The emerging role of targeted protein degradation to treat and study cancer

The evolution of cancer treatment has provided increasingly targeted strategies both in the upfront and relapsed disease settings. Small‐molecule inhibitors and immunotherapy have risen to prominence with chimeric antigen receptor T‐cells, checkpoint inhibitors, kinase inhibitors, and monoclonal antibody therapies being deployed across a range of solid organ and haematological malignancies. However, novel approaches are required to target transcription factors and oncogenic fusion proteins that are central to cancer biology and have generally eluded successful drug development. Thalidomide analogues causing protein degradation have been a cornerstone of treatment in multiple myeloma, but a lack of in‐depth mechanistic understanding initially limited progress in the field. When the protein cereblon (CRBN) was found to mediate thalidomide analogues' action and CRBN's neo‐targets were identified, existing and novel drug development accelerated, with applications outside multiple myeloma, including non‐Hodgkin's lymphoma, myelodysplastic syndrome, and acute leukaemias. Critically, transcription factors were the first canonical targets described. In addition to broadening the application of protein‐degrading drugs, resistance mechanisms are being overcome and targeted protein degradation is widening the scope of druggable proteins against which existing approaches have been ineffective. Examples of targeted protein degraders include molecular glues and proteolysis targeting chimeras (PROTACs): heterobifunctional molecules that bind to proteins of interest and cause proximity‐induced ubiquitination and proteasomal degradation via a linked E3 ligase. Twenty years since their inception, PROTACs have begun progressing through clinical trials, with early success in targeting the oestrogen receptor and androgen receptor in breast and prostate cancer respectively. This review explores important developments in targeted protein degradation to both treat and study cancer. It also considers the potential advantages and challenges in the translational aspects of developing new treatments. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Thalidomide and its analogues
Thalidomide was the first immunomodulatory drug to be developed, initially as an anti-emetic during pregnancy.Following widespread use in the 1950s, it was found to be teratogenic and subsequently withdrawn from use [1,2].Multiple theories were put forward, including thalidomide's generation of reactive oxygen species and antiangiogenic properties that would be particularly devastating during embryonic development [3].The latter was initially attributed to the inhibition of fibroblast growth factor and depletion of vascular endothelial growth factor receptors [3][4][5].More recent experimental evidence has implicated the developmental transcription factors p63 and SALL4both thalidomide targets known to cause limb defect syndromes when mutated [6,7].
Interest in repurposing thalidomide as a potential treatment for multiple myeloma (MM) emerged on the basis that bone marrow angiogenesis was increased in MM patients.A 1990s trial comprising 84 patients with relapsed MM found thalidomide to be effective at reducing the paraprotein burden arising from abnormal expansion of plasma cells that is pathognomonic of the disease process [8].Building on its initial success, thalidomide showed benefit in conjunction with a host of other regimens in the upfront and relapsed/refractory treatment of MM including cyclophosphamide, dexamethasone, melphalan, proteasome inhibitors, cisplatin, doxorubicin, and etoposide [9,10].Over time, thalidomide analogues' so-called immunomodulatory effect in the bone marrow microenvironment was shown to extend beyond inhibition of angiogenesis to the inhibition of NF-κB signalling, activation of caspases, and induction of apoptosis within the target cell [9,11].
Lenalidomide is a second-generation thalidomide analogue developed for use in MM.In combination with dexamethasone, lenalidomide showed response rates of 55-60% in patients with relapsed disease [12,13].Alongside dexamethasone and proteasome inhibition with bortezomib, lenalidomide quickly became the standard of care in the upfront treatment of MM following a 2010 trial that demonstrated 18-month progression-free and overall survival as 75% and 97% respectively [14].It was also utilised in the posttransplant setting as maintenance, with significant improvement in progression-free and overall survival in a randomised phase 3 trial where 85% of patients in the lenalidomide group versus 77% of patients in the placebo group were alive at 3-year follow-up [15].Pomalidomide, with its increased substrate specificity, became the most potent and least toxic thalidomide analogue available for treatment in MM [16,17].Its 10-fold increase in potency compared with lenalidomide is thought to be in part attributed to its degradation of the ARID2 subunit of the chromatin remodelling complex poly-bromo-associated BRG1/BRM-associated factor (PBAF), which upregulates the proto-oncogene MYC [18,19].ARID2 is a neo-substrate specific to pomalidomide and not targeted by lenalidomide.A progression-free survival benefit was demonstrated with pomalidomide alongside low dose dexamethasone in the relapsed disease setting, catalysing its use in a range of other pomalidomide-based regimens in relapsed myeloma [20][21][22].

Protein degradation and discovery of cereblonmediated thalidomide analogue action
Protein degradation is a highly regulated homeostatic process in response to abnormal protein folding, cell stress or to facilitate protein turnover to enable amino acid availability [23].Proteins marked for degradation are ubiquitinated through the sequential action of an E1 ubiquitin-activating enzyme, an E2 ubiquitin conjugating enzyme, and an E3 ubiquitin ligase [24].The tagged protein is then degraded by the 26S proteasome made up of a barrel-shaped 20S core particle with proteolytic active sites and the 19S regulatory particles that facilitate recognition, deubiquitination, and transfer of the protein to the core particle [25].
A ubiquitously expressed protein called cereblon (CRBN) acts as a substrate receptor for the CRL4 CRBN E3 ubiquitin ligase complex, which also comprises the ligase scaffold cullin-4 (CUL4), RING-finger protein RING-box1 (RBX1), and the adapter damage-specific DNA binding protein 1 (DDB1) [26].CRBN binds endogenous substrates via amino acid degrons and guides the E3 ubiquitin ligase complex to ubiquitinate specific proteins, which are then degraded by the 26S proteasome (Figure 1) [26,27].N-terminal targets are well established, and it was recently discovered that the C-terminal cyclic imide degron arising from post-translational modification of glutamine or asparagine amino acids was recognised by CRBN with high specificity [28,29].These C-terminal imide degrons are found in ageing proteins that have been rendered malfunctional following chain breaks and are therefore targeted for degradation [29].
In 2010, it was discovered that thalidomide analogues bound to CRBN to facilitate protein degradation, as illustrated by CRBN knockdown that conferred resistance to pomalidomide and lenalidomide but not to other standard myeloma treatments, including bortezomib, dexamethasone, and melphalan [30,31].Mechanistically, thalidomide analogues mimic the substrate C-terminal cyclic amide degron and can be functionally substituted to engage CRBN and enable proteolysis of 'neo-substrates' via a β-hairpin loop with a sentinel glycine residue [28,31,32].As molecular glues, thalidomide analogues therefore enable the E3 ligase complex to be in proximity with and ubiquitinate proteins that would not otherwise interact to be targeted for degradation in this way.
The widely used immunosuppressant cyclosporin was the earliest described molecular glue via its interaction with calcineurin and cyclophilin [33].The term molecular glue now encompasses a heterogenous group of compounds that facilitate protein-protein interactions.Since the discovery that thalidomide analogues operate as neomorphic substrate molecular glues, other molecular glues with differing mechanistic modalities have been described.These include molecular glues that act as neomorphic receptors and via covalent modification and polymerisation, with examples of these shown in Table 1 [34][35][36].
Multiple neo-substrates have been identified as amenable to thalidomide analogue-induced degradation including the zinc-finger-and non-zinc-finger-containing transcription factors and proteins listed in Figure 1 [32,37].Notable intrigue arose around the degradation of the developmental transcription factor SALL4 because SALL4 mutations in humans cause limb defect syndromes comparable to those seen in newborns exposed to thalidomide [7].The breakdown of SALL4 may therefore explain thalidomide's teratogenicity to a greater extent than theories around reactive oxygen species and inhibition of angiogenesis.Importantly, mice do not develop the same characteristic limb defects in response to thalidomide analogue exposure.This is due to species-specific differences in the sequences of CRBN and SALL4 that prevent binding, ubiquitination, and degradation of SALL4 in the presence of thalidomide analogues.
There are over 20 amino acid variations between mouse and human CRBN.The most significant is V388 in human CRBN, which predicts thalidomide-induced teratogenicity and corresponds to I391 in mice, which predicts a lack of response to thalidomide [38].The significance of this isoleucine residue is demonstrated by the induction of thalidomide sensitivity in Crbn I391V mice, enabling degradation of hallmark thalidomide targets, including Ikaros family zinc finger proteins Ikaros (IKZF1) and Aiolos (IKZF3) [38].For the GSPT1 targeting drug eragidomide (CC-90009, discussed below), an additional humanisation of CRBN, V380E, is required for substrate degradation in mouse models [39].

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Even in the presence of humanised CRBN, thalidomideinduced degradation of SALL4 is not seen in mice due to key mutations in the ZnF2 domain of SALL4.However, humanising the ZnF2 domain with a five amino acid substitution was sufficient to enable thalidomide-induced degradation of SALL4 in the presence of human CRBN [7,40].These species-specific differences exemplify a limitation of studying thalidomide analogues in mouse models and help to explain why the teratogenicity observed in humans was not foreseen by preclinical studies on thalidomide.
As outlined in Figure 2, molecular glues exhibit event-driven pharmacology from transient and iterative interactions with proteins of interest (POIs) to enable degradation (as opposed to inhibition).This differs from occupancy-driven pharmacology classically employed by small-molecule inhibitors that is dependent on a specific binding site to inhibit POIs [41].Not only does this render up to 85% of the proteome 'undruggable', but these active sites are prone to mutations that can confer drug resistance [42].Moreover, relatively high drug concentrations are required, which can further drive resistance and toxicity compared to the sub-stoichiometric concentrations required for degraders [43].

Thalidomide analogue neo-substrates as cancer drug targets
There has been widespread interest in IKZF1 and IKZF3 as neo-substrates [44,45].These transcription factors  The emerging role of targeted protein degradation 405 play an essential role in lymphocyte development and differentiation [46][47][48].Downstream targets include the lymphocyte-activating transcription factor IRF4, inhibition of which is toxic to myeloma cells [49].IRF4 also drives MYC expression, which promotes aberrant B-cell proliferation in myeloma [49].Accordingly, IKZF1 and IKZF3 have been harnessed as therapeutic targets across a host of malignancies.Phase I studies of patients with solid organ and haematological malignancies exposed to the CRBN modulating agent avadomide (CC-122) not only yielded promising safety data but also showed signs of response in the non-Hodgkin's lymphoma subgroups [50][51][52].A dedicated follow-up phase I study of patients with relapsed/refractory diffuse large B-cell lymphoma showed good tolerability and an overall response rate of 29% [53].
Non-zinc-finger-containing proteins with analogous β-hairpin loops and sentinel glycine residues were also found to be degraded by thalidomide analogues.For example, it was found that lenalidomide bound to CRBN targets the tumour suppressor gene casein kinase 1 alpha (CK1α), which is essential for normal haematopoiesis [54].Biallelic loss of CK1α results in stem cell failure, whereas heterozygous deletion results in stem cell hyperproliferation, as seen in myelodysplastic syndrome (MDS) [54].Because of the location of CK1α within the 5q deletion region of 5q deleted MDS [del (5q) MDS], lenalidomide has proven to be highly effective against haploinsufficient del(5q) MDS [55].In a cohort of 148 patients with del(5q) MDS, 67% achieved transfusion independence and 45% of evaluable patients achieved complete cytogenetic remission [56].The structural basis of lenalidomide-induced CK1α degradation via CRL4 CRBN is by CRBN and lenalidomide jointly providing the binding interface for a CK1α β-hairpin-loop with the kinase N-lobe [57].Conflicting studies have shown this process to be thalidomide analogue dependent and independent, with the latter proposed via activation of Wnt signalling that stimulates CRBN to associate with and degrade CK1α [58].
The process of CK1α degradation is p53 dependent, and patients with mutated p53 del(5q) MDS show resistance to lenalidomide [59,60].Unlike lenalidomide, pomalidomide does not degrade CK1α as part of p53-mediated apoptosis [55].This is relevant in the context of therapy-related myeloid neoplasms; TP53 mutations have been found to be associated with lenalidomide exposure driving clonal haematopoiesis and a selective advantage for p53-mutant haematopoietic stem and progenitor cells (HSPCs) [61].With pomalidomide not showing the same selective pressure on p53-mutated HPSCs, in the setting of MM there may be a shift in preferentially using pomalidomide over lenalidomide to reduce the risk of p53 mutant therapy-related myeloid neoplasms.
A breakthrough in targeted protein degradation occurred with the discovery of dual degraders of both CK1α and IKZF2: DEG-35 and DEG-77 [62].IKZF2 is central to myeloid leukaemic stem cell activity and functions to block myeloid differentiation, making it an attractive target in acute myeloid leukaemia (AML).DEG-35 and DEG-77 operate in a CRBN-dependent manner to induce myeloid differentiation and slow AML progression in human and mouse models [62].
Selective IKZF2 degraders have also been developed to target IKZF2's role in maintaining regulatory T-cell function, which contributes to immune evasion.NVP-DKY709 is a selective IKZF2 degrader currently being evaluated in phase I trials for patients with advanced solid tumour malignancies [63,64].Structure-guided drug development approaches such as these offer both the potential to expand the repertoire of druggable substrates and enable degradation of multiple relevant targets at nanomolar concentrations.
Another POI in AML is GSPT1, a β-hairpin loop but non-zinc-finger-containing protein that functions to regulate protein translation and the cell cycle [65].In 2016, a novel thalidomide analogue derivative (CC-885) was discovered to act in a CRBN-dependent manner to target GSPT1 with potent anti-tumour activity in cell lines including patient-derived AML cells [65].The clinical-grade GSPT1 targeting modulator eragidomide (CC-90009) demonstrated pro-apoptotic and anti-proliferative activity in a range of AML xenograft assays and promising anti-leukaemic activity in a phase I trial of patients with relapsed/refractory AML [66,67].CC-90009 is also being investigated in a phase Ib trial in combination with other anti-leukaemic agents, including venetoclax, azacitidine, and gilteritinib [68].The novel GSPT1-degrading modulator SJ6986 has also shown in vitro and in vivo efficacy against acute lymphoblastic leukaemia (ALL) [69].Beyond acute leukaemias, other novel GSPT1 degraders such as MRT-2359 have shown anti-proliferative activity

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MH Brodermann et al in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cell lines [70].MRT-2359 is now in phase I/II for patients with NSCLC, SCLC, highgrade neuroendocrine cancers, diffuse large B-cell lymphoma (DLBCL), and tumours with L-MYC or N-MYC amplification [71].A second GSPT1 degrader (ORM-5029) is also in phase I investigation for patients with HER2-expressing advanced solid tumours [72].A dual degrader of c-MYC/GSPT1 (GT19715) has been described with preclinical evidence of efficacy in leukaemia and lymphoma settings [73].
The cytotoxic mechanism of GSPT1 degradation is through disruption of translation termination and activation of the integrated stress response that leads to cell death [39].In a mouse leukaemia model with humanised CRBN, in vivo activity of GSPT1 degradation was demonstrated with relative sparing of haematopoietic stem cells [39].Despite significant structural and functional differences between GSPT1 and existing neo-substrates such as IKZF1, it was noted that there was a commonality in the degron responsible for recruiting the E3 ubiquitin ligase to its substrate [65].Indeed, using systematic proteomic screening assays, additional neo-substrates of CC-885 have been identified, including the proto-oncogene PLK1, cyclin-dependent kinase 4 (CDK4), and the proapoptotic protein BNIP3 [74].BNIP3 also has a role in mitophagy, and AML cell lines deficient in BNIP3 are sensitive to the mitochondria-targeting drug rotenone [75].

Overcoming the challenge of drug resistance
Resistance to protein-degrading drugs remains a significant barrier to effective treatment in relevant disease settings and prompts novel drug development to bypass resistance mechanisms.This challenge is exemplified by emergent resistance to thalidomide analogues, which can be divided into CRBN and non-CRBN mechanisms.Quantitative mass spectrometry demonstrated that CRBNmediated resistance mechanisms included the abundance of competing substrates for CRBN, the strength of the ligase-substrate interaction, and the level of CRL4 CRBN E3 ubiquitin ligase components [17].
The relative levels of CRBN and its substrates as well as the presence of CRBN mutations correlate with the efficacy of thalidomide analogue activity against myeloma cells [76][77][78][79].CRBN overexpression has also been shown to sensitise cell lines to thalidomide analogues that were previously resistant [17,80].This would support the hypothesis that malignant cells develop resistance by upregulating either competing unrelated substrates or by upregulating target substrates like IKZF1 and IKZF3, relying on increased CRBN availability [81].Thalidomide analogue concentration should also be an important consideration in the context of resistance: anti-proliferative activity and CRBN mRNA concurrently fell with increasing concentrations of lenalidomide in MM cell lines while pomalidomide efficacy was maintained [82].
Sequence analysis in a cohort of 50 refractory MM patients demonstrated that over 20% had an acquired mutation in CRBN, a component of the E3 ligase complex, or a downstream pathway [83].The six CRBN-mutated patients all had differing mutations at critical sites impairing thalidomide analogue binding.These patients, as well as 91% of the CRBN pathway-mutated patients, were unresponsive to thalidomide analoguebased treatment [83].Independently of CRBN expression or its sequence, increased IRF4 is present in MM cell lines generated to be resistant to thalidomide analogues in comparison to those that were sensitive [84].This could be overcome with molecules that target IRF4 signalling and indicates the importance of downstream targets as contributors to resistance.
In practice, there is a reliance on more potent thalidomide analogues such as pomalidomide when patients are refractory to first-line thalidomide analogue therapy.This has also resulted in the generation of novel cereblon E3 ligase modulators (CELMoDs) such as iberdomide (CC-220) and mezigdomide (CC-92480), which differ from thalidomide analogues in having additional phenyl and morpholino moieties to enable up to 20Â higher affinity binding with CRBN, which results in more potent degradation of IKZF1 and IKZF3 in comparison to lenalidomide and pomalidomide [85][86][87].This may be a result of effective degradation in the presence of lower levels of CRBN.Iberdomide could also maintain activity in pomalidomide resistant cell lines, and preliminary data from the first phase Ib/IIa trial of 58 patients with relapsed/refractory MM indicate that over 50% of patients achieve disease benefit with iberdomide in combination with dexamethasone [88,89].A phase I/II study of mezigdomide in combination with bortezomib and dexamethasone showed an overall response rate of 74% with acceptable tolerance and a median duration of 10 months in 19 patients with relapsed/refractory MM [90].
Non-CRBN-mediated mechanisms of resistance including immune and tumour microenvironmental interactions are increasingly being studied [91].The observation that co-administration of daratumumab with lenalidomide/ pomalidomide can overcome resistance in patients who are refractory to both drugs suggests a synergistic potentiation of drug activity, immune cell activation, change in CD38 expression, or other microenvironmental change independent of CRBN [92].CELMoDs have also been shown to expand and activate T-cells and natural killer (NK) cells as a possible contributing factor to overcoming thalidomide analogue resistance [93].Exploring the tumour microenvironment and indirect targets beyond the tumour cells is an exciting and comparatively underresearched area in protein degradation.

Proteolysis-targeting chimeras
Before the mechanism of thalidomide analogues had been discovered, targeted protein degraders such as proteolysis-targeting chimeras (PROTACs) were in The emerging role of targeted protein degradation development.PROTACs are heterobifunctional small molecules with a 'warhead' ligand bound to the POI joined by a linker molecule to a ligand that binds an E3 ligase (Figure 3).Together, these form a ternary complex that results in proximity-induced ubiquitination and degradation of the POI via the ubiquitinproteasome system (UPS) [94].Despite overlapping features, they differ in this way from monovalent thalidomide analogue 'molecular glues' that bind to and mediate a more direct interaction of an E3 ligase with a POI.
The concept of chimeric degraders was filed in a 1999 patent [95].The first PROTAC itself was described in 2001, binding the E3 ligase β-TRCP to target METAP2, which itself binds to the angiogenesis inhibitor ovalicin [96].Von Hippel Lindau (VHL) and CRBN have since become the predominant E3 ligases incorporated into PROTAC design.Their widespread expression facilitates systemic targeted protein degradation but also raises the possibility of off-target toxicity and side effects.Other E3 ligases used include MDM2, RNF4, RNF114, DCAF15, DCAF16, KEAP1, and cIAP, and with more than 600 E3 ligases encoded by the genome, the potential range of binding targets for PROTAC technology remains extensive [97,98].
Ubiquitination of surface lysine residues is a key component of proteasomal degradation.As such, the availability and distribution of lysine residues on a POI impacts the potency and selectivity of PROTACs [99].The site of the variable PROTAC linker influences which lysine residues can be ubiquitinated and is therefore of increasing interest in the structural design of PROTACs.The length of the linker is also an important consideration; inadequate linker length sterically impairs the engagement of the PROTAC components, whereas excessive linker length undermines the proximity requirement for PROTAC function [100].The linker attachment points therefore directly influence PROTAC selectivity and neo-substrate degradation [101].The combination of optimising the choice of E3 ligases and its ligand, and manipulating the structure of the linker molecule has allowed newer PROTACs to be more selective and up to a thousand-fold more potent than their predecessors [102].

Proteolysis-targeting chimera targets and clinical applications
Unlike small-molecule inhibitors, degraders including PROTACs can target both the enzymatic and scaffolding functions of proteins.The innate immune protein IRAK4 is a notable example showing the potential benefit of this.IRAK4 activates downstream inflammatory signalling both as a kinase via Toll-like and IL-1 family receptors and as a scaffold in the Myd88-IRAK4-IRAK1/2 complex [103,104].Simple inhibitors of IRAK4 kinase activity have shown limited efficacy in inflammatory diseases [105].By contrast, KT-474, a heterobifunctional degrader of IRAK4 that removes both kinase and scaffold functions, shows enhanced efficacy [106].
The first PROTACs to enter phase I clinical trials were ARV-471, which targets the oestrogen receptor (ER), and ARV-110, which targets the androgen receptor (AR).ARV-471 monotherapy was evaluated for ER + /HER2 À locally advanced or metastatic breast cancer and ARV-110 in metastatic castration-resistant prostate cancer [111][112][113][114].Both drugs bind CRBN as their E3 ligase and entered phase I clinical trials in 2019 before progressing to phase II trials by 2021 following promising safety data and demonstration of clinical activity [113,114].Orally bioavailable ARV-110 demonstrated in vivo degradation of AR at over 90% the observed maximum degradation (Dmax) [115].ARV-110 was also superior to the AR antagonist enzalutamide at reducing tumour growth and was efficacious in an enzalutamide-resistant model [116].
Resistance mechanisms including mutations in the AR and ER hormone receptors, especially in pretreated patients, may limit the efficacy of targeted drugs, necessitating the exploration of combination and novel treatments in ongoing trials.ARV-110 is being investigated in a phase Ib trial to see if co-administration with the anti-androgen drug abiraterone can reverse resistance in patients resistant to abiraterone [117].
Like the AR, the ER is a well-established target in the treatment of breast cancer, but drug resistance similarly limits existing hormonal therapy options [118].In xenograft models, ARV-471 inhibited tumour growth at over 90% (compared to 46% by the ER degrader fulvestrant) and at over 65% in a tamoxifen-resistant xenograft model [119].Combination therapies have also been tested with ARV-471 and the CDK4/6 inhibitor

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MH Brodermann et al palbociclib, together having a greater effect on tumour growth inhibition than either drug alone, prompting further evaluation in phase I/II trials of patients with advanced-stage ER + HER2 À breast cancer [119,120].A second-generation PROTAC-targeting the ER (AC682) has shown similarly promising initial results both alone and in combination with palbociclib [121].Other PROTACs following ARV-471, AC682, and ARV-110 into clinical development are listed in Table 2 [97,122].
Targets include B-cell malignancies, sarcomas, and autoimmune disease.Bruton's tyrosine kinase (BTK)-targeted PROTACs are among the fastest progressing in haematological malignancies.BTK affects B-cell receptor signalling through PI3K, MAPK, and NF-κB [123] and is the target of the breakthrough small-molecule BTK inhibitor (BTKi) ibrutinib that was first reported to be efficacious in B-cell lymphomas in 2010 [124].Ibrutinib and subsequent generations of BTKis have since enjoyed huge success across chronic lymphocytic leukaemia (CLL), B-cell lymphomas, and chronic graft versus host disease and remain under evaluation for use in autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus [123,[125][126][127]. PROTACs targeting BTK are in development, including CRBN-based NX-2127, which demonstrated efficacious BTK degradation across multiple B-cell malignancy cell lines (including those resistant to ibrutinib), as well as superiority to existing BTKis [128,129].A phase I study of NX-2127 showed promising initial data of 86% mean BTK degradation in patients with relapsed/refractory B-cell malignancies [130].In a CLL cohort, an overall response rate of 40.7% was observed [131].NX-5948 is a PROTAC that also targets BTK but, unlike NX-2127, was designed to do so independently of IKZF1/3 degradation.Preclinical data showed over 50% degradation in B-cell malignancy cell lines, and early phase I trial data are encouraging [132,133].
Specific and non-genetic inhibitors of apoptosis protein (IAP)-dependent protein erasers (SNIPERs) are chimeric molecules that also hijack endogenous proteolysis to cause targeted protein degradation.They differ from CRBN or VHL-based PROTACs in that they harness IAP ubiquitin ligases such as cIAP1 and XIAP, which are themselves degraded alongside the target protein [134].IAPs are overexpressed in tumour cells, influencing NF-κB signalling and inhibiting caspase activity, and can therefore contribute to drug resistance [134].This allows SNIPERs to play an elegant dual role as a targeted protein degradation tool.Effective SNIPERs have been developed for targets including AR, BCL-XL, BCR-ABL, BTK, BRD4, CDK, CRABPII, EGFR, ER, HDACs, H-PGDS, IRAK4, JAK, mHtt, NOTCH1, P97, PDE4, and RARα [134][135][136][137][138][139][140].SNIPERs may also play adjunctive roles as proven by a SNIPER developed against transforming acidic coiled-coil-3 (TACC3) using XIAP that showed synergism with bortezomib in cancer cell lines [141].SNIPERs are an exciting development in the targeted protein degradation field but share the same challenges as conventional PROTACs including optimising component choice and design.The emerging role of targeted protein degradation 409

Limitations to proteolysis-targeting chimeras
Despite their potential to access novel protein targets, there are multiple barriers to PROTAC development.
The first is that, despite orally and topically administered PROTACs progressing through clinical trials, their physiochemical properties, including relatively high molecular weight, mean they do not conform to the classical Lipinski 'Rule of 5' that would usually confer good oral absorption [122,142,143].Moreover, they are subject to the 'hook effect' of concentration informing PROTAC activity levels.Higher concentrations risk saturation of E3 ligase or POI binding sites, limiting ternary complex formation and, in turn, protein degradation [144].Moreover, as previously noted, there are a wide array of potential ligands, E3 ligases, and linker confirmations.While this offers broad scope, the optimal structural and functional combinations will take time to realise with a reliance on focused medicinal chemistry and structural biological studies.Alongside traditional crystallographyand spectroscopy-based approaches, there will be increasing use of artificial intelligence in the elucidation of binding structures.There are also tissue-specific considerations of E3 ligase expression.For example, one study of a BTK-directed PROTAC showed differential BTK degradation in the liver and spleen despite similar tissue uptake [140].Furthermore, although largely regarded as a benefit of PROTAC technology, whole protein degradation compared to inhibition means there are off-target and toxicity unknowns that require further enquiry.
Finally, PROTACs offer a solution to drug resistance but themselves are susceptible to acquiring resistance, for example, through mutations in POIs or E3 ligases [145,146].Altering constituent components of PROTACs such as the E3 ligase is already a potential solution to acquired PROTAC resistance [147].Certain targets, including BCL6 (a key B-cell transcriptional repressor), also appear to be more resistant to effective PROTAC degradation in comparison to novel small-molecule degraders that induce target polymerisation and degradation [148].

Protein degradation technology as a tool to study disease dTAG
Tagging proteins of interest with a degradation tag offers an alternative approach to targeted protein degradation.As shown in Figure 4, dTAG protein degradation employs a lentivirus or CRISPR/Cas9 to express the target protein in frame with mutant FKBP12 F36V , which is identified by a 'dTAG' comprising an E3 ligase ligand linked to an FKBP12 F36V ligand.Akin to PROTACs, this forms a complex that results in proximity-induced ubiquitination and degradation of the target protein [149].However, contrary to classical PROTAC or SNIPER heterobifunctional protein degraders, dTAG circumvents the requirement to identify a specific ligand binding to POIs.Its ability to tag endogenously enables the study of protein degradation when the protein is expressed at physiological levels.dTAG has proven effective in the degradation of BRD4 and treatment of leukaemia cell lines in vivo [149].

HaloTag
HaloTag technology tags a hydrophobic group via a fusion protein to a POI to give the appearance of partial denaturation, which marks the protein for degradation by the proteasome [150].HaloTags are effective at reducing tumour burden in vivo and have been incorporated into PROTAC technology (HaloPROTACs) that uses small-molecule VHL or cIAP ligands to successfully degrade HaloTag fusion proteins (Figure 4) [150][151][152].HaloTags have also been incorporated into transcription factor-targeting chimeras (TRAFTACs) that degrade transcription factors of interest.These comprise a chimeric oligonucleotide that binds to the transcription factor and HaloTag-fused dCas9 protein to mediate proteasome-induced degradation [153].(E) TRIM-Away with the E3 ligase TRIM21 bound to an antibody that is also bound to the protein of interest (POI), causing the entire complex to be degraded following E3 ligase-mediated ubiquitination.

Auxin-inducible degrons
Another approach is degron-induced degradation whereby a degron tag is bound to a POI, activated by the presence of a specific ligand, and degraded via the UPS [154].The most well characterised is the auxin-inducible degron (AID) system using the plant hormone auxin as the regulating ligand.Auxin bound to the SCF TIR1 ubiquitin ligase triggers the degradation of auxin-responsive transcriptional repressors, the degron sequence of which can be bound to the POI (Figure 4) [154][155][156].Administering auxin subsequently induces degradation of the bound protein.There is an incomplete understanding of how expressing a plant protein impacts mammalian cells; however, with no known mammalian targets, off-target effects are theoretically negated [155].Drawbacks include leaky degradation and the requirement for high doses of auxin, both of which have been comprehensively overcome with a modified secondgeneration version of the AID [157].

IKZF3 degrons
A further tractable degron-mediated degradation paradigm builds on IKZF3 as a known target of thalidomide analogues bound to the CRL4 CRBN E3 ubiquitin ligase complex.Koduri and colleagues mapped a thalidomide analogue-responsive IKZF3 degron of 25 amino acids that could be fused to a target protein for thalidomide analogue-induced degradation [158].Advantages include its small size and ease of incorporation, but further characterisation studies are required after only 30-40% of targeted proteins were degraded by this system [158].

Super-degrons
The use of degron-mediated degradation to target zinc fingers has evolved through identification of zinc finger subdomains that combine to form thalidomide analoguedependent 'super-degron' hybrids that enable degradation at significantly reduced ligand concentration [159].By incorporating super-degrons into chimeric antigen receptor (CAR)-cell technology, an on/off switch can be created as a safety mechanism in the setting of immune hyperactivation syndromes that carry significant morbidity and mortality [160].The on switch harnesses a lenalidomide-inducible dimerisation system with a split CAR that requires both CD19 and the presence of lenalidomide.The off switch is via introduction of a degron tag into the CAR to facilitate lenalidomide-induced degradation of the CAR.This reversible on/off switch-containing CAR was tested in CD19 + mantle cell lymphoma cells engrafted into mice and showed anti-tumour activity [159].

SMASh tags
Small-molecule-assisted shutoff (SMASh) tags have also been developed, where the POI is fused to a SMASh tag degron via a hepatitis C virus nonstructural protein 3 (NS3) protease [161].In the absence of an NS3 protease inhibitor, the degron removes itself via protease activity and the protein is left untagged.In the presence of an NS3 protease inhibitor, degron removal is blocked and the POI is subject to degradation in a dose-dependent manner [161].

TRIM-away
Unlike the aforementioned tagging tools, TRIM-Away technology (Figure 4) harnesses the ability of the E3 ligase TRIM21 to bind to antibody Fc domains [162].The antibody is also bound to the POI, causing the entire complex to be degraded following E3 ligase-mediated ubiquitination.However, not only must there be an antibody known to bind the POI, but the antibody-TRIM21 interaction is only possible by artificially overcoming the antibody's inability cross into the cell (e.g. by electroporation) [162].TRIM-Away degrades proteins in human and murine cell lines and offers a targeted protein degradation tool in nondividing cells without the requirement for genetic manipulation in dTAG or IKZF3 degron tools [162].

Protein degradation to unlock disease biology
These technologies exemplify the range of proteindegrading tools available to study the biology of disease.The acceleration of targeted protein degradation owes its success to the sophistication of proteomic analysis that enables target identification.For example, mass spectrometry-based approaches, such as stable isotope labelling with amino acids in cell culture (SILAC), have played a central role in discovering thalidomidedependent substrates of CRL4 CRBN such as ZFP91 [163].The paradigm in haematological and solid organ malignancies thus far has been to characterise and utilise targets in the context of new and existing protein degradation technologies in disease settings such as those discussed herein.
This review has focused predominantly on diseases that would benefit from targeted degradation of pathological proteins.However, many nonmalignant diseases are the result of abnormal protein folding or degradation leading to insufficient normal protein.An example is cystic fibrosis, where a single amino acid deletion of phenylalanine results in a misfolded chloride ion channel that is degraded by the proteasome [164].In analysing this, the seminal discovery was made that the defective cystic fibrosis transmembrane conductance regulator (CFTR) was temperature sensitive, and lower temperatures reduced protein misfolding and increased CFTR functionality [165].This prompted the development of corrector and potentiator small-molecule drugs lumacaftor and ivacaftor, which aid trafficking and opening of the CFTR [166].
This example exemplifies how advances in protein degradation provide a mechanism to study disease from a wider perspective.Beyond identifying novel clinical tools, there is the opportunity to gain insight into the The emerging role of targeted protein degradation biology of disease and understand how drugs with previously unknown mechanisms of action operate.An obvious example is thalidomide and its analogues for treating haematological malignancies, as discussed here.Another example is arsenic for treating acute promyelocytic leukaemia (APML), which was initially poorly understood and paradoxically known to be inherently carcinogenic.We now know that arsenic binds to free cysteine residues in the PML-RARα fusion protein, triggering ubiquitination and proteasomal degradation [164].Whether arsenic has a therapeutic role elsewhere remains to be seen; however, the underlying concept may be translatable to other disease settings.
Off-target effects are also being minimised with the development of novel molecular glue complexes.A phage-assisted continuous evolution platform has generated compact zinc finger degrons that are readily incorporated into target gene sequences, bind CRBN, and facilitate specific, tunable degradation via an otherwise inactive thalidomide analogue-derived small molecule [167].The wide-ranging applicability of this platform to selectively modulate endogenous proteins has enormous potential in the interrogation of disease biology.

Targeted protein degradation beyond the ubiquitin-proteasome system
The ubiquitin-proteasome system is the predominant system for homeostatic protein degradation.However, the lysosomal/autophagy pathways that act independently of the proteasome are also gaining interest in targeted protein degradation drug development.Autophagy is the intracellular protein degradation process by which cells harness lysosome-mediated destruction in response to cellular stress [168].PROTACs, dTAG, and TRIM-Away are only possible for proteins with ligand binding domains within the cytosol and are therefore ineffective for extracellular and certain membraneassociated proteins including important cancer targets [169].To overcome this, lysosome-targeting chimeras (LYTACs) have been developed that comprise a glycopolypeptide ligand conjugated to an antibody or small molecule to traffic secreted and membrane-associated proteins to the lysosome for degradation [169].In the proof-of-concept study, LYTACs successfully degraded cancer targets, including CD71 and EGFR across multiple cancer cell lines [169].LYTAC technology is a breakthrough in targeted protein degradation, unlocking new protein targets and utilising a UPS-independent pathway.
Autophagy-targeting chimeras (AUTACs) have also been developed as a tool to access targets cleared by autophagy rather than UPS breakdown.AUTACs are made up of a guanine derivative substrate tag and a target binding warhead.They have been shown to clear cytosolic substrates, including fragmented mitochondria [170].

Conclusion
Targeted protein degradation is offering new and improved therapeutic potential across a range of haematological and nonhaematological malignancies.The discovery that thalidomide analogues bound CRBN was a watershed moment that not only unlocked the understanding of thalidomide analogues but also enabled their incorporation into technology such as CELMoDs and PROTACs that are rapidly overcoming emergent drug resistance and enabling previously inaccessible targets to be engaged.
The field has advanced from degrading substrates with molecular glues by serendipity to a deep structural and mechanistic understanding that facilitates identification and design of molecules that tune in and out specific targets.PROTACs offer particularly attractive selectivity, event-driven pharmacology, and a potentially expansive range of targets throughout the proteome, including scaffolding proteins and transcription factors.Mechanisms of resistance and pharmacokinetic barriers owing to PROTACs' relatively high molecular weight remain important considerations to inform ongoing development.Novel degradation tags, LYTACs, and AUTACs are similarly exciting technologies.With rapidly increasing identification, application, and optimisation of various targeted protein degradation tools, we are likely to see emerging therapies in a range of disease settings in the years to come.

Figure 1 .
Figure 1.Protein degradation in untreated cells and in the presence of thalidomide analogues.(A) A Cullin-RING E3 ubiquitin ligase comprising a cullin protein scaffold, ubiquitin conjugating enzymes, a substrate-binding receptor, and a bridging adapter.Ubiquitination of the substrate results in proteasomal degradation.(B) CRL4 CRBN E3 ubiquitin ligase complex: ligase scaffold cullin-4 (CUL4), RING-finger protein RING-box1 (RBX1), the adapter damage-specific DNA binding protein 1 (DDB1), and cereblon (CRBN), which acts as a substrate receptor.Thalidomide analogue binding facilitates interaction with a neo-substrate, which is ubiquitinated and degraded by the proteasome.Degradation is inhibited by proteasome inhibitors carfilzomib and bortezomib.(C) Structure of thalidomide and lenalidomide with differing neo-substrate-binding moieties that dictate substrate specificity.Examples of zinc finger and non-zinc-finger-containing substrates are provided below.

Figure 2 .
Figure 2. Small-molecule inhibitor occupancy-driven pharmacology compared to molecular glue event-driven pharmacology.(A) Occupancy-driven pharmacology relies on target site or allosteric binding to inhibit the protein of interest (POI).Mutated binding sites render inhibitors ineffective.(B) Molecular glues such as thalidomide analogues show event-driven pharmacology by enabling the interaction between proteins of interest and E3 ligases to facilitate ubiquitination.

Figure 3 .
Figure 3. Structure of a proteolysis-targeting chimera.A linker molecule separates a warhead ligand that binds to the protein of interest (POI) from an E3 ubiquitin ligase adapter.

Figure 4 .
Figure 4. Protein degradation technology.(A) Using either lentiviral transduction of a fusion protein or CRISPR-mediated endogenous protein tagging enables proximity-induced ubiquitination and degradation of the target protein.This can be achieved via (B) dTag, (C) HaloTag, or (D) Auxin-inducible degrons (AID).(E)TRIM-Away with the E3 ligase TRIM21 bound to an antibody that is also bound to the protein of interest (POI), causing the entire complex to be degraded following E3 ligase-mediated ubiquitination.

Table 1 .
Differing modalities of molecular glues to enable targeted protein degradation.