Protein Kinases as Drug Targets

Bcr-Abl and Chronic Myeloid Leukemia

Genetics 144: Oncogenomics

Presented by: Shohreh Farzan

shohreh.farzan@dartmouth.edu

Paper presented: O'Hare et al., Blood. 104: 2532-2539, 2004.

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I. Background: Protein Kinases

Kinases are enzymes that catalyze phosphorylation reactions. They can transfer a phosphate group to other proteins by binding both ATP and the substrate protein that is to be phosphorylated. The ATP donates its terminal gamma phosphate group to the substrate, which is transferred by the kinase. This phosphorylation can either activate or inactivate the substrate and can also change the conformation of the substrate protein. Inhibitors can bind to various sites on the kinase, but most often bind to the ATP binding domain. Inhibitors can bind to the kinase in either its active, ATP-binding state or in its inactive state, non-ATP binding conformation. Inhibitors that bind to the inactive state are often thought to be more specific, as the ATP-binding conformation is similar from kinase to kinase, since all bind ATP in a similar manner. A good demonstration of binding is shown in this diagram of Bcr-Abl inhibition by imatinib mesylate and can be viewed here.

II. Cancer Therapy and Kinases as targets for inhibition

Protein kinase signaling is involved in many normal cellular functions. Recently, kinases have been identified as potential targets for therapeutic inhibition, as kinases are implicated in cellular processes such as mitosis, angiogenesis, replication checkpoints, as well as metabolism. When abberant signaling occurs during any of these processes, the cell gains the potential for oncogenic growth. Abnormal kinase activity has been observed in a variety of human cancers. Inhibition of kinases may prove to be a promising cancer therapy if potential inhibitors can be proven to be specific to their targets. Many small molecule inhibitors bind to the ATP binding pocket of kinases, which tends to be highly conserved across kinases. Thus, it is possible that one small molecule inhibitor may bind to several different kinases, in addition to the target, blocking normal signaling that is needed for normal cellular function. However, it is possible that the potential to block a multitude of kinases may prove to be useful in several types of cancers, as long as the inhibitor is specific to malignant or pre-malignant cells (1).

III. Chronic Myeloid Leukemia and the Bcr-Abl Kinase

Chronic myeloid leukemia (CML) is a malignancy caused by proliferation of hematopoetic myeloid precursor stem cells, which affects approximately 1 in 100,000 worldwide and accounts for approximately 20% of all cases of leukemia. This disorder is most often characterized by the presence of the Philadelphia Chromosome, which is present in approximately 95% of all CML patients. The translocation between chromosomes 9 and 22 fuses the Abl kinase gene to the Bcr gene, creating a 210kd fusion protein, which results in the constitutively activated Bcr-Abl protein kinase ( 2, 3, 4). The Bcr-Abl fusion protein thus acts as an oncogene and has been shown to be able to produce a CML-like disease in mice ( 5, 6, 7 ).
IV. Current Therapies for CML

The current standard of care for CML is bone marrow transplant, in combination with drug therapy. Until recently, standard chemotherapy consisted of treatment with Interferon-a. In 2001, a new drug, imatinib mesylate (Gleevec ) was approved to treat CML. Imatinib mesylate specifically inhibits the Bcr-Abl tyrosine kinase activity by binding to the inactive ATP binding conformation of the Bcr-Abl fusion protein. Unfortunately, many patients with advanced stage CML have no response to the drug and many others relapse after initial treatment. Ongoing research endeavors to elucidate the mechanisms by which inhibition occurs, as well as the mechanisms of relapse and drug resistance.

V. Bcr-Abl: Structure and Function
The structure of the Bcr-Abl fusion protein creates a constitutively active kinase ( 2) that is responsible for the cell's oncogenic progression. Alone, the cytoplasmic abl protein is a proto-oncogene and contains a tyrosine kinase domain, which acts in the Ras pathway. The function of Bcr is not well defined, but it is known that its oligomerization domain is critical to the function of the Bcr-Abl fusion protein (8). Bcr-Abl activates several pathways and results in cancerous grouth by altering mitotic progression and preventing apoptosis.

VI. Inhibition of Bcr-Abl by novel drug, AP23464

Previous studies and clinical trials have shown that imatinib mesylate (Gleevec) successfully inhibits Bcr-Abl kinase activity in CML patients, but many patients relapse due to drug resistance. Drug resistance occurs due to mutations in the Bcr-Abl ATP binding domain, which affects the ability of imatinib to bind and inhibit. The study described here endeavored to find an alternative to imatinib mesylate, which could inhibit Bcr-Abl kinase activity. The drug AP23464 was identified as an inhibitor of Abl and Src-family kinases and based on the similarity of Abl and Src-family kinases to Bcr-Abl, AP23464 was hypothesized to also be able to inhibit Bcr-Abl kinase activity. In addition, AP23464 was predicted to not only bind wt Bcr-Abl, but also bind and inhibit imatinib mesylate resistant Bcr-Abl mutant kinases.

VI. AP23464 binds specifically to Bcr-Abl and prevents cell proliferation

O'Hare et al. began by establishing that AP23464 acts specifically on cells expressing Bcr-Abl. A cellular proliferation assay, performed in K562 cells expressing wt Bcr-Abl protein, in the presence of either imatinib mesylate or AP23464 showed that AP23464 significantly inhibited cell growth in comparison to cells treated with imatinib (Figure 1.). HL60 cells, which do not express Bcr-Abl were not affected by AP23464 treatment, thus showing that AP23464 only acts on cells expressing Bcr-Abl.

Figure 1. also shows that treatment with increasing concentrations of AP23464 arrests K562 cells in G0/G1, while decreasing the number of cells in S-phase and G2/M. An apoptosis assay also showed that K562 cells expressing Bcr-Abl and treated with AP23464 were labeled with annexin V to a greater extent than HL60 cells that were also treated with AP23464, but do not express Bcr-Abl (Figure 1.).
VII. Inhibition of Bcr-Abl and its downstream targets by AP23464

In order to determine whether AP23464 has a downstream effect on targets of Bcr-Abl, cell lysates from K563 cells treated with increasing concentrations of AP23464 were analyzed using antiphospho- antibodies to downstream phosphorylation targets, STAT5 and Crkl. Figure 2 shows an overall decrease in phosphorylated targets, as well as a decrease in autophosphorylation. Phosphorylation decreased with increasing concentrations of AP23464 and phosphorylation is ablated at a concentration of approximately 300nm.

VIII. Inhibition of tyrosine phosphorylation of wt and mutant Abl-kinase domains.

A cell-free assay shown in Figure 3 used both wild-type and mutant GST-Abl fusion proteins, in order to determine whether AP23464 was capable of inhibiting Abl-kinase activity in vitro. Treatment with increasing concentrations of AP23464 showed that all phosphorylation was blocked at concentrations of 80-160nm, with the exception of the T315I mutant, which showed no change in level of phosphorylation. The IC50 values for inhibition of autophosphorylation are shown in column 3 of Table 1. and values for substrate phosphorylation are shown in column 4. Note that IC50 values are fairly low, indicating that a low dose of AP23464 is needed to cause inhibition of phosphorylation, in all cases except that of T315I.

IX. AP23464 induces apoptosis and inhibits proliferation in cells expressing imatinib-resistant Bcr-Abl

The in vivo assay shown in Figure 4 shows that kinase inhibition results in an antiproliferative effect on cells expressing wt Bcr-Abl and mutant Bcr-Abl. With increasing concentrations of AP23464, rapid growth inhibition was observed in cells expressing both wild-type and mutant forms of Bcr-Abl. Cells transfected with the vector only showed no growth inhibition and the mutant line expressing T315I also showed no growth inhibition.

In addition to inhibiting proliferation, AP23464 induces apoptosis in cells expressing wild-type and mutant Bcr-Abl as shown by Figure 5. Treatment with AP23464 at concentrations of 0, 50 and 100 nm induced apoptosis, as indicated by annexin V labeling, in Ba/F3 cells expressing wt and mutant Bcr-Abl, with the exception of the T315I mutants. Control Ba/F3 parental cells, which were not transfected with a Bcr-Abl construct, did not show a significant level of apoptosis and were comparable to untreated cells.

X. Bcr-Abl tyrosine phosphorylation is reduced in the presence of AP23464 in vivo.

Figure 6 shows that AP23464 successfully inhibits Bcr-Abl tyrosine phosphorylation with increasing concentrations, in all cases except that of mutant T315I, as detected by an anti-phosphotyrosine antibody. Column 2 of Table 1 shows that all IC50 values are reasonably low and within a similar range for all except the T315I mutant.

Part B of Figure 6 shows that AP23464 also affects phosphorylation levels of Src-family kinases. Treatment of cells expressing imatinib mesylate resistant mutant T315I with increasing concentrations of AP23464 decreased phosphorylation levels of Src-family kinases, while at the same time upregulation expression of unphosphorylated Lyn, also a Src-family kinase.

XI. Conclusions

AP23464 is a novel drug which has been shown to block proliferation of cells expressing wild-type and mutant forms of Bcr-Abl, as well as block cell cycle progression. AP23464 also induces apoptosis in expressing wild-type and mutant forms of Bcr-Abl and can reduce phosphorylation of Bcr-Abl downstream targets. Autophosphorylation and substrate phosphorylation of wt and mutant Bcr-Abl was blocked in all cases except that of the T315I mutant. In vivo experiments showed that AP23464 also inhibits proliferation of cells expressing wt and mutant Bcr-Abl (with the exception of T315I) with 25 fold higher potency than imatinib and IC50 values indicate that inhibitor enters cells efficiently.

Figure 6 also indicates that AP23464 has the potential to be a dual inhibitor of both Bcr-Abl and Src-family kinases, as AP23464 prevented Src-family kinase signaling in T315I mutants.
AP23464 may be a more effective drug than imatinib mesylate, as shown by the IC50 values in Table 1. AP23464 binds differently than imatinib mesylate, as demonstrated by the structural diagrams shown in Figure 7. AP23464 binds to the active conformation of Bcr-Abl, making it a more versatile inhibitor, as it does not need the induced fit of the inactive conformation. However, AP23464 appears to be somewhat specific in that it does not affect the growth of cells not expressing Bcr-Abl, meaning that normal tissues will not be affected by AP23464.
There are many possible directions that this study could follow. More detailed in vivo studies are needed to establish whether AP23464 is inhibiting other targets within the cell, in order to reduce the chance of toxicity. The data presented here shows that AP23464 also inhibits Src-family kinases. It is likely that other kinases are affected by AP23464, as imatinib mesylate has also has been shown to be useful in treating other types of cancers, because it inhibits other kinases such as c-kit and PDGFR. Following successful in vivo studies, it would be useful to test AP23464's efficacy in a CML-like mouse model.
Imatinib mesylate resistant Bcr-Abl mutant T315I has been shown to also be resistant to AP23464. Future studies to modify drug a to target T315I mutants would be useful. Gorre et al. showed in 2001 that the structure of the T315I mutant protein does not vary greatly in its binding conformation from the wild type conformation (3). A single threonine to isoleucine substitution drastically reduces the ability of imatinib mesylate to bind to the kinase domain, as the threonine creates a critical hydrogen bond with the drug. It is likely that the same hydrogen bond is needed for AP23464 to bind. This study, along with others, will help to elucidate the mechanisms by which inhibition of Bcr-Abl occurs, in order to prevent both relapse and resistance to current therapies..
References.

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2. . Deininger, M.W.N, Goldman J.M., and J.V. Melo. 2000. The molecular biology of chronic myeloid leukemia. Blood. 96: 3343-3356.

3. Gorre, M.E., Mansoor, M., Ellwood, K., Hsu, N., Paquette, R., Rao, P.N., and C.L. Sawyers. 2001. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science. 293: 876-880.

4. Konopka, J.B., Watanabe S.M., Singer J.W., Collins S.J., and O.N. Witte. 1985. Cell lines and clinical isolates derived from Ph1-positive chronic myelogenous leukemia patients express c-abl proteins with a common structural alteration. Proc Natl Acad Sci USA. 82: 1810.

5. Daley, G.Q., Van Etten R.A., and D. Baltimore. 1990. Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science. 247: 824.

6. Huettner, C.S., Zhang, P., Van Etten, R.A., and D.G. Tenen. 2000. Reversibility of acute B-cell leukemia induced Bcr-Abl1. Nat Gen. 24: 57-60.

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8. Zhao X., Ghaffari S., Lodish H., Malashkevich V.N., and P.S. Kim. 2002. Structure of the Bcr-Abl oncoprotein oligomerization domain. Nat Struct Biol. 9:117-20.

9. Wong S., McLaughlin J., Cheng D., Zhang C., Shokat K.M., and O.N. Witte. 2004. Sole BCR-ABL inhibition is insufficient to eliminate all myeloproliferative disorder cell populations. Proc Natl Acad Sci U S A. 101: 17456-17461.

10. O'Hare, T., Pollock, R., Stoffregen, E.P., Keats, J.A., Abdullah, O.M., Moseson, E.M., Rivera, V.M., Tang, H., Metcalf, C.A., Bohacek, R.S.,Wang, Y., Sundaramoorthi, R., Shakespeare, W.C., Dalgarno, D., Clackson, T., Sawyer, T.K., Deininger, M.W., and Druker, B.J. 2004. Inhibition of wild-type and mutant Bcr-Abl by AP23464, a potent ATP-based oncogenic protein kinase inhibitor: implications for CML. Blood. 104: 2532-2539.

CML Patient Resources and General Information.
A. Chronic Myeloid Leukemia. Novartis Pharmaceuticals Canada, Inc.

B. Chronic Myelogenous Leukemia. Medline Plus Medical Encyclopedia.