Evaluating PKN2 as a target in the host-tumour microenvironment.
Peter Parker (Crick) and Francesco Dazzi (King’s)
Apply to: King's
Summary:
The tumour microenvironment has a profound effect on the survival and dissemination of
tumour cells. This has been a longstanding area of investigation for the Dazzi laboratory and
the recent impact of immunomodulatory interventions targeting the microenvironment bring
this host-tumour interface into sharp focus. Targeting the microenvironment provides
particular opportunities both attacking the tumour and targeting the genetically stable cellular
environment, addressing a prevalent cause of tumour resistance. The collaboration established
here is directed at investigating the role of PKN2, a newly discovered regulator of stromal cells,
on the biology of the tumour niche. The results have huge translational potential aligned to
both an early drug development project, which has produced some chemical leads (Parker
laboratory). The advance of the programme will support a PhD project aimed at developing a
drug discovery process for clinical delivery.
This project is directed at assessing whether PKN2 plays a significant role in defining stromal
properties in the tumour microenvironment and hence whether PKN2 lends itself as a target
for intervention.
In initial phases of the study the Fellow will assess whether PKN2 in bone marrow derived
stromal cells, dermal fibroblasts or lung-derived fibroblasts (for simplicity referred to here as
stroma) influence ex vivo properties pertinent to the survival and immune-privileged position
of the tumour. In the very beginning, in order to be highly selective, the role of PKN2 will be
assessed by using murine stroma cells from a PKN2 conditional KO donor. Once we the
hypothesis will be confirmed, we will use human MSC in which PKN2 will be knocked-down by
shRNA. The Fellow will assess ex vivo how different stroma influence T-cell function in coculture systems; the immunosuppressive activity will be evaluated in vitro and in vivo. The in
vitro assay will consist of stimulating antigen-specific T cells with the cognate antigen in the
presence of serial concentrations of PKN2 KO or WT stroma (tamoxifen or vehicle treated).
Cultures will be assessed for T cell proliferation and compared with mono-cultures as controls.
To evaluate the activity in vivo, the first step will involve the use of a T-cell dependent hepatitis
model induced by concanavalin-A. This model has the advantage of being sensitive to human
MSC, short in duration and rapid in assessment (liver enzymes in serum), thus useful for future
screening of PKN2-directed drugs. A similar approach will be taken to assess the requirement
of PKN2 in stroma directed myeloid differentiation ex vivo. PKN2 KO and WT stroma will be
tested for their ability to induce the differentiation of immunosuppressive macrophages from
common myeloid progenitors. Finally, the role of PKN2 on the ability of stroma to protect
tumour cells from anti-cytotoxic agents will be investigated. The ex vivo assays will be run
initially in the KCL lab and transferred to the Crick lab to provide an efficient means of
completing these experiments. There will be an ex vivo follow up using pan-PKN tool
compounds from the drug development programme to evaluate the drug-induced inhibition
on the described stroma cell parameters.
References:
1. Quetier, I., Marshall, J. J., Spencer-Dene, B., Lachmann, S., Casamassima, A., Franco, C.,
Escuin, S., Worrall, J. T., Baskaran, P., Rajeeve, V., Howell, M., Copp, A. J., Stamp, G.,
Rosewell, I., Cutillas, P., Gerhardt, H., Parker, P. J.*, and Cameron, A. J.* (2016)
Knockout of the PKN Family of Rho Effector Kinases Reveals a Non-redundant Role for
PKN2 in Developmental Mesoderm Expansion. Cell Reports 14, 440-448
2. Lachmann, S., Jevons, A., De Rycker, M., Casamassima, A., Radtke, S., Collazos, A., and
Parker, P. J. (2011) Regulatory Domain Selectivity in the Cell-Type Specific PKNDependence of Cell Migration. PLoS One 6, e21732
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cells inhibit proliferation and apoptosis of tumor cells: impact on in vivo tumor growth.
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4. Vianello F, Villanova F, Tisato V, Lymperi S, Ho KK, Gomes AR, Marin D, Bonnet D,
Apperley J, Lam EW, Dazzi F. (2010) Bone marrow mesenchymal stromal cells non
selectively protect chronic myeloid leukemia from imatininb-induced apoptosis via the
CXCR4/CXCL12 axis. Haematologica. 95(7):1081-9