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New Frontiers in Cellular Reprogramming: Exploiting Cellular Plasticity

Periodic Reporting for period 4 - CELLPLASTICITY (New Frontiers in Cellular Reprogramming: Exploiting Cellular Plasticity)

Reporting period: 2020-05-01 to 2021-09-30

This project was based on the hypothesis that understanding cellular plasticity could yield new insights into cancer and ageing. Our unifying hypothesis is that cellular plasticity lies at the basis of tissue regeneration (“adaptive cellular plasticity”), as well as at the origin of cancer (“maladaptive gain of cellular plasticity”) and ageing (“maladaptive loss of cellular plasticity”). We have taken advantage of our diverse background and integrate the above processes. A key experimental system will be our “reprogrammable mice” (with inducible expression of the four Yamanaka factors), which we regard as a tool to induce cellular plasticity in vivo. During the completion of this project, we have made relevant contributions to the fields of cellular reprogramming, cellular senescence, cancer, and ageing.

The project has been divided in three major objectives:

Objective #1 – Cellular plasticity and cancer: role of tumour suppressors in in vivo de-differentiation and reprogramming / impact of transient de-differentiation on tumour initiation / lineage tracing of Oct4 to determine whether a transient pluripotent-state occurs during cancer.

The main conclusion of this objective has been that the induction of cellular plasticity by the Yamanaka factors is not very tumorigenic. The expression of these plasticity factors together with oncogenes or mutagens did not have a dramatic impact on tumorigenesis. This indicates that cellular plasticity per se is not tumorigenic because it may drive cells into differentiated states that oppose tumorigenesis. This conclusion is of relevance for the use of transient expression of the Yamanaka factors in rejuvenation and regeneration.

Objective #2 – Cellular plasticity in tissue regeneration and ageing: impact of transient de- differentiation on tissue regeneration / contribution of the damage-induced microenvironment to tissue regeneration / impact of transient de-differentiation on ageing.

In contrast to cancer, the Yamanaka factors are able to induce a reversion of aging molecular markers in vivo. We have found that a single burst of transient and reversible expression of the Yamanaka factors is sufficient to reverse markers of cellular aging, including the transcriptome and the epigenome.

Another important conclusion of our work is the discovery that the Yamanaka factors need to operate in a tissue microenvironment modified by injury. In other words, the Yamanaka factors require cytokines associated to injury to trigger plasticity and rejuvenation. This is the case of interleukin-6, which we discovered as a critical factor for the induction of cellular plasticity by the Yamanaka factors.

Objective #3: New frontiers in cellular plasticity: chemical manipulation of cellular plasticity in vivo / new states of pluripotency / characterization of in vivo induced pluripotency and its unique properties. We anticipate that the completion of this project will yield new fundamental insights into cancer, regeneration and ageing.

We have identified markers of intermediate reprogramming in vivo that are applicable to several tissues. This will allow to unequivocally define for the first time those cells that have reached the state of partial reprogramming, which is the state when epigenomic rejuvenation reaches its maximum.

We have also found a novel mechanism to manipulate the strength of the enhancers that maintain cellular identity. This is based on small compound that inhibit a protein kinase known as MEDIATOR-kinase (or CDK8). The chemical inhibition of this kinase boosts the strength of transcriptional enhancers and therefore opposes cellular plasticity. Therefore, this tool may serve to oppose or to extinguish cellular plasticity. This may be important to prevent the emergence of cancer from plastic cells.
The main achievements attained during this reporting period are enumerated below:

1) Tissue damage and senescence provide critical signals for cellular reprogramming in vivo (see Figure). The expression of the Yamanaka factors of reprogramming in vivo triggers two different cellular outcomes: reprogramming in a small fraction of cells, and damage and senescence in many other cells. Cellular senescence creates a tissue context that favours Yamanaka factors-driven reprogramming in neighbouring cells. The positive effect of senescence on reprogramming is mediated by secreted factors, of which interleukin-6 (IL-6) is a key player. Pharmacological manipulations that reinforce senescence, such as palbociclib (a CDK4 inhibitor) or recombinant IL-6, promote in vivo reprogramming.

2) Development of AAV vector-mediated technology for in vivo reprogramming into pluripotency. We accomplished full in vivo reprogramming using the Yamanaka factors, both with or without c- Myc. Our approach advances crucially in vivo reprogramming technology.

3) Demonstration that a single period of transient and reversible expression of the Yamanaka factors in vivo is able to reverse markers of cellular aging, including the transcriptome, the epigenome and the serum metabolome. (Figure 2).

4) We have discovered that natural killer (NK) cells are a main barrier for in vivo reprogramming. The transient depletion of NK cells strongly enhances reprogramming.

5) Identification of markers of intermediate reprogramming in vivo applicable to multiple tissues.

6) A new mechanism of regulation of the tumour suppressor p21 by the mTORC1/4E-BP1 pathway, revealing new tumour markers for the progression of head and neck cancer.

7) Discovery of RPAP1 protein as a novel RNA polymerase II regulator, that is essential for the communication between DNA enhancer elements and gene promoters. In particular, RPAP1 is a key subunit for the association of the MEDIATOR complex bound to enhancers with the RNA polymerase II bound to gene promoters. Loss of RPAP1 triggers a decrease in the association between Mediator and Pol II affecting cell identity and viability.

8) A novel chemical method to manipulate cellular plasticity by inhibiting the MEDIATOR-kinase. This reduces cellular plasticity and this has applications to stabilize some extremely plastic cell types and facilitate their propagation in vitro. The main example of this is the cultivation of naïve human pluripotent cells.

9) Design of a drug encapsulation system that selectively targets senescent cells. Our studies have uncovered the potential of beta-galactosidase for the activation of pro-drugs targeted to senescent cells. These have paved the way for therapeutic approaches to eliminate senescent cells in many diseases, such as pulmonary fibrosis and cancer.

All the above results have been published in scientific journals of high impact and have been presented in multiple scientific conferences. Also, they have been the topic of outreach activities for the general public and young students.
We have gone beyond the state of the art in several aspects:

- We have pioneered the use of beta-galactosidase to activate pro-drugs in senescent cells.

- We have discovered that some available pharmacological agents can be used to promote in vivo reprogramming, such as palbociclib, recombinant IL-6, or antibodies against Natural Killer cells.

- We have discovered a method to prevent cellular plasticity by using small chemical compounds that inhibit the MEDIATOR-kinase. This can be useful to stabilize some unstable cell states, notably due to their importance for cellular therapies the stabilization of naïve human pluripotent cells.
Figure 1. Tissue damage and ageing favour in vivo reprogramming.
Figure 3. Model for RPAP1 function.
Figure 2. Model for mTOR-mediated regulation of p21.
Figure 4. Senescent cells targeted drug delivery system.