UCL Cancer Institute

Signal Transduction and Drug Resistance in Leukaemia Cells


Group Leader: Dr Gitendra Wickramasinghe


Our group uses a biochemical approach to study signal transduction in leukaemia cells. The overall emphasis is to use insights provided by these studies to identify novel drugs and strategies which can be used to induce more effective apoptotic killing of chronic lymphocytic leukaemia (CLL) cells than is possible using conventional chemotherapy. Recent studies fall into two principle categories, as outlined below.


The first approach involves the p53 gene and its protein product which plays a key role in inducing apoptosis following treatment with conventional agents. Mutation of this gene frequently contributes to drug resistance in CLL and in other cancers, therefore drugs which kill cells independently of p53 are of potential value in the treatment of CLL patients who have become resistant to standards regimes. These studies have resulted in the identification of three novel drugs which induce CLL apoptosis regardless of the mutation status of the p53 gene, but which are relatively inactive against normal cells.

The second approach attempts to define biochemical signalling strategies used by CLL cells to avoid apoptosis induction following treatment with cytotoxic agents. Some of these strategies are cell-intrinsic and include constitutive activation of STAT3, MAP kinase and PI-3kinase signalling pathways, while others involve signalling to CLL cells from extrinsic cytoprotective cytokines including IL-4. Understanding of these mechanisms has resulted in the identification of highly selective small molecule inhibitors which antagonize the protective mechanisms and result in more efficient cell killing. An example of this type of strategy, directed against a cell-intrinsic mechanism of drug resistance, is shown in the figures below.




induction of apoptosis by p53


Fig 1  
The conventional model for induction of apoptosis by p53. Cytotoxic drugs damage cellular DNA. In response to DNA damage, the levels of the p53 protein increase. According to the conventional model, p53 functions exclusively as a transcription factor which upregulates expression of pro-apoptotic genes such as the PUMA gene. The resulting increase in PUMA protein then induces apoptosis.




Subcellular fractionation of CLL cells


Fig 2  
Subcellular fractionation of CLL cells. Control CLL cells (NA) or cells incubated with the cytotoxic drugs chlorambucil (Chl) or fludarabine (flu) were fractionated into cytosolic, mitochondrial and nuclear fractions. As expected, p53 levels were increased following treatment with both of the drugs. Some p53 was found in the nuclear fraction, consistent with its function as a transcription factor. However, the majority of p53 was found associated with mitochondria. Mitochondria are key elements in the induction of apoptosis. Bcl-2, a key inhibitor of apoptosis, was also found exclusively in the mitochondria. Therefore, these data suggest that p53 may be inducing cell killing by a non-transcriptional mechanism, possibly involving a direct biochemical interaction between pro-apoptotic p53 and anti-apoptotic bcl-2.





Association between p53 and bcl-2


Fig 3  
Association between p53 and bcl-2.A direct association between bcl-2 and p53 was demonstrated by immunoprecipitation of bcl-2 from CLL cells treated with chlorambucil or with nutlin 3a (another p53-elevating agent).





blockade of p53-mediated transcription


Fig 4  
Blockade of p53-mediated transcription by pifithrin. Pifithrin a is an inhibitor of p53’s transcriptional function. This figure shows that transcription of the p21 protein, a known transcriptional target of p53, was extensively upregulated in CLL cells treated with nutlin. This upregulation was markedly blocked by pifithrin. Note also that the pro-apoptotic PUMA protein was constitutively expressed in CLL cells and was only marginally augmented following nutlin treatment.




pifithrin augments apoptosis induction



Fig 5   Treatment of CLL cells with pifithrin augments apoptosis induction. Although pifithrin was shown to block p53’s transcription function (Figure 4), treatment of CLL cells with this agent surprisingly increased apoptosis induction by nutlin, chlorambucil or fludarabine. In this figure apoptosis was quantified by counting of the percentage of apoptotic cells (A) or by western blot analysis of the cleavage of the PARP protein, a well-known molecular measure of apoptotic cell killing.






novel model for apoptosis induction by p53



Fig 6   A novel model for apoptosis induction by p53. The data shown here are compatible with a novel model for p53-dependent apoptosis induction. We suggest that the major route for cell killing by p53 involves a direct biochemical interaction between p53 and bcl-2, resulting in the neutralization of bcl-2’s anti-apoptotic action, resulting in cell killing. The data also suggest that p53-mediated transcription actually augments the expression of anti-apoptotic proteins which serve to antagonize the non-transcriptional mechanism of apoptosis induction. This accounts for the surprising observation that PFT? actually enhances, rather than diminishes, apoptosis induction by p53-elevating drugs and predicts that inhibitors of p53’s transcriptional function may be of value in the treatment of CLL and possibly of other malignancies Our novel model has now been verified by two independent laboratories.

 

Group Members


•  Dr Andrew J. Steele BSc (Hons) PhD, Honorary Lecturer/Research Fellow
•  Dr. Archibald G. Prentice MB, FRCP,FRCPath, Consultant Haematologist
•  Dr. R.Gitendra Wickremasinghe BSc (Hons) PhD, Reader in Experimental Haematology

 



 

Selected Publications


Phenylacetylenesulfonamide (PAS) induces p53-independent apoptotic killing of B-chronic lymphocytic leukemia (CLL) cells. Steele AJ, Prentice AG, Hoffbrand AV, Yogashangary BC, Hart SM, Lowdell MW, Samuel ER, North JM, Nacheva EP, Chanalaris A, Kottaridis P, Cwynarski K, Wickremasinghe RG. Blood. 2009 Aug 6;114(6):1217-25.

Baou M, Jewell A, Muthurania A, Wickremasinghe RG, Yong KL, Carr R, Marsh P, Murphy JJ. Leukemia. 2009 May;23(5):986-9.

Why is CLL refractory to bortezomib? Wickremasinghe RG. Blood. 2008 Nov 1;112(9):3540-187.

p53-mediated apoptosis of CLL cells: evidence for a transcription-independent mechanism. Steele AJ, Prentice AG, Hoffbrand AV, Yogashangary BC, Hart SM, Nacheva EP, Howard-Reeves JD, Duke VM, Kottaridis PD, Cwynarski K, Vassilev LT, Wickremasinghe RG. Blood. 2008 Nov 1;112(9):3827-34.

The sesquiterpene lactone parthenolide induces selective apoptosis of B-chronic lymphocytic leukemia cells in vitro. Steele AJ, Jones DT, Ganeshaguru K, Duke VM, Yogashangary BC, North JM, Lowdell MW, Kottaridis PD, Mehta AB, Prentice AG, Hoffbrand AV, Wickremasinghe RG. Leukemia. 2006 Jun;20(6):1073-9.

Selective apoptotic killing of malignant hemopoietic cells by antibody-targeted delivery of an amphipathic peptide. Marks AJ, Cooper MS, Anderson RJ, Orchard KH, Hale G, North JM, Ganeshaguru K, Steele AJ, Mehta AB, Lowdell MW, Wickremasinghe RG. Cancer Res. 2005 Mar 15;65(6):2373-7.