Tousled-like kinase activity is required for transcriptional silencing and suppression of innate immune signaling

Background
The fate and development of tumour cells during early carcinogenesis can be heavily determined by the emergence of increased replication stress, changes in the DNA damage response, as well as chromosomal instability caused by conflicts between DNA replication and transcription. These alterations are regulated by ATR and CHK1 kinases (1). Moreover, the activity of ATR and CHK1 kinases are tightly linked to tousled-like kinases (TLK1 and TLK2) that subsequently regulate downstream clients (ASF1a and ASF1b) affecting histone arrangement in DNA replication, repair and transcription (2). The loss of functions of both TLKs and ASF1s have been attributed to chromosomal instability and replication fork stability. Loss of TLKs also specifically increase ssDNA and DNA double-strand break levels indicating increased replication stress. TLK activity is often maintained in cancer cells due to their amplification, which is associated with poor prognosis (3). In addition, TLK can also lead to the dampening of the cGAS-STING signalling pathway (Stimulator of Interferon Genes) which is essential for the innate immune response required for recognition of tumours. This is because TLKs act to maintain CIN through regulating histone deposition which can prove to be problematic in the case of tumour cells, as proliferation is induced by suppression of cGAS-STING signalling and thus the inflammatory and innate immune response. In this preprint, the authors seek to elucidate the mechanisms of increased toxicity caused by TLK depletion and to understand how TLK activity intersects with the DNA damage response pathway involving the STING inhibitors BLM, SAMHD1 and MRE11.

Main Findings
Replication stress signalling requires BLM, MRE11 and SAMHD1 nucleases in the absence of TLKs
Firstly, TLK activity was analysed in relation to the mechanism of the generation of ssDNA induced by replication stress signalling. Because ASF1 generates ssDNA through facilitating duplex unwinding, it was sought whether TLK also functioned in this manner. Intriguingly, it was found that following treatment of replication stress inducers in TLK depleted cells, replication stress signalling was amplified characterized by ssDNA generation, while this was not the case in ASF1 depleted cells. Subsequently, it was further asked whether TLK activity generates ssDNA by helicases or nucleases instead of unwinding. This was done by knocking out multiple proteins such as MRE11, CTIP, SAMHD1 and BLM. In MRE11, SAMHD1 and BLM1 depleted cells, ssDNA accumulation was discontinued. This indicated that TLK-dependent stress response signalling is also dependent on these enzymes.

Depletion of TLK reduces gene silencing and induces innate immune responses
To probe the effect of TLK depletion, and thus hindrance o heterochromatin formation, on the silencing of heterochromatic sites, RNA expression was analysed by aligning samples with genetic variants annotated with GENCODE. It was found that deletion of TLKs caused upregulation of antisense and long intergenic noncoding RNA, and double deletion of both TLK1 and TLK2 yielded an additive phenotype. Moreover, expression of only coding regions was also analysed in which the most up-regulated effect was found in interferons. Antiviral responses comprised the majority of genes belonging to the upregulated category in GO analysis upon loss of TLK. A mediator of interferon activation, STING/TMEM173, as well as other IFN-stimulated genes were also activated which are involved in antiviral responses.

TLK depletion correlates with enhanced innate immune signalling in human tumour cells
Immunostimulation activates the STING/IFN response pathway in tumour cells with unstable genomes and micronuclei (4). This pathway subsequently promotes tumour cell death. However, many tumours prevail through the silencing of the STING pathway which dampens immune responses (5). To test the case of this in human cells, expression signatures were obtained for the top 10 upregulated interferon genes upon TLK depletion. TLK1 and TLK2 expression opposed STING expression in most tumours in the study. Both TLK1 and TLK2 expression also correlated with chromosomal instability and aneuploidy in many tumour types. Thus, TLKs act as a bridge between chromatin maintenance, genome stability and innate immune responses by promoting suppression of immune responses in tumours with high chromosome instability.

Why I chose this preprint
This preprint is particularly interesting as the results ultimately reveal TLK to be a very potential biomarker of tumour cell progression and development. The complex connection between TLK-dependent genome maintenance and innate immune responses is elucidated which allows for more effective targeting of a specific signalling pathway.

Questions
How could selective treatment of only tumour cells via TLK-targeting be carried out? Are there other components or pathways within tumour cells in relation to the TLK pathway which are upregulated in tumour cells compared to healthy cells that could be exploited?

References

1. Murga, M., Campaner, S., Lopez-Contreras, A.J., Toledo, L.I., Soria, R., Montana, M.F., D’Artista, L., Schleker, T., Guerra, C., Garcia, E., et al. (2011). Exploiting oncogene-induced replicative stress for the selective killing of Myc-driven tumors. Nat Struct Mol Biol, 18, 1331-1335.
2. Carrera P., Moshkin Y.M., Gronke S., Sillje H.H., Nigg E.A., Jackle H., and Karch, F. (2003). Tousled-like kinase functions with the chromatin assembly pathway regulating nuclear divisions. Genes Dev, 17, 2578-2590.
3. Lee, S.B., Seguar-Bayona S., Villamor-Paya M., Saredi G., Todd M.A.M., Attolini C.S., Change T.Y., Stracker T.H., and Groth A. (2018). Tousled-like kinases stabilize replication forks and show synthetic lethality with checkpoint and PARK inhibitors. Sci Adv 4, 4985.
4. Bakhoum S.F., and Cantley L.C., (2018). The Multifaceted Role of Chromosomal Instability in Cancer and Its Microenvironment. Cell 174, 1347-1360.
5. Chen Y.A., Shen Y.L., Hsia H.Y., Tiang Y.P., Sung T.L., and Chen L.Y., (2017). Extrachromosomal telomere repeat DNA is linked to ALT development via cGAS-STING DNA sensing pathway. Nat Struct Mol Biol 24, 1124-1131.

 

doi: https://doi.org/10.1242/prelights.10554

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