Deciphering protein-kinase C beta signalling in blood cancers

Primary supervisor: Ingo Ringshausen, UCL

Secondary supervisor: Pavel Tolar, UCL

Collaborator: Neil McDonald, Francis Crick Institute

Project

The development of normal B cells is an error-prone process which can lead to autoimmunity and cancer [1]. Notably, normal B cell development as well as evolution of tumour B cells is strictly dependent and coregulated by non-malignant bystander cells, commonly referred to as the (tumour) microenvironment (TME). Our group has previously identified molecular mechanisms by which tumour cells actively reprogram mesenchymal stromal cells (aka Cancer-associated fibroblasts (CAFs)) in the TME. Interference with this activation is a powerful and orthogonal approach to current cancer therapies: In particular, we discovered that genetic or pharmacological interference with the activation of protein kinase C-beta (PKCb), expressed in mesenchymal stroma-cells in the TME, increases the efficacy of co-applied cancer therapies and inhibits disease progression in blood [2] – and solid cancers [3]. Mechanistically, this is related to an impaired activation of the transcription factor NF-kappaB by PKCb, suppressing tumour-induced inflammation in the TME [4] . Notably, previously developed small molecule inhibitors for PKCb have poor pharmacokinetic profiles and therefore, targeting alternative signalling molecules in this pathway is needed.

This PhD-project aims at deciphering the molecular mechanism underlying the activation of PKCb in TME stroma cells and to understand how PKCb regulates the survival of cancerous B cells:

Aim1: To identify protein-binding partners of PKC-beta in tumour-activated stroma cells

While some binding partners of PKCb in normal B cells have been described [5], PKCb-signalling remains entirely unknown in mesenchymal stroma cells. The student will employ proximity-ligation assays and mass spectrometry to identify protein-complexes interacting with PKCb in tumour-activated stroma cells. Validation of protein-interactions will be achieved through immunofluorescence (split-Fluorescence protein labelling & confocal microscopy) and co-immunoprecipitation assays, focusing on binding partners with a potential of pharmacological interference. In addition, in collaboration with the McDonald group, binding of identified proteins will be modelled in silico.

Aim 2: To functionally assess the role of PKCb-binding proteins for cancer cell survival

Functions of proteins identified in aim 1 will be assess for their role in activating stroma cells and providing survival support to cancer cells: Genetic deletion using CRISPR-Cas9 technology in combination with NFkappaB- reporter assays will be used to identify whether these proteins are important for inducing inflammation in CAFs. The student will use established 2D and 3D co-culture systems to test whether inhibition/ deletion of these proteins will affect tumour cell survival, proliferation and resistance to therapies.

Aim 3: To characterise subsets to TME-cells with enhanced PKCb activity: 

Our laboratory has developed a novel reporter mouse strain which allows the identification of PKCb-induction its expression in vivo. Using adoptive transfer of murine malignant B cells into syngeneic mice, tumouractivated stroma cells can be identified. Single cell RNAseq and confocal microscopy will be used to define the origin and spatial distribution of PKCb expressing stroma cells, allowing for the in vivo validation of targets
identified in aims 1 and 2.

In summary, this project will provide a comprehensive analysis of PKCb signalling in the TME and lay the foundation for developing novel therapies antagonising TME-signals.

References

1. Küppers R. Mechanisms of B-cell lymphoma pathogenesis. Nature reviews Cancer. 2005;5(4):251-262. doi:10.1038/nrc1589
2. Park E, Chen J, Moore A, et al. Stromal cell protein kinase C-β inhibition enhances chemosensitivity in cell malignancies and overcomes drug resistance. Science Translational Medicine. 2020;12(526):eaax9340. doi:10.1126/scitranslmed.aax9340
3. Wallace JA, Pitarresi JR, Sharma N, et al. Protein Kinase C Beta in the Tumor Microenvironment Promotes Mammary Tumorigenesis. Frontiers Oncol. 2014;4:87. doi:10.3389/fonc.2014.00087
4. Lutzny G, Kocher T, Schmidt-Supprian M, et al. Protein kinase c-β-dependent activation of NF-κB in stromal cells is indispensable for the survival of chronic lymphocytic leukemia B cells in vivo. Cancer cell. 2013;23(1):77-92. doi:10.1016/j.ccr.2012.12.003
5. Su TT, Guo B, Kawakami Y, et al. PKC-beta controls I kappa B kinase lipid raft recruitment and activation in response to BCR signaling. Nature immunology. 2002;3(8):780-786. doi:10.1038/ni823