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New Frontiers in Cellular Reprogramming: Exploiting Cellular Plasticity

Periodic Reporting for period 3 - CELLPLASTICITY (New Frontiers in Cellular Reprogramming: Exploiting Cellular Plasticity)

Reporting period: 2018-11-01 to 2020-04-30

"Our research group has worked over the years at the interface between cancer and ageing, with a strong emphasis on mouse models. We have now become interested in cellular reprogramming because we hypothesize that understanding cellular plasticity could yield new insights into cancer and ageing. We have already made relevant contributions to the fields of cellular reprogramming, cellular senescence, cancer, and ageing. We take now advantage of our diverse background and integrate the above processes. 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”). 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.

The project is divided as follows:

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.

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.

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."
The main achievements attained during this reporting period are enumerated below:

1) Tissue damage and senescence provide critical signals for cellular reprogramming in vivo (Figure 1)

1a) 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.
1b) Cellular senescence creates a tissue context that favours Yamanaka factors-driven reprogramming in neighbouring cells.
1c) The positive effect of senescence on reprogramming is mediated by secreted factors, of which interleukin-6 (IL-6) is a key player
1d) Biological conditions linked to senescence, such as tissue injury or ageing, favour in vivo reprogramming by the Yamanaka factors.
1e) Senescence induced by the Yamanaka factors requires the tumour suppressor Ink4a (but not its genetic companion Arf). In the absence of Ink4a, senescence is not implemented and IL-6 is not secreted, and the consequence of this is an impairment of reprogramming.
1f) Pharmacological manipulations that reinforce senescence, such as palbociclib (a CDK4 inhibitor) or recombinant IL-6, promote in vivo reprogramming.

2) A new mechanism of regulation of the tumour suppressor p21 by the mTORC1/4E-BP1 pathway (Figure 2), revealing new tumour markers for the progression of head and neck cancer.

3) The tumour suppressor p21 has an unprecedented role as a positive regulator of PPARα, a key transcription factor that orchestrates multiple aspects of fasting adaptation.

4) 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 (Figure 3).

5) Design of a drug encapsulation system that selectively targets senescent cells (Figure 4). Our studies have paved the way for therapeutic approaches to eliminate senescent cells in many diseases, such as pulmonary fibrosis and cancer.

6) 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.
We hope to go beyond the state of the art by providing:
- Technology for the selective and efficient delivery of senolytic drugs into senescent cells.
- Methodology for promoting in vivo reprogramming of somatic cells into induced pluripotent stem cells (iPSC), including small compounds (such as palbociclib), biologicals (such as recombinant IL-6), or safe viral vectors (such as AAVs carrying the Yamanaka factors).

As for the expected results:

Role of the immune system on in vivo reprogramming
The in vivo environment constitutes a more complex situation than in vitro systems. Understanding the interplay between cellular reprogramming and the immune system will provide insights to modulate and control cellular plasticity in vivo. Cell dedifferentiation and pluripotency involves the expression of embryonic antigens that may trigger an adaptive immune response. Other changes during reprogramming may activate the innate immune systems.

Impact of transient de-differentiation on tissue regeneration
To demonstrate that tissue regeneration/repair involves the transient acquisition of a pluripotent-like state and also that the reprogramming factors can enhance regeneration/repair. This may be tissue specific and/or damage-specific, and we will examine this in multiple tissues. If successful, we may be able to isolate the plastic cells responsible for the regeneration/repair of damaged tissues and characterise them in detail.

Contribution of the damage-induced tissue microenvironment to tissue regeneration
Integrate into a unified model damage-induced senescence and damage-induced pluripotent-like plasticity. This will advance our understanding of tissue regeneration and repair.

Chemical manipulation of cellular plasticity in vivo
We will test in vivo the known capacity of specific chemicals to promote reprogramming and determine if this enhances tissue repair and/or regeneration.

New states of pluripotency
CDK8 is a negative regulator of the MEDIATOR complex. We have discovered that pharmacological potentiation of MEDIATOR, by using a small compound inhibitor of CDK8, is sufficient to induce the naïve state of pluripotent stem cells, both mouse and human. The naïve state has been stabilized before by using a MEK inhibitor, however, this has deleterous effects on the genomic stability of the cells, on imprinting and on the inactivation of the X chromosome. We think that the use of the CDK8 inhibitor will circumvent all these problems while efficientlly stabilizing the naïve state.
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.