targeting ret-driven cancers: lessons from evolving ...download.xuebalib.com/y7xo4hlapbw.pdfof the...

Meaningful clinical benefit can be achieved for patients with a variety of oncogene-driven cancers by pair-ing each tumour with a therapy targeting a critical signalling node or pathway. Such targeted therapies include ABL1 tyrosine-kinase inhibitors (TKIs) in BCR–ABL1-rearranged chronic myeloid leukaemia; EGFR, ALK, and ROS1 TKIs in EGFR-mutant, ALK-rearranged, and ROS1-rearranged non-small-cell lung cancers (NSCLCs), respectively; and BRAF and MEK inhibitors in BRAFV600E-mutant melanomas and NSCLCs1–6. Improvements in objective response rates (ORRs) and progression-free survival (PFS) compared with those achieved using standard-of-care cytotoxic chemo therapy regimens have resulted in the regulatory approval of several of these molecularly targeted agents for the treatment of patients with advanced-stage disease. Many of these agents are now also being tested before or after definitive therapy in earlier-stage disease.

Multikinase inhibitors with activity against the receptor- tyrosine kinase (RTK) RET, such as cabo-zantinib, vandetanib, and lenvatinib, have demon-strated efficacy in certain tumour types, with confirmed

responses and durable disease control achieved in a proportion of patients with thyroid cancers and RET-rearranged NSCLCs in prospective trials7–12. The degree of overall clinical benefit achieved with these drugs is modest, however, compared with the outcomes of tar-geted therapy in patients with different molecular sub-types of NSCLC or other malignancies1–6,13–20. Several factors might account for the relatively low degree of clinical benefit from RET-directed therapy with multi-kinase inhibitors (despite the substantial activity of these agents against RET in the laboratory), including sub optimal pharmacokinetic properties and toxicities resulting from more-potent inhibition of non-RET kinases, such as VEGFR2 (REFS 21–23).

An increased appreciation of the effects of somatic or germ-line RET alterations in human cancers has fortunately led to the advent of new clinical strategies. Combination therapies designed to improve on the efficacy of single-agent multikinase inhibition have entered clinical testing24, followed in 2017 by potent and specific RET inhibitors25,26. These approaches lever age a deeper understanding of the biology

1Department of Medicine, Memorial Sloan Kettering Cancer Center, 885 2nd Avenue, New York, New York 10017, USA.2Department of Medicine, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10065, USA.3Department of Medicine, Icahn School of Medicine, Mount Sinai Health System, Mount Sinai West, 1000 10th Avenue, New York, New York 10019, USA.4National Cancer Center, 11 Hospital Drive, Singapore 169610, Singapore.

Correspondence to A.D. [email protected]

doi:10.1038/nrclinonc.2017.175Published online 14 Nov 2017; corrected online 28 Nov 2017

Targeting RET-driven cancers: lessons from evolving preclinical and clinical landscapesAlexander Drilon1,2, Zishuo I. Hu3, Gillianne G. Y. Lai4 and Daniel S. W. Tan4

Abstract | The gene encoding the receptor‑tyrosine kinase RET was first discovered more than three decades ago, and activating RET rearrangements and mutations have since been identified as actionable drivers of oncogenesis. Several multikinase inhibitors with activity against RET have been explored in the clinic, and confirmed responses to targeted therapy with these agents have been observed in patients with RET‑rearranged lung cancers or RET‑mutant thyroid cancers. Nevertheless, response rates to RET‑directed therapy are modest compared with those achieved using targeted therapies matched to other oncogenic drivers of solid tumours, such as sensitizing EGFR or BRAFV600E mutations, or ALK or ROS1 rearrangements. To date, no RET‑directed targeted therapeutic has received regulatory approval for the treatment of molecularly defined populations of patients with RET‑mutant or RET‑rearranged solid tumours. In this Review, we discuss how emerging data have informed the debate over whether the limited success of multikinase inhibitors with activity against RET can be attributed to the tractability of RET as a drug target or to the lack, until 2017, of highly specific inhibitors of this oncoprotein in the clinic. We emphasize that novel approaches to targeting RET‑dependent tumours are necessary to improve the clinical efficacy of single‑agent multikinase inhibition and, thus, hasten approvals of RET‑directed targeted therapies.

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under lying the responses to RET-directed targeted therapies in molecularly enriched cohorts of patients and an improved knowledge of the chemistry required to design better drugs.

RET biology and activationRET is a transmembrane glycoprotein RTK that is encoded by the proto-oncogene RET (rearranged dur-ing transfection) located on chromosome 10 (REF. 27). This kinase has important roles in the development of the kidneys and enteric nervous system during embryogenesis28–30. RET is likewise important in the homeostasis of several tissue types, including neural, neuroendocrine, haematopoietic, and male germ-cell tissues31. In contrast to other RTKs, RET does not bind directly to its ligands, artemin, glial cell-line- derived neurotrophic factor (GDNF), neurturin, and persephin32. These GDNF family ligands (GFLs) instead bind to GDNF family receptor-α (GFRα) co-receptors33–36; subsequently, the GFL–GFRα com-plex mediates RET homodimerization, resulting in trans-autophosphorylation of tyrosine residues within the RET intracellular domains, the recruitment of key signalling adaptors, and the activation of several sig-nal transduction cascades involved in cellular prolifer-ation, including the MAPK, PI3K, JAK–STAT, PKA, and PKC pathways37–43.

Oncogenic activation of RET can occur by two primary mechanisms: first, chromosomal rearrange-ments can produce hybrid proteins that fuse the RET kinase domain with a partner protein that often con-tains a dimerization domain44–47; second, mutations can directly or indirectly activate the kinase48–50. These somatic or germ-line alterations are implicated in the pathogenesis of several human cancers44–51. More importantly, activating RET rearrangements and muta-tions share features with oncogenes that are established therapeutic targets. For example, diverse RET fusion proteins and select RET-mutant proteins transform primary cells (fibroblasts and thyrocytes) in vitro and

in vivo, and activate downstream signalling pathways in a ligand-independent fashion52–55. Moreover, RET rearrangements and mutations promote cell prolifer-ation and survival when expressed in human cancer cell lines56–59, recapitulate the features of human can-cers (lung and thyroid carcinomas) in genetically engi-neered mouse models60–62, and occur in cancers in a pre dominantly mutually exclusive pattern with other bona fide drivers of tumorigenesis45–47,63–66.

Other mechanisms beyond RET rearrangement and mutation might be involved in RET-mediated onco-genesis. First, increased RET expression in the absence of defined genomic alterations in RET might contribute to the growth and survival of some human cancers. RET has been shown to be a direct transcriptional target of the oestrogen receptor (ER)67,68, a finding that is consistent with the possible ER-mediated increase in RET expression in rare medullary thyroid cancers (MTCs) from families with inactivating germ-line mutations in ESR2 (encod-ing ERβ, which represses RET activation)69, as well as the increased expression of RET in some ER-positive breast cancers that have acquired resistance to anti-oestrogen therapy70,71, and the re-sensitization of cells from such breast cancers to anti-oestrogen treatment through RET inhibition70–72. Second, RET has been identified as a strong negative regulator of major histocompatibility complex (MHC) class I expression in cancer cell lines of diverse histologies73, suggesting a possible role for RET inhibition in upregulating the anticancer immune response.

RET rearrangementsBiology of oncogenesis. The oncogenic potential of RET was first identified with the discovery in 1985 that an exogenous segment of rearranged human lymphoma DNA containing part of the RET coding sequence can transform mouse NIH/3T3 fibroblasts74 (FIG. 1). RET rearrangements were subsequently identified in tumours from patients with papillary thyroid carcinomas (PTCs) or NSCLCs, and are probably acquired early in carcinogenesis75. Chromosomal rearrangements involve the long arm of chromosome 10, resulting in fusion of the 3ʹ kinase-domain-encoding region of RET to various 5ʹ heterologous upstream partner genes45–47. In PTCs, these upstream partners were initially identified in a numeric fashion as follows: PTC1 (CCDC6), PTC2 (PRKAR1A), PTC3/4 (NCOA4), PTC5 (GOLGA5), PTC6 (TRIM24), PTC7 (TRIM33), PTC8 (KTN1), and PTC9 (RFG9)44. Historically, each of these fusion genes was labelled RET–PTCx, such as RET–PTC1 for the CCDC6–RET fusion; however, in this Review, we hereafter identify these rearrangements across all tumours using the latter, contemporary nomenclature.

Intrachromosomal rearrangements are frequently observed in RET-rearranged cancers; CCDC6–RET and NCOA4–RET are the most commonly identified RET fusions in papillary thyroid cancers76,77, while KIF5B–RET is the most commonly identified RET fusion in NSCLCs45–47; CCDC6, NCOA4, and KIF5B are located on chromosome 10, along with RET, and rearrangement of these genes occurs via reciprocal or nonreciprocal para-centric (CCDC6 and NCOA4) or pericentric (KIF5B)

Key points

• Activating alterations of the RET kinase are therapeutically actionable oncogenic drivers across a variety of cancers; in‑frame RET rearrangements occur in subsets of non‑small‑cell lung and papillary thyroid cancers, and germ‑line or somatic RET mutations are enriched in medullary thyroid cancers

• Confirmed and durable responses to multikinase inhibitors with activity against RET can be achieved in a proportion of patients with RET‑rearranged or RET‑mutant cancers; however, objective response rates are modest

• The outcomes observed with RET‑directed therapy in RET‑rearranged or RET‑mutant cancers might be explained, in part, by the limitations of multikinase inhibitors, with inhibition of other kinases resulting in ‘off‑target’ toxicities that preclude optimal dosing

• Novel approaches to RET‑directed targeted therapy are currently being explored; potent and specific RET inhibitors with minimal preclinical off‑target activity are being evaluated in early stage clinical trials, as are combination therapies

• Salient to the clinical development of potent RET inhibitors for patients of all ages, selective RET inactivation can affect the nervous, genitourinary, gastrointestinal, and haematopoietic systems during early development, but in adulthood, it leads to mild phenotypes

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inversion45,46,76. Intrachromosomal RET rearrangements are also observed with ACBD5, PARD3, FRMD4A, AFAP1L2, or KIAA1217 (REFS 78–81). By contrast, inter-chromosomal RET rearrangement or translocation is less-frequently detected; however, rare translocations can involve the partners TRIM24, TRIM27, TRIM33, TBL1XR1, FGFR1OP, CUX1, HOOK3, PCM1, FKBP15, ERC1, PICALM, GOLGA5, KTN1, AKAP13, MYH13, PRKAR1A, RFG9, KIAA1468, SPECC1L, SQSTM1, PPFIBP2, and BCR44,64,65,82–95. The frequency of indi-vidual RET fusions in specific cancers might be affected by cell-specific nuclear dynamics (for example, CCDC6 and RET are often juxtaposed within the nuclear chro-matin domains of nonmalignant thyroid cells) and cellular context96.

RET rearrangement is hypothesized to result from the illegitimate repair of DNA double-strand breaks (DSBs) via non-homologous end joining and break- induced replication97. DSBs can occur as a result of cell-extrinsic factors (for example, ionizing radiation98), or can be caused by cell-intrinsic mechanisms (such as replication stress at DNA fragile sites99). Breakpoints preferentially occur in defined regions of RET, most commonly within intron 11, creating fusions containing only the cytoplasmic domains of RET; however, some fusion breakpoints involve introns 7 and 10, resulting in the additional inclusion of the RET transmembrane domain100 (FIG. 2a). The breakpoints within the upstream genes, such as KIF5B, are more variable101. The type of

rearrangement might affect the subcellular localization of the oncoprotein generated, as specified by informa-tion conferred by the N-terminal fusion partner: the coiled-coil domain of NCOA4 is thought to have a dual role, mediating localization of the NCOA4–RET fusion protein to the plasma membrane in addition to provid-ing a dimerization motif 102, as opposed to the coiled-coil domain of KIF5B that presumably only provides a dimerization motif, resulting in the localization of select KIF5B–RET fusions to the cytoplasm45. The inclusion of the transmembrane domain in some RET fusions might result in a fusion protein that can localize to the plasma membrane.

RET rearrangement can lead to downstream path-way activation via two main mechanisms. First, the chimeric gene fusion can result in transcriptional control of the RET kinase by its partner genes45,62. Of note, RET expression is normally restricted to specific nonembryonic cell types, whereas its partner genes can be expressed ubiquitously. The RET-rearranged state can thus lead to increased RET expression, the levels of which might affect its oncogenic potential51. Second, upstream fusion partners can contribute dimerization domains (for example, coiled-coil domains; FIG. 2a) that result in ligand-independent dimerization and constitutive activation of the RET kinase45–47,64. Beyond these two mechanisms, rearrangement might rarely cause simultaneous gain-of-function activity of RET and loss of function of its tumour-suppressor gene

Nature Reviews | Clinical Oncology

1985 1990 1995 2005 2010 2012 2014 2016 2017

Identification of RET fusions in papillary thyroid cancer

Identification of RET as an oncogene: RET fusion found in human lymphoma DNA

Discovery of germ-line RET mutations associated with multiple endocrine neoplasia type 2 (MEN2) syndrome

Discovery of RET fusions in lung adenocarcinoma

RET fusions identified in chronic myelomonocytic leukaemia

RET fusions identified in Spitz nevi, Spitz tumours and Spitzoid melanomas

RET fusions identified in colorectal and breast cancers

Trials of RET-specific inhibitors for advanced-stage, RET-dependent solid tumours begin

Trials of RET-specific inhibitors begin

Trials of RET-specific inhibitors begin

Cabozantinib approved for medullary thyroid cancers based on the results of the phase III EXAM trial

Vandetanib approved for medullary thyroid cancers based on the results of the phase III ZETA trial

Sorafenib approved for differentiated thyroid cancers based on the results of the phase III DECISION trial

Lenvatinib approved for differentiated thyroid cancers based on the results of the phase III SELECT trial

Two separate phase II trials of vandetanib in RET-rearranged lung cancers published

High prevalance of RET fusions noted after radiation exposure

Structure of the RET kinase domain reported

Thyroid cancer

Lung cancer

RET biology and discoveries in other cancers

Phase II trial of cabozantinib in RET-rearranged lung cancers published

Figure 1 | Timeline of key developments in therapeutically targeting RET in the clinic. Milestones in our understanding of the pathobiology and prevalence of RET‑activating germ‑line and/or somatic alterations — RET mutations and RET rearrangements — in cancers (yellow) are shown. Key advances in the development of RET‑targeted therapies (purple), including landmark clinical trials performed to evaluate the efficacy of multikinase inhibitors with activity against RET in patients with thyroid cancer (green) or non‑small‑cell lung cancer (blue)199–203, are also depicted.

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partners. The upstream partner of the PRKAR1A–RET fusion, for example, is a tumour-suppressor gene (PRKAR1A) that is inactivated in patients with Carney complex, a multiple endocrine neoplasia syndrome that confers an increased risk of thyroid cancer103.

In-frame RET rearrangements that include the RET kinase domain are activating in vitro and in vivo; exo-genous expression of RET fusions in NIH/3T3 fibro-blasts45,46 and thyrocytes104,105 results in morphological transformation, anchorage-independent growth, and subcutaneous tumour formation when engrafted in nude mice. Furthermore, transgenic mice develop lung adenomas and adenocarcinomas when expression of RET fusions is restricted to lung alveolar epithelial cells62, whereas they develop thyroid hyperplasia and PTCs when the fusion gene is expressed in thyroid follicular cells106. In these models, increased signal-ling through pathways downstream of RET, including

the RAF–MEK–ERK and PI3K–AKT axes, has been observed, resulting in increased cell survival, invasion, and angiogenesis107.

Clinical features and diagnosis. RET rearrangements are identified in 1–2% of unselected patients with NSCLCs45–47,64,108,109. Patients with NSCLCs harbour-ing these aberrations share many clinical features with those who have tumours harbouring ALK or ROS1 rear-rangements; for example, patients with RET-rearranged NSCLC tend to be relatively young (≤60 years of age), with minimal or no prior history of tobacco smok-ing66,110. Whether prior radiation exposure is associated with the acquisition of RET rearrangements in lung cancers remains unclear, although RET rearrange-ment can be induced by radiation in human lung cells in vitro98. Histologically, most RET rearrangements are identified in lung adenocarcinomas45–47, although these


Coiled-coildomain

5′ partner

a RET fusion genes

b RET nonsynonymous point mutations

3′ partner: RET

Exon 12

Exon 11

TMdomain

Activation by ligand-independent dimerization

MEN2AFMTC

MEN2BFMTC

Monomericactivation

Kinasedomain

Extracellulardomain

Exon 13E768DL790FY791F

Exon 8G533C

Exon 10C609F/G/R/S/YC611F/G/S/Y/WC618F/R/SC620F/R/S

Exon 11C630R/YD631YC634F/G/R/S/W/YK666E Exon 14

V804M/LY806C

Exon 15A883FS891A

Exon 16M918T

Tyrosine kinasedomain

LisH domain

WDR domain

SAM domain

Alternate dimerizationmechanism

CCDC6, NCOA4, KIF5B, GOLGA5, ERC1, KTN1, HOOK3, PCM1, TRIM24, TRIM27, TRIM33, AKAP13, FKBP15, SPECC1L, ACBD5, MYH13, CUX1, KIAA1468, FRMD4A, AFAP1L2, PPFIBP2, KIAA1217

TBL1XR1, FGFR1OP, KIAA1468

TBL1XR1, EML4

PPFIBP2, EPHA5

PRKAR1A, SQSTM1, PARD3, PICALM

P P P P

P P P P

P

P

P

P

P

P

P

P

P

P

P

P

Covalent disulfide bonds in cysteine-rich region

Figure 2 | Mechanisms of RET activation in cancer. a | In‑frame RET rearrangements that result in fusion proteins containing the RET kinase domain can lead to the activation of oncogenic RET signalling. Activating RET fusions maintain the tyrosine kinase domain of the 3ʹ RET gene; breakpoints commonly occur within intron 11; however, introns 10 and 7 (not shown) are occasionally involved, resulting in the inclusion of the RET transmembrane (TM) domain. A variety of upstream, 5ʹ gene partners contribute different domains, such as coiled‑coil, LIS1 homology (LisH), tryptophan–aspartate repeat (WDR), and sterile alpha motif (SAM) domains, to RET fusion proteins. These motifs mediate ligand‑independent dimerization of the chimeric oncoprotein and, thus, autophosphory‑lation of the RET kinase domain, resulting in the activation of downstream signalling pathways that drive tumour‑cell proliferation. 5ʹ gene partners that contain two different dimerization domains, such as TBL1XR1, are listed twice in the figure. In general, 5ʹ partners are not thought to exclusively pair with specific 3ʹ partners with defined breakpoints at introns 7, 10, and 11. RET rearrangements are largely thought to be somatic events, as opposed to RET mutations that can occur in the germ line or be acquired somatically. b | Activating RET mutations can result in substitutions of extracellular cysteine residues for alternate amino acids, which disrupt intramolecular disulfide bridges, enabling the formation of novel intermolecular covalent disulfide bonds that lead to ligand‑independent dimerization. Such mutations are identified in the germ line of patients with multiple endocrine neoplasia type 2A (MEN2A) and familial medullary thyroid cancer (FMTC), which are associated with cancer predisposition. Mutations in the intracellular kinase domain of RET occur in multiple endocrine neoplasia type 2B (MEN 2B), and occasionally in FMTC, leading to monomeric (depicted) and/or dimeric (not depicted) activation of RET. In cases of ligand‑independent RET dimerization and activation, downstream signalling might be enhanced by ligand binding. Select extracellular and intracellular domain substitution mutations are listed, along with the exon in which the mutation occurs.

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have also been found in lung cancers of other histol-ogies, including adenosquamous, squamous-cell, and neuroendocrine tumours111; the ‘solid-predominant subtype’ of NSCLC and the presence of signet ring cells (≥10% of tumour cells) were frequently identified (in 64% and 36% of patients, respectively) in one series66. RET rearrangements tend to be mutually exclusive with other major lung-cancer drivers, such as KRAS mutations, sensitizing EGFR mutations, and ALK or ROS1 rearrangements95. In terms of response to cyto-toxic chemotherapy, these tumours can be sensitive to pemetrexed-based regimens (ORR of 45%, median PFS duration of 19 months), similar to ALK-rearranged or ROS1-rearranged lung cancers112.

RET rearrangements are also identified in ~5–10% of PTCs, although the authors of smaller studies have estimated a higher prevalence64,65,109. Of note, the preva-lence of RET rearrangement is substantially higher among patients with prior exposure to radiation113; this genomic aberration was detected in about 60–80% of patients who developed PTCs in the wake of high levels of radiation exposure after the Chernobyl nuclear acci-dent77,114. RET rearrangement is also more-commonly detected in children than in adults, regardless of prior radiation exposure, possibly owing to a higher rate of follicular-cell proliferation in the former. The application of more-comprehensive DNA and/or RNA sequencing techniques has led to the identification of RET rear-rangements in other tumour types, including colorectal cancer, breast cancer, Spitzoid neoplasms, MTC, chronic myeloproliferative neoplasms, anaplastic thyroid car-cinomas with a differentiated component, and poorly differentiated thyroid carcinomas64,88,90,94,95,115.

Fusions involving RET can be detected using several methods in the clinic, including reverse transcription PCR (RT-PCR), break-apart fluorescence in situ hybridization (FISH), broad hybrid-capture-based next- generation sequencing (NGS), and anchored multiplex PCR66,82. Plasma-based platforms have likewise been used to detect these alterations in circulating tumour nucleic acids116. Earlier clinical series primarily focused on RT-PCR and FISH of tumour samples as screening tools66,82,117, but more-comprehensive sequencing platforms (such as NGS of broad gene panels) afford the potential identifi-cation of RET rearrangements as in-frame events along with the detection of upstream partners and concurrent genomic alterations that involve genes other than RET118. Immunohistochemistry has not proven consistently use-ful as a screening tool for identification of patients with RET-rearranged tumours66,110.

RET mutationsNonsynonymous point mutations in RET are patho-gnomonic in subsets of familial and sporadic thyroid cancers51. From a phenomic perspective, germ-line missense mutations are intimately linked with sub-types of the autosomal dominant multiple endocrine neo plasia type 2 (MEN2) syndrome — specifically MEN2A, MEN2B, and familial medullary thyroid car-cinoma (FMTC), all three of which confer an increased risk of developing MTC119. These cancer predisposition

syndromes correlate closely with particular mutational variants; thus, the biology of these mutations is discussed together with clinical features in the following section of this Review. RET mutations can be detected in the clinic using several types of assays, including NGS of both tumour and plasma DNA, although testing of a matched nonmalignant tissue sample (such as plasma) can be required with some tests to reliably distinguish germ-line mutations from somatic alterations120.

Activating germ‑line mutations. Germ-line RET muta-tions are aetiologically involved in the pathogenesis of about a quarter of all MTCs43. MEN2A is characterized by the presence of MTC in combination with pheo-chromocytoma in ~50% of all individuals with this cancer predisposition syndrome, and with parathyroid hyperplasia or adenoma and/or lichen planus amy loidosis in ~33% of this population51,121. Germ-line RET muta-tions involving exon 10 or 11 are found in the majority (>95%) of patients with MEN2A, which in turn is the most- common subtype of MEN2 (REF. 49). Mutation hot-spots affect the cysteine-rich region of the extracellular domain of the protein, resulting in the replacement of cysteine with a variety of other amino acids48,122. These substitutions decrease the capacity of intramolecular disulfide-bond formation, thereby promoting the forma-tion of intermolecular covalent disulfide bonds between free cysteine residues on separate RET monomers, resulting in increased receptor dimerization and acti-vation123–125 (FIG. 2b). Mutations resulting in amino acid substitutions at codons for cysteine are implicated in this mechanism, namely, codons 609, 611, 618, and 620 in exon 10, and codons 630 and 634 in exon 11 (REF. 51). The most-common genetic defect associated with MEN2A, found in more than 85% of patients, is a missense muta-tion that results in substitution of an alternate amino acid for cysteine at codon 634 (REFS 122,126).

MEN2B is characterized by the presence of MTC in conjunction with pheochromocytoma and develop-mental anomalies, including delayed puberty, mar-fanoid habitus, and mucosal neuromas122. As opposed to the RET extracellular domain mutations that occur in MEN2A, MEN2B is associated with germ-line intra-cellular kinase domain mutations, such as M918T (iden-tified in most patients) or A883F127. Interestingly, the protein encoded by the RETM918T variant can signal as a monomer, owing to an increased ATP-binding affin-ity and altered protein conformation compared with wild-type RET, leading to a loss of autoinhibition, or as a dimer123,128. RETM918T is highly transforming in vitro, and the clinical phenotypes associated with this muta-tion are aggressive129, in contrast to the less-aggressive phenotypes associated with the A883F mutation. With the latter mutation, disease manifestations such as MTC and pheochromocytoma occur later in life, and 5-year and 10-year overall and disease-specific survival rates are more favourable than with the RETM918T mutation130.

FMTC is challenging to diagnose, and occurs when isolated MTC is the only consistent clinical feature observed in several family members131. FMTC is thought to lie at one end of a phenotypic spectrum; MEN2A

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that presents with additional features, such as pheo-chromocytoma and parathyroid abnormalities, lies at the other end of this spectrum131. A variety of extracellular and intracellular domain mutations are associated with FMTC, which might contribute to the decreased pene-trance of other phenotypic abnormalities. For example, mutations in the cysteine codons implicated in MEN2A are also involved in FMTC, but a higher frequency of non-C634 substitutions (involving C609, C611, C618, C620, and C630) and a lower frequency of C634 sub-stitutions are noted in FMTC than in MEN2A44. Correspondingly, non-C634 substitutions carry a lower risk of the development of pheochromocytoma, hyper-parathyroidism, and aggressive MTC compared with C634 substitutions129. Intracellular domain mutations, resulting in amino acid substitutions at codons that include V804, S891, and A883, also occur in FMTC44,132; several of these intracellular mutations are likewise asso-ciated with a lower incidence of pheochromocytoma and hyperparathyroidism129.

Somatic mutations. Somatic RET mutations are found in ~65% of all sporadic MTCs, which in turn comprise about three-quarters of all MTCs43. RETM918T muta-tions are identified in most of these patients, although other somatic RET mutations have also been described, including E768D and A883F44; beyond point mutations, small insertions and deletions are detected in some patients133. RET mutations are less-commonly asso-ciated with sporadic pheochromocytoma (frequency of

series of knockout studies have demonstrated a critical function for RET in normal immune function in the developing gastrointestinal tract. RET has been shown to be a key regulator of Peyer’s patch organogenesis, with Ret knockout during embryonic development preventing the aggregation of gut immune cells that normally initiate Peyer’s patch formation150,151. More recently, in the third study of tissue-restricted Ret deletion149, targeted abla-tion of Ret in a specific subpopulation of innate immune cells involved in mucosal immunity (group 3 innate lym-phoid cells) did not impair Peyer’s patch development, but resulted in a propensity towards chemical-irritant- induced gut inflammation and bacterial translocation or infection in postnatal animals.

In summary, while the developmental loss of RET function leads to a variety of potentially severe pheno-types, selective Ret knockout in haematopoietic, neuronal, or lymphoid tissues causes tissue-specific histological changes in adult mice, albeit with relatively mild phenotypes and symptomatology in the absence of additional environmental insults. These findings suggest that potent RET inhibition in the clinic will result in few clinically significant adverse effects in older children and in adults. Further studies with inducible Ret-knockout models in adult animals are likely to be instructive,

although the results of preclinical animal toxicity studies of selective RET inhibitors and a careful cataloguing of drug-related adverse events in patients of varying ages who receive selective RET inhibitors on clinical trials are best poised to confirm this hypothesis.

Multitarget inhibitors of RETSeveral multikinase inhibitors with activity against RET are currently approved by one or more regu-latory agencies for the treatment of a variety of solid and haematological malignancies. Notably, many of these agents — cabozantinib, vandetanib, lenvatinib, and sorafenib — are approved for the treatment of thyroid cancers, while others — including ponatinib, alectinib, sunitinib, nintedanib, and regorafenib — are approved for, or have been evaluated in, other indica-tions. To date, none of these drugs has been approved for a molecularly enriched cohort of patients with tumours harbouring an actionable alteration involving RET. Several other multi kinase inhibitors with activity against RET have been explored in investigations rang-ing from preclinical testing to ongoing clinical trials. These include RXDX-105 (REF. 91), sitravatinib152, apat-inib153, and dovitinib154, in addition to other agents155,156. These drugs can be classified by their mode of kinase


Intestinal aganglionosis(Ret–/–)

Decreased number of Peyer’s patches (Ret–/–)

Bowel inflammation and/or infection (Rorgt-Cre-Retfl/fl)

Hirschsprung disease(HSCR)

Decreased respiratory control(Ret–/–)

Dopaminergic neuron loss(Dat-Retlx/lx, Nes-Retlx/lx)

Loss of superior cervical ganglion (Ret–/–)

Congenital central hypoventilation syndrome (CCHS)

Decreased fetal HSCs and fetal liver cellularity (Ret–/–)

Decreased HSC maintenance and stress response (Vav1-Retfl/fl)

Spermatogonial stem-celldepletion (Ret–/–)

Kidney dysgenesis and/or agenesis (Ret–/–)

Congenital abnormalities of the kidney and urinary tract (CAKUT)

Figure 3 | Consequences of inactivating RET mutations or Ret knockout. The inactivation of RET leads to a variety of consequences that can affect organs including the genitourinary (green and yellow), gastrointestinal (blue), respiratory (orange), and haematopoietic (light blue) systems. Shown here are the human conditions associated with germ‑line inactivating RET mutations (CAKUT, CCHS, and HSCR), and the pathobiological effects observed in Ret‑null (Ret−/−) or tissue‑specific Ret‑knockout (Retfl/fl or Retlx/lx) mice. These findings illustrate that the deficiency or absence of RET can have substantial consequences on embryonic development; however, the phenotypic effects of RET deficiency in adult animals are typically mild, with a less‑severe symptomatology, indicating that potent and selective RET inhibitors might have a favourable safety profile in older children or adult patients. HSC, haematopoietic stem cell.

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inhibition157. Vandetanib and sunitinib are type I inhib-itors that bind within the ATP-binding pocket in the active conformation of the RET kinase158. By contrast, cabozantinib, sorafenib, ponatinib, and RXDX-105 are type II inhibitors that bind within the ATP-binding pocket in the inactive ‘DFG-out’ conformation of the RET kinase91,156,158. The specific binding mode of other multikinase inhibitors to RET is less well-defined.

In vitro and in vivo activityIn general, multitarget RET inhibitors are active against full-length, wild-type RET with varying poten-cies — with a biochemical half-maximal inhibitory concentration (IC50) in the range of 200 nM**K

d nmol/l

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* *

*

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*Cell-based assay

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Figure 4 | Multikinase inhibitor activity against RET and other kinases. The half‑maximal inhibitory concentration (IC50) of select multikinase inhibitors with varying levels of activity against RET are shown. The various colours represent a range of IC50 values, from 200 nM. Unless otherwise indicated, the IC50 values shown reflect the results of in vitro kinase assays. The presence of two or more colours within a given box indicate different IC50 values reported in separate publications. A white box indicates that biochemical data are not currently available. The activity of select agents in cellular models is indicated by an asterisk; the inclusion of all reports of in vitro activity in RET‑mutant tumour cells was not feasible. Many agents potently inhibit wild‑type RET; however, fewer drugs have documented activity against RET V804L or V804M substitution variants. In addition, many of these drugs potently inhibit non‑RET targets, such as VEGFR1, VEGFR2, VEGFR3, KIT, BRAF, and FGFR1, which increases the risk of ‘off‑target’ toxicities in patients. These drugs have been explored preclinically in models of thyroid or lung cancer, and several have been explored clinically in patients with these cancer types.

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http://www.nature.com/nrclinonc/journal/vaop/ncurrent/full/nrclinonc.2017.175.html#supplementary-information

Treatment results in decreased downstream signalling, and decreased cell proliferation or restrained tumour growth, similar to the aforementioned data established with RET rearrangements.

Fewer drugs have activity against the V804M/L gatekeeper mutations (FIG. 4), which result in decreased access of agents such as cabozantinib and vandetanib to the hydrophobic ATP-binding pocket of the RET kinase and potentially increase the binding affinity of the RET kinase for ATP173,174. Of the multikinase inhibi-tors that are currently being explored in the clinic, pon-atinib, alectinib, sorafenib, and sunitinib have reported preclinical activity (of varying potencies) against cells harbouring RETV804M/L mutations165–167,172. Lastly, not all RET mutations are activating and amenable to RET-directed targeted therapy — variants of undetermined significance have been identified175.

Off-target activityMultikinase inhibitors are characterized by sub-stantial activity against a variety of targets in addi-tion to RET; depending on the agent in question, these kinases can include EGFR, MET, KIT, BRAF, and/or VEGFR2 (REFS 21,23,91,159). In particular, the RET kinase domain has substantial homology with VEGFR2. Correspondingly, many agents that potently inhibit VEGFR2, including cabozantinib, vandetanib, and lenvatinib, also have considerable activity against RET23,159,170 (FIG. 4). Importantly, however, many of these agents are pharmacokinetically limited in their ability to target RET compared with VEGFR2; at clinically achiev-able plasma concentrations, cabozantinib and vandetanib more-effectively inhibit VEGFR2 than RET170.

The fact that many currently available RET inhibitors have substantial ‘off-target’ activity reflects how the ini-tial search for RET inhibitors relied on the interro gation of compounds for activity against RET, as opposed to specifically designing drugs to target RET while sparing other kinases158. For example, early reports on vande-tanib and cabozantinib were focused on the activity of these drugs against VEGFR2 and EGFR, and VEGFR2 and MET, respectively21,23. Ponatinib and alectinib were developed primarily as inhibitors of BCR–ABL1 (REF. 166) and ALK165, respectively. Subsequently, these drugs were swiftly repositioned with a focus on targeting RET.

Clinical efficacy as RET-directed therapiesThyroid cancer. RET-directed targeted therapy with multikinase inhibitors was first explored in patients with thyroid cancers. Cabozantinib and vandetanib are both approved for the treatment of patients with advanced-stage MTC, but these approvals are not predi-cated on the presence of a RET aberration. In the random-ized, phase III, registrational trials of cabo zantinib (EXAM7) and vandetanib (ZETA8) in patients with this disease, the ORRs were 28% and 45%, and median PFS durations were improved versus those observed with pla-cebo, by 7.2 and 11.2 months, respectively. The difference in the degree of benefit observed between these two trials was potentially due to the eligibility requirement of recent

tumour progression before enrolment in EXAM, but not in ZETA. Nevertheless, the difference in overall survival was not statistically significantly between the two arms in either study — a phenomenon observed in trials of differ-ent targeted therapies in other driver-positive subsets of patients, at least partially owing to the fact that patients in the placebo arms can crossover to the experimental arm (although crossover was not allowed in the EXAM trial), or eventually receive targeted therapy after coming off study3,176. Other multikinase inhibitors with activity against RET (such as axitinib, sunitinib, sorafenib, pazo-panib, and lenvatinib) have been studied in single-arm phase II trials in patients with MTC (TABLE 1), and most had comparable efficacy.

Each of the aforementioned studies enrolled patients irrespective of the presence of RET mutation, although several included subgroup analyses to examine the potential influence of RET mutational status on the degree of clinical benefit (TABLE 1). In both EXAM and ZETA7,8, a positive association was observed between the presence of a RET mutation (and particularly RETM918T) and specific clinical outcomes: prolonged PFS with cabozantinib and vandetanib, and increased ORRs with vandetanib, compared with those of patients lacking RET mutations. Results of an exploratory ana-lysis of the EXAM trial177 indicate that cabozantinib pro-vides the greatest PFS benefit over placebo in patients with RETM918T-mutant MTC178. By contrast, RET muta-tion status was not correlated with clinical outcomes in a phase II study of lenvatinib, although the number of patients analysed was small179. Thus, the possibility of an increased clinical benefit with multikinase inhibitors for patients with RET-mutant MTC remains to be con-firmed, and ongoing and future molecularly enriched trials of RET-directed targeted therapy are likely to be informative.

Lung cancer. The use of multitarget inhibitors of RET also results in clinical benefit for a subset of patients with RET-rearranged NSCLC. A phase II trial of cabozantinib was the first study to demonstrate the activity of a RET inhibitor in a molecularly enriched cohort of patients with advanced-stage, RET-rearranged NSCLC9,82. This trial was followed by two phase II trials of vandetanib in this disease context, one conducted in Japan (LURET)11 and one in South Korea10. Preliminary results from a multicentre, phase II trial of lenvatinib in patients with RET-fusion-positive NSCLC have likewise been reported12. In these trials9–11, overall ORRs ranged from 16% to 53%, median PFS durations ranged from 4.5 to 7.3 months, and median overall survival ranged from 9.9 to 11.6 months (TABLE 2); the duration of disease control with these agents ranged from a few months to >3 years in select patients.

These prospective results are complemented by retrospective data from a global registry of patients with RET-rearranged NSCLC. Among patients in this registry who received a multikinase inhibitor with activity against RET, the best responses to cabo-zantinib, vandetanib, or sunitinib were comparable to those reported in prospective trials (partial response

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rates ranging from 18% to 37%), while median PFS durations were shorter (ranging from 2.2 months to 3.6 months)83. Partial responses or stable disease have

been reported with alectinib, sunitinib, nintedanib, and RXDX-105 in separate reports83,91,180 (TABLE 2), and trials of several multikinase inhibitors in patients with

Table 1 | Clinical outcomes with multikinase inhibitor therapy in thyroid cancers

Drug Clinical trial or case series (n)

ORR Median PFS Median OS Dose‑reduction rate

Discontinuation rate

Vandetanib Phase III trial8 (n = 331 with MTC; 231 in treatment arm and 100 in placebo arm)

Treatment: 104/231 (45%)

Placebo: 13/100 (13%)

Treatment: 30.5 months

Placebo: 19.3 months

NA Treatment: 81/231 (35%)

Placebo: 3/99 (3%)

Treatment: 28/231 (12%)

Placebo: 3/99 (3%)

Phase I/II trial211 (n = 16)

Overall: 7/16 (44%) NA NA 3/16 (19%) 1/16 (6%)

Cabozantinib Phase III trial7,177 (n = 330 with MTC; 219 in treatment arm and 111 in placebo arm)

Treatment: 58/208 (28%)

• RET‑mutation‑positive: 32/101 (32%)

• RET‑mutation‑negative: 7/32 (22%)

• RET‑mutation‑unknown: 19/75 (25%)

• RET‑mutation‑unknown function: 1/13 (8%)

• RETM918T‑positive: 26/77 (34%)

• RETM918T‑negative: 14/69 (20%)

• Non-RETM918T-mutation‑positive: 6/25 (24%)

• RETM918T status unknown: 18/63 (29%)

Placebo: 0/104 (0%)


• RET‑mutation‑positive: 14 months

• RET‑mutation‑negative: 5.8 months

• RET‑mutation‑unknown: 11.2 months

• RET‑mutation‑unknown function: 5.6 months

• RETM918T‑positive: 14.2 monthss

• RETM918T‑negative: 5.8 months

• Non- RETM918T-mutation‑positive: 8.4 months

• RETM918T status unknown: 11.4 months

Placebo: 4.0 months


• RETM918T‑positive: 44.3 months


Placebo: 21.1 months

• RETM918T‑positive: 18.9 months


Treatment: 169/214 (79%)

Placebo: 10/109 (9%)

Treatment: 35/214 (16%)

Placebo: 9/109 (8%)

Sorafenib Phase II trial212 (n = 16 with MTC)

Overall: 1/15 (6%) Overall: 17.9 months NR NA 3/16 (19%)

Lenvatinib Phase II trial179 (n = 59 with MTC)

Overall: 21/59 (36%) Overall: 9.0 months Overall: 16.6 months

35/59 (59%) 14/59 (24%)

Sunitinib Phase II trial213 (n = 35; 7 with MTC)

Overall: 11/35 (31%)

• MTC: 3/6 (50%)

NA (median TTP: 12.8 months)

NR 21/35 (60%) 4/35 (11%)

Phase II trial214 (n = 71; 26 with MTC, 41 with DTC, and 4 with ATC)

Overall: 19/71 (27%)

• MTC: 10/26 (38%)• DTC: 9/41 (22%)• ATC: 0/4 (0%)

Overall: NA

• MTC: 16.5 months• DTC: 13.1 months• ATC: 9.8 months

Overall: NA

• MTC: 29.4 months• DTC: 26.4 months• ATC: NA

NA NA

Dovitinib Phase II trial154 MTCs and DTCs (n = 40, 39 evaluable for response; 12 with MTC and 28 with DTC)

Overall: 8/39 (20%)

• MTC: 2/12 (17%)• PTC: 4/23 (17%)• FTC: 2/5 (40%)

Overall: 5.4 months

• MTC: 4.5 months• PTC: 6.3 months• FTC: 3.2 months

NR 19/39 (44%) 8/39 (20%)

Motesanib Phase II trial189 (n = 91 with MTC)

Overall: 2/91 (2%)

• RET‑mutation‑negative: 1/13 (8%)

• RET‑mutation‑positive: 0/33 (0%)

Overall: 48 weeks NE NA 13/91 (14%)

The antitumour activity of multikinase inhibitors with activity against RET in patients with thyroid cancers of various histologies is summarized in this table, with a focus on MTC and PTC. The clinical outcomes of subsets of patients by histology or RET mutation status are listed when available. Percentages were rounded to the nearest whole number. ATC, anaplastic thyroid cancer; DTC, differentiated thyroid cancer (includes FTC and PTC); FTC, follicular thyroid cancer; MTC, medullary thyroid cancer; n, number of patients; NA, not available; NE, not evaluable; NR, not reached; ORR, objective response rate; OS, overall survival; PFS, progression‑free survival; PTC, papillary thyroid cancer; TTP, time to progression.

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RET-rearranged NSCLC are ongoing (these include alectinib (NCT03131206), sunitinib (NCT01829217), ponatinib (NCT01813734), apatinib (NCT02540824), and RXDX-105 (NCT01877811)).

Intracranial activity. The importance of the intra-cranial activity of RET-directed targeted therapies is highlighted by the frequency of brain metastases in patients with NSCLCs. In a retrospective series

Table 2 | Clinical outcomes with multikinase inhibitor therapy in RET‑rearranged NSCLC

Drug Clinical trial or case series (n)

ORR Median PFS Median OS Dose‑ reduction rate

Drug discontinuation rate

Cabozantinib Phase II trial9 (n = 26; 25 evaluable for response)

Overall: 7/25 (28%)

• KIF5B–RET: 3/15 (20%)• FISH+: 2/6 (33%)• Other: 2/4 (50%)

Overall: 5.5 months

• KIF5B–RET: 4.6 months• FISH+: 8.4 months• Other: 7.5 months

Overall: 9.9 months

• KIF5B–RET: 9.0 months

• FISH+: 40.2 months• Other: 37.6 months

19/26 (73%) 2/26 (8%)

Retrospective series83 (n = 19)

Overall: 7/19 (37%)* Overall: 3.6 months Overall: 4.9 months NA NA

Vandetanib Phase II trial10 (n = 17)

Overall: 3/17 (18%)

• KIF5B–RET: 0/5 (0%)• CCDC6–RET: 1/2 (50%)• MYO5C–RET: 0/1 (0%)• Unknown: 2/9 (22%)

Overall: 4.5 months Overall: 11.6 months 4/17 (23%) NA

Phase II trial11 (n = 19)

Overall: 9/19 (47%, intention‑to‑treat); 9/17 (53%, primary analysis)

• KIF5B–RET: 2/10 (20%)• CCDC6–RET: 5/6 (83%)• Unknown: 2/3 (67%)

Overall: 4.7 months

• KIF5B–RET: 2.9 months• CCDC6–RET:

8.3 months• Unknown: 4.7 months

Overall: 11.1 months

• KIF5B–RET: 11.1 months

• CCDC6–RET: NR• Unknown:

11.0 months

10/19 (53%) 4/19 (21%)




Overall: 0/3 (0%)

• KIF5B–RET: 0/3 (0%)

NA NA NA 1/3 (33%)

Lenvatinib Phase II trial12 (n = 25)

Overall: 4/25 (16%) Overall: 7.3 months NE 16/25 (64%) 5/25 (20%)


Overall: 1/2 (50%)* NA NA NA NA

Sorafenib Phase II trial216 (n = 3)

Overall: 0/3 (0%)


NA NA 1/3 (33%) NA



Sunitinib Retrospective series83 (n = 9)


Alectinib Retrospective series180 (n = 4)

Overall: 1/4 (25%)


NA NA 0/4 (0%) 1/4 (25%)



Ponatinib Retrospective series83 (n = 2)


RXDX‑105 Phase I trial190 (n = 22)

Overall: 6/22 (27%)

• KIF5B–RET: 0/14 (0%)• Non‑KIF5B–RET: 6/8 (75%)

Regorafenib Retrospective series83 (n = 1)


Nintedanib Retrospective series83 (n = 2)


The antitumour activity of multikinase inhibitors with activity against RET in patients with RET‑rearranged NSCLC is summarized in this table. The clinical outcomes of subsets of patients with specific RET rearrangements are likewise listed when available. Percentages were rounded to the nearest whole number. FISH+, positive for RET fusion by fluorescence in situ hybridization; unknown, upstream gene partner unknown; n, number of patients; NA, not available; NE, not evaluable; NR, not reached; NSCLC, non‑small‑cell lung cancer; ORR, objective response rate; OS, overall survival; PFS, progression‑free survival. *Refers to partial response rate and not ORR, as responses were not systematically confirmed in this retrospective series.

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of patients with RET-rearranged NSCLC181, the fre-quency of brain metastases at the diagnosis of stage IV disease ranged from 23% in a single-institution expe-rience to 27% in a global registry with a large num-ber of patients, and the lifetime prevalence of brain metastases in a single-institution experience was 49%. In patients with thyroid cancers, brain meta-stases occasionally occur, but the overall prevalence of intracranial disease is low. Intracranial disease con-trol has been achieved with cabozantinib, RXDX-105, and alectinib in patients with RET-rearranged NSCLC91,180,181; however, many multikinase inhibi-tors have not been specifically designed to efficiently penetrate the central nervous system (CNS). Data on patterns of disease progression (a comparison of iso-lated intracranial progression, isolated extra cranial progression, or a combination of intracranial and extracranial progression) in clinical trials of RET inhibitors will provide additional information on the CNS activity of these agents.

Limitations of multikinase inhibitionWhereas multikinase inhibitors are active in patients with RET-driven tumours, response rates achieved in prospective series are lower than those observed in other driver-positive, advanced-stage tumours with matched targeted therapies. For example, the ORRs of 60–80% and median PFS durations of 8–19 months achieved with molecularly targeted TKIs in patients with EGFR-mutant, ALK-rearranged, or ROS1-rearranged NSCLCs exceed the previously summarized outcomes of RET-directed multikinase inhibition in those with RET-rearranged NSCLC (FIG. 5; TABLE 2); several factors might account for this inferior activity.

Clinical consequences of off‑target inhibition. One possible explanation for the limited efficacy of RET-directed therapy with multikinase inhibitors relates to the inhibition of non-RET kinases, as well as non-kinase targets. As previously outlined, many multikinase inhibitors equally or more-potently inhibit these targets compared with RET (FIG. 4). For example, the IC50 values of cabozantinib for VEGFR2 and RET are 0.035 nM and 5.2 nM, respectively170. Drug-related adverse events resulting from ‘off-target’ activity include hyper tension, hand–foot syndrome, and proteinuria attributed to VEGFR2 inhibition7,182,183; rash owing to EGFR or BRAF inhibition184,185; hypo-pigmentation due to KIT inhibition186; QT prolon-gation as a result of potassium voltage-gated channel sub family H member 2 (hERG) inhibition187; and diarrhoea as a consequence of EGFR inhibition188. Drug-related toxicities have limited long-term dosing of cabozantinib, vandetanib, and lenvatinib in prospec-tive clinical trials, with dose- reduction rates of 23–79% and treatment-discontinuation rates of 6–21% (TABLE 1; TABLE 2); therefore, achieving optimal RET-inhibitory plasma concentrations of these agents is difficult.

Conversely, the ‘off-target’ activity of multikinase inhibitors, specifically the antiangiogenic effects of these drugs, has been hypothesized to contribute to

their clinical efficacy. With regard to thyroid can-cer, responses to multikinase inhibitors have been achieved both in patients with and in those without documented RET mutations (TABLE 1); however, the true contri bution of the antiangiogenic activity of these agents to patient responses remains unclear. In the context of MTC, the clinical experience with motesanib is noteworthy. Unlike most other multi-kinase inhibitors, motesanib not only possesses 30-fold higher inhibitory activity against VEGFR2 than wild-type RET in enzymatic assays, but also has minimal activity against RET variants with M918 or C634 substitutions (FIG. 4). Despite this greater potency against VEGFR2, results of a phase II trial of mote-sanib in patients with advanced-stage MTC (70% of whom had tumours that harboured RET mutations) demonstrated an ORR of only 2%189. Notwithstanding the caveats of cross-trial comparisons, the much lower ORR with motesanib than with other agents that pos-sess more-potent activity against RET suggests that RET inhibition, either alone or in combination with VEGFR2 inhibition, is necessary to achieve responses in a larger proportion of patients with MTC.

Intrinsic resistance. The possible existence of intrin-sic resistance mechanisms is a second explanation for the limited activity of targeted therapy in RET-dependent tumours compared with cancers with other drivers. In RET-rearranged NSCLC, the type of upstream RET fusion partner has been hypo thesized to modify responses to multikinase inhibitors. In a Japanese phase II trial of vandetanib involving patients with RET-rearranged NSCLC11, a lower ORR and a shorter median PFS duration were observed among patients with tumours harbouring KIF5B–RET ver-sus those with tumours harbouring CCDC6–RET (KIF5B–RET: ORR 20% and median PFS 2.9 months; CCDC6–RET: ORR 83% and median PFS 8.3 months). Similar results were obtained in a phase Ib study of RXDX-105 (KIF5B–RET: ORR 0%; non-KIF5B–RET: ORR 75%)190. Preclinically, the presence of KIF5B upstream of RET results in a substantial increase in RET transcription, possibly increasing the amount of chimeric RET oncoprotein that needs to be over-come by tyrosine-kinase inhibition45. By comparison, a lower degree of RET expression driven by other upstream RET fusion partners, such as CCDC6–RET and NCOA4–RET, has been detected in thyroid can-cers191. In patients with thyroid cancers harbouring non-gatekeeper RET mutations, the influence of muta-tion type on clinical response is less well character-ized beyond RETM981T, the presence of which has been associated with an ORR of 34% with cabozantinib177.

Acquired resistance. RET mutations that confer resistance of RET-dependent tumours to multikinase inhibitors have been identified in preclinical models. Examples are the gatekeeper mutations RETV804M and RETV804L (REF. 167), and the non-gatekeeper muta-tion RETI788N (REF. 156). The RETG810R and RETG810A solvent-front mutations, the former of which is

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paralogous to the ALKG1202R, ROS1G2032R, NTRK1G595R, and NTRK3G623R resistance mutations192, provide addi-tional putative mechanisms of resistance167. To date, however, these mutations have not been reported in tumour samples from patients with RET-rearranged or RET-mutant cancers who develop acquired resistance to multikinase inhibitors. In ALK-rearranged NSCLCs, the frequency of acquired ALK mutations is higher after treatment with more-potent second- generation (alectinib or ceritinib) versus first-generation (crizo-tinib) ALK inhibitors193. This finding suggests that the lack of clinical data on acquired RET mutations in RET-dependent tumours is indicat ive of suboptimal RET inhibition with multi kinase inhibi-tors. As a corollary to this hypothesis, these mutations might be more likely to emerge in tumours that develop acquired resistance to more-potent RET inhibitors193.

C onc ur rent genomic a l te rat ions and/or bypass-pathway activation might have a role in con-ferring resistance. For example, EGFR was found to functionally interact with RET in RET-rearranged thy-roid cancer cell lines171,194, and subsequent studies in

a RET-rearranged lung cancer cell line indicated that EGFR-mediated signalling potentially mediates resist-ance to multikinase inhibition195,196. In one study, which also included ALK-rearranged, ROS1-rearranged, and NTRK-rearranged models, ligand-mediated EGFR activation resulted in fusion kinase re-phosphorylation and destabilization of multikinase inhibitor binding, a shift in adaptor protein binding from fusion kinases to EGFR, and increased MAPK-pathway activation in the context of ligand-mediated EGFR activation195. MAPK pathway reactivation via the acquisition of KRAS or NRAS mutations has likewise been shown to engender resistance to multikinase inhibitors in RET-rearranged lung cancer cell lines168. Finally, acquired amplification of MDM2, which encodes the E3 ubiquitin-protein ligase MDM2, has been identified in RET-rearranged NSCLC samples from patients who developed resist-ance to cabozantinib197. MDM2 is also concurrently amplified in a substantial number of patients with RET-rearranged NSCLC197; thus, MDM2 amplifica-tion potentially underlies both intrinsic and acquired resistance to multikinase inhibition.


24

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Med

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sur

viva

l (m

onth

s)

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II

Phase ofclinical testing

III

ALK-rearrangedROS1-rearranged

EGFR-mutant

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VandetanibVandetanib

Erlotinib

Afatinib

Lenvatinib

Ceritinib

Gefitinib

Crizotinib

CeritinibOsimertinib

AlectinibCrizotinib

Cabozantinib

Figure 5 | Comparative efficacy of RET‑directed targeted therapy in RET‑rearranged lung cancers. The efficacy of kinase‑inhibitor therapy in molecularly enriched subgroups of tyrosine‑kinase‑inhibitor treatment‑naive patients with non‑small‑cell lung cancers (NSCLCs) is depicted as a bubble plot showing the objective response rate (ORR) on the x‑axis and median progression‑free survival (PFS) duration on the y‑axis. Each circle on the plot represents a clinical trial of kinase‑inhibitor therapy directed at a specific driver of oncogenesis. Circle sizes represent different phases of clinical testing, as indicated in the key on the right. In comparison with the outcomes with single‑agent targeted therapy in patients with EGFR‑mutant176,204–206, ALK-rearranged3,207,208, or ROS1‑rearranged NSCLCs4,209, and recognizing the limitations of cross‑trial comparisons, relatively lower ORRs and median PFS durations have been achieved with single‑agent multikinase inhibition in patients with RET‑rearranged NSCLCs9–12. Notably, however, the ORRs achieved in the latter group of patients still exceed the historical ORRs achieved with single‑agent chemotherapy administered after progression on platinum‑doublet chemotherapy (for example, the ORR with second‑line, single‑agent docetaxel is

Emerging approaches to targeting RETCombination therapyIn other molecular tumour subsets, combination ther-apy has been shown to improve clinical outcomes com-pared with those achieved with single-agent targeted therapy. Specifically, in patients with BRAFV600E-mutant melanoma or NSCLC, combination BRAF and MEK inhibitor therapy has been shown to improve ORRs and PFS in comparison with single-agent BRAF inhi-bition5,18. The concurrent inhibition of RET and medi-ators of downstream signalling pathways has been explored in several contexts. First, RET inhibition has been shown to induce activation of the MAPK pathway in preclinical models that harbour activating RET alter-ations; combined RET and MEK inhibition has been explored in vitro, resulting in the abrogation of MAPK signalling156. Second, the coadministration of RET and mTOR inhibitors can augment growth inhibition of RET-dependent thyroid tumour cell lines198, compared with that achieved using either agent alone. Moreover, combined mTOR inhibition has been postulated to enhance the entry of vandetanib into the CNS24. The combination of vandetanib and the mTOR inhibitor everolimus is being investigated in an ongoing phase I trial involving patients with RET-rearranged or RET-mutant cancers24 (NCT01582191); objective responses have been observed in patients with tumours harbour-ing either alteration, and intracranial disease control was also noted in one patient with RET-rearranged, AKT2-amplified lung adenocarcinoma metastatic to the brain24. Finally, RET-dependent tumours might rely, in part, on the activity of EGFR or MDM2, as detailed previously, and the concurrent inhibition of RET and EGFR or MDM2 has been explored preclinically; combination therapy was more effective than RET inhibition alone in decreasing tumour-cell prolifera-tion or restraining tumour growth in RET-rearranged models195,197. Further studies are needed to determine whether co-dependencies on these pathways or other pathways in cancers primarily reliant on RET signalling are meaningful and amenable to combination therapy.

RET-specific inhibitorsNovel and potent inhibitors have been developed to selectively target the RET kinase. Two of these

RET-specific inhibitors, BLU-667 (REF. 25) and LOXO-292 (REF. 26), have broad preclinical activity against various RET rearrangements and RET muta-tions, including RETV804M/L gatekeeper mutations. These drugs were designed to surpass the limitations of multikinase inhibitors by sparing non-RET targets (such as VEGFR2)21,22,159, with the intent of decreasing toxicities that limit chronic drug dosing in patients. Furthermore, the potential decrease in ‘off-target’ adverse effects might enable the administration of these novel agents at higher doses, thereby improv-ing RET inhibition. Both RET-specific inhibitors have entered early phase clinical testing (NCT03037385, NCT03157128); the results of these trials are likely to inform the debate regarding the utility — or lack thereof — of concurrent inhibition of RET and angio-genic kinases in lung and thyroid cancers that harbour activating RET alterations.

ConclusionsActivating RET mutations and RET rearrangements drive the pathogenesis of several cancers, notably thyroid and lung carcinomas. Inhibition of RET can abrogate the growth of various cell lines and tumour xenografts that harbour these alterations. More importantly, these drivers are clinically actionable, and in several prospective clinical trials, the use of multikinase inhibitors with activity against RET has been associated with confirmed responses and dura-ble disease control in select patients with RET-mutant or RET-rearranged cancers. Multikinase inhibitors are, however, characterized by the substantial inhibi-tion of non-RET targets, including VEGFR2, resulting in drug-related adverse events that can limit chronic dosing and full on-target inhibition of RET. On the other hand, the concurrent inhibition of multiple targets might contribute to the observed therapeutic response. The development of RET-specific inhibitors that have limited off-target effects are likely to eluci-date the utility of more-selective inhibition of RET in patients with RET-mutant and RET-rearranged tumours. In parallel with these efforts, combination therapies are being explored in tumours harbour-ing RET mutations or rearrangements to boost the activity observed with existing agents in the clinic.

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