Professor EmeritusRichard Christopherson

Research in our group focuses on the use of proteomics with mass spectrometry to develop new methods for classification of chronic lymphocytic leukemia (CLL) and for elucidation of the mechanisms of action of drugs effective in treating CLL, either singly or in combination. Ibrutinib and idelalisib are ‘small molecule’ inhibitors that have changed the ‘landscape’ for treatment of CLL. Ibrutininb inhibits Bruton’s tyrosine kinase, and idelalisib inhibits phosphoinositide 3-kinase δ. Our current research involves identification of proteins in samples of CLL whose levels of phosphorylation have changed (up or down) in cells treated with a kinase inhibitor. These changing patterns of phosphorylation (+ drug) should provide an explanation for how mitogenic signals pass from the B-cell receptor (BCR) on the plasma membrane of CLL cells to the nucleus to drive cellular proliferation, and how the kinase inhibitors block these signaling pathways. CLL has been attributed to tonic signaling by the BCR to stimulate proliferation that can be blocked by these kinase inhibitors. Identification of phosphorylated proteins is being done in collaboration with Prof Lee Graves (Department of Pharmacology, University of North Carolina, USA).

Fludarabine (2-FaraA) is a purine nucleoside analogue that is ‘front line’ treatment for CLL. In some cases of CLL, 2-FaraA may be effective as a single drug that may have up to 5 different mechanisms of action. One of these mechanisms is inhibition of DNA repair, resulting in strand breaks in DNA that activate several protein kinases (ATM, ATR and CHK1) that would change patterns of phosphorylation in CLL, and presumably also affect BCR signaling. Thus, the identification of proteins whose patterns of phosphorylation change has been extended to 2-FaraA, in collaboration with Prof Graves.

In a project with Dr Vivian Whittaker (University of Sydney, deceased), we have found more than 150 proteins in melanoma cells that are subject to AMPylation, the covalent attachment of AMP residues to tyrosine, threonine or serine. These AMPylated proteins may be produced by an enzyme called HYPE when cells are under stress, for example, in rapidly growing cancer cells where there are unfolded proteins in the endoplasmic reticulum (ER), called ER stress. AMPylation of proteins may produce the unfolded protein response that enables cancer cells to continue growing, rather than die.

In a separate project, we are developing engineered antibodies, called demibodies and conditional bispecific T cell engagers (cBITE), for use in the treatment of CLL. In collaboration with Prof Jacqui Matthews (SOLES, University of Sydney) and A/Prof Ashley Buckle (Protein Engineering and Design Lab, Monash University), we are constructing synthetic genes from cassettes that encode the multiple domains of these ‘engineered’ antibodies. The open reading frame encoding the whole protein is inserted into an expression plasmid, transfected into HEK293 cells, expressed and purified in milligram quantities. The efficacy of demibodies and cBITE will be tested in cultures of primary CLL cells from patients, using a novel culture system to maintain cell viability during the experiment in vitro.