Is Bcr-Abl expression relevant for the survival of cancer stem cells in chronic myeloid leukaemia

CML has an incidence of 1.3 per 100,000 of the population with 750 new cases per year in the UK. CML begins when a stem or founder bone marrow cell develops a genetic change during division. This change results in the Philadelphia chromosome and the Bcr-Abl cancer protein being found in all CML cells, including the stem cells. The Bcr-Abl protein is an enzyme called a tyrosine kinase and tyrosine kinase inhibitors (TKIs) have now been introduced for the treatment of CML. Imatinib mesylate (IM), the first of these drugs to reach the clinic, is now the standard of care for 85% of CML patients in the early stages of the condition. When started at diagnosis this drug has induced remission in 80% of cases, leading to absence of the Ph chromosome, and has reduced the annual rate of progression from 10-15%, before the introduction of IM, to only 2-4%, with patients now expected to survive at least 10 years from diagnosis and possibly much longer. However IM does not cure CML and all patients still have CML stem cells present. We have recently shown that the amount of Bcr-Abl cancer protein is greatly increased in CML stem cells and we were the first to show that CML stem cells are resistant to killing by IM. Our current work is investigating whether IM, or new generation stronger TKIs, achieve high enough levels inside the CML stem cells to effectively overcome the very high levels of cancer protein and whether drug pumps, on the surface of CML stem cells, limit the build up of these drugs inside cells. The question we wish to address in this proposal is whether the activity (whether it is on or off) of the Bcr-Abl cancer protein is the major determinant of whether these stem cells survive and divide to sustain leukaemia in patients or undergo a process called programmed cell death when the protein is switched off. We are confident we have the expertise to investigate and answer this question. If CML stem cell survival does not require Bcr-Abl to be on at all times then we need to develop new approaches, other than TKIs, to kill these cells. Our overall aim is to understand what keeps CML stem cells alive and find new ways to kill these cells so that patients may be cured and come off all drug therapies.

Chronic myeloid leukaemia (CML) arises in a haemopoietic stem cell targeted by the t(9;22)/Ph translocation. Although all Ph+ cells express Bcr-Abl transcripts and oncoprotein, we have recently shown both mRNA and oncoprotein to be greatly overexpressed at the stem cell level. Bcr-Abl is a constitutively active tyrosine kinase and a number of tyrosine kinase inhibitors (TKIs) have now been introduced for the treatment of CML. We were the first to show that CML stem cells are inherently insensitive to imatinib mesylate (IM), the first of these agents to reach the clinic. Furthermore, IM does not eradicate CML stem cells in vivo and the majority of patients have residual disease as detected by RT-PCR for Bcr-Abl transcripts. Our current work has investigated whether IM, or more potent TKIs, achieve high enough intracellular levels within CML stem cells to effectively inhibit the upregulated levels of oncoprotein and the role that influx and efflux drug pumps play in determining intracellular drug accumulation. The key question that will be addressed here is whether Bcr-Abl activity is relevant for survival of CML stem cells. One hypothesis is that by inhibiting Bcr-Abl in a stem cell you simply revert the malignant phenotype to normal. The alternative hypothesis is that at the time the stem cell acquires t(9;22), additional changes occur that confer a survival advantage and the cell becomes dependent on Bcr-Abl expression. We are now confident we have the expertise to investigate and answer this question. We will use alternative but complementary approaches. Primary CD34+ CML cells will be exposed to TKIs in vitro with surviving stem cells tracked over time, at the single cell level, for inhibition of Bcr-Abl activity, apoptosis, retention of primitive phenotype and function and proliferation. To maximise Bcr-Abl inhibition at the stem cell level the above approach will be combined with lentiviral delivered shRNA against Bcr-Abl. Finally to demonstrate the effect of Bcr-Abl inhibition within transplantable stem cells in vivo, a transgenic model that expresses Bcr-Abl from a tetracycline responsive element driven by the SCL3? enhancer (to allow induced expression of Bcr-Abl in the stem cell compartment), will be employed. If CML stem cell survival is proven to be independent of Bcr-Abl, then alternative approaches to selectively target and eradicate the cancer stem cell pool will be required. For example, these might exploit Bcr-Abl expression to facilitate delivery of a cytotoxic agent selectively to Ph+ CML cells.