Conference Report – Protein Kinase Inhibitors in Cancer Treatment: Mixing and Matching?

Introduction

With the success of imatinib in the treatment of patients with chronic myelogenous leukemia (CML), “targeted” or “rational” therapies have become a hot topic in oncology. Imatinib inactivates the kinase activity of BCR-ABL, the fusion protein that causes CML. Can other oncogenic kinases be targeted in a similar manner?

Protein kinases play important roles in regulating most cellular functions — proliferation/cell cycle, cell metabolism, survival/apoptosis, DNA damage repair, cell motility, response to the microenvironment — so it is no surprise that they are often themselves oncogenes. Kinases such as c-Src, c-Abl, mitogen activated protein (MAP) kinase, phosphotidylinositol-3-kinase (PI3K) AKT, and the epidermal growth factor (EGF) receptor are commonly activated in cancer cells, and are known to contribute to tumorigenesis. Many of these occur in the same signaling pathway — for example, HER-kinase family members (HER1 [EGFR], HER3, and HER4) transmit signals through MAP kinase and PI3 kinase to promote cell proliferation.

Research groups have developed several ways to target these enzymes therapeutically, such as with antibodies or small molecules that block kinase-substrate interaction, or that inhibit the enzyme's adenosine triphosphate (ATP) binding site. A number of kinase inhibitors have therefore already been developed and approved for cancer treatment. These include inhibitors of c-Abl (imatinib, for treatment of CML), HER2 (trastuzumab, for treatment of breast cancer), vascular endothelial growth factor receptor (bevacizumab, for treatment of metastatic colorectal cancer), and the EGF receptor (gefitinib, cetuximab, for treatment of lung and colorectal cancer). Many other drugs are currently being tested in cancer clinical trials for their ability to target kinases.

The potential for targeting kinases in the treatment of cancer was the theme of the Keystone Symposium “Kinases and Cancer: The Promise of Molecular Based Therapies” recently held in Tahoe City, California.

In the opening address, Robert Wittes,[1] of the Memorial Sloan-Kettering Cancer Center, New York City, NY, discussed the pros and cons of targeting kinases in cancer patients. Kinase inhibitors designed to block the enzyme's ATP binding site can have broad specificity — imatinib not only inhibits the tyrosine kinase c-Abl, but also c-Kit and the platelet-derived growth factor (PDGF) receptor tyrosine kinases. So it can be used to treat gastrointestinal stromal and other types of tumors associated with activation of these signaling molecules.

Additionally, unlike cytotoxic drugs, many kinase inhibitors have been found to have low levels of toxicity in preclinical and clinical studies. Exceptions have been seen with trastuzumab, which can cause cardiac damage; bevacizumab, which has been associated with pulmonary bleeding; and gefitinib, which can cause interstitial lung disease. Of note, the long-term side effects of kinase inhibition are not known. Resistance may also be an issue, as many patients who were treated with imatinib have since become resistant, due to mutations in c-Abl. Kinase inhibitors might therefore be most effective if administered in combination.[1]

Modulating Activity of the EGFR

Gefitinib is a small-molecular-weight, ATP-mimetic that specifically inhibits the EGF receptor tyrosine kinase. In May 2003, it received approval from the US Food and Drug Administration as a third-line therapy for nonsmall-cell lung cancer. A phase 2 trial of patients who had failed chemotherapy showed that about 13% of patients responded to the drug. In phase 3 trials, however, patients who received gefitinib plus chemotherapy did not have better outcomes than those who received chemotherapy alone.[2,3]

Carlos Arteaga,[4] of the Vanderbilt-Ingram Cancer Center, Nashville, Tennessee, is one of several researchers who have been investigating why this drug is not successful in treating all tumors that overexpress the EGF receptor.[5,6] Breast cancer cells express this receptor, but its activity is not required for tumor development, since gefitinib does not block breast cancer cell proliferation. There are several mechanisms by which cancer cells can escape dependence on specific signaling pathways and therefore become resistant to therapeutics.

Arteaga's group took A431 squamous cells, which overexpress the EGF receptor and normally undergo apoptosis when exposed to gefitinib, and selected for resistant cells, by continually carrying them in the presence of the drug. They found that gefitinib-resistant cells activated alternative signaling pathways that mediated their survival. The EGF receptor usually transmits proliferative signals through activation of the Ras-Raf-MAP kinase signaling pathway. Arteaga showed, however, that a separate signaling pathway that involved activation of PI3K and AKT was upregulated in these cells. Treating gefitinib-resistant cells with PI3K or AKT inhibitors induced apoptosis, indicating they are dependent upon this alternative signaling pathway for proliferation. Conversely, transgenic expression of constitutively active forms of PI3K or AKT in wild-type A431 cells made them resistant to gefitinib therapy.[4]

This indicates that PI3K and AKT are preferential pathways of resistance to gefitinib in squamous cancer cells, and Arteaga's group provided evidence that multiple tumor types use the AKT escape pathway. So, the most effective way to treat cancer may be with a combination of kinase inhibitors. Another issue surrounding the use of “targeted therapies” is patient selection. Based on Arteaga's data, it should be feasible to preselect patients whose tumors are not likely to respond to gefitinib therapy — patients whose tumors have upregulated PI3K/AKT signaling.

Is it possible to predict the ability of a tumor to respond to certain drugs based on its “molecular profile” — that is, by looking for certain molecules that are up- or downregulated? Arteaga and colleagues investigated this question in EGF receptor-expressing primary human gastric tumor cells. Treatment of these cells with gefitinib reduced EGF receptor activation (as measured by receptor phosphorylation), but only reduced proliferation by 40% to 50%. They compared cells with low levels of AKT activation (measured by AKT phosphorylation) with those with high levels of AKT activation. This revealed that proliferation of cells with low levels of AKT phosphorylation were completely inhibited by gefitinib treatment, whereas cells with high levels of AKT activation did not respond to treatment.[4] Thus, measurement of phospho-AKT levels could be useful in determining which patients are most likely to respond to gefitinib therapy.

ErbB2/HER2 Inhibitors

Mark Slikowski,[7] of Genentech, discussed the methods his group is using to determine which patients are most likely to respond to ErbB2/HER2 inhibitors. The HER family of transmembrane tyrosine kinase receptors is composed of 4 members. HER2 is a ligand-orphan receptor that is expressed in many human tumors and overexpressed in 25% to 30% of breast cancers. It signals by forming heterodimers with other members of the HER family, and anti-HER2 monoclonal antibodies have been developed for cancer therapy.

The humanized antibody trastuzumab has antitumor activity against HER2-overexpressing human breast tumor cells and is used in the treatment of breast cancer.[8] Trastuzumab induces HER2 receptor downmodulation and, as a result, inhibits the signaling pathways (both RAS-RAF-MAP kinase and PI3K/AKT) that mediate cell proliferation. A limitation of trastuzumab is that its activity is largely restricted to breast cancers that overexpress high levels of HER2, although there is a large population of breast cancers that have low or moderate HER2 expression. This is another example of the benefits of prescreening patients to determine which are most likely to respond to therapy.

In patients who expressed low levels of HER2, HER2 functions as a coreceptor that interacts with HER1 (EGF receptor), HER3, or HER4. Slikowski's group has been investigating an antibody called 2C4, which binds to a different epitope of the HER2 ectodomain from trastuzumab and prevents it from forming heterodimers with these other HER receptors. 2C4 could therefore be used to treat patients whose tumors do not express high levels of HER2.[9] Slikowski demonstrated that 2C4 inhibits signaling by HER2-based heterodimers both in Calu3 cells, which have high levels of HER2 expression, and in MaxF449 cells, which have low levels of HER2 expression. The drug slowed the growth of both of these tumor types in experimental xenograft models.

In a phase 1 trial, 21 patients with advanced solid malignancies received intravenous 2C4 every 3 weeks at escalating doses (0.5 mg/kg–15.0 mg/kg). These patients had been previously treated unsuccessfully with various types of chemotherapy. 2C4 was apparently well-tolerated — there were no adverse events, and 3 of 21 patients (with ovarian, pancreatic, and prostate cancer) showed partial responses.[7] Phase 2 trials are underway in patients with a variety of solid tumor types.

Targeting RAF Kinases

In addition to drugs that target growth factor receptors themselves, their downstream signaling molecules are also viable therapeutic targets. Ligand binding to receptors such as the EGF receptor or HER2 binds activates receptor autophosphorylation, leading to activation of the small GTP-binding protein RAS. RAS binds and activates RAF, which phosphorylates and activates MEK1 and MEK2, and these in turn phosphorylate and activate MAP kinases (also known as extracellular signal-regulated kinases ERK1 and ERK2).

Overexpression or constitutive activation of this pathway has been shown to be important for the pathogenesis and progression of breast and other cancers, making the components of this signaling cascade potentially important as therapeutic targets.

Richard Marais,[10] of The Institute of Cancer Research, London, United Kingdom, and colleagues have been investigating the role of RAF in cancer. The 3 RAF genes code for cytoplasmic serine/threonine kinases (ARAF, BRAF, and CRAF) that are activated by binding to RAS. While screening 546 cell lines and 375 primary tumor samples, they observed that, overall, B-RAF was mutated in about 8% of the samples, but mutations were detectable in more than 70% of the malignant melanoma samples tested. A total of 90% of these mutants contained a V599E amino acid change.[11,12]

Amino acid 599 lies in the kinase domain of BRAF, and the V599E mutation makes the enzyme 5-fold more active than wild-type BRAF. When Marais transfected a mouse melanoma cell line (Melan-a cells) with the V599E mutant BRAF gene, the cells became highly proliferative and underwent dramatic morphologic changes, losing dendrites and halting melanin production. Evidence of RAF activity can be measured through quantification of levels of ERK phosphorylation. In cells that expressed V599E BRAF, ERK was constitutively phosphorylated. Marais showed that in human melanoma cell lines (A375, Colo829, and WM-266.4 cells), inhibition of BRAF by siRNA was sufficient to prevent ERK phosphorylation, as well as to inhibit DNA synthesis and induce apoptosis.[10]

BAY43-9006 is a small-molecule inhibitor of CRAF (IC₅₀=6–0;12nM). Marais showed that BRAF is also a target for this drug (IC5₅₀=43nM). His group found that BAY43-9006 suppressed growth of human melanoma tumor xenografts that express oncogenic BRAF, by blocking ERK signaling. In phase 1 and 2 studies, the drug has been shown to be well-tolerated and to reduce ERK phosphorylation levels in peripheral blood cells taken from trial participants.[10]

Marais's group has also solved the crystal structure of the BRAF kinase domain bound to BAY43-9006, and this has revealed important information about how the drug inhibits RAF kinase activity.

Because of Marais's report that BRAF is activated in such a large percentage of melanoma samples, BAY43-9006 is being tested in phase 2 trials for patients with this cancer. It is also in a phase 3 clinical study in patients with advanced kidney cancer, as well as in single-agent phase 2 clinical trials for patients with kidney, melanoma, liver, and other cancers. In addition, it is being tested in combination with a range of standard chemotherapeutics.

Developing MEK Kinase Inhibitors

Judith Sebolt-Leopold,[13] of Pfizer Inc., and colleagues have also been investigating inhibitors that act downstream of RAF in the MAP kinase signaling pathway. One of these, CI-1040, is an orally active, highly potent and selective inhibitor of MEK that ultimately blocks phosphorylation of ERK and continued signal transduction through this pathway. CI-1040 is the first MEK inhibitor to enter clinical trials for cancer.[14] This drug had been shown to cause tumor regression in a variety of experimental xenograft models, including inhibition of human colon carcinoma growth by up to 80%.

A phase 1 trial involved 78 patients who received 100–1600 mg doses for treatment of colon carcinoma or nonsmall-cell lung cancer. The drug was well tolerated, with 98% of adverse events being only grade 1 or 2. Phosphorylation levels of ERK were reduced by 50% or more in 25% of the tumor biopsies taken from the patients. Antitumor activity was also observed, with a partial response in 1 patient with pancreatic cancer who had received prior therapies, and stable disease in 24% of the patients 3 months after therapy. In a successive phase 2 trial, however, there were no complete or partial responders.[13]

A CI-1040 analog has therefore been developed with better pharmacokinetic properties — it has improved solubility, is metabolically more stable, and has improved bioavailability. It is now going through phase 1 trials, and Sebolt-Leopold suggested that it might be more effective to select patients for such trials according to the phosphorylation/activation levels of ERK in their tumors, as only those patients whose tumor cells have activated MEK activity are likely to respond to this drug.[13]

Targeting c-ABL

The EGF receptor-ERK signaling pathway is not the only way to inhibit kinase signaling in cancer cells. One of the most successful kinase inhibitors developed so far, imatinib, was first tested in the clinic for its ability to inhibit the kinase activity of c-ABL, and was therefore used to treat patients with BCR-ABL-associated CML. According to Charles Sawyers,[15] of the University of California, Los Angeles, 95% of patients with CML respond to therapy and remain in remission for years. When imatinib is administered to patients in blast crisis, however, only 70% of patients respond, and remission only lasts weeks to months. Resistance is primarily mediated by mutations in the c-ABL kinase domain, which validates BCR-ABL as a therapeutic target. One of these mutations occurs at amino acid 315, which lies in the ATP binding pocket of ABL. Forms of BCR-ABL that carry a T315I mutation cannot bind the drug and are therefore resistant to inhibition.[16,17]

Because imatinib also inhibits the PDGF receptor tyrosine kinase, it can also be used to treat patients with hypereosinophilic syndrome. This syndrome was recently found to be caused by a fusion of the Fip1-like 1 (FIP1L1) gene to the PDGFR (PDGFRA) gene through a deletion on chromosome 4q12. The resulting FIP1L1-PDGFR fusion protein is a constitutively activated tyrosine kinase that transforms hematopoietic cells and is inhibited by imatinib (IC₅₀ = 3.2 nM).[18] Sawyers reported that patients with hypereosinophilic syndrome can also become resistant to this drug by acquiring mutations in the ATP binding pocket of PDGFA.[15]

Tinkering With Kinase Translation and Folding

Finally, kinases do not always have to be targeted directly. Drugs that modify the translation and folding of kinase enzymes also have therapeutic potential. Paul Workman,[19] of The Institute of Cancer Research, Sutton, United Kingdom, and colleagues have been investigating proteins that maintain stability of kinases and other proteins. One of these chaperones, Hsp90, regulates the folding, stability, and function of many oncoproteins — primarily kinases, hormone receptors, and transcription factors. Because of its broad scope of functions, its inhibition provides a means to simultaneously attack multiple cancer pathways. Cancer cells may be particularly sensitive to Hsp90 inhibition as a result of their greater dependence on these pathways and the fact that they contain “superchaperone” complexes that are sensitive to inhibition.[19–22]

Most Hsp90 inhibitors developed to date are ATP nucleotide mimetics that block the essential ATPase activity of the chaperone. This leads to degradation of kinases and other proteins that interact with Hsp90. One Hsp90 inhibitor, geldanamycin (17AAG), has been shown to stop the growth of A2780 human ovarian cancer cell xenografts in nude mice. Markers of Hsp90 inhibition include depletion of the cyclin-dependent kinase CDK4 and RAF. The effect of 17AAG was reversible: when the drug was removed, the tumors grew back in the treated mice.[19]

Phase 1 clinical studies with 17AAG have shown that the drug is well tolerated. In patients with melanoma who were given 450mg/m²/week of 17AAG, RAF, and CDK4, protein levels immediately declined in tumor biopsy samples. One 64-year-old patient with chemotherapy-resistant malignant melanoma began 17AAG treatment in February 2001 and achieved stable disease by 3 months after therapy started. Analysis of a biopsy sample revealed that RAF and CDK4 protein levels were reduced in tumor cells. The patient's tumor has remained the same size for more than 3 years.[19] Further studies are in progress to evaluate efficacy of 17AAG in other patients and in other tumor types.

Conclusions

Because kinases mediate many of the signaling pathways by which cancer cells promote their own proliferation and survival, inhibitors of these enzymes have the potential to be developed as potent anticancer agents. Because kinases are also required for the function of normal cells, toxicity is an important issue that will have to be carefully monitored.

Some of the most important issues surrounding the development of these drugs is selection of patients that are most likely to respond to therapy — kinase inhibitors do not function like chemotherapeutic agents and destroy all rapidly dividing cells, but rather target specific signaling alterations of cancer cells. So only patients whose tumors depend on upregulation of these specific signaling pathways are likely to respond to treatment.

In addition, as cancer cells use multiple pathways to promote their own survival and proliferation, combination therapies (of multiple targeted therapeutics, or of targeted drugs plus chemotherapy) are likely to be required to completely eradicate a tumor and prevent resistance or relapse.