Understanding how kinase-targeted therapies work

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Mar 6, 2008 - leads to the development of a clinically successful anti-kinase drug is ... School; S.P. 162, Rm. 3.95; Candiolo 12060 Italy; Email: a.bardelli@.
[Cell Cycle 7:11, 1560-1563; 1 June 2008]; ©2008 Landes Bioscience

Perspective

Understanding how kinase-targeted therapies work Sabrina Arena1 and Alberto Bardelli1,2,* 1Laboratory of Molecular Genetics; The Oncogenomics Center; Institute for Cancer Research and Treatment (IRCC); University of Torino Medical School; Candiolo; and 2FIRC Institute of Molecular Oncology (IFOM); Milan, Italy

Key words: kinase, cancer, off-target effect, inhibitor, target validation, MET

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receptor protein tyrosine kinases (NRPTKs). There are around 40 lipid kinase genes in the human genome. Kinases are central switch in the signal transduction network controlling cell proliferation, differentiation, migration and many other cellular processes.1-4 Accordingly, the activity of kinases is tightly regulated at multiple levels. Genetic alterations affecting kinase genes, often leading to constitutively active protein, is central to the development of many tumor lineages. The most common molecular mechanisms leading to the deregulation of kinase activity include: genomic rearrangements,6 point mutations, deletions1,2 and overexpression2,7,8 (Table 1). A prototypic example is the lipid kinase PIK3CA which is genetically altered in many tumor types and is one of the most commonly mutated human oncogenes.9 The finding that kinase genes are often deregulated in cancer and the fact that they encode for ‘druggable’ enzymes makes them attractive targets for the development of novel therapeutics.12 The road that leads to the development of a clinically successful anti-kinase drug is currently fairly long and extremely costly. The reasons for this are not the subject of this review and have been previously analyzed.13-15 It is clear from these analyses however that two critical steps must be overcome during the road to new drugs targeting kinases. The first is associated with the identification and validation of a given kinase as valuable therapeutic target in tumor cells. Before embarking into the costly development of an anti-kinase compound investigators must unequivocally establish the contribution of the enzymatic activity of an individual kinase to the oncogenic properties of cancer cells. Although RNAi-mediated gene knock-down can be used to address this issue, this technology has drawbacks. These include the incomplete downregulation of the target protein and the well known off-target effects of the RNAi technology16. Furthermore, many proteins, including kinases and phosphatases have multiple independent functions that are associated to specific domains. Clearly, the RNAi technology does not allow to abrogate only one specific function (in this case the catalytic activity of a kinase), while leaving the rest of the protein (and its additional functions) intact in the target cells. The second step which often generates attrition in the development of anti-kinase compounds is ensuring that a drug of interest exerts its effect by inhibiting its putative kinase target. Protein kinases can be targeted by different approaches that exploit different classes of drugs; the most frequently used are monoclonal antibodies and small molecules inhibitors. The first approach leads to highly

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Kinases are central nodes in the cellular pathways that control differentiation, proliferation, apoptosis, motility and invasion. Most if not all human tumors are thought to bear alterations in one or multiple kinase genes which therefore represent attractive therapeutic targets. Accordingly, intense drug discovery programs have led to the development of clinically effective kinase inhibitors. The road to generate a kinase inhibitor requires, in the initial phase, validation of the oncogenic potential of the corresponding kinase gene in cancer cells. As the catalytic domains of kinases are highly homologous, most inhibitors are predicted to affect multiple kinases. It is therefore important to ensure that a drug of interest acts by direct inhibition of its putative target. To address these issues, we devised a strategy to genetically inactivate the catalytic activity of a given kinase in human cells. This approach generates isogenic cells in which a certain kinase gene is expressed but is devoid of enzymatic activity thus mimicking the chronic pharmacological treatment of cancer cells with a specific and selective kinase inhibitor. This strategy is not limited to kinase genes but could be broadly applicable to any drug/protein combination in which the target enzymatic domain of a gene is known.

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Protein kinases are key regulators of cell homeostasis and as such they represent a significant fraction (1.5–2.5%) of all eukaryotic genes.1-5 Kinases are generally characterized by the presence of a conserved catalytic domain and share the common ability to transfer the gamma phosphate group from the energy-carrying molecule ATP to the target substrate. On the basis of residue specificity, kinases can be classified into four main groups: tyrosine kinases, tyrosine kinase-like, serine-threonine kinases and lipid kinases. The total number of genes predicted to encode protein kinases in the human genome (the kinome) is 551. Serine-threonine kinases (STKs) constitute the large majority of kinases, accounting for roughly 400 members; while tyrosine kinases (TKs) include about 90 elements, that can be furthermore subdivided into receptor protein tyrosine kinases (RPTKs) and non*Correspondence to: Alberto Bardelli; Laboratory of Molecular Genetics; The Oncogenomics Center; Institute for Cancer Research and Treatment; University of Torino Medical School; S.P. 162, Rm. 3.95; Candiolo 12060 Italy; Email: a.bardelli@ ircc.it Submitted: 03/06/08; Accepted: 03/30/08 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/6031 1560

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Understanding how kinase-targeted therapy work

Table 1  Molecular alterations of kinase genes in human cancers Molecular alteration

Gene

Chromosome location

Disease

Amplification

EGFR

7p12

Glioma



ERBB2

17q21

Breast and ovarian cancer



MET

7p31

Gastric cancer Ovarian cancer

PIK3CA

3q26

Translocation

BCR-ABL

t(9;22) (q34;q11)

CML, ALL

Point mutation

BRAF

7q34

Melanoma, colorectal cancer



JAK2

9p24

ALL, AML



KIT

4q112

GIST



MET

7p31

HPRCC, HCC, HNSCC, lung cancer



PDGFRA

4q11

GIST



PIK3CA

3q26

Colorectal, brain, breast, endometrial cancer

7p12 17q21

MET

7p31



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EGFR ERBB2

Glioma, NSCLC Breast cancer

Gastric cancer

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CML, chronic myelogenous leukemia; ALL, acute lymphocytic leukaemia; AML, acute myelogenous leukaemia; GIST, gastrointestinal stromal tumour; HPRCC, hereditary papillary renal-cell carcinoma; HCC, hepatocellular carcinoma; HNSCC, head and neck squamous cell carcinoma; NSCLC, non small cell lung cancer.

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The derivative isogenic cells are identical to their parental counterpart except for the expression of a kinase-inactive MET receptor (MET-KD cells), which is unable to transduce the signals triggered by the Hepatocyte Growth Factor (HGF), its specific ligand. Then, we exploited this isogenic cell model to test the oncogenic properties of the Met receptor in vitro and in vivo, and to dissect which of these are strictly dependent on its kinase activity. We found that although the proliferative potential of targeted cells in basal conditions is almost unaffected, the invasive properties of KD-cells are completely impaired when stimulated with HGF, as demonstrated with the ancorage-independent growth assay and with the branching morphogenesis assay. Other experiments have shown that also HGF-mediated protection from apoptosis is abolished in MET-KD cells. Overall, these results indicate that the kinase activity of the MET receptor is required to mediate the biological effects of HGF and this information has obvious implications for the therapeutic targeting of the HGF-Met ligand-receptor pair. An exciting aspect of the approach we have developed is its application to assess whether drugs exert their effects by inhibition of their putative target enzymatic domain. As described before, due to the homology among the active sites of kinases, it is very difficult to ensure that a compound of interest interferes with any biological property by direct inhibition of its putative target. In vitro systems cannot provide definitive answers as to whether a kinase inhibitor is specific. Similarly, systems in which kinases are expressed exogenously and are not physiologically regulated can provide misleading results. As a result current approaches can not unequivocally identify the “off-target” effects of a given kinase inhibitors. Our genetic strategy provides a solution to this problem. We reasoned that any effects exerted by a kinase inhibitor in cells in which the ­corresponding kinase activity had been genetically inactivated should be associated with the inhibition of cellular activities independent from the target enzymatic domain. As a test case to show the relevance of our methodology to the field of pharmacogenomics, we treated wild-type and KD cells with

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specific drugs but can only be applied to kinases endowed with an ­extracellular domain (RPTKs). Paradigmatic examples of kinasetargeted monoclonal antibodies validated in clinical use are those against the extracellular domain of the EGFR (also called Erbb1) (cetuximab and panitumumab)17,18 ERBB2 (trastuzumab)19,20 and VEGFR (avastin).21 The second strategy used to target kinases involves small molecules that antagonize their catalytic activity (for example by blocking the binding of ATP). Gefitinib (Iressa) and Erlotinib (Tarceva) are two examples of drugs that have been developed to target the catalytic domain of the EGFR.22,23 Although the second approach can be broadly applied to any protein and lipid kinase, it also has limitations such as incomplete selectivity (Fig. 1A, reviewed in refs. 24 and 25). This arises from the high homology between the catalytic domain of kinase genes. In particular, the region encoding catalytic loop and the ATP binding site are extremely conserved among the various kinases. In summary, the validation of individual kinases as relevant targets in tumor cells and the specificity of kinase inhibitors are limiting steps in the road to the development of new anticancer drugs. To overcome these limitations we have recently developed an innovative genetic strategy to permanently abrogate the catalytic activity of kinases by stable modification of the corresponding genomic locus.26 This strategy generates human somatic cells in which a given kinase gene is expressed but is catalytically inactive and closely recapitulates the chronic pharmacological treatment of the target enzymatic domain with a specific and selective inhibitor. As a model to test this approach we chose the receptor tyrosine kinase MET which is molecularly altered in multiple cancers and is the focus of several therapeutic efforts.27-31 Met is activated by its cognate ligand Hepatocyte Growth Factor (HGF) and triggers “invasive-growth”, a complex biological programme whose deregulation leads to uncontrolled cell proliferation and metastasis.30,31 We exploited targeted homologous recombination to permanently delete the exon encoding the ATP binding site of MET from the genome of colorectal, endometrial and bladder cancer cells. www.landesbioscience.com

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Figure 1. (A) Kinases and the corresponding inhibitors. Relative IC50 (24) or Kd (25) are indicated. Grey boxes highlight the intended targets for each compound. (B) MET-KD cells were generated by targeted deletion of the ATP-binding site of the MET receptor in cancer cells. Any effect of the MET kinase inhibitor (SU11274) on MET-KD cells is independent (off-target) from the MET kinase activity.

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the reportedly specific MET kinase inhibitor SU11274.32 SU11274 was found to affects the proliferation of cells carrying kinase-inactive MET alleles indicating that this compound affects one or more MET kinase-independent’s cellular functions. This strategy was also used to identify the “off-target” effects of SU11274 (Fig. 1B). In conclusion, we have devised a genetic strategy to selectively inactivate a given kinase activity in cancer cells, thus mimicking the pharmacological treatment with a specific kinase inhibitor. This strategy can be broadly applied to assess the specific contribution of any given enzymatic activity to the biology of human cells. Furthermore, it can be used to evaluate the specificity of any drug/ protein combinations in which the target enzymatic domain is known.

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Acknowledgements

This work was supported by grants from Italian Association for Cancer Research (AIRC), Italian Ministry of Health, Regione Piemonte, Italian Ministry of University and Research, Association for International Cancer Research (AICR-UK), EU FP6 contract 037297. We thank Drs. Miriam Martini and Fonnet Bleeker for critically reading the manuscript. References

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