Antiangiogenic Tyrosine Kinase Inhibitors and Their Diverse Spectra of Inhibitory Activity: Implications for Personalized Therapy in Renal Cell Carcinoma

Key Points

• Introduction of multiple VEGFR TKIs with slightly different inhibition profiles lends itself to increasingly personalized treatments
• Tumor heterogeneity may complicate this approach
• Multiple biopsies may better determine the overall mutational burden of an RCC tumor, but this strategy is hampered by practical limitations and cost of technology
• We are in a unique position to potentially derive therapeutic benefit for RCC using detailed molecular tumor profiling
  

Tumor angiogenesis is the biologic process by which tumors induce host production of functional blood vessels. Antiangiogenic therapy exploits a critical need for angiogenesis because, in its absence, solid tumors cannot grow or metastasize. Despite this, antiangiogenic monotherapy has provided little or no clinical benefit in many solid tumor types.

One notable exception is renal cell carcinoma (RCC) – a highly vascularized tumor. The unique vascularization of RCC allows easy escape from the kidney to establish metastases throughout the body and accounts for the high morbidity and mortality rates associated with this tumor type. This phenotype is driven by mutation of the von Hippel-Lindau (VHL) tumor suppressor gene that is present in the clear cell histological subtype, which constitutes approximately 85% of RCCs.^1,2 VHL inactivates hypoxia-inducible factor – a transcription factor that, when induced, upregulates potent proangiogenesis genes, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF).^3,4 Hypoxia-inducible factor is activated in response to hypoxia, ultimately triggering vascular development and elevating hemoglobin levels and blood flow. Thus, when mutated, VHL loses its counterbalancing function, and hypoxia-inducible factor activity leads to polycythemia and the vascularized RCC phenotype.

Multiple pathways contribute to angiogenesis in vascularized tumors, either by directly stimulating vascular endothelial cell (EC) function or indirectly by recruiting/stimulating cells that support angiogenesis. For example, VEGF and angiopoietin-Tie (Ang-Tie) signaling directly stimulates ECs, whereas PDGF and fibroblast growth factor (FGF) pathways predominantly affect the pericytes and stromal components, respectively, to induce angiogenesis.

Because of the importance of angiogenesis in RCC, multiple antiangiogenic tyrosine kinase inhibitors (TKIs) are approved for treatment or are in clinical development. Although VEGF inhibition is the key contributor to the clinical activity of these agents, TKIs also target additional angiogenic pathways.

Proangiogenic Signaling Pathways With Importance in Tumor Angiogenesis
VEGF is one of the most potent angiogenic pathways and consists of 6 ligands (VEGFA, VEGFB, VEGFC, VEGFD, VEGFE, and placental growth factor) and 3 receptors (VEGFR-1, VEGFR-2, and VEGFR-3).⁵ Of these, VEGFA and VEGFR-2 appear to have the most dominant roles in tumor angiogenesis. VEGFRs are expressed on the surface of ECs and promote downstream intracellular signaling through the mitogen-activated protein kinase (MAPK) and phosphoinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathways when activated.⁶ This signaling ultimately promotes vascular permeability, EC migration, and cell growth and survival.7 Development of agents targeting VEGF signaling represented a major breakthrough in antiangiogenic therapy, and this pathway remains the primary focus of antiangiogenic research (Figure 1).

Figure 1

The Ang-Tie pathway is critical for normal angiogenic processes in embryos and adults and also contributes to tumor angiogenesis. Ang-Tie signaling involves 4 angiopoietin growth factors (Ang1, Ang2, Ang3, and Ang4) and their receptors (Tie1 and Tie2).⁸ Ang1 and Ang2 both bind to Tie2 but mediate different effects. Ang1, which is mainly expressed by perivascular cells, seems to primarily induce vascular maturation and homeostasis, whereas Ang2, which is expressed with Tie2 primarily by ECs, appears to mediate vascular destabilization and vessel sprouting. In contrast to the VEGF pathway, which mainly operates early in vessel development, Ang-Tie signaling functions in late vessel maturation and stabilization.⁸ Its functions in tumor vasculature, however, are incompletely understood (Figure 1).

The FGF pathway has a larger biological footprint: 18 to 23 ligands and 4 receptors (FGFRs 1-4) that can undergo alternative splicing to generate multiple isoforms.9 FGF signaling modulates bone growth, wound healing, and nerve regeneration, and its role in angiogenesis and tumor growth is well established (Figure 1). MAPK, PI3K, and protein kinase C are the primary transducers of FGF signaling and modulate downstream gene expression. ECs express high levels of FGFRs 1 and 2, which are activated primarily by FGFs 1 and 2, resulting in EC proliferation and migration, extracellular matrix degradation, alteration of intercellular adhesion, and communication. Unlike VEGFR, which is fairly restricted to the endothelial compartment, FGFRs are also found on stromal cells and can participate in remodeling of the host microenvironment.

PDGF initially achieved prominence for its role in remodeling the vascular wall during atherosclerosis. PDGF signaling consists of 5 isoform ligands (PDGFA, PDGFB, PDGFC, PDGFD, and PDGF­­AB) and 2 receptors (PDGFR-? and -?) and plays an indirect role in angiogenesis by recruiting pericytes to vascular sprouts.^10,11 Pericytes, which constitute an encircling layer of smooth muscle around developing vasculature, establish and maintain the integrity of vascular function by modulating ECs (Figure 1).

Although these and other pathways uniquely act to promote angiogenesis, they may also act synergistically. For instance, a positive feedback mechanism appears to exist between FGF2 and VEGF.^12,13 Therefore, alternative pathways can potentially “rescue” tumor cells that have had their dominant angiogenesis pathway blocked by a TKI. Because of the emerging importance of these pathways in resistance to RCC therapy, this review highlights the diversity of pathways inhibited by these TKIs, with particular focus on non-VEGF “angiogenic” pathways. Although these agents primarily function through VEGF inhibition, their targeting of multiple kinases may provide an opportunity to simultaneously inhibit multiple pathways. This diversity was traditionally considered a negative “off-target” effect; however, we present the case that this diversity provides an opportunity to personalize the care of patients with RCC by targeting additional pathways based on the molecular characteristics of individual tumors. Whereas the RCC literature to date has been appropriately dominated by a VEGF-centric approach, this article discusses the potential to maximize response to primary therapy or, once resistance develops, to salvage treatment failure.

Targeting Proangiogenic Pathways
A number of antiangiogenic therapies are approved or are under investigation for various tumor types. Many strategies to block angiogenesis have been explored, including VEGF pathway inhibitors that directly bind to and inactivate circulating VEGF or that inhibit VEGFR signaling. Another strategy inhibits the ability of mTOR, a master molecular switch, to downregulate the synthesis of several angiogenic factors. These approaches have been extensively studied in advanced RCC and are now considered the standard of care for this cancer. Although it is thought that these agents mainly act through antiangiogenic avenues, they may also directly inhibit tumor cell proliferation.¹⁴

Seven agents are currently approved by the FDA to treat advanced RCC.15 Four of these agents – sunitinib, sorafenib, pazopanib, and axitinib – are TKIs, for which the primary clinical mechanism of action is assumed to be VEGFR inhibition (Table). The anti-VEGF monoclonal antibody bevacizumab is approved in combination with interferon. Everolimus and temsirolimus are mTOR inhibitors. Additional agents are under investigation, most notably a number of TKIs. The remainder of this review will focus on approved and emerging TKI therapies for advanced RCC. Detailed reviews for other targeted agents in RCC were published previously.^16-18

Table

Approved TKI-Targeted Treatments for RCC
Generally, TKIs bind reversibly to or adjacent to the ATP-binding site within its target, thereby inhibiting kinase activity through steric hindrance. Given that these domains are structurally similar from one kinase to another, and because inhibition is partly related to the 3-dimensional shape of the TKI, it is not unexpected that these agents may also target conserved ATP-binding sites in other kinases (Figure 2). In contrast, allosteric inhibitors, which bind to fairly unique sites outside of the kinase domain, tend to be target specific.¹⁹ Therefore, TKIs may simultaneously block kinases in multiple angiogenic pathways to delay or circumvent acquired resistance to therapy.

Figure 2

Sunitinib inhibits VEGFRs 1-3, PDGFR-? and -?, colony-stimulating factor 1 receptor, stem cell factor receptor (c-KIT), FMS-like tyrosine kinase 3 receptor (FLT3), and neurotrophic factor receptor.20 Sunitinib was evaluated in a phase 3 trial with 750 treatment-naive patients randomized to either sunitinib or interferon-? therapy.²¹ Compared with interferon-?, sunitinib demonstrated superior progression-free survival (PFS) (11 vs 5 months), overall survival (OS) (26.4 vs 21.8 months), and quality of life.^21,22 Sunitinib has also demonstrated antitumor activity in patients with RCC who progressed after cytokine therapy.²³ Sunitinib is approved and recommended for first-line use and second-line use after cytokine therapy in advanced RCC.¹⁵ Sunitinib strongly inhibits c-KIT, an oncogene involved in gastrointestinal stromal tumors (GISTs), and is also approved as GIST therapy.

Sorafenib inhibits VEGFRs 1-3, PDGFR-?, c-KIT, FLT3, and RAF – an MAPK pathway intermediate.24 A phase 3 trial compared second-line sorafenib with placebo in 903 patients.²⁵ An interim PFS analysis demonstrated the superiority of sorafenib over placebo (5.5 vs 2.8 months); thus, the protocol was amended to permit placebo-assigned patients to cross over to sorafenib. OS with sorafenib was similar to that of placebo (17.8 vs 15.2 months, respectively); however, a secondary analysis censoring crossover patients revealed an OS benefit favoring sorafenib (17.8 vs 14.3 months).²⁶ Sorafenib was also active in the third-line setting after first-line sunitinib and second-line temsirolimus or everolimus.²⁷ In the first-line setting, a phase 2 trial demonstrated greater tumor shrinkage and a higher quality of life with sorafenib versus interferon-?. However, median PFS was similar between arms (5.7 months for sorafenib, 5.6 months for interferon-?).²⁸ Sorafenib therapy is approved and recommended for use as second-line treatment after progression on cytokine or TKI therapy in advanced RCC15 and is also approved for use in unresectable hepatocellular carcinoma (HCC). Importantly, in HCC, the clinical activity of sorafenib may result from both antiangiogenic activity and inhibition of RAF-dependent and -independent pathways.²⁹

Pazopanib inhibits VEGFRs 1-3, PDGFR-? and -?, and c-KIT.30 A phase 3 trial compared the efficacy of pazopanib with that of placebo in treatment-naive (n=233) and cytokine-pretreated (n=202) patients with advanced RCC.31 Pazopanib demonstrated superior PFS over placebo in the overall population (9.2 vs 4.2 months), the treatment-naive population (11.1 vs 2.8 months), and the cytokine-pretreated population (7.4 vs 4.2 months). Although OS was similar in both arms as a result of patient crossover, analyses to censor for crossover suggested an OS benefit with pazopanib.³² Pazopanib is approved in advanced RCC and is recommended in the first-line setting and in the second-line setting after cytokine therapy.¹⁵ Pazopanib is also approved for patients with advanced soft tissue sarcoma after chemotherapy.³³

Axitinib inhibits VEGFRs 1-3, with one of the highest in-class potencies, and also inhibits PDGFR-? and -? and c-KIT.³⁴ For these reasons, the use of axitinib as a salvage TKI was thought to be a promising strategy. A phase 3 trial evaluated second-line axitinib versus sorafenib in 723 patients with RCC.³⁵ Compared with sorafenib, axitinib significantly prolonged median PFS (6.7 vs 4.7 months) and was associated with a greater delay in time to symptom worsening. These results suggest that a more potent inhibition of VEGFR may induce a more robust clinical outcome in the salvage setting. Another phase 3 study compared axitinib with sorafenib in 288 treatment-naive patients and showed a nonsignificant increase in PFS for axitinib (10.1 vs 6.5 months).³⁶ Axitinib is currently approved for advanced RCC after the failure of a prior systemic therapy.

In summary, a detailed analysis of these agents showed a cross-section of inhibition involving not only different classes of angiogenesis-related receptors but different classes of kinases in general (Figure 2); individual agents differed in their avidity toward target kinases.³⁷ This diverse spectrum of activity most likely accounts for the differences in efficacy against different cancers, independent of antiangiogenic effects. This focus on target diversity carries through to the agents under discussion below.

TKIs Under Investigation in RCC
Dovitinib (TKI258) inhibits VEGFRs 1-3, PDGFR-?, colony-stimulating factor 1 receptor, c-KIT, FLT3, and FGFRs 1 and 3.38 A phase 1/2 trial investigated dovitinib in patients with advanced RCC or metastatic RCC (mRCC) previously treated with a VEGFR (sunitinib and/or sorafenib) and/or an mTOR inhibitor.^39,40 This trial is particularly interesting because it tested a major working hypothesis regarding resistance to antiangiogenic therapy, ie, that alternative angiogenic pathways such as FGF underlie tumor escape from anti-VEGF therapy. Indeed, baseline basic plasma FGF levels were elevated in patients who previously received anti-VEGF therapies. Preliminary analysis of 59 patients revealed the antitumor efficacy of dovitinib; 3.4% achieved a partial response (PR), and 49.2% and 27.1% achieved stable disease lasting ?2 and ?4 months, respectively.40 Preliminary median PFS and OS of 5.45 and 11.79 months, respectively, were reported. A phase 3 trial comparing dovitinib with sorafenib after the failure of antiangiogenic therapy is under way.⁴¹ Ongoing trials are studying dovitinib for HCC, endometrial cancer, adenoid cystic carcinoma, GIST, glioblastoma, non–small cell lung cancer (NSCLC), breast cancer, gastric cancer, and prostate cancer.⁴² The activity of dovitinib in tumors that are not strong candidates for anti-VEGF therapies but instead rely on FGF signaling may be interesting.

Tivozanib (AV-951) is a potent inhibitor of VEGFRs 1-3 and also targets c-KIT and PDGFR-?.⁴³ In a development plan mimicking the potent inhibitor axitinib, a phase 3 trial compared tivozanib versus sorafenib in treatment-naive and pretreated patients with advanced RCC. Tivozanib significantly improved median PFS over sorafenib (11.9 vs 9.1 months).⁴⁴ A phase 2 discontinuation study in 272 patients with advanced RCC or mRCC also demonstrated the activity of second-line tivozanib, yielding 1 complete response (CR), and 51 PRs throughout the study. Median PFS was 11.7 months.⁴⁵ Tivozanib is also under investigation in NSCLC, metastatic breast cancer, colorectal cancer, and soft tissue sarcoma.⁴²

Nintedanib (BIBF 1120) inhibits VEGFRs 1-3, PDGFR-? and -?, and FGFRs 1-3.⁴⁶ A phase 1 study demonstrated antitumor activity; 1 CR and 1 PR were observed in 10 patients with advanced RCC.⁴⁷ Seven patients with RCC remained on therapy for ?5 months; 2 remained on therapy for >1 year. A phase 2 trial comparing nintedanib versus sunitinib in first-line RCC is under way (NCT01024920).⁴² Nintedanib is also under investigation in HCC, glioblastoma, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, and endometrial cancer. Phase 3 studies are ongoing in NSCLC and ovarian cancer.⁴²

Vandetanib (ZD6474) is an inhibitor of VEGFRs 2 and 3, endothelial growth factor receptor (EGFR), and neurotrophic factor receptor; it was recently approved for the treatment of medullary thyroid cancer and has also demonstrated antitumor activity in a murine RCC model.^48,49 Vandetanib is under investigation in a phase 2 trial of patients with VHL-related kidney cancer (NCT00566995).42 Clinical trials are ongoing in several tumor types, including glioblastoma, breast cancer, head and neck cancer, and pancreatic cancer.⁴²

The Diversity of Pathways Targeted by Individual TKIs May Influence Patient Care
The clinical benefit currently gained from anti-angiogenic TKIs is varied. Some patients appear to be intrinsically resistant to these agents and gain no objective benefit from treatment. In contrast, impressive response rates were observed in clear cell RCC, which is dependent on the VEGF pathway. However, the clinical benefit is transient, and progression typically occurs after an initial response.⁵⁰

The emerging TKIs described herein hit novel targets in different combinations and may have improved utility in the clinic because their inhibitory spectra expand beyond targeting angiogenic receptors to also inhibit intracellular protumor signaling. In other tumor types, the non-VEGF inhibitory activities of TKIs are important and appear to contribute to clinical benefit. For instance, patients with specific GIST genotypes have shown improved outcomes with sunitinib treatment. This highlights the utility of sunitinib in also attacking the “off-target” c-KIT pathway.⁵¹ Patients with HCC whose tumors expressed high levels of activated extracellular signal-regulated kinase, an MAPK intermediate, achieved a longer time to tumor progression with sorafenib than did patients with low levels of activated extracellular signal-regulated kinase, which suggests an improved response to sorafenib when it can inhibit the MAPK pathway.⁵²

In addition, the diversity of TKI targeting is notably important within nonvascular compartments of malignant tumors. Tumor cells do not readily express VEGFRs but do rely on growth pathway signaling. One working model predicts that mTOR inhibitors function both in tumor and vascular ECs. Thus, TKIs that inhibit multiple pathways might target both cell types.

The hypothesis that the diversity of tyrosine kinase inhibition affects clinical benefit may be applied to RCC and suggests that the efficacy of an agent in RCC may not depend solely on its specificity for the VEGF pathway. Comparison of a “strong” VEGFR inhibitor (eg, axitinib) with a “weak” one (eg, sorafenib), as was done in the AGILE 1051 study, would provide insight about this hypothesis. In this randomized phase 3 trial, 280 treatment-naive patients with advanced RCC were assigned to either sorafenib or axitinib. Recently reported results show that the primary end point, PFS, was not significantly different between these therapies.36 These findings suggest that factors other than the potency of VEGF inhibition contributed to clinical efficacy in this setting.

The use of a TKI with the capability to inhibit “off-target” proto-oncogenic pathways may also be advantageous in settings of acquired resistance. Resistance to anti-VEGF therapies is not likely due to the accumulation of mutations in the EC VEGF pathway, because ECs are host-derived, end-differentiated, nonclonal cells that possess neither genomic instability nor the promiscuous gene expression inherent in tumor cells. Instead, adaptive resistance likely occurs through other means, including activation of alternative proangiogenic pathways within tumor cells. Targeting alternative pathways may help delay acquired resistance or treat resistant disease. For example, a retrospective evaluation of 216 patients with mRCC who progressed on first-line anti-VEGF therapy demonstrated that some patients still obtained benefit after receiving a second-line VEGF TKI.⁵³ This suggests that the non-VEGF inhibitory activities of TKIs are important and may contribute to the benefit observed. Moreover, resistance to one TKI may not imply cross-resistance to another TKI with a different “off-target” profile. Likewise, the concept of switching TKI therapies can extend even further into the treatment time line. For instance, third-line sorafenib has demonstrated activity after first-line sunitinib and second-line temsirolimus or everolimus.²⁷ Unfortunately, limited clinical trial data exist concerning the effective sequencing of TKIs.¹⁵ Thus, sequencing decisions are driven by anecdotes and user experience, putting a heavier onus on basic and translational science data to drive the field.

To maximize the targeting of multiple pathways and gain further clinical utility, one rational strategy involves combining a TKI with another biological inhibitor or combining TKIs with complementary inhibition profiles. In support of this idea, cediranib – a VEGF TKI that is not currently under investigation in RCC – in combination with the EGFR inhibitor gefitinib yielded PRs in 6 of 18 patients with RCC in a phase 1 trial.⁵⁴ It is important to note, however, that toxicity may limit these combination approaches.^55,56

Another therapeutic strategy combines TKIs with other treatment modalities. Although bevacizumab has been added to chemotherapy without increasing toxicity,^57,58 combining TKIs with chemotherapy may be more difficult and may require extensive clinical evaluation to determine optimal dose levels and schedules. Notably, several clinical trials exploring these regimens have been discontinued.⁴² Combining TKIs with surgical resection may also provide additional benefit. However, this approach has raised concern, because VEGF inhibition could negatively affect perioperative wound healing. Nonetheless, recent work has demonstrated that surgery after VEGF-directed therapies can be safe if an appropriate amount of time passes between treatment and surgical procedures.⁵⁹ Furthermore, this combination may provide substantial benefit – a subgroup of patients from the phase 2 trial of tivozanib in RCC detailed above, who had undergone nephrectomy, had the highest median PFS and overall response rate (ORR).⁴⁵ Additional results may be obtained from an ongoing phase 3 trial of adjuvant axitinib versus placebo in high-risk patients with RCC (NCT01599754).⁴²

A final approach for consideration combines immunotherapy with TKIs. Larger tumors are exquisitely dependent on blood supply and are sensitive to antiangiogenic therapy, whereas small tumors appear to survive by direct diffusion and are insensitive to antiangiogenic therapy. The opposite is true of immunotherapy, for which the antigen-excess and other immune-suppressive factors generated by large tumors result in immune tolerance, whereas smaller tumors may be more immune sensitive. Thus, TKI-induced debulking followed by immunotherapy may be advantageous and, indeed, has demonstrated notable preclinical antitumor activity.^60,61 Furthermore, recent work in melanoma showed that a BRAF TKI enhanced antigen presentation and may increase immunoresponsiveness.⁶² Despite promising preclinical results, translating these strategies into the clinic may prove difficult. For example, severe cardiovascular toxicity was observed in patients with RCC who received high-dose interleukin-2 immunotherapy shortly after VEGF TKIs.⁶³

The introduction of multiple VEGFR TKIs with slightly different inhibition profiles into the RCC pharmacopeia lends itself to a near future in which treatments will become increasingly personalized. However, tumor heterogeneity may complicate this approach. A recent study using RCC as a model showed that genomic analyses from single biopsies grossly underestimated the mutational burden of RCC tumors.⁶⁴ In biopsies of multiple areas within the same tumor and of metastatic sites, up to 69% of somatic mutations were not detected across all samples. Furthermore, 26 of 30 tumor samples from 4 tumors harbored divergent allelic imbalance profiles, indicating extensive intratumor heterogeneity. Multiple biopsies may better determine the overall mutational burden of an RCC tumor, but this strategy is currently hampered by practical limitations in the delivery and cost of technology. Despite these drawbacks, VEGF TKIs may be a ready-made solution to the problem of tumor heterogeneity. As the understanding of RCC mutational burden matures, TKIs with primary activity against VEGF or mTOR may be selected for treatment on the basis of their secondary inhibition of other kinase targets.

In conclusion, the plethora of drugs in the RCC pharmacopeia presents a unique situation in which agents are closely related through their dominant mechanism of action but have diverse “off-target” effects that may be useful in clinical targeting. Therefore, we are in a unique position to potentially derive therapeutic benefit for RCC using detailed molecular tumor profiling.

Acknowledgments
We thank Michelle Boehm, PhD, and Melanie Vishnu, PhD, for their medical editorial assistance with this manuscript.

Disclosure Statement
Financial support for medical editorial assistance was provided by Novartis Pharmaceuticals. MKKW received honoraria from Novartis Pharmaceuticals, Merck Pharmaceuticals, and Pfizer Pharmaceuticals. SSA has no conflicts to report.

Dr Agarwala is Professor of Medicine at Temple University School of Medicine in Philadelphia and Chief of Oncology & Hematology at St. Luke’s Cancer Center in Bethlehem, Pennsylvania. He received his degree and completed an internship and residency at the G.S. Medical College and King Edward Memorial Hospital in Bombay, India. He also completed residency training in internal medicine and a fellowship in hematology-oncology at the University of Pittsburgh School of Medicine. He is a nationally recognized expert in the treatment of metastatic melanoma.

Dr Wong is Professor of Medicine, Head of the Solid Tumors Section, and leads the Melanoma Program at USC Norris Comprehensive Cancer Center and Hospital in Los Angeles, California. He has a PhD in experimental pathology and was a National Cancer Institute of Canada Postdoctoral Scholar in the molecular biology of angiogenic cytokines. His research has focused on angiogenesis, immunomodulation, and tumor-matrix interactions.

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