conflicts of interest to declare.