Therapeutic Targeting of Cancer Cells

Era of Molecularly Targeted Agents

Shivaani Kummar, Anthony J. Murgo, Joseph E. Tomaszewski and James H. Doroshow

• Molecularly targeted anticancer agents (MTAs) are those that selectively target specific molecular features of cancer cells such as aberrations in genes, proteins, or pathways that regulate tumor growth, progression, and survival.

• Molecular targets include the following: products of activating mutations and translocations, growth factors and receptors, aberrant signal transduction and apoptotic pathways, factors that control tumor angiogenesis and microenvironment, dysregulated proteins, DNA repair machinery, and aberrant epigenetic mechanisms.

• The development of MTAs requires innovative strategies that differ from those traditionally applied to nontargeted conventional chemotherapy.

• Successful development of an MTA depends largely on the importance of the target in controlling tumor cell proliferation and survival and effective modulation of the target in the tumor at clinically achievable concentrations.

• Primary objectives of clinical trials of MTAs differ from those used in trials of conventional chemotherapy. An important objective in phase 1 trials of MTAs should be to determine a phase 2 dose based on optimal target modulation (i.e., a biologically effective dose) rather than on maximum tolerated dose. In addition, objective tumor response may not be an adequate end point for efficacy evaluations of MTAs that have a primarily cytostatic effect. Alternate end points, such as progression-free survival, may be more appropriate.

• Functional and molecular imaging plays an increasingly important role in the development of MTAs.

• Patient selection is critical for trials with MTAs.

Introduction

Improved knowledge of cancer biology and synthetic chemistry along with advances in biotechnology have generated extraordinary opportunities for the development of molecularly targeted cancer therapeutics. Molecularly targeted anticancer agents (MTAs) are defined here as agents that selectively target specific molecular features of cancer cells such as aberrations in genes, proteins, or pathways that regulate tumor growth, progression, and survival. By identifying ways that cancer cells differ from normal healthy cells at the molecular level, scientists can exploit these differences to develop drugs that selectively target cancer cells while sparing normal cells. Consequently, an increasing number of MTAs are being developed with the goal of producing more effective and minimally toxic anticancer therapeutics. Furthermore, progress in the development of MTAs can shape cancer therapeutics into a more individualized form of cancer medicine. This chapter will review the principles of molecularly targeted therapy, including strategies for preclinical and clinical development.

Molecular Targets

The increasing number and assortment of molecular targets can be broadly categorized according to genetic or functional properties, including products of activating gene mutations and translocations; growth factors and receptors; aberrant signal transduction and apoptotic pathways; factors that control tumor angiogenesis and microenvironment; dysregulated proteins; DNA repair machinery; and aberrant epigenetic mechanisms (Tables 28-1 and 28-2).

Table 28-1

U.S. Food and Drug Administration–Approved Molecularly Targeted Agents

FDA Approved Agents(s)	Target	Disease Indication
Imatinib mesylate	BCR-ABL, KIT, PDGFR-β	Ph+ CML, Ph+ ALL, chronic monomyelocytic leukemia, dermatofibrosarcoma protuberans
Dasatinib	BCR-ABL, Src family kinases	Ph+ CML
Nilotinib hydrochloride monohydrate	BCR-ABL	Ph+ CML
All-trans retinoic acid	PML-RAR	Acute promyelocytic leukemia
Sunitinib malate	VEGFR, KIT, PDGFR-α	GIST, pancreatic neuroendocrine tumors, kidney cancer
Vemurafenib	BRAF^V600E mutation	BRAF^V600E-positive melanoma
Crizotinib	ALK tyrosine kinase	ALK-positive non–small cell lung cancer
Erlotinib	EGFR	Non–small cell lung cancer
Panitumumab	EGFR	Colorectal cancer
Cetuximab	EGFR	Head and neck cancer, colorectal cancer
Trastuzumab	ErbB-2	HER2 overexpressing breast cancer
Lapatinib	ErbB-2	HER2 overexpressing breast cancer
Vismodegib	Hedgehog pathway	Advanced/metastatic basal cell carcinoma
Brentuximab vedotin	CD30	Hodgkin lymphoma, anaplastic large cell lymphoma
Ofatumumab	CD20	CLL
Ibritumomab		Non-Hodgkin lymphoma
Rituximab		B-cell non-Hodgkin lymphoma
Alemtuzumab	CD52	B-cell chronic lymphocytic leukemia
Bevacizumab	VEGF	Colorectal cancer, non–small cell lung cancer, renal cell carcinoma
Vandetanib	VEGFR	Medullary thyroid cancer
Sorafenib	VEGFR, B-Raf kinase	Kidney cancer, hepatocellular cancer
Pazopanib	VEGFR	Soft tissue sarcomas, kidney cancer
Axitinib	VEGFR	Kidney cancer
Temsirolimus	mTOR	Renal cell carcinoma
Everolimus	mTOR	Renal cell carcinoma, advanced pancreatic neuroendocrine tumors
Azacitidine	DNA methyltransferase	Myelodysplastic syndrome
Decitabine	DNA methyltransferase	Myelodysplastic syndrome
Vorinostat	Histone deacetylase	Cutaneous T-cell lymphoma
Romidepsin	Histone deacetylase	Cutaneous T-cell lymphoma
Bortezomib	Proteosome	Multiple myeloma
Ipilimumab	Cytotoxic T-lymphocyte–associated antigen 4	Metastatic melanoma

ALK, Anaplastic lymphoma receptor tyrosine kinase; ALL, acute lymphocytic leukemia; CLL, chronic lymphocytic leukemia; CML, chronic myelogenous leukemia; EGFR, epidermal growth factor receptor; GIST, gastrointestinal stromal tumor; HER2, human epidermal growth factor receptor-2; mTOR, mammalian target of rapamycin; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.

Table 28-2

Classes of Molecularly Targeted Anticancer Agents under Clinical Development

Target	Role of Target	Disease Indication	References
FMS-like tyrosine kinase-3 (FLT-3)	Regulates cell cycle progression, proliferation, and survival	Acute myeloid leukemia	61
Poly (ADP-ribose) polymerase (PARP)	Single-strand DNA break repair	Various solid tumors, BRCA-positive breast and ovarian cancer	62
O⁶-Alkylguanine DNA alkyltransferase	Prevents intrastrand DNA cross-links		63
Insulin-like growth factor-1 receptor (IGF-1R)	Regulates cell proliferation, differentiation and survival	Various adult and pediatric tumors	64
Insulin-like growth factor binding protein-3 (IGFBP3)	Regulates cell proliferation, differentiation and survival	Various adult and pediatric tumors	64
Hypoxia-inducible factor-1α (HIF-1α)	Regulates tumor cell response to oxygen deprivation	HIF-1α–expressing solid tumors	65
α_vβ₃ integrin receptor	Involved in cell adhesion	Glioma and various solid tumors	66, 67
Syk	Inhibits differentiation and induces growth factor–independent proliferation of pre–B cells	B-cell lymphomas	68
MET	Regulates cell growth, anti-apoptosis, altered cytoskeletal function	Solid tumors	69
MEK1/2	Regulates tumor cell proliferation and survival	Various solid tumors, melanoma	70, 71
RAS	Promote cell proliferation and survival	Acute myeloid leukemia	72
RAF	Regulates tumor cell proliferation and survival	Melanoma and other solid tumors	73
RET	Regulates tumor cell proliferation and survival	Medullary thyroid cancer	74
Akt kinase	Regulator of cell cycle and apoptotic pathway	Variety of solid and hematologic cancer	75
Clusterin	Promotes cell survival	Variety of solid tumors	76
HSP-90	Chaperone for several oncogenic proteins and growth factors	Myeloid leukemia and solid tumors	77
Bcl-2	Antiapoptotic	Lymphomas, solid tumors	78
Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)	Apoptotic mechanism	Various types of cancer	79
Angiopoietin 1 and 2	Reduces blood flow to tumor	Solid tumors	80
DNA methyltransferase inhibitors	Reexpression of genes silenced by methylation	Myelodysplastic syndrome, acute myeloid leukemia, various solid tumors in combination with chemotherapy or radiation	58, 59
Src inhibitors	Src family kinases that interact with other growth, proliferation, and angiogenic pathways	Solid tumors	81
VEGFR, VEGF	Reduces blood flow to tumor	Solid tumors	11
CD105 (Endoglin)	Reduces blood flow to tumor	Solid tumors	82
HDAC	Reexpression of silenced genes	T-cell lymphoproliferative disorders, adenoid cystic cancer, solid tumors	83
Retinoid receptor	Increase in reactive oxygen species and dihyceramide levels resulting in cytotoxicity	T-cell lymphoproliferative disorders	84, 85
Wee 1	DNA repair and cell cycle (abrogates the G2 cell cycle checkpoint)	Solid tumors	86
BTK inhibitor	Bruton tyrosine kinase	Lymphoma, chronic lymphocytic leukemia	87
Gamma secretase inhibitor	Notch pathway	Solid tumor	88
CDK inhibitors	Cyclin-dependent kinases (cell cycle, proliferation)	Solid tumors	89
CHK1 inhibitors	Checkpoint kinases (cell cycle)	Solid tumors	90
DNA minor groove	DNA damage	Solid tumors	91
SMAC mimetic, IAP inhibitor	Induction of apoptosis	Solid tumors	92

The most promising molecular targets are those solely responsible for sustaining tumor growth and survival because agents that potently and selectively inhibit these critical targets are likely to have a major clinical impact. Probably the best example of a critical target is BCR-ABL in patients with chronic myelogenous leukemia (CML). BCR-ABL is a fusion protein formed by the reciprocal translocation of chromosomes 9 and 22. Knowledge that this dysregulated tyrosine kinase played a causal role in the pathogenesis of essentially all cases of CML spurred preclinical studies, which led to the development of a potent and selective ABL tyrosine kinase inhibitor (TKI), imatinib mesylate (previously known as STI571).¹ Subsequent clinical trials established imatinib mesylate as the first highly effective molecularly targeted therapy for CML and a prototype for the development of others in the class. Imatinib mesylate is also a potent inhibitor of other tyrosine kinases including PDGFR and KIT, and it is highly effective in the treatment of gastrointestinal stromal tumors (GISTs) bearing activating c-KIT mutations and in some GISTs bearing activating PDGFR mutations.^1,2

Unfortunately, most human tumors, including the most common types, are genetically complex and do not have a single critical target, and the relative critical importance of a target in different tumors may vary. Most tumor types have various genetic and molecular abnormalities driving their growth and survival. The existence of multiple abnormalities in one or more molecular pathways helps explain resistance to molecularly targeted therapy and provides a rationale for treatment strategies combining two or more targeted agents.3,4 However, cancer cells may become “addicted” or physiologically dependent on the sustained activity of specific oncogenes for maintenance of a malignant phenotype and for survival. This dependence mechanism, termed oncogene addiction^7–7 (Fig. 28-1), is associated with differential attenuation rates of prosurvival and proapoptotic signals stemming from the oncoprotein, with predominant apoptotic signals resulting in cell killing. The latter process, termed “oncogenic shock,”⁸ could explain the remarkably rapid clinical responses to TKIs in some patients with solid tumors, including those typically having complex molecular abnormalities. Other possible factors controlling sensitivity or resistance to molecularly targeted therapy include increased expression of the target due to gene amplification or transcription, emergence of resistant target gene mutations, and overexpression of multidrug transporter membrane proteins.^4,7

Figure 28-1 **Models of oncogene addiction. A,** The “genetic streamlining” theory postulates that nonessential pathways (*top, light grey*) are inactivated during tumor evolution, so that dominant, addictive pathways (*red*) are not surrogated by compensatory signals. Upon abrogation of dominant signals, a collapse in cellular fitness occurs and cells experience cell-cycle arrest or apoptosis (*bottom, red to gray shading*). B, In the “oncogenic shock” model, addictive oncoproteins (e.g., RTKs, *red triangle*) trigger at the same time prosurvival and proapoptotic signals (*top, red* and *blue* pathways, respectively). Under normal conditions, the prosurvival outputs dominate over the proapoptotic ones (*top*), but after blockade of the addictive receptor, the rapid decline in the activity of survival pathways (*dashed lines, bottom*) subverts this balance in favor of death-inducing signals, which tend to last longer and eventually lead to apoptotic death. C, Two genes are considered to be in a synthetic lethal relationship when loss of one or the other is still compatible with survival but loss of both is fatal. In the top panel, biochemical inactivation of pathway A (*gray*) has no effect on cell viability because pathway B (*red*), which converges at some point on a common substrate or effector (*yellow*), has compensating activity. When the integrity of pathway B is disrupted (*bottom*), the common downstream biochemical function is lost and again cancer cells may experience cell cycle arrest or apoptosis. (Reprinted with permission from Torti D, Trusolino L. Oncogene addiction as a foundational rationale for targeted anti-cancer therapy: promises and perils. EMBO Mol Med 2011;3(11):623–36.)

Interest in molecular therapy directed at factors controlling angiogenesis has increased since the United States Food and Drug Administration (FDA) approved several agents that target vascular endothelial growth factor (VEGF) and its receptor (VEGFR). The VEGF pathway, involving the VEGF family of proteins and their receptors, is an important regulator of both physiological and pathological angiogenesis. Through its signaling pathways, VEGF/VEGFR activation contributes to increased vascular permeability, mobilization of bone marrow–derived endothelial cell precursors, degradation of the extracellular matrix, and endothelial cell division, differentiation, migration, and survival.9,10 VEGF overexpression occurs in most types of cancers, including colorectal, renal, gastric, pancreatic, liver, lung, breast, thyroid, and genitourinary; it also occurs in glioma and other intracranial tumors, as well as in hematologic malignancies, and it is associated with tumor growth and a worse clinical outcome in a number of these tumor types.^10,11

Potential therapeutic strategies to inhibit signaling through VEGF and VEGFR pathway activation include monoclonal antibodies directed against VEGF or VEGFR, TKIs, and antisense strategies (antisense oligodeoxynucleotides, antisense RNA, and small interfering RNAs). In 2004, the FDA approved bevacizumab, a humanized murine monoclonal antibody directed against VEGF, for treating metastatic colorectal cancer in combination with fluorouracil-based chemotherapy. The FDA subsequently approved sunitinib, sorafenib, and axitinib, three small-molecule TKIs with activity against VEGFR, for the treatment of advanced renal cancer.¹¹ In addition, sorafenib is effective for treating hepatocellular carcinoma, a tumor against which standard cytotoxic chemotherapy has little or no activity.^11,12 The experience with these agents has established the VEGF/VEGFR pathway as a valid target for cancer therapeutics.¹¹

Mammalian target of rapamycin (mTOR) has also emerged as a validated target with the demonstration that the small-molecule mammalian target of rapamycin inhibitors temsirolimus and everolimus are effective in the treatment of renal cell carcinoma.¹³ This activity of temsirolimus is attributed to the downregulation of factors that control cell growth and angiogenesis.¹³

Preclinical Development of MTAs

The discovery and development of molecularly targeted therapies requires closely aligned laboratory and clinical research, integrating drug discovery, development, and clinical investigation. In such a cooperative setting, researchers can effectively take rational and iterative steps from target identification to clinical evaluation (Box 28-1) (Fig. 28-2). A crucial early step in developing a molecularly targeted therapy is target validation, defined as experimental evaluation of the role of a given gene or protein.^14,15 The target validation process involves a variety of preclinical approaches, including genetic, cell-based, and animal models.¹⁶ Validation and prioritization of molecular targets for therapeutic development depends on a variety of criteria, taking into consideration chemical, biological, clinical, and practical factors¹⁵ (Box 28-2). The fundamental goal is to provide evidence that the target is valid (i.e., that affecting the target inhibits tumor growth, progression, or survival) and that making drugs that hit the target is feasible. The next major step in the development of molecularly targeted therapy is finding/synthesizing compounds directed against that target.