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Implications of VHL-HIF Pathway Dysregulation
in Renal Cell Carcinoma: Current Therapeutic
Strategies and Challenges

 

 

Eric Jonasch, MD

Professor, Department of
    Genitourinary Medical Oncology

Director, The Von Hippel Lindau
    Clinical Center

The University of Texas
    MD Anderson Cancer Center

Houston, TX

 

 

 

 

Keywords: renal cell carcinoma, VHL-HIF pathway, loss of VHL, HIFs dysregulation, Mutational landscape of RCC, William Kaelin's discovery, therapeutic targets

 

Corresponding Author: Eric Jonasch, MD, Department of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.  Email: ejonasch@mdanderson.orgu

 

 

Introduction

Kidney cancer is among the 10 most common cancers in both men and women, leading to approximately 74,000 new cases and to more than 14,000 deaths annually in United States alone.1 Early stage, localized renal cell carcinoma (RCC) has a significant cure fraction and a survival rate of 92%, whereas the treatment of late stage recurrent metastatic RCC remains highly challenging, with a minority of patients with metastatic RCC surviving past 5 years.2 Given that RCC is chemo-resistant and radiation-resistant, novel targeted therapies were required for the prevention and management of advanced and/or metastatic RCC.

Studies found that the majority of localized and advanced clear cell RCCs (ccRCCs) are characterized by mutational inactivation and allelic loss of the von Hippel-Lindau (VHL) tumor-suppressor gene.3 The groundbreaking discoveries made by William G. Kaelin Jr., Sir Peter J. Ratcliffe and Gregg L. Semenza on the involvement of the VHL gene in various fundamental processes, including but not limited to sensing and adapting to the changing oxygen environment eventually led to the Nobel prize in physiology and medicine in 2019. These key insights not only paved the way for our understanding of a key factor in ccRCC tumorigenesis, but also provided the basis for the development of VHL-hypoxia pathway-targeted therapies that includes tyrosine kinase inhibitors (TKIs) for treatment of RCC and other diseases.

In this review, we outline key aspects of VHL-hypoxia inducible factor (HIF) pathway and their impact on tumorigenesis in VHL disease and sporadic ccRCC. We then explore the current status and future challenges for the RCC treatment landscape in the context of VHL loss and other biological factors.

 

Implications of VHL Loss in
VHL Disease and Sporadic ccRCC

von Hippel-Lindau disease is a rare autosomal dominant hereditary neoplastic disorder triggered by germline mutations in the VHL tumor-suppressor gene with an incidence of roughly 1 in 36,000 births.4 Individuals with VHL disease are at increased risk of recurrent and bilateral kidney cysts and ccRCC, as well as retinal, cerebellar and spinal hemangioblastomas, pheochromocytomas, pancreatic cysts, serous cystadenomas and neuroendocrine tumors, endolymphatic sac tumors and epidymal and round ligament cysts.5  The discovery of the VHL gene in 19936 was driven by a desire to understand and treat VHL disease.  The impact of this seminal discovery on our understanding of disease manifestations in patients with VHL disease and on individuals with sporadic ccRCC cannot be overstated.  We now know that the majority of sporadic ccRCC cases also exhibit somatic loss-of-function mutations in the VHL gene,3 loss of 3p chromosome, or hypermethylation of the VHL locus.7,8

The mechanistic understanding of VHL protein (pVHL) function, driven by Kaelin’s group and others formed the cornerstone of our current understanding of ccRCC biology. Through additional work performed by a number of investigators and organizations including The Cancer Genome Atlas (TCGA), we now know VHL loss serves as the initiating truncal event for ccRCC tumorigenesis, eventually followed by additional mutational and chromosomal copy number altering changes that foster tumor growth and lethality.8-11

 

The VHL- HIF Pathway

Bill Kaelin and colleagues were instrumental in characterizing the VHL gene and its function. In 1995, Iliopoulos, Kibel, Gray and Kaelin showed that the reintroduction of a wild-type but not a mutant VHL cDNA into the 786-0 VHL(-/-) RCC cell line abrogated its ability to form tumors in nude mouse xenograft assays, reinforcing the concept that VHL is a bona fide tumor suppressor gene.12 In the same year, the Kaelin group showed that pVHL interacts with with elongins C and B to form the VBC complex.13  In 1996, Iliopoulos et al demonstrated that pVHL was involved in negatively regulating hypoxia-inducible genes.14  Over the next few years, further refinement of the VBC complex,15 and the solution of the crystal structure of the VBC complex, led to a broader understanding of pVHL function.

The next major step was the identification of HIF as the substrate for the VBC complex.  In 1991, Greg Semenza reported that HIF bound to enhancers near the human erythropoietin gene.16 Over the following decade Dr. Semenza and his colleagues further characterized HIF function, demonstrating its dimerization, DNA binding, and transactivation properties.17 In 1996 Jiang et al showed that vascular endothelial growth factor was HIF regulated.18

The third piece in the overall puzzle was the mechanism of oxygen sensing, elegantly discovered by Peter Ratcliffe and colleagues. Dr. Ratcliffe’s lab had been working on elucidating the key factors in erythropoietin gene activation since the early 1990s.19  In 1999, Max-well et al reported that pVHL was required to degrade HIF in an oxygen and iron-dependent manner,20 and in 2001 Jaakola et al reported this interaction was prolyl hydroxylation dependent.21

Further modeling showed that overexpression of a VHL- binding defective HIF2a variant was sufficient for tumorigenesis in a mouse model, suggesting that HIF overexpression is one of the major drivers of the malignant phenotype.  A review of the myriad functions of HIF1a and HIF2a show that each HIF isoform has both unique and over- lapping target genes, including angiogenesis, metabolism and glycolysis22 (Figure 1).

 

Figure 1. Mechanisms of VHL-mediated regulation of HIFα under normoxic, hypoxic and dysregulated VHL settings. Under normoxia, HIFα is hydroxylated by prolyl-4-hydroxylases enzymes (PHDs) via an oxygen-dependent enzymatic mechanism. The von Hippel Lindau (VHL) protein-E3 ligase complex recognizes HIFα and eventually HIFα are polyubiquitylated and degraded by the proteasome. Under hypoxia, prolyl hydroxylation of HIFα is impaired. As a consequence, HIFα accumulates in the cytoplasm, also forming complex with constitutionally expressed HIFβ. Similarly, in the presence of a mutated pVHL or in the absence of functional pVHL, HIF1α and HIF2α escape from such degradation, leading to stabilization of HIFα. Accumulated HIFα then dimerize with HIFβ to generate transcriptionally active HIF complex in cytoplasm. This complex eventually translocates into the nucleus where it binds to HIF response element (HRE) to initiate the repertoire of hypoxia-induced genes eg: of VEGF-A and PDGF like growth factors related to adaptation to a low oxygen environment. The resulting transcriptional stimulation of the HRE and downstream overexpression of numerous genes involved in angiogenesis (eg. VEGF), proliferation (eg. EGFR), cell migration and invasion (eg. CXCR4), erythropoiesis (eg. EPO), which ultimately facilitate ccRCC tumorigenesis.

Abbreviations: PI3K, phosphoinositide 3-kinase; HIFα, Hypoxia inducible factor alpha; Ub, Ubiquitin; VEGF, vascular endothelial growth factors; VEGFR, vascular endothelial growth factor receptor; PDGF, platelet derived growth factor; PDGFR, platelet derived growth factor receptor; CXCR4, C-X-C Motif Chemokine Receptor 4.

 

Putting all of these elements together, today we know that pVHL recognizes prolyl hydroxylated HIFa subunits in an oxygen dependent manner. Prolylhydroxylated HIF1a and HIF2a associate with the VBC complex, consisting of pVHL, elongin B, elongin C, cullin 2, and Rbx1.23 HIFa subunits are polyubiquitylated and degraded by the proteasome, thereby tightly regulating cytoplasmic HIFa protein levels. Conversely, hypoxic conditions impair the hydroxylation of HIFa and its subsequent degradation, leading to accumulation of HIFa, heterodimerization with ARNT (HIF1b) and translocation to the nucleus to enable transcription of HIFa dependent genes. Similarly, in the presence of a mutated pVHL or in the absence of any pVHL expression, HIF1a and HIF2a are not degraded. Interestingly, HIF1a and HIF2a were found to exhibit contrasting roles in ccRCC xenograft mice models. HIF2a reduction diminished tumor formation, whereas restoration of HIF2a level resulted in a more pronounced tumor burden.24,25  Conversely,  HIF1a expression was associated with decreased xenograft tumor growth in mice models,26 and  knockdown of HIF1a enhanced cell proliferation and tumor burden in animal model.24,27 These studies demonstrate that HIF1a behaves as a tumor suppressor in RCC and HIF2a acts as an oncogenic driver. Taken together, HIF2a is predominantly implicated in the pathogenesis of VHL-associated vascular tumors and pharmacologic blockade of HIF2a may be an attractive therapeutic strategy for RCC treatment.

 

Mutational Landscape of RCC

Intriguingly, biallelic loss of VHL is not sufficient to generate tumors in model systems and additional genetic events are required to predispose VHL deficient cells to develop into ccRCC.28 Studies using mouse embryo fibroblast cells or nonmalignant human tubular cells have shown that loss of VHL induces senescence.  This finding suggests  that additional events are needed for the malignant transformation of VHL-mutant proximal tubular cells.29 This concept is supported by the observation that in addition to deletions in VHL, ccRCCs harbor mutations in a number of chromatin remodeling genes found on chromosome 3p, including Polybromo-1 (PBRM1)30, SETD231, and BAP1.32 Additionally, loss of 9p and 14q chromosomal regions is associated with increased probably of tumor lethality.9   How these mutations and copy number changes impact ccRCC biology and subsequent response to therapy is an area of active research.

 

VHL-HIF Axis Based Targeted
Therapies for ccRCC

The development of agents targeting the consequences of VHL loss shifted the treatment landscape from cytokine based immunotherapeutics, such as IFNα and IL-2 towards targeted therapeutics fifteen years ago.33,34 Given that ccRCC are highly vascular tumors with overexpression of angiogenic vascular endothelial growth factor (VEGF) which is a downstream target of HIF, currently approved therapies include inhibitors of VEGF35,36 and VEGFR tyrosine kinases (TKIs).33,34,37-41 Patients with VHL disease also demonstrated some benefit from these agents, with a 33% objective response rate (ORR) in ccRCC after sunitinib treatment42 and a 51% ORR in ccRCC after pazopanib treatment.43 The key challenge with all of these agents is that there is significant on and off target toxicity, and a near inevitable failure to cure or ultimately control tumor growth.  There is no clear explanation for these findings, but there is undoubtedly room for a further refinement of VHL-HIF axis blocking agents.

There is a cogent mechanistic rationale for targeting the VHL-HIF pathway proximally to inhibit as many downstream branchpoints as possible. In preclinical models, inhibition of HIF2a appeared to be both necessary and sufficient to suppress ectopic blood vessel formation and decrease tumor growth.44  Targeting HIF2a is a very attractive but potentially daunting goal. Transcription factors are notoriously hard to develop small molecule inhibitors against due to their tight conformation, and the HIF isoforms were initially regarded as undruggable.  Nonetheless, a series of small molecule inhibitors were recently developed against HIF-2a. PT2399, a preclinical research compound, induced tumor regression in a VHL-defective ccRCC preclinical model.45 PT-2399 displayed on-target antitumor activity against a significant percentage of VHL-mutated or deficient ccRCC lines and patient-derived xenografts.45 PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumors, and was better tolerated45 (Figure 2).

 

Figure 2. Mechanism of action of VHL-HIF axis based targeted therapies interfering critical molecular pathways in ccRCC. Pharmacological agents inhibit a variety of therapeutic targets of signaling cascades in both ccRCC and endothelial/stromal cells. The signal pathways in ccRCC can be interfered/blocked in different ways, including but not limited to (i) inhibition of VEGF (by bevacizumab); (ii) inhibition of tyrosine kinase activity of RTKs (by sunitinib, pazopanib, and sorafenib); and (iii) inhibition of mTOR (by temsirolimus and everolimus); (iv) blocking MET/AXL/VEGFR2 by a TKI – (by cabozantinib).

Abbreviations: vascular endothelial growth factor (VEGFR); mTOR, mammalian Target of Rapamycin, MAPK, mitogen-activated protein kinase; TKI, tyrosine kinase inhibitor. PI3K, phosphatidylinositol 3-kinase, AKT, protein kinase B; RTKs, receptor tyrosine kinases.

 

The first-in-class clinical HIF-2aα inhibitor PT-2385 caused dramatic tumor regressions in patient-derived xenografts.46 Clinical data from PT-2385 in pretreated patients with metastatic clear cell renal carcinoma  (mRCC) were encouraging in a Phase I, dose-escalation trial, and demonstrated a favorable safety profile.47  PT2977/MK-6482 is the second generation of the HIF2 inhibitor and was tested in a 55 patient phase Ib-II study.48  This study, which was presented at the European Society of Medical Oncology meeting in the fall of 2019, described 55 patients with advanced cc RCC who had received at least one prior therapy and who were treated with 120 mg orally once daily dose of PT2977/MK6482. We found that PT2977/MK6482 was well tolerated and had a favorable safety profile. The most common Grade 3 adverse events and on-target effects of HIF2α inhibition were found to be anemia in 26% of patients and hypoxia in 15%, and only 2 patients experienced grade 4 toxicities. Despite having a study population treated with a median of three prior therapies, the ORR was 24%, the median progression-free survival (PFS) was an impressive 11 months (95% CI 6-17), and the 12-month PFS rate was 49%. PT2977/MK6482 is currently being tested in a randomized phase III study in patients with treatment refractory metastatic ccRCC (NCT04195750).

P2977/MK6482 is also being tested in patients with VHL disease (NCT03401788).  This study has completed accrual, and the data are maturing.  As there is currently no Food and Drug Administration approved therapy for VHL disease, we are anxiously awaiting the outcome of this trial to see if there is a potential registrational path for this agent in the treatment of VHL disease.

Recently, the approval of combination TKI- checkpoint blocking antibody therapy has resulted in a new treatment paradigm for many patients with ccRCC. 49,50 Tissue based studies suggest antiangiogenic agents are capable of increasing T-cell recruitment to the tumor microenvironment,51 providing a mechanistic rationale for this type of combination therapy.  Further investigations into the way VHL-HIF targeting agents can synergize with checkpoint blocking antibodies will undoubtedly further improve the treatment of patients with RCC.

 

Concluding Remarks

The seminal work by Drs. Semenza, Ratcliffe and Kaelin have fundamentally changed our understanding of ccRCC biology and ushered in a completely new treatment paradigm for this disease and others.  Elucidating the functional consequences of VHL loss has not only shed light on how cells sense and adapt to a hypoxic environment but has also paved the way for the development of a new class of target based therapeutic strategies to treat ccRCC. Although therapeutic agents targeting VEGF and VEGF receptors have demonstrated robust efficacy in clinical trials, few people have been cured. We await further development of HIF2 targeting agents to see whether they can move the treatment of ccRCC to the next level, either as monotherapy or in combination with other novel therapeutics.  It is imperative we continue to strive for a better understanding of how these agents impact tumor biology and the surrounding microenvironment to allow us to develop even better treatments for patients with RCC.

 

References

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5. Ho TH, Jonasch E. Genetic kidney cancer syndromes. Journal of the National Comprehensive Cancer Network : JNCCN. 2014;12(9):1347-1355.

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10. Turajlic S, Xu H, Litchfield K, et al. Deterministic Evolutionary Trajectories Influence Primary Tumor Growth: TRACERx Renal. Cell. 2018;173(3):595-610 e511.

11. Network CGAR. Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature. 2013;499(7456):43-49.

12. Iliopoulos O, Kibel A, Gray S, Kaelin WG, Jr. Tumour suppression by the human von Hippel-Lindau gene product. Nat Med. 1995;1(8):822-826.

13. Kibel A, Iliopoulos O, DeCaprio JA, Kaelin WG, Jr. Binding of the von Hippel-Lindau tumor suppressor protein to Elongin B and C. Science. 1995;269(5229):1444-1446.

14. Iliopoulos O, Levy AP, Jiang C, Kaelin WG, Jr., Goldberg MA. Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein. Proc Natl Acad Sci U S A. 1996;93(20):10595-10599.

15. Lonergan KM, Iliopoulos O, Ohh M, et al. Regulation of hypoxia-inducible mRNAs by the von Hippel-Lindau tumor suppressor protein requires binding to complexes containing elongins B/C and Cul2. Mol Cell Biol. 1998;18(2):732-741.

16. Semenza GL, Nejfelt MK, Chi SM, Antonarakis SE. Hypoxia-inducible nuclear factors bind to an enhancer element located 3’ to the human erythropoietin gene. Proc Natl Acad Sci U S A. 1991;88(13):5680-5684.

17. Jiang BH, Rue E, Wang GL, Roe R, Semenza GL. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J Biol Chem. 1996;271(30):17771-17778.

18. Forsythe JA, Jiang BH, Iyer NV, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol. 1996;16(9):4604-4613.

19. Pugh CW, Ebert BL, Ebrahim O, Maxwell PH, Ratcliffe PJ. Analysis of cis-acting sequences required for operation of the erythropoietin 3’ enhancer in different cell lines. Ann N Y Acad Sci. 1994;718:31-39; discussion 39-40.

20. ­­Maxwell PH, Wiesener MS, Chang GW, et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature. 1999;399(6733):271-275.

21. Jaakkola P, Mole DR, Tian YM, et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science. 2001;292(5516):468-472.

22. Keith B, Johnson RS, Simon MC. HIF1alpha and HIF2alpha: sibling rivalry in hypoxic tumour growth and progression. Nat Rev Cancer. 2011;12(1):9-22.

23. Mandriota SJ, Turner KJ, Davies DR, et al. HIF activation identifies early lesions in VHL kidneys: evidence for site-specific tumor suppressor function in the nephron. Cancer Cell. 2002;1(5):459-468.

24. Raval RR, Lau KW, Tran MG, et al. Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-associated renal cell carcinoma. Mol Cell Biol. 2005;25(13):5675-5686.

25. Kondo K, Klco J, Nakamura E, Lechpammer M, Kaelin WG, Jr. Inhibition of HIF is necessary for tumor suppression by the von Hippel-Lindau protein. Cancer Cell. 2002;1(3):237-246.

26. Jaakkola P, Mole DR, Tian YM, et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science. 2001;292(5516):468-472.

27. Shen C, Beroukhim R, Schumacher SE, et al. Genetic and functional studies implicate HIF1alpha as a 14q kidney cancer suppressor gene Cancer Discov. 2011;1(3):222-235.

28. Rankin EB, Tomaszewski JE, Haase VH. Renal cyst development in mice with conditional inactivation of the von Hippel-Lindau tumor suppressor. Cancer Res. 2006;66(5):2576-2583.

29. Young AP, Kaelin WG, Jr. Senescence triggered by the loss of the VHL tumor suppressor. Cell Cycle. 2008;7(12):1709-1712.

30. Varela I, Tarpey P, Raine K, et al. Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature. 2011;469(7331):539-542.

31. Dalgliesh GL, Furge K, Greenman C, et al. Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes. Nature. 2010;463(7279):360-363.

32. Pena-Llopis S, Vega-Rubin-de-Celis S, Liao A, et al. BAP1 loss defines a new class of renal cell carcinoma. Nat Genet. 2012;44(7):751-759.

33. Escudier B, Eisen T, Stadler WM, et al. Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med. 2007;356(2):125-134.

34. Motzer RJ, Hutson TE, Tomczak P, et al. Sunitinib versus interferon alfa in metastatic renal-cell carcinoma. N Engl J Med. 2007;356(2):115-124.

35. Escudier B, Pluzanska A, Koralewski P, et al. Bevacizumab plus interferon alfa-2a for treatment of metastatic renal cell carcinoma: a randomised, double-blind phase III trial. Lancet. 2007;370(9605): 2103- 2111.

36. Rini BI, Halabi S, Rosenberg JE, et al. Bevacizumab plus interferon alfa compared with interferon alfa monotherapy in patients with metastatic renal cell carcinoma: CALGB 90206. J Clin Oncol. 2008; 26(33):5422-5428.

37. Rini BI, Escudier B, Tomczak P, et al. Comparative effectiveness of axitinib versus sorafenib in advanced renal cell carcinoma (AXIS): a randomised phase 3 trial. Lancet. 2012;378(9807):1931-1939.

38. Choueiri TK, Escudier B, Powles T, et al. Cabozantinib versus everolimus in advanced renal cell carcinoma (METEOR): final results from a randomised, open-label, phase 3 trial. Lancet Oncol. 2016; 17(7): 917-927.

39. Motzer RJ, Hutson TE, Cella D, et al. Pazopanib versus sunitinib in metastatic renal-cell carcinoma. N Engl J Med. 2013;369(8):722-731.

40. Sternberg CN, Davis ID, Mardiak J, et al. Pazopanib in locally advanced or metastatic renal cell carcinoma: results of a randomized phase III trial. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2010;28(6):1061-1068.

41. Motzer RJ, Hutson TE, Glen H, et al. Lenvatinib, everolimus, and the combination in patients with metastatic renal cell carcinoma: a randomised, phase 2, open-label, multicentre trial. Lancet Oncol. 2015;16(15):1473-1482.

42. Jonasch E, McCutcheon IE, Waguespack SG, et al. Pilot trial of sunitinib therapy in patients with von Hippel-Lindau disease. Ann Oncol. 2011;22(12):2661-2666.

43. Jonasch E, McCutcheon IE, Gombos DS, et al. Pazopanib in patients with von Hippel-Lindau disease: a single-arm, single-centre, phase 2 trial. Lancet Oncol. 2018;19(10):1351-1359.

44. Zimmer M, Doucette D, Siddiqui N, Iliopoulos O. Inhibition of hypoxia-inducible factor is sufficient for growth suppression of VHL-/- tumors. Mol Cancer Res. 2004;2(2):89-95.

45. Wallace EM, Rizzi JP, Han G, et al. A Small-Molecule Antagonist of HIF2alpha Is Efficacious in Preclinical Models of Renal Cell Carcinoma. Cancer Res. 2016;76(18):5491-5500.

46. Chen W, Hill H, Christie A, et al. Targeting renal cell carcinoma with a HIF-2 antagonist. Nature. 2016;539(7627):112-117.

47. Courtney KD, Infante JR, Lam ET, et al. Phase I Dose-Escalation Trial of PT2385, a First-in-Class Hypoxia-Inducible Factor-2alpha Antagonist in Patients With Previously Treated Advanced Clear Cell Renal Cell Carcinoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2018;36(9):867-874.

48. Jonasch E, Klaassen Z. A First-in-Human Phase 1/2 Trial of the Oral HIF-2a Inhibitor PT2977 in Patients with Advanced RCC (Clinical trial identification: NCT02974738). ESMO 2019; 2019; Barcelona, Spain.

49. Motzer RJ, Penkov K, Haanen J, et al. Avelumab plus Axitinib versus Sunitinib for Advanced Renal-Cell Carcinoma. N Engl J Med. 2019;380(12):1103-1115.

50. Rini BI, Plimack ER, Stus V, et al. Pembrolizumab plus Axitinib versus Sunitinib for Advanced Renal-Cell Carcinoma. N Engl J Med. 2019;380(12):1116-1127.

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Kidney cancer is among the 10 most common cancers in both men and women, leading to approximately 74,000 new cases and to more than 14,000 deaths annually in United States alone.1 Early stage, localized renal cell carcinoma (RCC) has a significant cure fraction and a survival rate of 92%, whereas the treatment of late stage recurrent metastatic RCC remains highly challenging, with a minority of patients with metastatic RCC surviving past 5 years.2 Given that RCC is chemo-resistant and radiation-resistant, novel targeted therapies were required for the prevention and management of advanced and/or metastatic RCC.

Studies found that the majority of localized and advanced clear cell RCCs (ccRCCs) are characterized by mutational inactivation and allelic loss of the von Hippel-Lindau (VHL) tumor-suppressor gene.3 The groundbreaking discoveries made by William G. Kaelin Jr., Sir Peter J. Ratcliffe and Gregg L. Semenza on the involvement of the VHL gene in various fundamental processes, including but not limited to sensing and adapting to the changing oxygen environment eventually led to the Nobel prize in physiology and medicine in 2019. These key insights not only paved the way for our understanding of a key factor in ccRCC tumorigenesis, but also provided the basis for the development of VHL-hypoxia pathway-targeted therapies that includes tyrosine kinase inhibitors (TKIs) for treatment of RCC and other diseases.

von Hippel-Lindau disease is a rare autosomal dominant hereditary neoplastic disorder triggered by germline mutations in the VHL tumor-suppressor gene with an incidence of roughly 1 in 36,000 births.4 Individuals with VHL disease are at increased risk of recurrent and bilateral kidney cysts and ccRCC, as well as retinal, cerebellar and spinal hemangioblastomas, pheochromocytomas, pancreatic cysts, serous cystadenomas and neuroendocrine tumors, endolymphatic sac tumors and epidymal and round ligament cysts.5  The discovery of the VHL gene in 19936 was driven by a desire to understand and treat VHL disease.  The impact of this seminal discovery on our understanding of disease manifestations in patients with VHL disease and on individuals with sporadic ccRCC cannot be overstated.  We now know that the majority of sporadic ccRCC cases also exhibit somatic loss-of-function mutations in the VHL gene,3 loss of 3p chromosome, or hypermethylation of the VHL locus.7,8

The mechanistic understanding of VHL protein (pVHL) function, driven by Kaelin’s group and others formed the cornerstone of our current understanding of ccRCC biology. Through additional work performed by a number of investigators and organizations including The Cancer Genome Atlas (TCGA), we now know VHL loss serves as the initiating truncal event for ccRCC tumorigenesis, eventually followed by additional mutational and chromosomal copy number altering changes that foster tumor growth and lethality.8-11

Bill Kaelin and colleagues were instrumental in characterizing the VHL gene and its function. In 1995, Iliopoulos, Kibel, Gray and Kaelin showed that the reintroduction of a wild-type but not a mutant VHL cDNA into the 786-0 VHL(-/-) RCC cell line abrogated its ability to form tumors in nude mouse xenograft assays, reinforcing the concept that VHL is a bona fide tumor suppressor gene.12 In the same year, the Kaelin group showed that pVHL interacts with with elongins C and B to form the VBC complex.13  In 1996, Iliopoulos et al demonstrated that pVHL was involved in negatively regulating hypoxia-inducible genes.14  Over the next few years, further refinement of the VBC complex,15 and the solution of the crystal structure of the VBC complex, led to a broader understanding of pVHL function.

The next major step was the identification of HIF as the substrate for the VBC complex.  In 1991, Greg Semenza reported that HIF bound to enhancers near the human erythropoietin gene.16 Over the following decade Dr. Semenza and his colleagues further characterized HIF function, demonstrating its dimerization, DNA binding, and transactivation properties.17 In 1996 Jiang et al showed that vascular endothelial growth factor was HIF regulated.18

The third piece in the overall puzzle was the mechanism of oxygen sensing, elegantly discovered by Peter Ratcliffe and colleagues. Dr. Ratcliffe’s lab had been working on elucidating the key factors in erythropoietin gene activation since the early 1990s.19  In 1999, Max-well et al reported that pVHL was required to degrade HIF in an oxygen and iron-dependent manner,20 and in 2001 Jaakola et al reported this interaction was prolyl hydroxylation dependent.21

Further modeling showed that overexpression of a VHL- binding defective HIF2a variant was sufficient for tumorigenesis in a mouse model, suggesting that HIF overexpression is one of the major drivers of the malignant phenotype.  A review of the myriad functions of HIF1a and HIF2a show that each HIF isoform has both unique and over- lapping target genes, including angiogenesis, metabolism and glycolysis22 (Figure 1).

The development of agents targeting the consequences of VHL loss shifted the treatment landscape from cytokine based immunotherapeutics, such as IFNα and IL-2 towards targeted therapeutics fifteen years ago.33,34 Given that ccRCC are highly vascular tumors with overexpression of angiogenic vascular endothelial growth factor (VEGF) which is a downstream target of HIF, currently approved therapies include inhibitors of VEGF35,36 and VEGFR tyrosine kinases (TKIs).33,34,37-41 Patients with VHL disease also demonstrated some benefit from these agents, with a 33% objective response rate (ORR) in ccRCC after sunitinib treatment42 and a 51% ORR in ccRCC after pazopanib treatment.43 The key challenge with all of these agents is that there is significant on and off target toxicity, and a near inevitable failure to cure or ultimately control tumor growth.  There is no clear explanation for these findings, but there is undoubtedly room for a further refinement of VHL-HIF axis blocking agents.

The first-in-class clinical HIF-2aα inhibitor PT-2385 caused dramatic tumor regressions in patient-derived xenografts.46 Clinical data from PT-2385 in pretreated patients with metastatic clear cell renal carcinoma  (mRCC) were encouraging in a Phase I, dose-escalation trial, and demonstrated a favorable safety profile.47  PT2977/MK-6482 is the second generation of the HIF2 inhibitor and was tested in a 55 patient phase Ib-II study.48  This study, which was presented at the European Society of Medical Oncology meeting in the fall of 2019, described 55 patients with advanced cc RCC who had received at least one prior therapy and who were treated with 120 mg orally once daily dose of PT2977/MK6482. We found that PT2977/MK6482 was well tolerated and had a favorable safety profile. The most common Grade 3 adverse events and on-target effects of HIF2α inhibition were found to be anemia in 26% of patients and hypoxia in 15%, and only 2 patients experienced grade 4 toxicities. Despite having a study population treated with a median of three prior therapies, the ORR was 24%, the median progression-free survival (PFS) was an impressive 11 months (95% CI 6-17), and the 12-month PFS rate was 49%. PT2977/MK6482 is currently being tested in a randomized phase III study in patients with treatment refractory metastatic ccRCC (NCT04195750).

Recently, the approval of combination TKI- checkpoint blocking antibody therapy has resulted in a new treatment paradigm for many patients with ccRCC. 49,50 Tissue based studies suggest antiangiogenic agents are capable of increasing T-cell recruitment to the tumor microenvironment,51 providing a mechanistic rationale for this type of combination therapy.  Further investigations into the way VHL-HIF targeting agents can synergize with checkpoint blocking antibodies will undoubtedly further improve the treatment of patients with RCC.

The Official Journal of the Kidney Cancer Association

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Vol 17, No 3    2019