Figure from Src-family kinases in the development and therapy of Philadelphia chromosome-positive chronic myeloid leukemia and acute lymphoblastic leukemia. Shaoguang Li. Leuk Lymphoma. 2008 January; 49(1): 19–26
The figure shows: src family kinases (SFKs) directly interact with BCR-ABL resulting in (1) activation of SFKs and (2) augmentation of BCR-ABL kinase activity. Activated SFKs work cooperatively with BCR-ABL in facilitating the growth and progression of leukemia. Several downstream effectors of SFKs have been proposed to mediate the proleukemic effects, such as (3) STAT5, which is known to activate genes involved in growth factor independence, differentiation, adhesion, and DNA repair and (4) AKT, which is key in regulating cell proliferation and survival in BCR-ABL-dependent cells. (5) Active SFKs also phosphorylate certain tyrosine residues on BCR-ABL to create a binding site for GRB-2. This adaptor protein may link the BCR-ABL pathway to Ras, which is known to activate the MEK/ERK oncogenic signaling cascade.
The most startling and important development in targeted therapy has been the use of Glivec (or Gleevec) to treat chronic myeloid leukemia. Even when I was at medical school CML was characterized by the presence of the Philadelphia chromosome. When I was a junior doctor I delighted in correcting a very eminent hemato-oncologist who hadn’t kept up with the news that the Philadelphia chromosome was formed by a translocation between chromosomes 9 and 22 that involved putting two genes, bcr and abl, so close together that they were translated as a single protein. We discovered later that abl was a tyrosine kinase that was switched on and off as the white cell proliferated at the first sign of infection and then died down again when the danger was past. In its elongated form, bcr-abl is a tyrosine kinase fixed in the ‘on’ position. The white cells proliferate without the ‘go’ signal and can’t be switched off. At least they couldn’t until Brian Druker from Portland, Oregon invented STI571, which came to be known as Glivec or imatinib to give it its proper name. Imatinib works by denying bcr-abl its energy supply. In cells, energy is carried around as ATP which needs to plug itself into bcr-abl in order for the tyrosine kinase to do its work. Imatinib blocks the socket where ATP plugs in.
It was hoped that imatinib would be the first of many such inhibitors and that this technology could be adapted to treat other cancers. To be honest that have been other successes, but they have all been in small print diseases or rare variants of common cancers. So far they don’t work for really important diseases. Then came the bad news. Patients with CML started getting resistant to imatinib. It turns out that point mutations in the abl molecule allow the ATP to fuel the molecule again (it sort of widens the socket to allow the fuel cell to plug in. The chemists started messing around with the molecule and came up with AMN-107 (or nilotinib), which works in some cases of imatinib resistance but not in others. It seems to deal with all the abl mutations except one called T315I. BMS-354825 or Dasatinib is more useful, it deals with all the abl mutations except T315I but being a rather different molecule it has a broader spectrum and inhibits other tyrosine kinases than bcr-abl and in particular inhibits src family kinases.
Evidence is accumulating that imatinib resistance in accelerated phase CML and Ph-positive ALL is caused by the activity of src family kinases. Upregulation of Lyn and Hck (see previous article) has been observed in blasts from patients with imatinib-resistant CML. It was later found that while imatinib effectively reduced activation of BCR-ABL and consequently downstream activation of SFKs in specimens derived from patients with imatinib-sensitive CML, this agent had no effect on SFK activation in samples from resistant patients despite BCR-ABL inhibition.
Dasatinib is the the only dual SFK/BCR-ABL inhibitor approved in the United States and Europe for the treatment of patients with imatinib-resistant or -intolerant CML and Ph+ ALL. This orally available tyrosine kinase inhibitor is structurally unrelated to imatinib and has a 325-fold greater activity against native BCR-ABL in vitro as compared to imatinib. Several animal studies have indicated that dual SFK/BCR-ABL inhibition with dasatinib is advantageous in CML and Ph+ ALL. Dasatinib was able to recover the antitumor activity lost with imatinib treatment as a result of BCR-ABL–independent Lyn activation. It also induces complete remission of Ph+ ALL, and significantly prolongs survival of CML in mice. It was found in mice that SFKs are required for the progression of CML to lymphoid blast crisis, suggesting that treatment with dasatinib could potentially delay the transition of CML from chronic phase to lymphoid blast crisis. Although dasatinib does not kill leukemic stem cells, studies in mice with ALL suggest that the cytostatic effects of dasatinib on this cell population could prevent leukemic transformation and afford long-term control of the disease.
Clinical trials have shown that dasatinib induces rapid and deep responses in imatinib-resistant patients across all phases of CML and Ph+ ALL. While it is clear that the more potent activity of dasatinib against native and mutant variants of BCR-ABL is partly responsible for its clinical efficacy in imatinib-resistant CML, BCR-ABL–independent effects appear to play a role as well. Dasatinib has shown activity in imatinib-resistant patients with no detectable abl mutations.
Could its activity against SFKs be useful in other diesases? In particular, could it be useful in CLL? A paper just published online from Veldurthy and colleagues which include Michael Hallek suggests that dasatinib might have a role in the treatment of CLL.
It was already known from a JCI paper published in 2005 that CLL cells express Lyn protein between 2.5- and 5-fold more than normal B cells while in contrast, Src, Fyn, Fgr, and Lck levels were similar in normal and leukemic cells. Lyn distribution analyzed in normal B cells by confocal microscopy appears punctate and random across the plasma membrane whereas a more intense and coalescent fluorescence revealed that an abnormal amount of Lyn was present in the plasma membrane of leukemic cells, seemingly concentrated in lipid rafts. In contrast to normal B cells, Lyn tyrosine kinase is constitutively (ie switched on all the time) active in CLL cells, as demonstrated by remarkable kinase activity in the anti-Lyn immunocomplexes. These findings did not correlate with the expression of ZAP-70 or with the presence or absence of somatic mutations. In contrast, tyrosine kinase activity was undetectable in immunoprecipitates obtained in parallel experiments with anti-Src, anti-Fyn, anti-c-Fgr, and anti-Lck antibodies. Whereas stimulation of the BCR of normal B cells increases the amount of Lyn ninefold, no such increase is seen in CLL. It appears that the increased amount of Lyn is at least in part responsible for the defective apoptosis of CLL cells.
Veldurthy et al show that incubation of CLL cells with dasatinib markedly reduces tyrosine phosphorylation and also the downstream signalling pathways involving MAP kinase, Erk1/2, Akt and p38. Incubation with dasatinib increased apoptosis, an effect that was not inhibited by p53 deficiency or functional loss, but the effect of dasatinib was greater in CLLs with unmutated rather than mutated IGHV genes. The combination of fludarabine and dasatinib was additive.
One caveat to this exciting news. When CLL cells were co-cultivated with the bone marrow stromal cell line, HS5, a degree of protection was provided against dasatinib induced apaotosis, suggesting that the CLL cells might be safe from such an attack when residing in lymph nodes or bone marrow.