The Developing Diversity of Targeted Therapy in Oncology, Presented by by Gordon Mills, MD, PhD; Funda Meric-Bernstam, MD
Introduction
The relatively narrow therapeutic window characteristic of the vast majority of traditional chemotherapeutics has led to pursuit
of targeted therapies with the hope of providing both improved efficacy and reduced toxicity. These targeted therapies use
agents that impact specific factors or cellular pathways that have been shown to have abnormal activity in neoplastic cells
and that drive cancer progression. The diversity of potential targets is currently most impressive, with an astounding number
of new possibilities emerging. However, translation of these targets into useful approaches for clinical drug development
has provided both exciting successes and disappointing failures. Some of the most attractive targets for molecular-targeted
strategies for the treatment of cancer include the family of receptor and non-receptor tyrosine kinases (TKs) and more specific
areas of the growth factors and epidermal growth factor receptor (EGFR) inhibitors; the Ras/Raf/MAPK pathway; thephosphatidylinositol-3
kinase (PI3K)/Akt/PTEN pathway, gene expression modification with antisense oligonucleotides and RNAi; and endothelial cell and angiogenesis-associated
factors such as the vascular endothelial cell growth factor (VEGF).[1-3]
Although some of these approaches have produced clinically useful agents, primarily due to the nature of the abnormality targeted,
other approaches have not been as successful because of the redundancies and overlapping nature of complex signal transduction
pathways. In many cases, although the aberration is present in cancer cells, it does not drive tumor behavior. Indeed, one
of the most significant challenges is to distinguish molecular events that drive tumor behavior from epiphenomena that arise
secondarily to an important event or due to the inherent genetic instability in tumors.[4] Although single-agent therapy remains a goal, experience with conventional cytotoxic therapy and with experimental agents
suggests that combining these new, targeted agents with either conventional therapies or one another is likely to provide
the best efficacy. Indeed, given the complex crosstalk between signaling pathways, it is likely that it will be necessary
to hit severalcritical signaling nodes to attain optimal outcomes. However, as combinations of targeted therapeutics may be
more effective, they may also be more toxic, a process that can be quantitated only in patient studies.[4]
Signal Transduction Kinases
-
Kinases are enzymes that regulate the function of other proteins/enzymes and other target molecules. Tyrosine kinases accomplish
signal transduction by transferring an activating gamma phosphate group from ATP to a tyrosine residue on the substrate molecule,
usually another protein. Serine and threonine kinases are a separate group of transduction enzymes that target serine and
threonine residues rather than tyrosine residues on substrate molecules. In addition to proteins, a number of other intracellular
signaling molecules, such as membrane lipids or intracellular inositol rings, are regulated by their phosphorylation status
through the action of specific kinases. Phosphatases remove phosphates on the substrate molecules and thus oppose kinase activity
and inhibit the signal transfer. These 2 enzyme classes act as switches; they activate and deactivate pathways according to
the signals received by the cell. Although in general, phosphorylation events positively regulatesignaling, there are circumstances
in which phosphorylation inhibits function. So depending on the cell system and the target, kinases and phosphatases can be
either positive or negative regulators. Cellular signaling frequently involves phosphorylation of all 3 amino acid residues
(tyrosine, threonine, and serine), sometimes at multiple sites on one protein, or of other signaling molecules such as lipids
and on multiple components in a pathway, suggesting that multiple kinases and kinase classes function collaboratively to orchestrate
the final outcome of signal transduction. Figure 1 is a simplified example of a model signal transduction pathway. An extracellular
signal activates a membrane-bound receptor, such as EGFR, which forms either homodimers with another EGFR or heterodimers
with other family members to initiate the signal relay across the cytoplasm, culminating in changes of gene transcription
in the nucleus and subsequent regulation of important cellular functions such ascell proliferation, migration, or survival.[5]
Some kinases are membrane bound, and others reside in the cytoplasm. Thus, development of agents to modify kinase activities
has included antibodies targeted to the extracellular portion of the membrane-bound kinase, eg, trastuzumab, as well as small
molecules that act in the cytoplasm, eg, imatinib mesylate.[1,2] Inhibition of intracellular TK activity has been achieved using small molecules whose structures are based on the structure
of ATP. This characteristic allows these molecules to affect not only tumors that overexpress TK activity, but also those
with normal TK expression or mutated, constitutively activated forms of TK. These competitive inhibitors of the ATP-binding
site on the kinase can be specific for individual kinases or have a broader spectrum inhibiting multiple kinases, such as
the effects of imatinib mesylate on Abl, Kit (CD 117), and platelet-derived growth factor (PDGFR), and have the clinical benefit
of oral bioavailability.[6]
-
Figure 1.
Simplified signal transduction cascade through binding of a ligand to the membrane receptor to final effects on gene expression
in the nucleus.[5]
The Bcr-Abl Tyrosine Kinase and Inhibition by Imatinib
-
Imatinib mesylate (STI571) was the first agent in the class of small-molecule inhibitors of TK activity proven to be clinically
useful, establishing intracellular kinases as "druggable" targets. Imatinib was designed to inhibit the constitutively activated
cytoplasmic Bcr-Abl kinase created by the Philadelphia chromosome abnormality in chronic myeloid leukemia (CML). Downstream
proliferative signal transduction and the inhibition of apoptosis were thus prevented (Figure 2).[7] The agent also inhibits 2 other TKs, Kit and PDGFR. Because of this activity, imatinib is also used for the treatment of
Kit-positive unresectable or metastatic gastrointestinal stromal tumors (GISTs), PDGFR-driven hypereosinophilic syndrome,
and dermatofibrosarcoma protuberans.[8-10] Indeed, imatinib mesylate appears to demonstrate activity in cells with mutations or rearrangement in Abl, Kit, or PDGFR,
but to have limited activity in tumors without genetic aberrations in these targets.This suggests that the presence of a genetic
aberration provides an indication that the target is indeed driving tumor progression. The high rates of response to imatinib
in these diseases are exciting; however, they may not represent the results to be expected in most epithelial tumors. This
set of tumors may represent early disease in which the tumors have not yet acquired the multiple genetic aberrations that
are found in most epithelial tumors.
Studies with GIST have proven particularly informative. Patients with perimembrane-activating mutations in Kit have shown
high-frequency, durable responses. Patients with mutations at other sites in Kit have shown both a lower frequency of response
and short duration of responses.[11] This observation suggests that, while the presence of mutations may signal tumors driven by a particular enzyme, not all
mutations are created equal. Even more intriguing is the observation that positron emission tomography (PET) imaging of FDG-glucose
uptake can identify patients destined to respond to therapy early in the course of treatment.[9] Indeed, a decrease in FDG-glucose uptake within the first week of therapy indicates eventual responsiveness, which may not
be evidenced using other technologies, including computed axial tomography (CAT) scans, for several months. Strikingly, a
failure to normalize FDG-glucose uptake indicates that a patient is unlikely to respond totherapy. Thus, molecular imaging
can predict responsiveness and allow persistence of therapy or early change to more effective approaches. Every effort must
be made to identify molecular imaging approaches applicable to the plethora of targeted therapeutics under development.
-
Figure 2.
Inhibition of Bcr-Abl by imatinib. The aberrant phosphorylation and activation of downstream signal transduction by the Bcr-Abl
fusion protein is inhibited by the competitive binding of imatinib in the ATP-binding site.[7]
The ErbB Receptor Tyrosine Kinases
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The ErbB family of receptor tyrosine kinases (RTKs) encompasses 4 closely related transmembrane receptors: erbB-1 (EGFR or
HER-1), erbB-2 (HER-2 or neu), erbB-3 (HER-3), and erbB-4 (HER-4).[12,13] These receptors exhibit similar structures with 3 distinct regions: an extracellular ligand-binding domain, a transmembrane
region, and an intracellular region with tyrosine kinase activity. Receptor activation occurs after a ligand binds to the
extracellular domain, which triggers dimer formation. The dimers can be either homodimers or heterodimers of the various erbB
proteins. Indeed, erbB-2 does not appear to have a specific ligand, but rather functions as a dimerization partner for other
family members. When overexpressed, it appears to form constitutively active homodimers. Dimerization causes activation of
the intracellular kinase via autophosphorylation of tyrosine moieties in its intracellular domain and transmission of downstream
signaling molecules.Intriguingly, erbB-3 appears to be kinase inactive and instead functions as a linker molecule, altering
or amplifying the spectrum of molecules phosphorylated by members of this family. The potential for dimerization with another
erbB family member or self-association magnifies or redirects the downstream impact of elevated or aberrant erbB-RTK function.
-
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Studies have shown that abnormal EGFR signaling can result from increased EGFR expression, mutation of EGFR, or decreased
activity of opposing phosphatases. Downstream pathways activated by EGFR signaling include the Ras/Raf/MAPK cascade, which
is implicated in proliferation, migration, and differentiation. EGFR also signals through the PI3K/Akt pathway, which is involved
in proliferation and anti-apoptosis/survival.[1,2,14] These membrane-bound receptor kinases offer 2 opportunities for regulation of signal transduction: 1) through the extracellular
domain, especially with monoclonal antibody blockade; and 2) through small-molecule inhibition of the intracellular kinase
activity (Figure 3).[13]
The overexpression of erbB-2/HER-2 in many cancers and, in particular, in approximately 25% of breast cancers, is well established.
It is also an indicator of poor prognosis for breast, colorectal, gastric, non-small cell lung, ovarian, and prostate cancers
and melanoma.1 erbB-2/HER-2 is the target for monoclonal antibody therapy with trastuzumab for patients with metastatic breast
cancer overexpressing erbB-2/HER-2.[6,15,16] Success of trastuzumab, a monoclonal antibody therapy directed toward an erbB RTK, stimulated interest in antibody-targeted
therapy for EGFR.
EGFR overexpression and/or abnormal EGFR-TK activity is associated with many human malignancies, including head and neck,
colorectal, non-small cell lung, gastric, pancreatic, ovarian, breast, and prostate cancers and glioma.[1,2] In these malignancies, overexpression of EGFR has been shown to be an indicator of advanced disease and poor prognosis.[14] Blockade of the receptor with mAb 225 (C225 or cetuximab) or ABX-EGF competitively inhibits the activation of the EGFR-TK.[17] Cetuximab has recently been approved for the treatment of advanced colorectal cancer in patients whose tumors express EGFR.
In a study of cetuximab combined with irinotecan, tumor shrinkage was observed in 22.9% of patients and tumor progression
was delayed by approximately 4.1 months. Monotherapy with cetuximab showed a tumor response rate of 10.8% and a tumor progression
delay of 1.5 months.[18]
-
Figure 3.
EGFR signal transduction and EGFR domain targeting by monoclonal antibodies and small-molecule tyrosine kinase inhibitors.[2]
-
A number of small-molecule inhibitors of the EGFR-TK have been approved for cancer treatment, and others are in various stages
of clinical development.[1,6] These agents are classed by their selectivity (monofunctional, erbB-1/EGFR-specific versus multifunctional, pan-erbB activity)
and the reversibility of their inhibition (Table 1). Generally, these small molecules block the enzyme activity of transmembrane
kinase, autophosphorylation, and subsequent signal transduction. They are given orally and generally show only mild to moderate
toxicities, predominantly a class-associated acneiform skin rash in 50% to 70% of patients and diarrhea in approximately 40%
of patients due to the normal presence of high levels of EGFR in these sites.[1,6] Although somewhat controversial, several studies demonstrate a correlation between these side effects and patient outcomes.
This has been posited to be due to delivering a "biologically relevant dose" or, alternatively, torepresent an indication
of immunological competence. Of these agents, gefitinib and erlotinib are the most advanced in clinical trials.
In preclinical studies, gefitinib has shown antitumor activity in breast, lung, and ovarian cancers. Gefitinib has been approved
in the United States for monotherapy of patients with advanced non-small cell lung cancer (NSCLC) whose cancer has progressed
after treatment with platinum-based and docetaxel chemotherapies. In a study of patients with NSCLC (N = 216), including 142
with refractory disease, the response rate (at least 50% tumor shrinkage lasting for 1 month) was about 10%.25 For some patients,
the response was more durable, with a median duration of 7 months. Since the use of the agent is third line (when no other
viable therapy exists), the low response rate was thought to confer some clinical benefit. However, in 2 controlled, randomized
clinical trials (N = 2130) of first-line treatment for NSCLC, gefitinib failed to confer any benefit when added to standard,
platinum-based therapy.[26,27] Similar results have been seen with other chemotherapy agents suchas gemcitabine and paclitaxel.[28] As a result, gefitinib is not indicated for use in this setting. Moreover, an approximate 1% occurrence of interstitial lung
disease has been reported, with approximately 30% of these occurrences resulting in deaths.19 In part due to the observation
that cell lines expressing erbB-2 are sensitive to gefitinib and that in combination with trastuzumab, gefitinib exhibits
a synergistic inhibition in cells lines co-expressing erbB-1 and erbB-2, this agent has been studied in patients with advanced
breast cancer.[2,29] Although the participating patients (N = 63) were not screened for EGFR, 43% had tumors that were estrogen-receptor positive
and 27% overexpressed erbB-2. The objective response was low in this heavily pretreated population, and only 14.3% of patients
achieved a partial response or stable disease that endured for up to 6 months. Gefitinib is currently being studied in a wide
diversity of clinical trials, includingmonotherapy for refractory ovarian cancer and combination therapy for metastatic breast
cancer and bladder cancer.[30]
Recent studies have demonstrated that individuals with marked response to gefitinib express a unique pattern of activating
mutations in the intracellular domain of EGFR.[31,32] Strikingly, the frequency of these activating mutations is higher in women, non-smokers, Asians, and adenocarcinomas, replicating
the clinical observations. This observation suggests once again that the presence of mutations in the target is an indication
that the molecule is driving tumor behavior. Further, it suggests that methods to identify patients whose tumors are driven
by the target should be developed concurrently with targeted therapeutics if we are to achieve their optimal utility.
Another agent, erlotinib, has also shown inhibitory activity in multiple tumor cell lines both as a single agent and in combination
with chemotherapeutic agents, radiation therapy, or other targeted agents.[33] Initial phase II results for treatment of patients with advanced head and neck cancer (N = 115) showed an objective response
in 4.3% in these heavily pretreated patients, regardless of their EGFR status, with a median overall survival duration of
6 months.[34] In this study, as well as in phase II trials in NSCLC and ovarian cancer, the subgroup of patients developing the classic
rash showed a significant association with improved survival.[34,35] Phase III trials of erlotinib are underway in NSCLC and pancreatic cancer as a single agent or in combination therapy.[20,21] Preliminary data indicate that, like gefitinib, erlotinib will be most active in patients with specific mutations in EGFR.
Development of irreversible inhibitors of the erbB family, such as CI-1033 and EKB-569, was pursued to some extent because
of the high levels of reversible erbB inhibitors necessary for continuous inhibition of phosphorylation. Covalent bonding
of these irreversible inhibitors to the TK domain might sustain inhibition more effectively and for a longer duration at a
lower drug level. Additional impetus came from the speculation that reversible inhibitors would not overcome the high intracellular
concentrations of ATP. Furthermore, there are as yet unproven expectations that drug resistance emergence might be avoided.
Combination therapy with traditional chemotherapeutics and other drug classes also presents an attractive option. However,
these agents are in the early stages of investigation, and clinical benefits are unproven.[2,14] Moreover, the long half-life of orally delivered gefitinib or erlotinib suggests that there may be only limited advantages
to theirreversible inhibitors. Perhaps 1 reason for the lack of high response rates among these small-molecule EGFR-TK inhibitors
is the absence of screening for patients with tumors expressing the EGFR target molecules. Trastuzumab studies did select
for patients overexpressing erbB-2 and were able to demonstrate survival benefit.[36] Indeed, had the studies been done in all breast cancer patients without screening for erbB-2 overexpression, the activity
of trastuzumab would have been missed due to the confounding effects of the large number of nonresponders. Imatinib also is
specific for patients expressing defined kinase abnormalities, such as Bcr-Abl, PDGFR, and Kit. As indicated above, EGFR inhibitors
appear to exhibit maximal activity in patients with activating mutations in the intracellular domain. This suggests that selection
of patients whose tumors are driven by particular genetic aberrations will be necessary for optimal clinical benefit. Moreover,
delay of tumorprogression may be a more appropriate endpoint for clinical trials with these agents, since that was an important
finding in preclinical studies.[2] On the other hand, many of these agents are of interest due to their additive and synergistic potential in combination with
traditional therapies or other emerging therapies.
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Table 1.
HER-1/EGFR-TK Small-Molecule Inhibitors
Ras/Raf/MEK/MAPK Pathway
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Ras proteins occupy a pivotal role in signal transduction, from RTKs and external growth stimuli to a variety of intracellular effector pathways, including the downstream targets of Raf and MEK (a mitogen-activated protein kinase kinase), leading to cell proliferation (see Figure 3). As a response to growth-inducing signals, the inactive membrane-bound Ras-guanosine 5´-diphosphate (Ras-GDP) is switched to the activated membrane-localized Ras-GTP, which subsequently acts on downstream components.[5,37] Normal control of Ras signaling involves GTP hydrolysis by GTPase-activating proteins, which rapidly convert Ras to the inactive
Ras-GDP state. To perform signal transduction, the Ras protein also undergoes a series of post-translational modifications
that facilitate its localization to the inner surface of the cell membrane and subsequent activity. The first of these modifications
requires farnesylation (lipid prenylation) of the Ras protein at a C-terminaltetrapeptide sequence (the CAAX motif) in a step
catalyzed by the enzyme farnesyltransferase (FTase). The introduction of this hydrophobic modification allows the Ras protein
to more effectively associate with the membrane lipid bilayer. Other protein prenyltransferases, the geranylger-anyltransferases
(GGTase) also catalyze prenylation of several proteins, such as Ras.[5,37]
Mammalian cells have at least 3 ras proto-oncogenes: K-ras, H-ras, and N-ras, which, upon activating mutation, result in constitutive proliferation and anti-apoptotic signaling. Overall, ras mutations have been identified in about 30% of all human cancers, such as the K-ras mutations in NSCLC, colorectal cancer, and pancreatic cancers; H-ras mutations in bladder and kidney cancers; and N-ras mutations in melanomas and hematologic cancers.[38] Ras signaling is also influenced by aberrant upstream signaling, as Ras functions to transmit extracellular signals from
growth factors and their receptors to intracellular pathways. With the established involvement of Ras in cancer and the necessity
of prenylation for Ras activity, targeting of the FTases with inhibitors presented an attractive strategy to block this pathway.
-
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Several classes of FTase inhibitors exist. Although many have been studied preclinically, few have progressed far in clinical
development (Table 2). Early clinical trials of both orally active and parenterally administered FTase inhibitors show similar
toxicities, including schedule-dependent myelosuppression and gastrointestinal effects. Studies in solid tumors have also
been disappointing. Monotherapy showed no objective responses in tumors, including NSCLC, SCLC, pancreatic, colorectal, and
prostate.[1,5] However, preliminary results suggest that R115777 might have some benefit in hematologic malignancies.[42]
The lack of specificity of the FTase inhibitors suggests that these agents might also inhibit farnesylation of some of the
300 known proteins possessing a CAAX motif. This inhibition might account for some of the antiproliferative activity attributed
to these agents.[1,5] Additionally, redundant pathways might subvert the impact of the FTase inhibitors on Ras. Geranylgeranylation of K-Ras and
N-Ras after blocking farnesylation may offer a means for continued signal transduction. Thus, although the exact mechanism
of action of these agents is not as clear as once thought, preclinical data still suggest that FTase inhibitors might play
a role in therapy if properly used.[1,5]
The downstream MAPK effector pathway, which includes Raf and MEK, also offers sites for drug targeting. B-Raf mutations have
been documented in a number of human cancers, including early ovarian cancers and melanomas, and the MAPK pathway is activated
in approximately 30% of human malignancies.[44-46] Raf is a family of serine-threonine kinases. Because they can also be activated by protein kinase C alpha and are known to
contribute to multidrug resistance expression, they play a large role in oncogenesis.[2] The only known substrates for MEK are the MAPK kinases Erk1 and Erk2 (extracellular signal-regulated kinases), whose substrates
are cytosolic and nuclear proteins (probable transcription factors).[5] These downstream kinases have only recently garnered the interest that had previously been reserved for Ras; therefore, inhibitors
of these kinases are not as far along in clinical investigations. BAY 43-90006 is an orally available potentinhibitor of Raf-1
that targets the ATP-binding site and is in clinical trials. CI-1040, an inhibitor of MAPK at a non-ATP site, is also in early
phase II clinical trials in pancreatic, breast, renal, and colorectal cancers.[1,5] The response rates in renal cell cancers to BAY 43-9006 may, however, represent a rather promiscuous activity of this inhibitor,
blocking for example the Kdr VEGF receptor.
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Table 2.
Farnesyltransferase Inhibitors[5,32]
PI3K/Akt/PTEN Pathway as an Emerging Target
The PI3K pathway plays a role in cell growth; cell cycle progression (both during the G1 phase of the cell cycle and at the
G2/M transition); protein translation; sensing the environment, particularly available nutrients and growth factors; motility;
neovascularization; metastasis; drug resistance; and cell survival, particularly apoptosis and anoikis (matrix deprivation-induced
apoptosis), as well as in transformation induced by many different oncogenes and tumor suppressor genes.[47] Given the importance of these processes in transformation and tumor progression, the PI3K pathway is a potential driver of
tumor behavior. Various elements of the PI3K/Akt/PTEN pathway are activated in at least 70% of all cancers, resulting in constitutive activation of the pathway. Indeed, the PI3K
pathway is targeted for mutagenesis in human tumors more frequently than any other pathway, with the potential exception of
the p53 pathway.[48-50] However, the p53 and thePI3K pathways interact at multiple levels, suggesting that these 2 pathways are really a network
and any attempt to distinguish between the pathways is moot. The PI3K pathway is targeted by amplification of the catalytic
subunit of PI3K, Akt and S6K1, activating mutations in the catalytic subunit of PI3K and the regulatory subunit of PI3K, inactivating
mutations in TSC1/2, LKB1, and PTEN and rearrangements of forkhead and TCL1. In addition, activation of Akt can bypass the effects of radiation and chemotherapy and also of targeted therapeutics such
as gefitinib and erlotinib. This has not escaped the attention of the pharmaceutical and biotech industries, where there are
multiple programs targeting many of the components of the pathway. CCI-779, RAD001, and AP23573, which target mTOR, a downstream
component of the pathway, are in clinical trials and have demonstrated remarkable responses in a subset of patients with glioblastoma
multiforme,metastatic melanoma, malignant glioma, mantle cell non-Hodgkin's lymphoma, small cell lung cancer (SCLC), and renal
cell carcinoma. Objective response rates have been as high as 10% to 20% in several of the tumors, with stable disease in
a significant fraction of patients. Mantle cell lymphoma is particularly intriguing, as response rates may be as high as 44%
(see accompanying article by S. Faivre and E. Raymond for a discussion of clinical results with these agents). Why mantle
cell lymphoma is highly sensitive is under extensive investigation. Ongoing clinical trials are evaluating mTOR inhibitors
in combination with other targeted therapeutics and also in combination with chemotherapy.
Apoptosis Modulation and Antisense Oligodeoxynucleotides
The use of antisense oligodeoxynucleotides (AS-ODNs) involves synthetically producing a short sequence (usually an oligomer
of about 20 nucleotides) of chemically modified DNA that will hybridize with a specific mRNA sequence to prevent the translation
of the targeted mRNA into protein. It is thought that the mRNA in the AS-ODN complex is subjected to degradation by ribonuclease
H, thus removing the mRNA from the pool of translatable RNAs.[51] The AS-ODN enters the cell through endocytosis and, possibly, a receptor-mediated process. The AS-ODN approach has been validated
with the approval of the antisense drug fomivirsen for the treatment of cytomegalovirus retinitis in patients with AIDS. A
number of AS-ODNs are in clinical development and target proteins, such as H-Ras, c-Raf, and Bcl-2.1 The oncogene Bcl-2 is
overexpressed in more than 50% of human cancers.[52] It is known to code for an anti-apoptotic protein that can block cancer cells from undergoingprogrammed cell death (apoptosis)
on exposure to anticancer therapies, such as traditional cytotoxic chemotherapy, radiotherapy, and monoclonal antibody therapy.
Oblimersen sodium is an 18-mer oligodeoxynucleotide antisense sequence that hybridizes with a key region of Bcl-2 mRNA to
block expression.[52,53] Clinical trials of oblimersen in combination with cytotoxic therapies and biologic/immunotherapies are underway in metastatic
breast cancer, SCLC, and hematologic malignancies.[30] Initial reports from a phase I study in advanced SCLC showed that oblimersen use in combination with carboplatin and etoposide
was well tolerated and produced encouraging results.[54] However, follow-up studies have not been as encouraging.[2] Whether this represents a problem with the approach, failure to deliver stable antisense oligodeoxynucleotides, or selection
of a tumor type not dependent on Bcl-2 will require additional investigation.
Small interfering RNAs (siRNA) have demonstrated an ability to downregulate gene expression by targeting RNAs to the RISC
complex with subsequent degradation.[55] Although in an earlier stage of evolution, siRNA appears to be a natural gene expression regulator and may have more generalizability
than antisense. Small interfering RNAs has shown the ability to regulate gene expression and tumor growth in animal models.
Tumor Angiogenesis Inhibition
Based on evidence that angiogenesis plays a crucial role in tumor growth and metastasis, development of agents that prevent
formation of new blood vessels is focused on a number of approaches, of which the following are furthest along: inhibition
of VEGF; inhibition of the tyrosine kinase activity associated with the VEGF receptor (VEGFR) on endothelial cells; and inhibition
of endothelial cell proliferation. Vascular endothelial cell growth factor is the most frequently upregulated angiogenic growth
factor and has been associated with both metastasis and/or poor prognosis in numerous human solid tumors.[56,57] In tumor tissues, VEGF can be constitutively upregulated through both autocrine and paracrine loops with involvement of a
wide variety of oncogenes, such as K-ras, p53, WTI, and v-Raf, as well as the growth factors LPA PDGF and IGF-1 and the PI3K pathway described above.[56,57] With recent data supporting the validation ofangiogenesis as an exciting therapeutic opportunity, the National Cancer Institute
(NCI) and the Eastern Cooperative Oncology Group (ECOG) have initiated a number of clinical trials for the treatment of melanoma
and myeloma, as well as breast, colon, lung, renal, and head and neck cancers, with antiangiogenesis therapies such as bevacizumab
and thalidomide as monotherapy and in combination with standard therapies.[58,59]
Bevacizumab (rhuMAB-VEGF) is a recombinant humanized monoclonal antibody directed to bind and neutralize VEGF, thus preventing
VEGF from binding to and activating VEGFR on endothelial cells. A report from a phase II study of bevacizumab monotherapy
in patients with metastatic renal cell cancer showed that the treatment significantly prolonged time to progression, but not
overall survival compared with placebo (P < .001) in patients receiving high-dose antibody (10 mg/kg every 2 weeks).[60] Also promising was the report that bevacizumab (at low [5 mg/kg] or high [10 mg/kg] doses) in combination with fluorouracil/leucovorin
as first-line therapy of metastatic colorectal cancer provided higher response rates, longer median time to disease progression,
and longer median survival (control, 13.8 months; low-dose bevacizumab, 21.5 months; high-dose bevacizumab, 16.1 months) compared
with fluorouracil/leucovorin alone.[61] Although a phase III study ofbevacizumab combined with capecitabine showed a near doubling of the response rate (19% to 30%),
the response was not durable and there was no difference in progression-free survival.[62] Since it is thought that earlier use of bevacizumab might provide better outcomes as seen in the trial in colorectal cancer,
a new phase III trial has been initiated to investigate bevacizumab combined with paclitaxel in patients newly diagnosed with
metastatic breast cancer.[2] Indeed, recent clinical results showing efficacy of bevacizumab in colorectal cancer have resulted in its approval by the
US Food and Drug Administration (FDA), making this the first agent to target the vasculature to achieve approval.
Whereas bevacizumab acts directly on VEGF, other small molecules can modulate VEGF activity through selective inhibition of
VEGF membrane RTKs. The compounds currently being investigated are competitive tyrosine kinase inhibitors that block the ATP-binding
site, preventing downstream signaling. Although most of these molecules have rather broad receptor specificity, the multiplicity
of pro-angiogenic factors implicated in tumor angiogenesis suggests that monotherapy will not be sufficient. As a result,
these agents are commonly evaluated in combination with conventional chemotherapy.
Vatalanib (PTK787/ZK222584) inhibits all 3 VEGF receptors, as well as PDGFR-beta, c-Kit, and CSF-1R.[63,64] Encouraging results from phase I/II trials in metastatic colorectal, recurrent glioblastoma multiforme, and metastatic renal
cell cancer supported further clinical development of this compound. Several phase II studies in a variety of tumor types
are ongoing, as well as 2 phase III trials in metastatic colorectal cancer.[28,65-68]
The multi-kinase inhibitors SU11248 and ZD6474 are also progressing in clinical development. In addition to all 3 VEGF receptors,
these small molecules inhibit a wide variety of other membrane protein kinases and have demonstrated anti-angiogenic and antitumor
effects in animal models.[69] SU11248 is being evaluated in a number of clinical trials in metastatic beast, renal, and colorectal cancers.[30] The demonstrated objective responses after therapy with SU11248 in patients with metastatic GISTs resistant to imatinib supported
evaluating this compound in a phase III trial in this patient population.[70] ZD6474 is being evaluated in several phase II trials in both non-small cell and SCLC.
Another area of investigation is vascular targeting agents (VTAs). Rather than inhibiting the growth of new blood vessels,
VTAs target the already formed vasculature of tumors, causing the tumor blood vessels to shut down. The ensuing ischemia can
cause extensive tumor cell death.[71] However, a thin rim of tumor cells adjacent to normal tissue often survive. Thus, curative regimens using these agents will
most likely also include conventional anticancer therapies.
Compared with normal tissues, vascular architecture in solid tumors is abnormal, chaotic, and inadequate in both structure
and function.[72] Although these characteristics often impede the effectiveness of conventional chemotherapeutic agents, they provide a unique
target for the developing novel antitumor agents.[73] Examples of tumor vascular endothelial targets include prostate-specific membrane antigen (PSMA), VEGF receptors, fibronectin
ED-B domain, alphavbeta3 integrin, E-selectin, vascular cell adhesion molecule-1 (VAM-1), and phosphatidyl serine. A number of biological molecules
have been developed that target tumor endothelium directly. These molecules are currently being evaluated in preclinical and,
in the case of PSMA, early clinical testing.[72,73]
Small-molecule VTAs are further along in clinical development. Two classes of small molecule VTAs are currently being evaluated.
The first is flavone-8-acetic acid (FAA) and its derivatives. The mechanism of action of these molecules appears to be indirect
through the induction of various modulators of the immune system, such as nitric oxide, serotonin, and tumor necrosis factor
(TNF)-alpha.[74] 5,6- dimethylxanthenone-4 acetic acid (DMXAA) is one of the most promising FAA derivatives. In preclinical studies DMXAA
demonstrated antitumor activity in a wide variety of murine tumors and induced extensive tumor necrosis in human tumor xenografts.[75] In a phase I study of 63 patients with advanced cancers, a patient with metastatic cervical carcinoma achieved a partial
response, and 22 patients achieved stable disease. In 5 patients, the duration of stable disease exceeded 12 weeks. The antitumor
activity of DMXAA was observed at well-tolerateddoses.[76]
The second class of small-molecule VTAs consists of tubulin-binding agents. Colchicine, vincristine, and vinblastine are tubulin-binding
molecules that have been developed as anticancer agents. The antitumor effects of these agents were uniformly very close to
their maximum tolerated dose (MTD). "Second-generation" tubulin destabilizing agents have been developed that disrupt tumor
blood vessels at doses well below their MTD.[73] These newer tubulin-binding agents consist of combretastatins and their analogs. Combretastatin A-4 disodium phosphate (CA4DP),
the lead compound in this series, has shown potent antivascular and antitumor effects in a wide variety of preclinical tumor
models.[77] Three phase I clinical trials of CA4DP involving a total of 96 patients with advanced cancers have been reported.[78-80] Tumor pain was a unique side effect of combretastatin in these studies. In one study, 1 patient with anaplastic thyroid cancer
had a completeresponse.[78] Dose dependent reductions in tumor blood flow have been shown by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI)
and PET at doses that were reasonably well tolerated by patients.[78,81-82]
Several other tubulin-binding agents are also in phase I clinical development, including AVE8062A, ZD6126, and ABT-751.[66] The early clinical evidence of these VTAs warrants further evaluation.
Conclusions
The explosion of new therapeutic targets associated with signal transduction pathways has led to the development of rationally
designed drugs and the emergence of biologic-based therapies that have provided both encouraging advances in outcomes and
dramatic disappointments. However, interest has not waned, and this area of investigation remains in the early stages of discovery
in terms of both the basic science of cellular control and appropriate regimen and patient selection for clinical study. Efforts
continue to explore opportunities in this area for medical value. The optimal implementation of these drugs will require the
concurrent development of targeted therapeutics with molecular markers able to identify tumors driven by particular targets
and molecular imaging approaches able to identify patients who are responding early in therapy.
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