Abstract
The association between RET and multiple endocrine neoplasia type 2 was established in 1993 and remains one of the very few oncogenes for which distinct phenotypes (medullary thyroid cancer or pheochromocytoma) are associated with the same hot-spot variants occurring in either germline or somatic DNA. Somatic RET fusion events have also been described in several cancers, including papillary thyroid cancer, non-small-cell lung cancer, breast cancer, salivary gland cancer and pancreatic cancer. Highly selective RET inhibitors have improved outcomes in RET-altered cancers and have been well-tolerated. Nevertheless, primary and acquired drug resistance has been observed, arising from distinct genomic alterations either in RET (on-target resistance) or via alternate oncogenic pathways (bypass resistance). The same mechanisms of resistance have been observed across multiple cancer types, which implies RET-altered cancers evolve away from RET addiction via stochastic subclonal events. Understanding these mechanisms is crucial for identifying therapeutic opportunities to overcome resistance. Successful treatment targeting bypass oncogenes has been reported in several instances, at least for short-term outcomes; in contrast, although several compounds have been reported to overcome on-target RET alterations, none have yet been translated into routine clinical practice and this remains an area of urgent clinical need.
Introduction
After the cloning of rearranged during transfection (RET) proto-oncogene on chromosome 10q11.2 (Takahashi et al. 1985), its association with various human malignancies was progressively uncovered: first, with finding somatic RET rearrangements (as fusion genes) in up to 20% papillary thyroid cancers (PTC) (Grieco et al. 1990, Viglietto et al. 1995) and then establishing RET germline mutations as the cause of multiple endocrine neoplasia type 2A (MEN2A) (Donis-Keller et al. 1993, Mulligan et al. 1993) and MEN2B (Carlson et al. 1994, Eng et al. 1994, Hofstra et al. 1994). Since then, hot-spot mutations in RET exons 8, 10, 11 and 13–16 and rarely 1 and 5 have been found in nearly all cases of MEN2A and 2B syndromes (Gild et al. 2023). Remarkably, the same mutations (most commonly M918T; this review will use amino acid terminology commonly used by clinicians throughout, i.e. M918T instead of p.Met918Thr) also occur somatically in around 25–50% of sporadic medullary thyroid cancers (MTC) (Romei et al. 2016). Somatic RET rearrangements (as fusion genes with CCDC6, NCOA4, KIF5B, PRKAR1A, TRIM33 and other rarer partners) have now been found in 1–2% of non-small-cell lung cancers (NSCLC) and at lower prevalence in many other cancers including pancreatic, colon, breast and salivary gland (Ou & Zhu 2020). Intriguingly, RET fusions also develop as a mechanism of acquired resistance to EGFR inhibitor treatment in NSCLC (Piotrowska et al. 2018, Wang et al. 2022).
The protein RET is a single transmembrane tyrosine kinase, which normally forms a cell-surface homodimer complex, which on binding glial-derived neurotrophic factor family neurotrophins triggers transautophosphorylation of tyrosine residues in the intracellular kinase domain (Manie et al. 2001) (Fig. 1A). These phosphotyrosines then recruit several adaptor proteins to mediate the activation of downstream signaling cascades including phospholipase Cγ/protein kinase C, c-Jun N-terminal kinases, Src-related kinases and nuclear factor κB, P13 kinase/AKT and the MAP kinase pathways (Li et al. 2019).
Normal and pathological RET signaling: (A) RET is normally activated after binding of glial cell line-derived neurotrophic factor (GDNF) ligand dimers to GDNF family receptor-alpha (GFRα) co-receptors, triggering transautophosphorylation of tyrosine residues in the RET cytoplasmic domain (starbursts), (B) mutations in RET cysteine-rich domain associated with MTC lead to ligand-independent homodimerization via interchain disulfide bridges, (C) mutations at RET M918T or A883F associated with MTC lead to monomeric activation of the kinase domain and (D) RET fusion partners associated with PTC, non-small-cell lung cancers or other cancers provide a dimerization domain for RET homodimers to form intracellularly and activate downstream signaling cascades. Data from Drilon et al. 2018a,b, Hu et al. 2023. Created with BioRender.com.
Citation: Endocrine-Related Cancer 32, 2; 10.1530/ERC-24-0224
RET mutations cause constitutive kinase activation by one of the three main mechanisms (Drilon et al. 2018a): (a) ligand-independent homodimerization via interchain disulfide bonds (classically seen in RET mutations at cysteine residues in codons 610 and 634; Fig. 1B), (b) monomeric activation of the kinase domain (e.g., RET M918T and A883F; Fig. 1C) and (c) for RET fusions, dimerization or oligomerization via coiled-coil or leucine-zipper domains in the fusion partner (Fig. 1D). RET gain-of-function by amplification is also seen in a few cancer types (Kato et al. 2017).
This strong genotype–phenotype relationship has driven the search for RET kinase inhibitors for treating RET-altered cancers. These inhibitors have targeted mainly the ATP-binding pocket within the catalytic domain (Santoro & Carlomagno 2006). Modest success of multikinase inhibitors (MKIs) paved the way for more selective RET inhibitors, which have now become first-line therapies for RET-altered cancers ((Kurzrock et al. 2011), Wells et al. 2012, Wirth et al. 2020). However, these successes have been moderated by the emergence of drug resistance, which continues to challenge clinical practice and inform further efforts in novel drug design.
This review examines mechanisms of resistance to RET inhibitors in the context of their discovery in clinical cases and experimental systems, including on-target and bypass mechanisms but also sanctuary sites (brain) and dose-limiting tolerability. The focus will be on clinical trials of RET inhibitors in thyroid cancer, drawing on the extensive insights gained from their use in NSCLC.
Defining resistance
Drug resistance is defined as the progression of disease despite maximally tolerated doses. Resistance may be primary or acquired; primary resistance is defined where best oncological response is progressive disease, whereas acquired resistance is defined when progressive disease occurs after a period of objective response. Mechanisms of resistance can be divided broadly into on-target (variation in the target that prevents drug response), off-target or bypass (activation of an alternate oncogenic pathway), or reduced drug accumulation at the target site (either by the lack of penetration (e.g., cerebral) or by the upregulation of drug-efflux pumps) (Lin & Shaw 2016). Lineage plasticity – where adenocarcinomas develop resistance through epithelial-to-mesenchymal transition, seen in some cases of NSCLC treated with EGFR inhibitors (Schoenfeld et al. 2020) – has been reported in two recent cases of resistance to RET inhibition (Gazeu et al. 2023, Peng et al. 2023) although this mechanism of resistance does not appear to be common (Lin et al. 2020, Rosen et al. 2022). Progressive dedifferentiation and increased proliferative rate has been noted in subjects who developed resistance to RET inhibition (Hadoux et al. 2023b).
To confirm a mechanism of resistance, it is not sufficient for an oncogenic variant to be present simply at the time resistance emerges; for instance, PI3K signaling activation is not uncommonly observed in RET-altered cancers but does not appear to be associated with resistance to RET inhibitors (Rosen et al. 2022). Rather, the putative resistance mechanism must be shown to correlate with disease progression across multiple sites of disease and in multiple patients, be validated in experimental systems and, ideally, be shown to respond to retargeted therapy where available.
Discovering mechanisms of resistance to targeted therapies has accelerated rapidly through routine use of next-generation sequencing (NGS), initially on tissue biopsies but more recently using liquid biopsies of cell-free and circulating tumor DNA (ctDNA), which can monitor emergence of a range of oncogenic pathways including genetic variation in the target at reasonable sensitivity. Limitations of liquid biopsy need also to be considered – limit of detection is generally around 1% variant allele frequency, which may miss resistance variants at an early stage, and NGS panels may not include all possible variations associated with resistance (Subbiah et al. 2021d). Nevertheless, the noninvasive nature of liquid biopsies has facilitated personalized cancer care adapted to treatment-emergent resistance pathways.
RET inhibition: MKIs
Similarity of the ATP-binding domain in many tyrosine kinase receptors led to the therapeutic development of MKIs with activity against multiple targets. Initial MKIs were developed focusing on the inhibition of VEGF receptors with vandetanib emerging as a highly potent inhibitor of Flt-1 (VEFGR-1) and KDR (VEGFR-2) and of RET kinase activity including oncogenic RET mutants and fusion isoforms (Carlomagno et al. 2002, Vidal et al. 2005). In the ZETA trial, patients with locally advanced or metastatic MTC treated with vandetanib had superior progression-free survival (PFS) compared to placebo (19.3 vs 30.5 months, respectively; hazard ratio (HR) 0.46; 95% CI 0.31–0.69) (Wells et al. 2012). Vandetanib received FDA approval for treatment of non-resectable advanced or metastatic MTC in 2011.
Cabozantinib is a potent inhibitor of c-MET, VEGFR2 and other kinases including RET, FLT3, TRK and KIT (Yakes et al. 2011). In patients with progressive MTC in the EXAM study, cabozantinib improved PFS compared to placebo (11.2 vs 4 months), although objective response rate (ORR) was only 28% (Elisei et al. 2013). The median overall survival after long-term follow-up (minimum 42 months) in MTC harboring RET M918T on cabozantinib was 44.3 vs 18.9 months for those on placebo (HR, 0.60; 95% CI, 0.38–0.94) (Schlumberger et al. 2017). Treatment-related adverse events were frequent and often dose-limiting including diarrhea, fatigue, hypertension and other toxicities (Elisei et al. 2013). Cabozantinib was FDA-approved for treatment of MTC in 2012. Cabozantinib has also been studied in other RET-altered cancers including renal cell carcinoma (Choueiri et al. 2014) and NSCLC (Drilon et al. 2016).
Other MKIs have been studied in RET-altered cancers including lenvatinib, which had modest success in MTC with ORR 36% and PFS of 9 months (Schlumberger et al. 2016). Lenvatinib was FDA-approved for radioiodine-refractory differentiated thyroid cancer in 2015, some of which cases will be associated with RET fusions (Schlumberger et al. 2015).
Dose-limiting tolerability of these MKIs emphasizes two points: first, since tolerated doses are often below that required for complete RET inhibition, their efficacy may in fact depend on inhibiting VEGF receptors or other kinases; second, incomplete RET inhibition may increase the risk of treatment resistance emerging (Tiedje & Fagin 2020). Nonetheless, these MKIs remain the cornerstone of managing patients with non-RET-driven advanced, progressive MTC.
Early pre-clinical data showed naturally occurring RET V804L/M gatekeeper mutations conferred resistance to vandetanib via steric hindrance to drug binding (Carlomagno et al. 2004). Notably, these mutations occur spontaneously at the germline or somatic level in MEN2A or sporadic MTC, respectively, and cause constitutive ligand-independent activation of RET signaling (Iwashita et al. 1999). Subsequently, these gatekeeper mutations were shown to emerge in patients with RET-altered MTC or NSCLC after treatment with either vandetanib or cabozantinib (Dagogo-Jack et al. 2018, Subbiah et al. 2018a,b, Wirth et al. 2019) (Supplementary Table (see the section on Supplementary materials given at the end of the article)). Another mechanism of vandetanib resistance was reported in a patient with NSCLC and CCDC6-RET, in which acquisition of RET S904F resulted in increased basal kinase activity (Nakaoku et al. 2018).
Other non-gatekeeper RET mutations may be resistant to these MKIs; RET D898_E901del was shown to be insensitive to cabozantinib and partially sensitive to vandetanib (Porcelli et al. 2023). Nevertheless, some patients with MTC associated with this variant had short-term tumor control on cabozantinib possibly due to its antiangiogenic effects (Porcelli et al. 2023).
Some MKIs including vandetanib may have poor cerebral penetration leading to low response rates in patients with brain metastases (Drilon et al. 2018a,b).
RET inhibition: highly specific inhibitors
First-generation selective inhibitors were designed to bind the RET kinase domain but avoiding steric clash with the gatekeeper V804L/M mutations described above. Unlike MKIs, which access the front and back clefts of RET kinase domain via the ‘gate’ at V804, selpercatinib (LOXO-292) and pralsetinib (BLU-667) avoid the gate by a wrap-around access to front and back clefts (Subbiah et al. 2021a). These drugs are also far more selective for the RET kinase, with less off-target kinase inhibition and therefore better tolerability (Brandhuber et al. 2016).
Promising preclinical and early clinical studies of these two selective RET kinase inhibitors (Subbiah et al. 2018, Wirth et al. 2019) led to phase 1/2 trials LIBRETTO-001 (selpercatinib) (Drilon et al. 2020a, Wirth et al. 2020) and ARROW (pralsetinib) (Gainor et al. 2021a, Subbiah et al. 2021b). These clinical trials recruited patients who were naïve to MKIs, and those who had been pre-treated. Both selpercatinib and pralsetinib showed durable responses not only in RET-altered thyroid cancers and NSCLCs but also in other malignancies associated with RET mutations (Drilon et al. 2020a, Wirth et al. 2020, Gainor et al. 2021a, Subbiah et al. 2021b, 2022a,b). For instance, in treatment-naïve MTC, ORR was 73% for selpercatinib and 71% for pralsetinib; in pretreated MTC, ORR was 69 and 60%, respectively; and in RET fusion PTC, ORR was 79 and 89%, respectively (Wirth et al. 2020, Subbiah et al. 2021b). Median PFS rates are not yet defined although complete responses were uncommon. Cerebral penetration was also evident for each inhibitor with responses of intracranial metastases from NSCLC (Drilon et al. 2020a, Gainor et al. 2021a). Treatment-related adverse events were usually mild and most commonly hypertension, elevated transaminases and fatigue/asthenia; more serious but less common adverse events included hypersensitivity or QT prolongation (selpercatinib) and pneumonitis or anemia/neutropenia (pralsetinib). Dose reductions for toxicity were seen in 30% of patients on selpercatinib and 46% on pralsetinib, with discontinuation rates of only 2–4% for either (Wirth et al. 2020, Subbiah et al. 2021b). Efficacy of these two targeted treatments despite previous MKI therapy again emphasizes the importance of achieving maximal inhibition of RET kinase when treating these cancers. Selpercatinib is now being studied in neoadjuvant settings for advanced thyroid cancers (NCT04759911).
Recently, results from a phase 3 randomized trial (LIBRETTO-531) reported superior progression-free survival with selpercatinib as compared with physician’s choice of either cabozantinib or vandetanib in treatment-naive patients with progressive, advanced RET mutant medullary thyroid cancer; hazard ratio for disease progression or death was 0.28 (95% CI, 0.16–0.48; P < 0.001), and PFS at 12 months was 86.8% (95% CI, 79.8–91.6) in the selpercatinib group and 65.7% (95% CI, 51.9–76.4) in the control group (Hadoux et al. 2023a). ORR was 69.4% (95% CI, 62.4–75.8) in the selpercatinib group – including 11.9% with complete responses – and 38.8% (95% CI, 29.1–49.2) in the control group. Treatment discontinuation was only 4.7% in the selpercatinib arm compared to 26.8% in the control (cabozantinib or vandetanib) arm.
Importantly, both selpercatinib and pralsetinib efficacy appears independent of the mechanism by which RET is activated – response rates have been similar in cancers containing RET M918T, other missense RET variants associated with MEN2A and RET fusions, emphasizing the central role RET kinase activity plays in these cancers. Although selpercatinib and pralsetinib are generally well-tolerated, real-world surveillance identified small bowel edema, chylous effusions and erectile dysfunction as potential adverse effects of selpercatinib (Kalchiem-Dekel et al. 2022, Tsang et al. 2022, Matrone et al. 2024). Despite the success of these highly selective RET inhibitors, complete responses are seen in only 10% patients and the presence of residual disease allows for the development of treatment resistance.
Primary resistance
Primary resistance to highly selective RET inhibitors is rare, occurring in around 2% of MTC patients on selpercatinib in clinical trials (Wirth et al. 2020, Hadoux et al. 2023a,b). A molecular cause for primary resistance to selpercatinib was reported for two patients with RET fusions, in whom pre-treatment plasma sequencing revealed KRAS mutations (G12D and G12V), the allele fractions of which increased on treatment (Rosen et al. 2022) (Supplementary Table). Interestingly, in each case, the RET fusion remained suppressed on treatment raising the possibility multiple tumor clones with distinct drivers (either RET or KRAS) might co-exist at the baseline and behave differently under selective pressure of RET inhibition (Rosen et al. 2022). In other cases, the cause of primary resistance to selpercatinib has not been established (Pitoia et al. 2024).
Acquired resistance: on-target
On-target RET mutations causing resistance to selective kinase inhibitors occur at the solvent front, hinge region or roof of the ATP-binding pocket (Fig. 2). These mechanisms of RET resistance were foreshadowed by preclinical studies. Using random mutagenesis of KIF5B-RET vectors transduced into Ba/F3 murine B cells and treated with cabozantinib, lenvatinib or vandetanib, several RET kinase domain mutations were identified as pan-resistance markers including solvent front G810S and roof L730I (Liu et al. 2018), which would later be identified as mediating resistance to selpercatinib and pralsetinib.
On-target RET resistance. Ribbon diagram of RET kinase–selpercatinib complex (7JU6:chain B) (Subbiah et al. 2021a). Key residues mediating on-target resistance to selpercatinib are shown as red dots: solvent front (G810), hinge (Y806), gatekeeper (V804) and roof (L730). The commonest codon mutated in MTC, RET M918, is also shown in proximity to the activation loop. Created with BioRender.com after importing structure from the Protein Data Bank (PDB).
Citation: Endocrine-Related Cancer 32, 2; 10.1530/ERC-24-0224
The first clinical description of solvent front mutations was in a 61-year-old man with metastatic KIF5B-RET NSCLC progressing after carboplatin, pemetrexed, pembrolizumab and lenvatinib who commenced selpercatinib and had rapid clinical improvement until progression at 6 months and death at 7 months. Serial ctDNA analyses found RET G810S emerged some 3 months before clinical progression became apparent. Remarkably, tissue samples obtained at rapid autopsy were heterogeneous for distinct metastatic subclones containing G810R or G810S or G810C (Solomon et al. 2020). This report was extended by finding acquired mutations at RET G810 in three additional cases with RET-altered cancers progressing on selpercatinib: CCDC6-RET fusion NSCLC, RET fusion NSCLC and RET mutant MTC, respectively (Solomon et al. 2020) (Supplementary Table).
Since then, RET solvent front mutations have been reported in many other RET-altered NSCLC and MTC treated with either selpercatinib or pralsetinib, accounting for around 20–25% cases of acquired resistance (Lin et al. 2020, Subbiah et al. 2021a, Gainor et al. 2021b, Rosen et al. 2022, Hadoux et al. 2023b) (Fig. 3) (Supplementary Table).
Range of mechanisms for resistance to RET inhibitors. On-target (solvent front and hinge) and off-target (KRAS mutations, oncogene fusion, MET amplification, ‘other’) resistance mechanisms are shown for (A) MTC and (B) NSCLC. Data derived from cases where molecular mechanism for resistance to highly specific RET inhibitor is reported (see Supplementary Table). Created with BioRender.com.
Citation: Endocrine-Related Cancer 32, 2; 10.1530/ERC-24-0224
Extensive experimental evidence supports mutation at RET G810 as a mechanism of resistance to selpercatinib. RET G810S was shown to evolve in a patient-derived xenograft model using a lung cancer sample with CCDC6-RET in mice treated orally with selpercatinib (Solomon et al. 2020). In vitro experiments confirmed RET G810S/R/C mutants were resistant to selpercatinib, pralsetinib, cabozantinib and vandetanib (Solomon et al. 2020, Subbiah et al. 2021a). BaF3 cells containing KIF5B-RET and subject to a random mutation library or prolonged culture with selpercatinib acquired RET V738A, Y806C/N and G810S; of note, V738A is outside coverage of clinical cell-free DNA assays and therefore may be an undetected cause of on-target resistance. RET G810 substitutions had minor effects on ATP affinity, consistent with direct inhibition of drug binding rather than increased kinase activity (Subbiah et al. 2021a). Structural modeling showed these acquired RET mutations caused steric hindrance to selpercatinib binding (Solomon et al. 2020, Subbiah et al. 2021a).
Rosen et al. (2022) extended the spectrum of on-target RET resistance describing a novel mutation in the hinge region (Y806C) when in cis with V804M; in combination, these mutations were shown to induce steric hindrance to selpercatinib binding. Intriguingly, the double V804M/Y806C mutation has been reported in an MEN2B-like family and shown in vitro to increase RET-transforming activity by 8–13-fold higher than that of single RET mutants (Iwashita et al. 2000). Aside from this unique case, and in contrast to the gatekeeper mutation RET V804M, which accounts for 26% cases of MEN2 (Elisei et al. 2019), available evidence suggests that no other naturally occurring RET mutants are intrinsically resistant to selpercatinib (Szymczak et al. 2023); IC50 in vitro is mildly higher for some mutants, such as KIF5B::RET and RET E632_L633del compared with RET M918T, but preclinical evidence suggests complete RET inhibition is achieved at concentrations of selpercatinib likely to be achieved in vivo, regardless of the underling RET mutation.
There may be differences in RET resistance to selpercatinib or pralsetinib; for instance, L730V/I mutations in the roof of the solvent front are resistant to pralsetinib but not apparently to selpercatinib (Shen et al. 2021) (Fig. 2). A preliminary analysis of pralsetinib resistance occurring in the ARROW trial reported these RET L730 mutations in ctDNA samples (Gainor et al. 2021b).
RET mutations mediating on-target resistance to highly specific inhibitors will be resistant to MKIs as well (Fancelli et al. 2021), emphasizing the urgent need to find new therapies to overcome this mechanism of resistance. This has so far proven challenging. Promising preclinical data have been reported for several compounds including TPX-0046, a potent RET/SRC inhibitor with preclinical potency against RET G810 solvent front mutations, which was in phase 1 testing in patients with advanced RET-altered solid tumors (NCT04161391) (Drilon et al. 2020b), and vepafestinib (TAS0953/HM06), a next-generation RET inhibitor, which inhibited RET variants at codons L730, V804 and G810 with a unique binding mode and additionally showed improved brain penetration in an intracranial model of RET-driven cancer (Miyazaki et al. 2023) and which is being investigated in an ongoing phase 1/2 clinical trial (NCT04683250).
Acquired resistance: bypass
Bypass resistance is defined by progressive disease occurring despite ongoing suppression of RET and accompanied by the activation of one or more alternate oncogenic pathways. Indeed, most of the cases progressing on RET-selective inhibitors are driven by these off-target, RET-independent mechanisms of resistance (Lin et al. 2020, Hadoux et al. 2023b) (Fig. 3). Bypass resistance has been associated with the reactivation of the MAPK pathway via oncogene gain by amplification (MET, KRAS, FGFR1 or HER2), activating mutations (KRAS, HRAS, NRAS, BRAF or MAP2K), fusion events (NTRK or ALK) or tumor suppressor loss (CDKN2) (Table 1) (Fig. 3).
Mechanisms of resistance to RET inhibitors.
| Target | Mechanism | Mutation | Resistant to | Susceptible to | References |
|---|---|---|---|---|---|
| Primary | |||||
| On-target | Gatekeeper: Steric hindrance | RET V804L/M | Cabozantinib, vandetanib | Selpercatinib, pralsetinib | Subbiah et al. (2018a,b), Wirth et al. (2019) |
| Bypass | Oncogene mutation | KRAS G12D/V | Selpercatinib | - | Rosen et al. (2022) |
| Acquired | |||||
| On-target | Gatekeeper: Steric hindrance | RET V804L/M | Cabozantinib, vandetanib | Selpercatinib, pralsetinib | Subbiah et al. (2018a,b), Wirth et al. (2019) |
| On-target | Solvent front: Steric hindrance | RET G810S/C/R/V | Selpercatinib, pralsetinib | - | Solomon et al. (2020), Lin et al. (2020), Subbiah et al. (2021b), Rosen et al. (2022), Hadoux et al. (2023b) |
| On-target | Hinge: Steric hindrance | RET Y806C (in cis with V804M) | Selpercatinib, pralsetinib | - | Rosen et al. (2022), Hadoux et al. (2023b) |
| On-target | Roof: Steric hindrance | RET L730I | Pralsetinib | - | Gainor et al. (2021b) |
| Bypass | Oncogene amplification | MET | Selpercatinib, pralsetinib | Crizotinib, capmatinib | Lin et al. (2020), Rosen et al. (2021), Zhu et al. (2021), Leite et al. (2023) |
| KRAS | 〃 | - | Lin et al. (2020) | ||
| FGFR1 | 〃 | - | Rosen et al. (2022) | ||
| HER2 | 〃 | Trastuzumab | Vakkalagadda & Patel (2023) | ||
| Bypass | Oncogene activating mutation | KRAS G12A/R/V, G13D, A59del | Selpercatinib, pralsetinib | - | Rosen et al. (2022) |
| KRAS G12D | 〃 | - | Hadoux et al. (2023b) | ||
| NRAS G13E, Q61R | 〃 | - | Rosen et al. (2022) | ||
| BRAF | 〃 | - | Rosen et al. (2022) | ||
| HRAS A59T | 〃 | - | Hadoux et al. (2023b) | ||
| MYCN P44L | 〃 | - | Hadoux et al. (2023b) | ||
| MAP2K E102-I103 del | 〃 | Pishdad et al. (2024) | |||
| Bypass | Oncogene fusion | NTRK3 | Selpercatinib, pralsetinib | Larotrectinib | Subbiah et al. (2021d) |
| ETV6::NTRK3 | Larotrectinib | Subbiah et al. (2024) | |||
| EML4::ALK | Entrectinib | Subbiah et al. (2024) | |||
| Bypass | Tumor suppressor loss | CDKN2A, CDKN2B | Selpercatinib, pralsetinib | - | Porcelli et al. (2023) |
MET amplification is a common off-target signaling pathway in NSCLC bypassing drivers such as EGFR mutations or ALK fusions (Engelman et al. 2007, Piotrowska et al. 2018, Dagogo-Jack et al. 2020, Schoenfeld et al. 2020). Similarly, MET amplification has been found in patients with RET fusion positive NSCLC progressing on selpercatinib or pralsetinib (Lin et al. 2020, Rosen et al. 2021, 2022, Zhu et al. 2021) (Fig. 3). In one case, the addition of crizotinib (a MET inhibitor) to selpercatinib extended treatment response by 10 months (Rosen et al. 2021). In vitro data showed MET overexpression-induced selpercatinib resistance, which could be overcome by crizotinib (Lin et al. 2020). Another case of selpercatinib resistance associated with MET amplification achieved durable response after adding capmatinib (Leite et al. 2023).
Subbiah et al. (2024) have recently reported a remarkable example of multiple bypass resistance mechanisms emerging on RET inhibition, several of which were targeted successfully with sequential combination therapy. A patient presenting with metastatic MTC associated with somatic RET D898_E901del was treated with selpercatinib after thyroidectomy and neck dissection; disease progression at 24 months associated with ETV6::NTRK3 fusion was treated with larotrectinib in addition to selpercatinib; further disease progression after another nine months associated with EML4::ALK fusion was treated by switching from larotrectinib to entrectinib; unfortunately, further progression associated with NTRK3 G623R ultimately led to treatment failure. This report also emphasized the utility of using liquid biopsy to detect these bypass mechanisms and guide personalized treatment changes. NTRK3 fusion had previously been shown to induce resistance to selpercatinib in KIF5B-RET-transformed BaF3 cells, which was responsive to the combination of selpercatinib and larotrectinib (Subbiah et al. 2021d).
Similar success in targeting bypass mechanisms of RET inhibitor resistance was seen using trastuzumab to target HER2 amplification (Vakkalagadda & Patel 2023).
Interestingly, HRAS mutations at Q61 and G13, which are the most frequent non-RET mutation in sporadic MTC (and mutually exclusive to RET mutations) (Xu et al. 2024), have not yet been described as a mechanism of resistance to RET inhibitors.
Resistance due to other mechanisms
Although favorable CNS responses have been reported in patients treated with selpercatinib or pralsetinib (Drilon et al. 2020a, Gainor et al. 2021a), not all patients show response in the brain. A recent report demonstrated around one-third of patients with baseline brain metastases suffered from CNS progression while on therapy with selpercatinib (Subbiah et al. 2021c). Variable expression of ATP-binding cassette (ABC) transporters, especially the multidrug resistance protein 1 (MDR1, also known as P-glycoprotein encoded by ABCB1), is known to confer resistance to cytotoxic and targeted chemotherapy and may be particularly relevant for brain penetration (Robey et al. 2018). Animal models have shown P-glycoprotein (ABCB1/MDR1) and BCRP (ABCG2) limit brain accumulation of selpercatinib (Wang et al. 2021) and further research is required to determine whether this may account for some cases of RET inhibitor resistance.
Limitations of existing data and areas for future research
Despite progress in understanding the mechanisms of RET resistance, many cases of both primary and acquired resistance remain unexplained (Rosen et al. 2022, Pitoia et al. 2024). There is clearly more to be learned about pathways to resistance and ways to block these. Fibroblast growth factor receptor (FGFR) signaling may play a role both as a driver of some thyroid cancers and as a mechanism of resistance to RET inhibition (Rosen et al. 2022, Sabbagh et al. 2024), and FGFR inhibition has been shown to mitigate resistance to a selective RET inhibitor in CCDC6-RET containing TPC1 cells and a transgenic zebrafish model (Raman et al. 2022). In MTC cell lines, pralsetinib resistance was found to be accompanied by aberrant activation of the Hedgehog/GLI pathway which was susceptible to mitigation by Hh/GLI inhibitors (Trocchianesi et al. 2023). Undoubtably, multiple mechanisms of resistance are likely to evolve and co-exist with prolonged treatment on selective inhibitors.
At this stage, it seems unlikely immune evasion represents a mechanism of resistance to RET inhibition. In a retrospective study of 74 patients with RET-rearranged lung cancers, both PD-L1 expression and tumour mutation burden (TMB) were low, and outcomes in 16 patients who had received immunotherapy were poor (Offin et al. 2019). Whether RET inhibition alters this immunologically cold phenotype is unknown.
The biological basis for determining whether a cancer develops on-target or bypass resistance remains unclear. In some cases, resistance-causing mutations may be preexisting (Lin & Shaw 2016, Rosen et al. 2021, 2022). Polyclonal resistance has also been observed: solvent front RET mutations co-occurring with KRAS G12 mutations have been reported in two cases, suggesting that independent clonal trunks had emerged on selpercatinib treatment (Rosen et al. 2022, Hadoux et al. 2023b). Although most cases of bypass resistance are associated with persistent suppression of RET (evidenced on liquid biopsy), in at least some cases the bypass resistance variant emerges in parallel with increasing RET allele frequency, and it is unclear whether this represents simply a passenger event to the bypass loop or loss-of-target engagement by RET inhibitors (Zhu et al. 2021, Lin et al. 2020). Whether upfront combination treatments might prevent acquired resistance needs further study. A recent report highlighted the utility of cabozantinib targeting simultaneously both MET and RET in a case of NSCLC in which acquired resistance to MET exon 14 skipping arose via emergence of a RET fusion on treatment with a MET-specific inhibitor (Torrado et al. 2024).
The rapid uptake of liquid biopsies has accelerated clinical translation of known mechanisms of resistance but is less sensitive than tissue biopsies to clonal heterogeneity and may impede discovery of the full range of genetic evolution under selective pressure of kinase inhibitors (Lin & Shaw 2016). Diagnostic accuracy of liquid biopsies depends on shedding of tumor-derived DNA (in turn, dependent on cancer type, extent and location of disease) and coverage and depth of the assay; whereas older assays for ctDNA had limited sensitivity for some RET variants and fusions (Allin et al. 2018, Ciampi et al. 2022), newer NGS assays have improved concordance with tissue testing and can detect RET mutations or fusions at very low allelic frequencies (Belli et al. 2021). Nevertheless, even using an ultra-deep sequencing targeted cancer gene panel, Rosen et al. (2022) detected RET alterations on liquid biopsy in 95% of RET mutant cases but only 74% of RET fusion cases, and in cases who had developed resistance, longitudinal plasma samples were able to detect a genomic mechanism of resistance in only 61% cases. Moreover, it is still crucial to know which genes are covered to properly interpret a ctDNA result; for instance, in the case of advanced MTC reported by Subbiah et al. (2024) mentioned above, an ETV6::NTRK3 fusion was not covered on a liquid biopsy panel and only detected on tissue biopsy using a combined DNA/RNA-based NGS assay.
More broadly, there is no clear consensus on when and how to perform genetic reassessment when resistance to RET inhibition is suspected in clinical practice, particularly in resource-constrained healthcare – acknowledging responses of RET-altered cancers to highly selective inhibitors such as selpercatinib are often durable for years (Hadoux et al. 2023a). The European Society of Medical Oncology has published recommendations for methods to diagnose RET fusions and mutations at initial presentation, but this did not include guidance for re-sampling on diagnosis of progression on RET inhibitor therapy (Belli et al. 2021). The many research studies cited herein highlight the range of mechanisms by which resistance is acquired and also emphasize finding a result may not lead to change in management. Detection of a druggable target may be worthwhile for some patients but requires assays to cover a higher number of potential bypass oncogenes, whereas finding a solvent front or hinge RET mutation can be achieved using more focused sequencing but offers little solace in the absence of an efficacious second-generation RET inhibitor. A cost-effective approach may be to use targeted sequencing on biopsies of radiologically progressing lesions where possible, reserving higher coverage NGS assays when ‘hot-spot’ mutations (e.g., RET solvent front or KRAS G12X) are absent.
More data for acquired resistance come from lung cancer cases than from thyroid cancer cases at this stage (Supplementary Table). Moreover, clinicopathological risk factors associated with acquired resistance are yet unclear: specifically, whether specific tumor features (e.g., cancer type, Ki-67, tumor mutation burden, metastatic burden and duration of disease), pre-treatment with other therapies or duration and/or dose of selective inhibitor contribute most strongly to acquisition of resistance. A recent study reported outcomes from 46 patients with MTC on RET inhibitors, of whom 16 progressed and four died; notably, serial biopsies were available from some cases and showed increasing Ki-67 and more poorly differentiated histology as progressive disease emerged on treatment (Hadoux et al. 2023b).
Conclusions
The discovery of the RET proto-oncogene and its association with cancer has led to the development of highly specific RET inhibitors, which have transformed the therapeutic landscape for RET-altered cancers, including thyroid and NSCLCs. This success is tempered by the emergence of resistance to these inhibitors with prolonged treatment; whether resistance is inevitable in every case remains to be seen. Molecular mechanisms have been defined for primary and acquired resistance, including on-target and bypass oncogenic activation. Strategies for preventing or treating resistance are beginning to be identified, particularly when bypass resistance is mediated by a druggable oncogene (e.g., NTRK, ALK, MET or HER2); in such cases, it is recommended to also continue RET inhibitor therapy. Conversely, solventfront mutations may be susceptible to second-generation RET inhibitors currently in development but account for only 20–25% cases of resistance. There is still an urgent need to find new therapies for patients who otherwise succumb rapidly once resistance develops. Future research will define optimal timing for starting targeted therapies, stratifying those at greatest risk of developing resistance, and targeting patient-specific resistance with next-generation RET inhibitors or combination therapies either up-front or sequentially.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/ERC-24-0224.
Declaration of interest
The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of the work.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Acknowledgements
The author is grateful to Dr Matti Gild for critical review of the manuscript.
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