Incidence of grave pathologies including neurodegeneration and cancer increases in today’s aging European populations and new approaches to battle these diseases are required. Shared by these pathologies and premature aging syndromes is the enhanced DNA damage and genomic instability, likely causal phenomena that can be better understood by functional elucidation of the cellular DNA damage response (DDR) machinery and its defects. The central hypothesis of this project is that ‘nucleolar genome’ that contains many copies of rDNA genes, the repetitive and most highly transcribed genomic sequences essential for ribosome biogenesis and protein synthesis, may represent an exceptionally vulnerable ‘Achilles heel’ of our genome whose instability fuels aging and tumorigenesis. This project proposes to approach this problem by a combination of innovative cellular models and techniques including gene editing, proteomics and live cell imaging, to assess rDNA damage and the ensuing genomic instability in human cells exposed to exogenous (radiation, chemotherapy drugs) and endogenous (replication and transcription stress, ribosome biogenesis stress, activated oncogenes) insults and identify and functionally characterize signalling and ‘repair’ factors that guard nucleolar integrity and function. This proposal is timely due to the emerging concept of nucleolus as a sensor of diverse stresses and the fact that technological advances now allow analysis of the difficult-to-assess rDNA genes. The applicant is experienced in the DDR field and modern technologies, the receiving institute is among the world leading centers in DNA damage, cancer and cell stress research fields. These aspects, together with the large body of preliminary unpublished data makes this ambitious project feasible. The results will provide novel insights into genome integrity maintenance and cell stress responses, and may inspire novel strategies to treat or even prevent age-related diseases, especially cancer.

Accurate chromosome distribution during mitosis requires that each pair of sister chromatids attaches to microtubules from opposite spindle poles via centromere-based kinetochores. However, incorrect kinetochore-microtubule attachments can be formed. These errors must be corrected to prevent chromosome missegregation and the resulting aneuploidy, an abnormal chromosome count characteristic of most human cancers. Error correction relies on the centromere/kinetochore-localised kinase Aurora B, which promotes the depolymerisation of kinetochore-binding microtubules in response to lack of tension within erroneous attachments. Tension locally modulates the accessibility of Aurora B to its substrates, which include the microtubule-depolymerising motor MCAK. Paradoxically, Aurora B suppresses MCAK activity and should, therefore, stabilise erroneous tensionless attachments. This contradiction nurtures the ambiguity of the long-sought error correction mechanism. Findings from the host laboratory provide new insights into this process. Microtubule poleward flux, driven by motor-mediated sliding of interpolar microtubules, suppresses MCAK depolymerising activity, potentially by generating tension within attachments. My goal is to provide a precise and integrative mechanism on how motor proteins modulate the error correction mechanism. I will focus on three key objectives: (1) identifying the flux-generating motors that create tension within attachments, (2) determining the role of tension in MCAK-mediated error correction, and (3) dissecting the tension-related interplay between Aurora B and MCAK during error correction. To achieve this, I will integrate super-resolution live cell imaging with acute protein depletion using the auxin-degron system, delivered at endogenous loci through CRISPR-Cas9. This approach will enable the monitoring of multiple motors in error correction with unprecedented spatiotemporal resolution, providing new insights into how cells prevent aneuploidy.

Faithful eucaryotic cell division requires spatio-temporal orchestration of multiple sequential events. Among the crucial steps to providing the daughter cells with identical set of chromosomes is the DNA replication. During this phase, cells coordinate the speed of DNA synthesis with the length of the cell cycle to ensure genome integrity. To do so, growth factors and metabolic signals are integrated primarily by D-type Cyclins. Indeed. their deregulation can directly lead to some of the hallmarks of cancer by causing proliferation that is independent of normal extracellular cues. We previously demonstrated that aberrant accumulation of Cyclin D1 results in a faster cell cycle, with uncontrolled speed of DNA replication fork progression and genome instability (Maiani & Milletti et al., 2021). Despite, frequently altered in many tumors (Musgrove et al., 2011), a unifying theory that clarify how Cyclin D1 promote cancer transformation is still lacking. In the lab of Prof. Jiri Bartek, it was previously shown that PARP1 inhibition increases replication fork speed (Maya-Mendoza et al., 2018). PARP1i uncouples the leading and lagging DNA synthesis resulting in fast fork speed and genome instability. However, it is currently unknown whether the aberrant accumulation of Cyclin D1 has similar effect on DNA synthesis as PARPi. Furthermore, it is also undetermined whether aberrant levels of Cyclin D1 could trigger metabolic changes that accelerate the speed of DNA synthesis. Taking advantage of our previous observations, we aim with this proposal to: i) identify metabolic signatures that can predict dis-regulated DNA synthesis in response to cell cycle alterations; ii) define the molecular mechanism of how cyclin D1 accumulation induces accelerated fork speed; iii) identify new metabolic genes involved in the control of S phase progression and the speed of DNA synthesis by CRISPR-Cas9 screening technology; iv) suggest druggable targets that could be used in cancer therapy.