The emerging role for Cullin 4 family of E3 ligases in tumorigenesis

Abstract

As a member of the Cullin-RING ligase family, Cullin-RING ligase 4 (CRL4) has drawn much attention due to its broad regulatory roles under physiological and pathological conditions, especially in neoplastic events. Based on evidence from knockout and transgenic mouse models, human clinical data, and biochemical interactions, we summarize the distinct roles of the CRL4 E3 ligase complexes in tumorigenesis, which appears to be tissue- and context-dependent. Notably, targeting CRL4 has recently emerged as a noval anti-cancer strategy, including thalidomide and its derivatives that bind to the substrate recognition receptor cereblon (CRBN), and anticancer sulfonamides that target DCAF15 to suppress the neoplastic proliferation of multiple myeloma and colorectal cancers, respectively. To this end, PROTACs have been developed as a group of engineered bi-functional chemical glues that induce the ubiquitination-mediated degradation of substrates via recruiting E3 ligases, such as CRL4 (CRBN) and CRL2 (pVHL). We summarize the recent major advances in the CRL4 research field towards understanding its involvement in tumorigenesis and further discuss its clinical implications. The anti-tumor effects using the PROTAC approach to target the degradation of undruggable targets are also highlighted.

Introduction

The ubiquitin-proteasome system (UPS)

The ubiquitin-proteasome system (UPS) is an evolutionarily conserved apparatus that serves as a major regulator of proteostasis in eukaryotic cells¹. The UPS generally consists of ubiquitin, ubiquitination enzymes and the 26S proteasome, which synergistically form an enzymatic cascade to transfer ubiquitin in a substrate-specific manner to promote subsequent proteolysis and degradation of the target protein². The machinery of the ubiquitin-proteasome cascade has been reviewed extensively^3,4. Briefly, the ATP-dependent activation of ubiquitin by the E1 activating enzyme is indispensable for the initiation of the enzymatic cascade⁵. As a result, activated ubiquitin is then transferred to an E2 conjugating enzyme, which then assist in the recruitment of an E3 ligase into a complex with the ubiquitin moiety. Subsequently, E3 ligase determines the substrate specificity and facilitates the formation of a covalent isopeptide bond between the ubiquitin and lysine residues of target proteins, leading to substrate ubiquitination. Additionally, E3 ligases also mediate the attachment of the ubiquitin moiety to existing ubiquitin chains on a substrate protein, resulting in poly-ubiquitination and diverse consequences to the target protein, such as degradation^2,6, altered activity, or subcellular localization of substrates⁷.

Physiological significance and pathological roles of the UPS

Consistent with a broad central role that the UPS plays in proteostatic control, nearly every aspect of cellular biology is regulated in some manner by the UPS pathway, especially the cell cycle, cell growth, immune homeostasis, and metabolic stability⁶. For instance, proteasomal degradation of cell cycle regulators, such as p21 and p27, is a critical to control cell cycle progression⁶. In addition, proteolytic activities provide necessary raw materials for intracellular recycling and rebuilding, such as generation of amino acids^6,8. Meanwhile, via proteasomal cleavage of endogenous proteins, the UPS governs the production of MHC (major histocompatibility complex) Class I antigens, which are critical for the functions of the immune response^9,10. Dysregulation of the UPS is involved in the pathogenesis of multiple disorders, especially neurodegeneration, neoplastic transformation and autoimmune diseases¹¹. Pathologically, neurodegenerative disorders often feature the accumulation of misfolded proteins, such as tau aggregates and Aβ plaques in Alzheimer’s disease^12,13. Autoimmune diseases are often induced by the mis-recognition of endogenous proteins as exogenous antigens¹⁴, which was tightly controlled by the UPS. For instance, increased generation of MHC Class I antigen HLA (human leukocyte antigen)-B27 closely correlates to Ankylosing Spondylitis^15,16.

Cancer relevant roles of the UPS

Dysregulation of the UPS is associated with tumorigenic events^17,18. E3 ligases are involved in the final enzymatic activity leading to ubiquitination and dictates the specificity of substrate selection. Hence, depending on the tumorigenic properties of specific substrates, E3 ligases play a context-dependent role by degrading tumor suppressors or oncoproteins^19,20. It is noteworthy that E3 ligase may also cause altered activity or subcellular re-localization of target proteins. For example, MDM2 (mouse double minute 2 homolog), serving as an E3 ligase to destabilize tumor suppressor p53, has been regarded as one of the most frequently mutated oncoproteins in lung and breast carcinoma^21–23. On the other hand, SPOP (speckle-type POZ protein) is frequently mutated adaptor protein in prostate cancer serving as a tumor suppressor by targeting TRIM24 (tripartite motif-containing 24) and the androgen receptor^24,25. Due to their critically important role in substrate selection and ubiquitin transfer during the UPS cascade, E3 ligases are broadly acknowledged as a key target for anti-cancer therapeutics, thus the use of small molecules targeting UPS for cancer treatment has been developed for diverse malignancies, especially multiple myeloma and lymphoma^26,27,28, which will be discussed in the following sections.

DUBs: the antagonizing force in counter-balancing the UPS

Similar to other post-translational modifications, there are also mechanisms in place to reverse and therefore antagonize the role of ubiquitination and thus maintain protein stability, which is largely mediated by a cluster of proteins termed deubiquitinating enzymes (DUBs)^29,30. Currently, nearly 100 DUBs have been identified in humans³¹, which are divided into 7 subgroups by a specific functional domain, including ubiquitin C-terminal hydrolase (UCH) DUBs, ubiquitin specific protease (USP) DUBs, ovarian tumor (OTU) DUBs, Josephin DUBs, JAB1/MPN+/MOV34 (JAMM) DUBs³² and another two non-canonical DUBs, MINDY DUBs³³ and ZUFSP DUBs^34,35. Consistent with the roles for the UPS participating in critically important cellular biological processes, DUBs are likewise involved in nearly every aspect of cellular biology^17,36. For instance, USP9X (ubiquitin specific peptidase 9) is found to be negatively correlated with carcinogenesis, which deubiquitinates and functionally stabilizes its substrate LATS1 (large tumor suppressor kinase 1), leading to the inactivation of the Hippo pathway³⁷. USP2 (ubiquitin specific peptidase 2) triggers malignant progression in prostate cancer cells via deubiquitination and stabilization of fatty acid synthase (FAS), which positively correlates with aggressiveness and negatively predicts survival³⁸.

CRL4 E3 ligases in tumorigenesis

E3 ligases and the Cullin-RING family

There are hundreds of E3 ligases identified in mammalian ubiquitination cascades^39,40. Based on structural and functional differences, E3 ligases are subdivided into two groups, namely the HECT (homologous to the E6AP carboxyl terminus) family and the RING (really interesting new gene) family⁴¹. Specifically, RING E3 ligases bind to ubiquitin-E2 heterodimer and protein substrates in a simultaneous manner, facilitating an efficient transfer of ubiquitin onto selected substrates. Whereas, ubiquitination mediated by HECT E3 ligases is separated into two steps, formation of a thioester bond with ubiquitin followed by substrate recognition and ubiquitin transfer^40,42. In addition, the RING ligases have been further categorized into several subfamilies, including BARD1 (BRCA1-associated RING domain 1), c-Cbl (Casitas B-lineage lymphoma), anaphase-promoting complex (APC), heterodimer of BRCA1 (breast cancer 1) and Cullin-RING ligases (CRLs), each harboring a RING catalytic domain⁴⁰.

Characterized by being composed of multi-subunit complexes, the Cullin-RING ligases are the most extensively studied subfamily of RING ligases. Structurally, a Cullin-RING ligase complex is composed of four essential subunits, including a Cullin scaffold, RING-finger protein, adaptor protein, and a substrate recognition protein⁴³. As a central coordinator of complex formation, the Cullin scaffold provides the platform for both the RING-finger protein and adaptor protein. The RING-finger protein serves as a docking site for both the ubiquitin-E2 complex and the substrate recognition module to facilitate the transfer of ubiquitin to the recruited substrate. The substrate recognition module that associates within the complex through its interaction with the adaptor protein and dictate substrate specificity^43,44. To date, eight Cullin scaffolds have been characterized in mammalian cells (Cullin 1, Cullin 2, Cullin 3, Cullin 4A, Cullin 4B, Cullin 5, Cullin 7, and Cullin 9) as well as two RING-finger proteins (RBX1 and RBX2), four adaptor proteins (SKP1, ElonginB, ElonginC, and DDB1) and more than 400 substrate recognition receptor proteins⁴⁵. These subunits are assembled into hundreds of unique Cullin-RING ligase complexes that are responsible for nearly 20% of ubiquitination events mediated by the UPS^46,47.

Introduction of Cullin 4 E3 ligases

The Cullin 4-RING ligases (CRL4s) contain two homogenous scaffolds, defined as Cullin 4A (CUL4A) and Cullin 4B (CUL4B), respectively. Commonly, both scaffolds bind to damaged DNA binding protein 1 (DDB1) and the catalytic subunit RING-finger protein RBX1 through their N- and C-terminus respectively to support the structure of entire complex⁴⁸. However, despite of 82% identity with regard to their genomic sequences, each scaffold targets a unique set of substrates. Compared to CUL4A, CUL4B features a longer N-terminus, which contains an extra nuclear localization signal (NLS) that governs its targeting to the nucleus, in addition to interaction with DDB1. On the other hand, the majority of CUL4A resides in the cytoplasm to regulate substrate ubiquitination⁴⁹, although a small fraction is found in the nucleus to target nuclear proteins⁵⁰ (Table 1). CUL4 scaffolds are also neddylated by the ubiquitin-like protein NEDD8 (neural precursor cell expressed, developmentally down-regulated 8), which is essential to stimulate the activity of CRL4s complexes⁵¹. Moreover, RBX1 acts as a regulatory subunit of the CRL4 complex through recruitment of ubiquitin E2 complex and transferring the ubiquitin moiety. The adaptor DDB1 serves as the bridging factor between the Cullin scaffold and substrate recognition subunit, DDB1-CUL4-associated factor (DCAF) (Figure 1)^51,52.

Table 1.

Pathological involvements of mutated components of CRL4

Gene	Component category	Alteration	Consequence
Cul4b	Scaffold	Mutation	X-linked mental retardation77,78; Cerebral malformations80
Dcaf8	Substrate recognition protein	Mutation	Axonal hereditary motor and sensory neuropathy (HMSN2)81
Dcaf14	Substrate recognition protein	Mutation	Developmental retardation, intellectual defects, obesity and dysmorphic features82
Dcaf17	Substrate recognition protein	Mutation	Woodhouse Sakati syndrome83
Ddb2	Substrate recognition protein	Mutation	Xeroderma pigmentosum84
Gnb3	Substrate recognition protein	Mutation	Hypertension85
Wdr62	Substrate recognition protein	Mutation	Cerebral malformations80

Figure 1. Overview of neoplastic roles of CRL4 components.

Physiological roles of Cullin 4 E3 ligases -evidence from knockout mouse models

According to the phenotypic studies derived from knockout mouse models on CRL4 components, CRL4s participate in embryonic development as well as maturation and homeostasis of multiple tissues and organs. Unlike the essential roles of most Cullins, germline knockouts of Cul4a or Cul4b are both viable and shows no overt growth abnormalities^48,53–55, which is likely due to redundancy between CUL4A and CUL4B. Abrogation of both CUL4A and CUL4B in mouse embryonic fibroblasts and tumor cell lines led to growth arrest and loss of viability⁵⁵. Moreover, germline knockout of the Ddb1 adaptor is embryonic lethal^56,57, highlighting the essential role of the CRL4 ubiquitin ligase in maintaining growth and survival of mammals. Although a previous study suggested an embryonic requirement of Cul4a⁵⁸, other studies have shown that systemic ablation of Cul4a does not lead to embryonic lethality^55,59, and the embryonic phenotype seen in the earlier study may be attributed to an unintended deletion of the adjacent Pcid2 gene on the complementary strand of DNA around the Cul4a locus⁵⁵.

The hematopoietic lineage is another system where CRL4 activity regulates protein homeostasis to control biological outcome. Earlier proteomic/yeast two-hybrid assays identified the HOX Homeodomain transcription factors to be targeted by the CRL4 ubiquitin ligase for ubiquitination and degradation^49,60. As the hematopoietic stem cells (HSCs) and progenitors undergo differentiation, HOX genes are transcriptionally downregulated^61–63 and the HOX proteins are targeted for degradation via the CRL4 ubiquitin ligase to ensure proper differentiation⁴⁹. Importantly, a conserved LXCXE motif was identified in the Helix I region of HOX HD which serves as the CRL4 degron motif that is conserved among all 39 HOX family members⁶⁰. Failure of HOXA9 degradation by CRL4 blocks granulocytic differentiation⁴⁹, while transduction of an engineered degradation-resistant HOXB4 into adult HSCs could effectively promote ex vivo expansion of HSCs and multipotent progenitors, and enhance bone marrow engraftment of transduced human adult CD34+ HSCs⁶⁰.

The potential significance of CRL4 in the reproductive system was further revealed in the Cul4a and Cul4b knockout animals: Cul4a-null mice exhibit male infertility, yet had no major effect on reproduction of females was observed, revealing the essential roles of CRL4A in male reproduction^64–66. Cul4b^−/Y males are also infertile, while no Cul4b^−/− females can be derived as CUL4B is x-linked. While the redundancy of the two CUL4s accounts for the viability and normal development of Cul4a^−/− or Cul4b^−/Y mice, CUL4A and CUL4B genes are differentially expressed at distinct stages of male meiosis^64–66. As such, Cul4a^−/− spermatocytes are arrested at pachytene to diplotene transitions of Meiosis I, while Cul4b^−/Y sperms are defective at the later stage of spermiogenesis^64–66. Accumulation of CRL4 target CDT1, as well as p53 was observed, consistent with the increased apoptosis among germ cells^64–66. While female reproduction remains normal in individual Cul4a^−/− mice, abrogation of the entire CRL4 ubiquitin ligase led to infertility in females⁶⁷. Interestingly, conditional deletion of Ddb1 or CRL4 substrate receptors VprBP/DCAF1 or DCAF2 cause female infertility possibly via diverse mechanisms including, massive DNA damage and disruption of the cell cycle, TET (Ten-eleven translocation methylcytosine dioxygenase) inactivation-mediated ovulation defects, oocyte loss, or repression on PI3K/Akt pathway^68–70.

Another core system that CRL4 regulates is the central nervous system. Conditional deletion of Ddb1 in the murine brains leads to neonatal lethality by increased accumulation of p53, although p53 is not considered a direct substrate of CRL4⁵⁷. Meanwhile, specific ablation of Ddb1 in the hippocampus and cerebral cortex generates an epileptic phenotype in experimental mice, mechanistically via changes in restriction on the activity of the BK (Ca²⁺ and voltage-activated K⁺) channel, which further indicates the potentially pathological role of CRL4 in neuroelectrophysiological disorders⁷¹.

CRL4s are also found to function in other organ systems. For instance, hepatocyte-specific deletion of Ddb1 disables liver gluconeogenesis in part due to increased accumulation of the CRL4 substrate CRY1 (cryptochrome 1), which in turn leads to downregulation of the FOXO1 (forkhead box protein O1)-driven gluconeogenic responses⁷². However, CRL4s seem to play contradictory roles towards hepatic cell expansion under various circumstances. Deletion of Ddb1 accelerates liver regeneration and trigger spontaneous onset of hepatic carcinogenesis, suggesting a role in controlling cellular proliferation⁷³. This phenotypic disparity may be explained by DDB1 engaging in other regulatory networks besides CRL4, which could cloud our understanding of the effect of DDB1 loss due to reduction in CRL4 activity. Therefore, the phenotypic information from Cul4a knockout mouse models could be more informative. Moreover, CRL4s may have a role in regulating metabolism, since depletion of Cul4b in adipocytes and pancreatic δ cells produces opposing outcomes, namely enhanced insulin sensitivity by stabilizing PPARγ (peroxisome proliferator-activated receptor γ), or glucose intolerance by blocking PRC2 (polycomb repressive complex 2)-induced somatostatin secretion, respectively^74,75. Meanwhile, CRL4s also demonstrate neoplastic involvement in skin carcinogenesis through restricting the capacity of nucleotide excision repair^55,76, which will be further discussed in subsequent sections (Table 2).

Table 2.

Summarized phenotypes of knockout mouse models on CRL4 components

Gene	Component category	Knockout mode	Phenotype
Cul4a−/−	Scaffold	Germline	Normal development/lifespan. No gross abnormalities55. Male infertility64,66; mild Cardiac hypertrophy and hypertension in male but not female mice85,249
		Conditional (skin)	Resistant to UV-induced skin carcinogenesis55
		Conditional (liver)	Reduced hepatocyte proliferation after exposure to liver toxins59
Cul4b−/Y	Scaffold	Germline (epiblast)	Normal development/lifespan; No obvious gross abnormalities54 ; Infertility65
		Conditional (pancreatic δ cell)	Decreased glucose intolerance and insulin secretion75
		Conditional (adipocyte)	Enhanced expansion of adipose tissue; Increased glucose tolerance and insulin sensitivity74
		Conditional (germ cell)	Male infertility65
		Conditional (hematopoietic cell)	Increased accumulation and activity of MDSCs post LPS250
		Conditional (nervous system)	Elevated amount of astrocytes251
		Conditional (myeloid system)	Enhanced peritonitis induced by LPS administration252
		Systematic	Embryonic lethality resulting from defective extra-embryonic development54; Growth retardation of embryos253
		Germline	Embryonic lethality57,254
Ddbr−/−	Adaptor	Conditional (brain and lens) Conditional (skin)	Neonatal lethality57 Postnatal lethality56
		Conditional (hematopoietic cell)	Bone marrow failure254
		Conditional (oocyte)	Ovulation defect69,70
		Conditional (liver)	Impaired hepatic gluconeogenesis72; Liver regeneration and spontaneous development of hepatocellular carcinoma73
		Conditional (hippocampus and cerebral cortex)	Epilepsy71
		Germline	Embryonic lethality255
Dcaf1−/−	Substrate recognition protein	Conditional (B cell)	Impaired B cell maturity and development256
		Conditional (T cell)	Insufficiency of T cells257
		Conditional (oocyte)	Oocyte death and infertility69,70
		Conditional (oocyte)	Female infertility68
Dcaf2−/−	Substrate recognition protein	Germline	Susceptible to UV-induced skin carcinogenesis76

Abbreviations: MDSCs: myeloid-derived suppressor cells; LPS: lipopolysaccharide; UV: ultraviolet;

Pathological involvement of Cullin 4 E3 ligases-evidence from clinical studies

Consistent with their diverse physiological roles in mouse models, mutations of CRL4 components have also been linked to multiple human pathological conditions, especially in the nervous system.

X-linked mental retardation (XLMR), also known as Cabezas Syndrome, is genetically characterized by specific mutations in the X chromosome, which causes short stature, hypogonadism, learning disability, obesity, aggressive outbursts, intentional tremor, pes cavus and seizures among adolescents^77,78. Current studies have confirmed that mutations in X-linked Cul4b gene account for the onset of XLMR, which may be partially attributed to the stability of its substrate WDR5 (WD repeat-containing protein 5) that facilitates the epigenetic silencing of neuronal genes and thus inhibits neurite outgrowth^77–79, although WDR5 accumulation was not observed in Cul4b^−/− MEFs⁵⁴. Genetic rescue of Cul4b-mutant mice significantly decreases the occurrence of XLMR, suggesting a potential of Cul4b-targeted therapy⁵³. In addition, mutation of Cul4b also leads to cerebral malformations, possibly through dysregulation of the function of WDR62 (WD repeat-containing protein 62), a substrate recognition receptor (DCAF) of CRL4 and also a protein whose mutation is frequently detected in patients with microcephaly⁸⁰. Additionally, a specific mutation in Dcaf8, leading to the amino acid substitution R317C, disrupts its binding to the DDB1 adaptor and the formation of a functional CRL4 complex, leading to axonal hereditary motor and sensory neuropathy (HMSN2) and giant axons⁸¹. Moreover, patients bearing a mutant of Dcaf14 feature developmental retardation, intellectual defects, obesity and dysmorphic characteristics⁸². Dcaf17 is the causative gene of Woodhouse Sakati Syndrome (WSS), which is characterized by progressive extrapydamidal symptoms, together with hearing loss, hypogonadism, diabetes and learning disability⁸³. This experimental evidence verifies the close correlation between CRL4 mal-function and pathogenesis in the central nervous system.

Meanwhile, mutated Gnb3, encoding a substrate recognition receptor GNB3 (G protein subunit beta 3), pathologically associates with hereditary hypertension while mutation on Ddb2 results in xeroderma pigmentosum, which features impaired DNA repair upon UV exposure and higher susceptibility to skin carcinogenesis^84,85. Considering the diverse physiological roles of CRL4, we may predict that more pathological correlations between CRL4 dysregulation and human disorders may be identified in the near future (Table 3).

Table 3.

Major mammalian substrates of CRL4 and their biological impacts

Substrate recognition protein	Scaffold	Substrate	Modification	Biological impact
DCAF1	CUL4A	LATS1	Degradation	Oncogenic contribution139,140
	CUL4A	LATS2	Altered activity	Oncogenic contribution139
	CUL4A	HLTF	Degradation	Triggering HIV replication110
	CUL4A	Dicer1	Degradation	Enhanced HIV infection258; Colon cancer progression109
	CUL4A/B	FOXM1	Degradation	Cell cycle arrest136
	CUL4A/B	PP2A	Degradation	Crucial for oocyte meiosis and female fertility67
	CUL4A/B	MyoD	Degradation	Halted differentiation of skeletal muscle137
	CUL4A	MCM10	Degradation	Cell cycle arrest259,260
	CUL4B	TR4	Degradation	Resistant to high fat caused fatty liver261
	CUL4A/B	SAMHD1	Degradation	Facilitating HIV replication262
	CUL4A/B	TET1/2/3	Altered activity	Essential for ovary maturity69; Tumor263 suppression
	CUL4A/B	RORα	Degradation	Transcriptional repression138
	CUL4A	UNG2	Degradation	Cell cycle arrest264,265
	CUL4A	SMUG1	Degradation	Cell cycle arrest264
	CUL4A	NF2	Degradation	Activation of oncogenic pathways141
DCAF2	CUL4A	CRY1	Degradation	Promotion of liver gluconeogenesis; Homeostasis of molecular circadian behavior72,147
	CUL4A/B	CDT1	Degradation	Stimulating the proliferation of melanoma150,152
	CUL4A/B	p21	Degradation	Stimulating the proliferation of melanoma150; Regulation of replication licensing98
	CUL4A	p53	Degradation	Cell cycle control266
	CUL4A/B	SET7	Degradation	Maintaining genomic integrity143–145
	CUL4A/B	SET8	Degradation	Stimulating the proliferation of melanoma150; DNA damage control145,267
	CUL4A/B	PCNA	Altered activity	Enhanced translesion DNA synthesis268
	CUL4A/B	p12	Degradation	DNA repair control269
	CUL4A	CHK1	Degradation	Cell cycle progression146
	CUL4A/B	MMSET	Degradation	Cell cycle arrest270
	CUL4A/B	XPG	Degradation	DNA repair271
	CUL4A/B	TDG	Degradation	Homeostasis of DNA replication272
	CUL4A	SDE2	Degradation	Protection of genomic stability against replication stresses273
	CUL4A/B	CDC6	Degradation	Cell cycle regulation274
	CUL4A	Tob	Degradation	Apoptotic response to DNA damage275
	CUL4A	GCN5	Degradation	Transcriptional suppression148
DCAF4L2	CUL4A	PPM1B	Degradation	Increased invasion of colorectal cancer276
DCAF7	CUL4A/B	LigI	Degradation	NS277
DCAF8	CUL4A	H3	Altered activity	Postnatal liver maturation278
	CUL4B	CDC25A	Degradation	NS170
DCAF9	CUL4A/B	H2A	Altered activity	Suppressed adipogenesis279
DCAF11	CUL4A	NRF2	Degradation	More sensitive to chemotherapies171
	CUL4A	SLBP	Degradation	Cell cycle homeostasis280,281
	CUL4B	p21	Degradation	Inhibiting cell cycle progression172
DCAF15	CUL4A/B	CAPERα	Degradation	Anti-growth effect in cancer cells173
AhR	CUL4B	ERα	Degradation	NS282
COP1	CUL4A	ETV5	Degradation	Lung homeostasis and tumor suppression166
	CUL4A	c-Jun	Degradation	Inhibition on oncogenic transcriptions167
COPS8	CUL4A	CENP-A	Subcellular relocalization	Homeostasis of mitosis283
CRBN	CUL4A/B	IKZF1/3	Degradation	Anti-tumor impacts against multiple myeloma cells162
	CUL4A	ZFP91	Degradation	NS284
	CUL4A	GSPT1	Degradation	Anti-tumor impacts against leukemic cells163
	CUL4A/B	APP	Degradation	Alleviation of neurodegeneration159
	CUL4A	GS	Degradation	NS185
	CUL4A/B	CK1α	Degradation	Inhibition on myelodysplastic syndrome164
	CUL4A/B	CLC-1	Degradation	Homeostasis of membrane excitability160
	CUL4A	SLO1	Altered activity	Preventing epileptogenesis71
DDB2	CUL4A	p27	Degradation	Cell cycle progression156
	CUL4A	AR	Degradation	Suppression of prostate cancer proliferation154,285
	CUL4A/B	H2A	Altered activity	DNA repair84
	CUL4A/B	HBO1	Degradation	Suppressed cell proliferation158
	CUL4A	DDB2	Degradation	Regulation of DNA damage286,287
FBXO44	CUL4B	RGS2	Degradation	NS288
FBXW5	CUL4A	DLC1	Degradation	Growth of lung cancer cells289
	CUL4A/B	TSC2	Degradation	Homeostasis of cell growth290
GNB2	CUL4A	GRK2	Degradation	Cardiovascular protection249
GNB3	CUL4A	GRK2	Degradation	Cardiovascular homeostasis85
HBx	CUL4A/B	SMC5/6	Degradation	Increased replication of HBV128
HOXB4	CUL4A	Geminin	Degradation	Elevated proliferation of hematopoietic stem and progenitor cells291
RBBP7	CUL4A/B	CENP-A	Subcellular relocalization	Homeostasis of mitosis292
STRAP	CUL4A	PNKP	Degradation	Susceptible to oxidative DNA damage293
WDR70	CUL4A/B	H2B	Altered activity	Stability of cell division294
Other substrates*	CUL4A/B	γ-tubulin	Degradation	Stability of centrosome295
	CUL4B	HUWE1	Degradation	Reduced apoptosis after DNA damage296
	CUL4A/B	AMBRA1	Degradation	Autophagy termination297
	CUL4B	p53	Degradation	Impeding stress-induced cellular senescence298
	CUL4A	PEX7P	Degradation	Control of peroxisome biogenesis299
	CUL4A	HOXB4	Degradation	Decreased proliferation of hematopoietic stem cells60
	CUL4B	CSN5	Degradation	Regulation of bone morphogenetic signaling300
	CUL4A	p73	Altered activity	Transcriptional repression301
	CUL4A	GRK5	Degradation	NS302
	CUL4A	ORCA	Degradation	Cell cycle regulation303
	CUL4B	WDR5	Degradation	Promoting neurite outgrowth79
	CUL4B	Peroxiredoxin III	Degradation	Increased production of cellular reactive oxygen species304
	CUL4A	RASSF1A	Degradation	Cell cycle progression305
	CUL4B	cyclin E	Degradation	Cell cycle progression306
	CUL4A	CHK1	Degradation	Genomic instability307
	CUL4B	H3/H4	Altered activity	Facilitating cellular response to DNA damage308
	CUL4A	p27	Degradation	Cell proliferation50
	CUL4A	HOXA9	Degradation	Myelocytic maturation49
	CUL4A	REDD1	Degradation	Activation of mTOR signaling309

^*:Those proteins are assumed to be the substrates of CRL4 without detailed description of binding motif and interactions with substrate recognition proteins.

Abbreviations:COPS8: COP9 signalosome subunit 8; CENP-A: centromere protein A; DCAF2: alias CDT2; CRY1: cryptochrome 1; TR4: nuclear receptor subfamily 2 group C member 2 (alias NR2C2); CRBN: cereblon; ZFP91: zinc finger protein 91; NS: not specified; DCAF11: alias WDR23; CAPERα: coactivator of activating protein-1 and estrogen receptor α; NRF2: nuclear factor erythroid 2 like 2; LATS1: large tumor suppressor 1; FOXM1: forkhead box M1; ETV5: ETS variant 5; AR: androgen receptor; H3: histone H3 protein; ERα: estrogen receptor α; SDE2: SDE2 telomere maintenance homolog; HBx: hepatitis B virus regulatory protein X; SMC5: structural maintenance of chromosome 5; LigI: DNA ligase I; GNB3: G protein subunit beta 3; GRK2: G protein-coupled receptor kinase 2; GSPT1: G1 to S phase transition 1; CDT1: chromatin licensing and DNA replication factor 1; SET8: lysine methyltransferase 5A (alias KMT5A); APP: amyloid precursor protein; SLBP: stem-loop binding protein; PPM1B: protein phosphatase, Mg2+/Mn2+ dependent 1B; WDR70: WD repeat domain 70; GS: glutamine synthetase; HLTF: helicase-like transcription factor; MMSET: nuclear receptor binding SET domain protein 2 (alias NSD2); HBO1: lysine acetyltransferase 7 (alias KAT7); PP2A: phosphatase 2A; CK1α: casein kinase 1A1; MCM10: minichromosome maintenance 10 replication initiation factor; CLC-1: chloride voltage-gated channel 1; FBXO44: F-box protein 44; RGS2: regulator of G protein signaling 2; TET: tet methylcytosine dioxygenase; XPG: Xeroderma pigmentosum group G protein; TDG: thymine DNA glycosylase; SLO1: potassium calcium-activated channel subfamily M alpha 1 (alias KCNMA1); CDC6: cell division cycle 6; FBXW5: F-box and WD repeat domain containing 5; DLC1: DLC1 Rho GTPase activating protein; CHK1: checkpoint kinase 1; RORα: RAR related orphan receptor A; STRAP: serine-threonine kinase receptor associated protein; PNKP: polynucleotide kinase 3’-phosphatase; UNG2: uracil DNA glycosylase 2; SMUG1: single-strand-selective monofunctional uracil-DNA glycosylase 1; GCN5: lysine acetyltransferase 2A (alias KAT2A); PCNA: proliferating cell nuclear antigen; β-TRCP: beta-transducin repeat containing E3 ubiquitin protein ligase; REDD1: regulated in development and DNA damage responses 1; TSC2: TSC complex subunit 2; NF2: neurofibromin 2; AhR: aryl hydrocarbon receptor; ERα: estrogen receptor α; COP1: ring finger and WD repeat domain 2 (RFWD2); CDC25A: cell division cycle 25A; HUWE1: HECT, UBA and WWE domain containing 1; AMBRA1: autophagy and beclin 1 regulator 1; PEX7P: peroxisomal biogenesis factor 7p; HOXB4: homeobox B4; CSN5: COP9 signalosome subunit 5; ORCA: origin recognition complex subunit 1; RASSF1A: RAS association domain family 1 isoform A; CHK1: checkpoint kinase 1; SKP2: S-phase kinase associated protein 2; HOXA9: homeobox A9; SAMHD1: SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1.

Cullin 4 E3 ligases in tumorigenesis

According to the canonical definition of cancer, there are six hallmarks of cancer pathology, including sustaining proliferative signaling, resisting cell death, evading growth suppression, activating invasion and metastasis, inducing angiogenesis, and enabling replicative immortality⁸⁶. Any regulators that effects these hallmarks are believed to be involved in tumorigenesis, either in a positive or negative manner⁸⁷. Due to the participation of CRL4 in multiple cellular processes, including cell cycle progression and regulation of apoptotic death, its potential role in tumorigenesis has drawn much attention. Current studies have verified a close, but complicated, and context-dependent correlation between CRL4 activity and malignancies, on basis of the evidence from genetic mouse models, clinical pathological specimens and cellular and molecular experiments, which will be discussed in detail below (also see a recent review on pathological role of the CRL4 ubiquitin ligase⁸⁸).

Cullin 4 scaffold protein in tumorigenesis

As described above, there are two homologous CRL4scaffold proteins, namely CUL4A and CUL4B, which have shared substrates but possess non-overlapping activities. Current investigations implicate that both scaffold proteins exert oncogenic functions in a variety of malignancies, suggesting that the entire CRL4 system may play a role in tumorigenesis despite having distinct interactions towards cellular substrates, since the scaffold protein exclusively corresponds to the activity of the whole complex^{89–92,51,93}.

Mounting evidence suggests critical roles that CUL4A plays in cellular response to DNA damage. Following genotoxic stress, CUL4A targets the CDT1 DNA replication licensing factor, the PR-Set7/Set8 histone methyltransferase, and the p21 cyclin dependent kinase inhibitor for ubiquitin-proteasomal degradation to ensure that damaged DNA is not replicated^94–100. However, CUL4A also restricts DNA repair capacity post UV. Conditional knockout of Cul4a in skin rendered the mice hyper-resistant to UV-induced dermatological carcinogenesis⁵⁵. In response to UV irradiation, cells activate the nucleotide excision repair (NER) pathway for removing UV-induced cyclobutane pyrimidine dimers and 6, 4-photoproducts, and the G1/S DNA damage checkpoint pathway to stop the cell cycle until DNA lesions are repaired by NER. Liu et al showed that CUL4A is a potent inhibitor of DNA damage response by ubiquitin-dependent degradation of DDB2 and XPC, two rate-limiting NER factors responsible for recognition of DNA damage, as well as the p21 effector of the G1/S DNA damage checkpoint pathway. As such, Cul4a^−/− cells displayed dramatically enhanced DNA repair and DNA damage checkpoint activities, and Cul4a knockout mice are hyper-resistant to UV-induced skin carcinogenesis⁵⁵. Therefore, blocking CUL4A activity represents an attractive new strategy for cancer prevention.

Genetic and pathological evidence suggests that CUL4A is frequently dysregulated in neoplastic events. Gene amplification and transcriptional upregulation are both shown to account for CUL4A overexpression in tumors (e.g. CUL4A and CUL4B are both targets of LEF/TCF transcription factors of the canonical Wnt signaling)¹⁰¹ Overexpression of CUL4A leads to initiation and progression of lung cancer in mice^89–91. Consistently, numerous pathological studies have detected upregulation of CUL4A in various human cancer specimens, such as gastric cancer^102–104, breast cancer^104–106, colorectal cancer^{104,107–109} and lung cancer^93,110,111, which is also negatively correlated with prognostic survival^{93,110,111,112–116}. As the core component of the CRL4 complex, the tumorigenic effects of CUL4A overexpression mainly depend on its interactions with downstream targets (details will be discussed in subsequent sections), which trigger cell cycle progression and/or epithelial mesenchymal transition, leading to tumor proliferation, invasion or drug resistance^106,107,117.

CUL4B has also been recognized as a tumorigenic protein in many malignancies. Transgenic upregulation of Cul4b induces spontaneous hepatic carcinogenesis⁹². Analyses from clinical samples show that aberrant expression of CUL4B has been observed in a wide spectrum of malignant tumors, especially lung cancer^93,118, colorectal cancer^115,116 and pancreatic cancer¹¹⁹, serving as a negative indicator of patient survival as well^116,120. Analogous to CUL4A, abnormal interactions between CRL4B and substrates directly link to activation of proliferative pathways (such as the Wnt/β-catenin pathway) and epigenetic silencing, participating in nearly every aspect of tumor progression^121,122. On the other side, downregulation of certain miRNAs such as miR-194 and miR-300 may explain, in part, the mechanisms inducing the overexpression of CUL4B overexpression in several cancers, although its upstream mechanisms remain largely unknown^118,119 (Table 4).

Table 4.

Neoplastic roles of CRL4 components

Component	Neoplastic role	Physiological evidence (knockout or transgenic mouse models)	Pathological evidence (cancer relevant human specimens)
CUL4A	Oncogenic	Lung cancer initiation and progression (transgenic overexpression of Cul4a)89–91;Resistant to UV-induced skin carcinogenesis (skin-specific knockout of Cul4a)55	Overexpressed in multiple myeloma310, cholangiocarcinoma113,114, gastric cancer102–104, colorectal cancer104,107–109, ovarian cancer104,112,311, lung cancer93,110,111, breast cancer104–106, osteosarcoma312,313, hepatocellular cancer117,314, malignant pleural mesothelioma315,316, pituitary adenoma317, prostate cancer318
CUL4B	Oncogenic	Spontaneous development of liver cancer (transgenic overexpression of Cul4b)92	Overexpressed in cholangiocarcinoma120, pancreatic carcinoma119, lung cancer93·118, colorectal cancer115,116, glioma319, liver cancer122, cervical cancer121, esophageal cancer320, ovarian cancer112
RBX1	Oncogenic	N/A	Overexpressed in ovarian cancer123, lung cancer124, gastric cancer125, bladder cancer126
DDB1	Context-dependent	Spontaneous development of liver cancer (hepatocyte-specific knockout of Ddbl)73	Overexpressed in ovarian cancer123

The RING-finger protein RBX1 and adaptor DDB1 in tumorigenesis

Similar to the Cul4A and Cul4B scaffold proteins, the catalytic component, RING-finger RBX1 is also regarded as an oncoprotein in ovarian cancer¹²³, lung cancer¹²⁴, gastric cancer¹²⁵, and bladder cancer¹²⁶. Nevertheless, both genetically engineered mouse models and mechanistic studies are still limited.

The adaptor DDB1, it displays a context-dependent role in tumorigenesis as well. Results from knockout mouse models show that hepatocyte-specific ablation of Ddb1 facilitates spontaneous development of liver cancer⁷³, whereas overexpression of DDB1 is detected in ovarian cancer, consistent with the oncogenic role of other CRL4 complex components¹²³. This context-dependent role of DDB1 may reflect its diversity on neoplastic contributions of the CLR4 complexes, or is possibly a result of other non-CLR4 related activities, since DDB1 is not an exclusive protein for CRL4 and could involve other regulatory networks in cancer cells. In this regard, DDB1 was initially identified as a component of the UV-DDB complex that surveys the chromosomes for UV-induced DNA lesions and recruits the nucleotide excision repair apparatus to sites of DNA damage (Table 4)¹²⁷.

Cullin 4 substrate proteins in tumorigenesis

Oncogenic members

Similar to the oncogenic functions of the CUL4A/B scaffolds, the majority of the CRL4 substrate recognition receptors play tumorigenic roles. HBx (hepatitis B virus regulatory protein X) is a recombinant protein encoded by the HBV genome (hepatitis B virus) and expressed following infection in human hepatocytes. This protein can function as a substrate recognition receptor of CRL4 leading to the destabilization of SMC5/6 (structural maintenance of chromosome 5/6) to increase the replication of HBV¹²⁸. Transgenic overexpression of Hbx induces liver carcinogenesis in rodent models^129,130, which is also found to be upregulated in human liver cancer specimens¹³¹. Increased HBx expression is also observed in intrahepatic cholangiocarcinoma¹³² and adenoid cystic carcinoma¹³³, suggesting a general involvement of HBx in promoting tumorigenesis. Mechanistically, apart from its involvement in HBV replication, SMC5/6 also has a vital role in regulating cell division and proliferation while HBx could interact with apoptotic machinery to stimulate downstream oncogenes^134,135, which together explain the oncogenic impact of HBx in human cancers (Table 3 and Table 5).

Table 5.

Major oncogenic substrate recognition proteins

Substrate recognition protein	Physiological evidence (knockout or transgenic mouse models)	Pathological evidence (cancer relevant human specimens)	Biochemical evidence (cancer relevant substrates)
DCAF1	NA	Overexpressed in ovarian cancer136, colon cancer109	LATS1/2139,140, Dicer1109, NF2141
DCAF2	NA	Overexpressed in head and neck squamous cell carcinoma149, melanoma150, ovarian cancer123, Ewing sarcoma151	CDT1150,152, p21150, SET8150
DCAF4L2	NA	Overexpressed in colorectal cancer276	PPM1B276
DCAF6	NA	Overexpressed in prostate cancer154	NA
DCAF13	NA	Overexpressed in hepatocellular carcinoma155	NA
AhR	NA	Overexpressed in liver cancer321, thyroid cancer322	NA
COPS8	NA	NA	CENP-A323
FBXO44	NA	Overexpressed in breast cancer324	NA
FBXW5	NA	NA	DLC1289, TSC2290,325
HBx	Hepatocarcinogenesis in transgenic Hbx mice129,130	Overexpressed in liver cancer131, intrahepatic cholangiocarcinoma132, adenoid cystic carcinoma133	NA
RBBP7	NA	NA	CENP-A292,323
STRAP	NA	Overexpressed in colon cancer326, lung cancer326	NA
HOXB4	NA	Overexpressed in acute myeloid leukemia327, nephroblastoma328	Geminin329

DCAF1 is one of the primary and well-defined substrate recognition receptors of CLR4 complexes, where more than 15 proteins have been identified as substrates of DCAF1. By interacting with target substrates, resulting in either degradation or non-degradation outcomes, DCAF1 participates in multiple physiological and pathological processes, including cell cycle arrest¹³⁶, germ cell meiosis⁶⁷, virus replication¹¹⁰, skeletal muscle differentiation¹³⁷ and transcriptional repression¹³⁸. As for its neoplastic roles, DCAF1 has been regarded as an important oncogenic component of the CRL4 complex, where it is found to be upregulated in both ovarian cancer¹³⁶ and colon cancer specimens¹⁰⁹. This oncogenic role may be mechanistically attributed to its destabilizing effects on substrates such as LATS1 (large tumor suppressor 1)^139,140, Dicer1¹⁰⁹, and NF2 (neurofibromin 2)¹⁴¹, which normally act as tumor suppressors to inhibit the activity of multiple downstream oncogenic pathways, including Hippo/YAP^139,140 and JAK/STAT3¹⁰⁹. Interestingly, the tumor suppressor Merlin (NF2) is a direct inhibitor of CRL4-DCAF1/VPRBP, and tumor-associated mutations of Merlin either fail to bind DCAF1 or are unable to move into the nucleus to block DCAF1 ubiquitin ligase activity¹⁴². However, genetically engineered mouse models are still necessary to fully appreciate the oncogenic roles of DCAF1 (Table 3 and Table 5).

Besides DCAF1, DCAF2 is also a well-investigated substrate receptor of CRL4, with more than 15 defined downstream ubiquitin substrates. By destabilizing or activating specific substrates, it is involved in the regulation of various biological events, including genomic stability^143–145, cell cycle progression¹⁴⁶, gluconeogenesis^72,147 and transcriptional suppression¹⁴⁸. As for a role in tumorigenesis, DCAF2 has been recognized as a major oncogenic receptor of CRL4. Elevated levels of DCAF2 are frequently detected in head and neck squamous cell carcinoma¹⁴⁹, melanoma¹⁵⁰, ovarian cancer¹²³ and Ewing sarcoma¹⁵¹. Mechanistic studies have confirmed that degradation of CDT1, p21, and SET8 (set-domain histone methyltransferase-8) by CRL4 (DCAF2) regulates histone methylation and stimulates proliferation of melanoma, serving as a potential target of anti-melanoma therapeutics^150,152. However, biochemical evidence of a role for DCAF2 in other malignancies remains limited, let alone in rodent models (Table 3 and Table 5). Recently, WDR4 (WD repeat 4-containing cullin-RING ubiquitin ligase 4) has been reported as an oncogenic receptor of CRL4 by promoting the degradation of the tumor suppressor PML (Promyelocytic leukemia) via ubiquitination to re-modulate an immunosuppressive tumor microenvironment in lung cancer setting¹⁵³.

In addition to the aforementioned receptors, there are CRL4 substrate recognition receptors responsible for triggering the onset and progression of malignancies, including overexpression of DCAF6 in prostate cancer¹⁵⁴, DCAF13 in hepatocellular carcinoma¹⁵⁵. The majority of substrate-mediated mechanisms are centered on cell cycle progression, genomic instability, and regulation of apoptosis, suggesting therapeutic potential in targeting CRL4 ubiquitin ligase (Figure 2, Table 3 and Table 5).

Figure 2. Major oncogenic substrates of CRL4.

Tumor suppressive members

Despite mounting evidence of an oncogenic role of CRL4, many substrate receptors of CRL4 have demonstrated tumor suppressive effects, adding diversity and complexity into the regulatory landscape of CRL4 in tumorigenesis.

DDB2 (damaged DNA binding 2)

DDB2 is recognized as one of the most consequential tumor suppressors in human cancer. Physiologically, the majority of CRL4 (DDB2)-substrate interactions lead to alterations of cell cycle regulation and DNA repair, hinting at an underlying role in neoplastic control^84,156. Genetic ablation of Ddb2 sensitizes rodent models to UV-induced skin tumorigenesis⁷⁶ while downregulated expression of DDB2 is commonly observed in cases of human prostate cancer¹⁵⁴, skin cancer⁸⁴ and colorectal cancer¹⁵⁷, which support the tumor suppressive role of DDB2. Moreover, studies have shown that destabilization of oncogenic substrates, including HBO1 (histone acetyltransferase bound to ORC 1)¹⁵⁸, may contribute to the cancer inhibitory effects of DDB2 (Table 3 and Table 6).

Table 6.

Major tumor suppressive substrate recognition proteins

Substrate recognition protein	Physiological evidence (knockout or transgenic mouse models)	Pathological evidence (cancer relevant human specimens)	Biochemical evidence (cancer relevant substrates)
DCAF8	NA	NA	CDC25A170
DCAF11	NA	NA	NRF2171, p21172
DCAF15	NA	NA	CAPERα173
COP1	NA	Downregulated in renal cell carcinoma165	ETV5166, c-Jun167
CRBN	NA	Downregulated in multiple myeloma161	IKZF1/3162, GSPT1163, CK1α164
DDB2	Susceptible to UV-induced skin carcinogenesis (systemic Ddb2 knockout)76	Downregulated in prostate cancer154, skin cancer84, colorectal cancer157	AR154,285, HBO1158
WDR70	NA	Loss-of-function mutation in ovarian cancer169	H2B294,330

Cereblon (CRBN)

Besides DDB2, cereblon (CRBN) is another vital substrate receptor of CRL4 found negatively correlated with tumorigenic behaviors. Owing to the variety of downstream substrates, CRL4 (CRBN) has multiple impacts on cellular biology, especially in neurodegeneration¹⁵⁹, anti-epileptogenesis⁷¹ and homeostasis of membrane excitability¹⁶⁰. The anti-tumor role of CRBN is usually centered on hematological malignancies, whose expression level is dramatically decreased in multiple myeloma cells¹⁶¹. Although IKZF1/3 (Ikaros family zinc finger protein 1/3), GSPT1 (G1-to-S phase transition 1) and CK1 (casein kinase 1α) may not be traditional substrates of CRBN, the induced turnover of IKZF1/3¹⁶², GSPT1¹⁶³ and CK1α¹⁶⁴ by CRL4 (CRBN) accounts for the inhibitory effects of thalidomide-based molecules (thalidomide, lenalinomide, and pomalidomide) against multiple myeloma, leukemia, and myelodysplastic syndrome, respectively, which may mechanistically correlate to the inhibition on cell cycle progression (Table 3 and Table 6). The role of thalidomide-based molecules inducing CRBN-dependent degradation of various key tumorigenic factors will be discussed further below.

COP1 (constitutive photomorphogenesis 1)

Expression of COP1 is suppressed in renal cell carcinoma¹⁶⁵. Moreover, biochemical studies suggest that by destabilizing its substrate ETV5 (ETS variant 5) and c-Jun, CRL4 (COP1) suppresses multiple oncogenic transcription factors in lung tumorigenesis, suggesting a tumor suppressive function^166,167. These results have indicated a role for COP1 in cancer regulation. Cop1 knockout mice are embryonic lethal, whereas Cop1^+/− mice are viable and fertile¹⁶⁸. Through the generation of the Cop1 hypomorphic alleles, in which the Cop1 protein level was reduced by 90%, shows a 15–20% reduction in body weight compared to wild-type mice, coupled with decreased organ sizes¹⁶⁸ (Table 3 and Table 6).

WDR70 (WD repeat domain 70)

Loss-of-function mutations of WDR70 have been identified in ovarian cancer¹⁶⁹. WDR70 mediates mono-ubiquitination of histone H2B, thereby ensuring H2B stability during cell division and preventing malignant transformation¹⁶⁹. DCAF8, DCAF11 and DCAF15 have been identified as potential tumor suppressors due to their effects on regulating the stability of CDC25A (cell division cycle 25A)¹⁷⁰, NRF2 (nuclear factor erythroid 2-related factor 2)¹⁷¹, p21¹⁷² and CAPERα (coactivator of activating protein-1 and estrogen receptor α)¹⁷³, respectively (Figure 3, Table 3 and 6).

Figure 3. Major tumor suppressive substrates of CRL4.

Context-dependent members

Unlike those members with specific neoplastic contributions, context-dependent substrate recognition receptors are rarely identified. Both GNB2 (G protein subunit beta 2) and GNB3 are substrate receptors for CRL4, which degrade GRK2 (G protein-coupled receptor kinase 2) resulting in cardiovascular protective effects. However, due to the inconsistent roles of GRK2 in tumorigenesis, it is thought to be an oncoprotein in breast cancer¹⁷⁴ and tumor suppressor in hepatocellular carcinoma¹⁷⁵, these two receptors are accordingly considered as context-dependent members, despite a lack of clinical or mouse models studies^175–177 (Table 3 and Table 7).

Table 7.

Major context-dependent substrate recognition proteins

Substrate recognition protein	Pathological evidence (cancer relevant human specimens)	Biochemical evidence (cancer relevant substrates)
GNB2	NA	GRK2175–177
GNB3	NA	GRK2175–177

Degrons recognized by Cullin 4 substrate receptors

Degrons, specific short amino acid within a target protein, is critical for the interaction of target protein with their E3 ligase receptors¹⁷⁸. Degrons have been identified within many E3 ligase receptor substrates, such as the ETGE degron motif for KEAP1¹⁷⁹; and the D-box, the KEN box and the ABBA motif for the APC/C E3 ligase complex¹⁸⁰. More importantly, regulation of degron recognition and/or availability including through post-translational modifications such as phosphorylation, methylation, acetylation and hydroxylation, has been reported to promote or inhibit substrate recognition by E3 ligases in response to environmental stimuli. For instance, the substrate recognition receptor β-TRCP recognizes a phosphorylated DSG degron¹⁸¹, and pVHL utilizes the prolyl hydroxylated degrons¹⁸². Conceivably, inherited/somatic mutations have been identified in degrons contributing to the accumulation of oncoproteins through escaping E3 ligase-mediated degradation, leading to various diseases, including cancer¹⁷⁸. Thus, defining the degrons within substrates of E3 receptors is not only crucial to validate the direct interaction of a target protein with its E3 ligase receptor, but also highlight the potential to design therapeutics to target the oncoproteins for degradation.

CRL4 substrate receptors also display recognition of their substrates in a degron dependent manner. For instance, COP1 has been characterized to bind degrons within substrates with the consensus amino acid sequence EExxxVP[D/E] (Figure 4A)¹⁸³, and Cdt2, also termed DCAF2, recognized its substrates containing a PCNA-interacting peptide (PIP) degron (Figure 4B)¹⁸⁴. Interestingly, DCAF1, harboring a putative chromo domain, has been demonstrated to read a “monomethyl degron” (Figure 4C)¹³⁸, whereas, Cereblon (CRBN), a direct thalidomide teratogenicity target protein, recognizes an “acetylated degron” in the presence of high glutamine (Figure 4D)¹⁸⁵. However, due to limited number of substrates identified for CRL4 receptors, more effort is necessary in identifying CRL4 substrates, and degrons within those substrates, to clarify the exact degron requirements. These will benefit biomedical studies and further improve development of cancer treatments, especially in utilizing the PROTAC technology¹⁸⁶, which has been considered a novel mechanism targeting what have been long thought of as “undruggable” targets.

Figure 4. A schematic illustration of degrons that are recognized by CRL4 receptors.

Harnessing the ubiquitin ligase for targeted degradation of cellular proteins

During the past two decades, targeted therapy has become the most promising strategy in modern medicine through the identification and development of small therapeutic molecules targeting mutated or modified factors driving cancer. In addition, biologics such as antibody-based therapeutics, RNA interference, and CRISPR-mediated genetic modifications, have gained much interest recently in drug discovery.

The primary mechanism behind antibody-based biologics is based on antigen-antibody interactions that may specifically and potently block the activity of pathogenic antigens to achieve therapeutic goals, which has been successful in cases such as bevacizumab for VEGFR (vascular endothelial growth factor receptor) and cetuximab for EGFR (epidermal growth factor receptor)¹⁸⁷. However, since their ionic properties are naturally rejected by the non-ionic cell membrane, these antibody-based biologic drugs are only effective to extracellular or membrane proteins and also costly in nature, which greatly limit their clinical applications¹⁸⁶. In addition, the RNAi (RNA interference) technology has emerged as a vital addition to antibody-based regimens, which focuses on degrading intracellular mRNA to restrict the translation and expression of specific proteins via pre-designed siRNA. Nevertheless, because of the instability of siRNA (small interfering RNA) under physiological circumstances, RNAi treatment displays unfavorably oral bioavailability and often poor tissue distribution, which has largely limited its usage thus far to liver diseases¹⁸⁸. In consideration of these challenges, a better small molecule with intracellular permeability, increased bioavailability, and widespread distribution may therefore provide wider bioavailability and efficacy.

Protein Knockout

Most intracellular proteins are directed to the ubiquitin machinery for proteolytic destruction. Owing to the outstanding selectivity and degradative proficiency of the ubiquitin machinery, nature has chosen ubiquitin ligases as a prime apparatus for eliminating key target proteins, thereby altering a desired cellular process or pathway (Figure 5). In 1993, Scheffner and colleagues described the first case of viral hijacking of the ubiquitin ligase to promote tumorigenesis: the high-risk human papillomavirus (HPV) types 16 and 18 utilize their oncogenic E6 oncoprotein to recruit p53 tumor suppressor to the cellular E6AP ubiquitin ligase for ubiquitination and proteasomal degradation¹⁸⁹. Here E6 functions as a bridging peptide that brings E6AP and p53 in close proximity to facilitate ubiquitin transfer onto p53.

Figure 5. Timeline illustrating crucial discoveries of the protein knockout and PROTAC techniques.

Given that ubiquitin ligases are modular proteins and substrate specificity is dictated by protein-protein interactions, and inspired by the hijacking mechanism of the HPVs, Zhou et al. set out to engineer SCF^β-TRCP ubiquitin ligase to target cellular proteins which are otherwise not substrates of β-TRCP or escaped recognition by β-TRCP due to oncogenic mutations of the DSG degron^190,191. This is achieved by covalent attachment of specific binding peptide of the desired target to β-TRCP or truncated β-TRCP deleted of the substrate-binding WD40 domain, thereby enabling recruitment of desired target protein to the SCF machinery for ubiquitination and proteasomal degradation. This engineered ubiquitination machinery, designated protein knockout (PKO), was employed to successfully deplete a variety of intracellular or membrane proteins of interest, including pRB, p107, p130, β-catenin, c-Myc, EGFR, ErbB2/HER2, ErbB3/HER3, as well as viral oncoproteins (HIV16 E1)^191–197. Moreover, the use of PKO demonstrated the remarkable versatility in selective targeting of post-translationally modified subpopulation of desired cellular proteins, a unique function that are not attainable by CRIPSPR-Cas9, RNAi and antisense technologies^191–197. Furthermore, PKO can be integrated with RNAi to block protein synthesis and speed up protein destruction simultaneously, thereby achieving more rapid and effective depletion of stable proteins¹⁹⁸. Taken together, these studies set the stage for harnessing ubiquitin ligases in degrading cellular proteins of interest, and opened up new avenues to design and develop therapeutic strategies in drug discovery.

PROTAC (PROteolysis TArgeting Chimera)

PROTAC technique has been established as an alternative to current small molecule therapies. PROTACs feature a bimodal molecule that simultaneously connects an E3 ligase and the target protein, leading to ubiquitin-dependent degradation on specific substrates¹⁹⁹. This method creates an opportunity to degrade specific proteins that currently could not be pharmaceutically targeted by small molecules or without characterized E3 ligases, and could theoretically lead to the proteolysis of any substrate via only a single E3 ligase. Therefore, identifying appropriate binding ligands for E3 ligases and substrates is the most critical and challenging step for PROTAC design (Figure 5).

The first successful model of PROTAC was reported in 2001 by Sakamoto and colleagues, who creatively designed a chimeric molecule with a small ligand for MetAP2 (methionine aminopeptidase 2) and a 10-amino-acid phosphopeptide for β-TRCP, a substrate recognition receptor for CRL1²⁰⁰. Later, both the Ivan and Jaakkola groups described a VHL (von Hippel-Lindau)-based PROTAC design which promoted the degradation of FKBP12 (the 12-kDa FK506-binding protein) and AR via the VHL E3 ligase²⁰². The HIFα (hypoxia inducible factor α) moiety, which was a natural substrate for CRL2 (VHL), was therefore incorporated into the PROTAC molecule as a ligand for VHL, a substrate receptor of CRL2. The hydroxylation of Proline-564 on HIFα was pre-requisite and necessary for recruiting VHL^182,201. Therefore, this hydroxyproline-containing chimeric molecule became a classical representative of peptide-based PROTACs. Nonetheless, due to the peptidic nature of HIFα moiety in this PROTAC, its inefficient intracellular delivery limits in vivo applications. The hydroxyproline core is actually a non-ionic subunit of the HIFα moiety, and a re-designed VHL ligand featuring a sole hydroxyproline core greatly reduces the peptidic nature, while still retains binding affinity, which is therefore defined as small molecule-based PROTAC and more amenable for delivery to intracellular targets²⁰³ (Details will be discussed in subsequent sections) (Figure 6 and Table 9).

Figure 6. Major CRL4 targeted drugs and pharmacological mechanisms.

Table 9.

Major peptide PROTACs and their biological impacts

Name	Target	Target ligand	E3 ligase	E3 ligase ligand	Biological impacts
Api-PROTAC	AHR	Apigenin	CRL2 (VHL)	A pentapeptide sequence derived from HIF1α	Potential roles against AHR positive disorders332,333
Tri_a-PROTAC	AKT	PCC	CRL2 (VHL)	A heptapeptide derived from HIF1α	Potential roles against AKT positive disorders227
PROTAC-3	AR	DHT	CRL1 (β-TRCP)	A phosphopeptide derived from IκBα	Potential roles against AR positive disorders228
PROTAC-5	AR	DHT	CRL2 (VHL)	A heptapeptide derived from HIF1α	Potential roles against AR positive disorders202
PROTAC-2	ER	Estradiol	CRL1 (β-TRCP)	A phosphopeptide derived from IκBα	Potential roles against ER positive disorders228
E2-SMPI	ER	Estradiol	CRL2 (VHL)	An octapeptide or pentapeptide derived from HIF1α	Inhibition of endothelial cell differentiation220–222
PROTAC-4	FKBP12	AP21998	CRL2 (VHL)	A heptapeptide derived from HIF1α	Potential roles against FKBP12 positive disorders202
phosphoPROTAC-1	FRS2α	Tyrosine phosphorylation sequences of TrkA	CRL2 (VHL)	A heptapeptide derived from HIF1α	Inhibition of neuronal differentiation223
HBx-PROTAC	HBx	The oligomerization domain of HBx	CRL2 (VHL)	ODD domain of HIF1α	Potential roles against HBV infection and development of liver cancer225
Fu-SMPI	MetAP-2	Fumagillol	CRL2 (VHL)	An octapeptide derived from HIF1α	Potential roles against MetAP-2 positive disorders220
phosphoPROTAC-2	PI3K	Tyrosine phosphorylation sequences of ErbB3	CRL2 (VHL)	A heptapeptide derived from HIF1α	Loss of viability of breast cancer cells223
Smad3-PROTAC	Smad3	SMC	CRL2 (VHL)	A pentapeptide derived from HIF1α	Potential roles against the development of renal fibrosis226
TH006	Tau	Sequences derived from β-tubulin	CRL2 (VHL)	A heptapeptide derived from HIF1α	Reduced proteotoxic effects among AD mice224

Abbreviations: NS: not specific; AHR: aryl hydrocarbon receptor; PCC: protein-catalyzed capture agent; DHT: dihydroxytestosterone; FRS2α: fibroblast growth factor receptor substrate 2 α; TrkA: tropomyosin receptor kinase A; PI3K: phosphatidylinositol-3-kinase; ErbB3: erythroblastosis oncogene B3; MetAP-2: methionine aminopeptidase-2; SMC: small molecule compound; ERK: extracellular regulated protein kinase; ODD: oxygen-dependent degradation; E2-SMPI: Estradiol-Small molecule proteolysis inducer; SNIPER: specific and nongenetic IAPs-dependent protein erasers; AR: androgen receptor; ER: estrogen receptor; AD: Alzheimer’s Disease.

Small molecule-based PROTACs

CRL2 (VHL)-based PROTACs

The peptidic features of PROTACs limited its clinical application, which led to the design of less ionic and therefore a more bioavailable type of PROTAC, later defined as small molecule PROTACs¹⁸⁶. The first report of a CRL2 (VHL)-based small molecule PROTAC emerged in 2012, which featured a hydroxyproline core inside the ligand for VHL with the unnecessary residues of HIFα moiety trimmed to minimize the molecular weight and peptide nature, allowing greater penetration of the cell plasma membrane^204–206. In 2015, the degrading effects of this small molecule CRL2 (VHL)-based PROTAC was demonstrated in preclinical cancer models where this PROTAC displayed marked degradation of specific proteins of more than 90%, including RIPK2 (receptor interacting serine/threonine kinase 2), ERRα (estrogen-related receptor α) and HaloTag fusion proteins^203,207. Therefore, these results demonstrate potential for PROTACs as a novel therapeutic option against numerous diseases, especially cancers. Currently, more preclinical data is necessary before assessing therapeutic efficacy in clinical trials (Table 10).

Table 10.

Major small molecule PROTACs and their biological impacts

Name	Target	Target ligand	E3 ligase	E3 ligase ligand	Biological impacts
SNIPER-1	AR	DHT	cIAP1	BE04	Potential roles against AR positive disorders216
SARM-nutlin PROTAC	AR	SARM	MDM2	Nutlin	Potential roles against AR positive disorders214
TKI-PROTAC-1	BCR-ABL	TKI moiety (bosutinib and dasatinib)	CRL4 (CRB)	Pomalidomide	Reduced viability of chronic myelogenous leukemic cells238
Compound MZ1	BRD4	JQ1	CRL2 (VHL)	VHL binders	Suppression on oncogenic transcriptions associated with BRD4235
ARV-771	BRD4	OTX015	CRL2 (VHL)	HIF1α-derived and hydroxyproline-contained ligand	Tumor suppression in CRPC mouse models233; Induced apoptosis in MCL cells334
Compound 23	BRD4	Azacarbazole	CRL4 (CRB)	Thalidomide	Growth suppression against xenograft leukemic tumors234
dBET1	BRD4	JQ1	CRL4 (CRB)	Phthalimide	Delayed leukemia progression in mice211
CLIPTAC-1	BRD4	TCO-tagged JQ1	CRL4 (CRB)	Thalidomide	Potential roles against BRD4 positive disorders237
ARV-825	BRD4	OTX015	CRL4 (CRB)	Pomalidomide	Suppressed proliferation and enhanced apoptosis in Burkitt’s lymphoma210; Induced apoptosis in MCL cells334
TKI-PROTAC-2	c-ABL	TKI moiety (dasatinib)	CRL2 (VHL)	HIF1α-derived and hydroxyproline-contained ligand	Potential roles against c-ABL positive disorders238
TKI-PROTAC-3	c-ABL	TKI moiety (bosutinib and dasatinib)	CRL4 (CRB)	Pomalidomide	Potential roles against c-ABL positive disorders238
CDK9-PROTAC	CDK9	Aminopyrazole analog	CRL4 (CRB)	Thalidomide	Potential roles against CDK9 positive disorders213
Compound 4	CRABP I/II	ATRA	cIAP1	MeBS	Inhibited cell migration of neuroblastoma cells218,219
brequinar-PROTAC	DHODH	Brequinar	CRL2 (VHL)	HIF1α-derived and hydroxyproline-contained ligand	Cytotoxicity against cancer cells239
SNIPER-2	ER	Estrone	cIAP1	BE04	Potential roles against ER positive disorders216,217
CLIPTAC-2	ERK1/2	Probe 1	CRL4 (CRB)	Thalidomide	Potential roles against ERK1/2 positive disorders237
PROTAC_ ERRα	ERRα	Thiazolidinedione-based ligand	CRL2 (VHL)	HIF1α-derived and hydroxyproline-contained ligand	Potential roles against ERRα positive disorders203
HT7-PROTAC-1	FKBP12	NS	ß-TRCP-HT7	Chloroalkane	Potential roles against FKBP12 positive disorders230
dFKBP	FKBP12	Steel factor	CRL4 (CRB)	Phthalimide	against FKBP12 positive disorders211
HT7-PROTAC-2	FKBP12	NS	Parkin-HT7	Chloroalkane	Potential roles against FKBP12 positive disorders230
SNIPER-3	RAR	Ch55	cIAP1	BE04	Potential roles against RAR positive disorders216
PROTAC_ RIPK2	RIPK2	Vandetanib	CRL2 (VHL)	HIF1α-derived and hydroxyproline-contained ligand	Potential roles against RIPK2 positive disorders203
SirReal-PROTAC	Sirtuin 2	SirReal	CRL4 (CRB)	Thalidomide	Enhanced acetylation of the microtubule network212
SNIPER (TACC3)	TACC3	KHS101	APC/C (CDH1)	Bestatin	Cytotoxic effects against cancer cells229
PROTAC 3i	TBK1	Aminopyrimidine chemotype	CRL2 (VHL)	HIF1α-derived and hydroxyproline-contained ligand	Potential roles against TBK1 positive disorders335
Homo-PROTAC	VHL	VH032	CRL2 (VHL)	VH298	Self-degradation of E3 ligases231

Abbreviations: NS: not specific; ODD: oxygen-dependent degradation; E2-SMPI: Estradiol-Small molecule proteolysis inducer; SNIPER: specific and nongenetic IAPs-dependent protein erasers; AHR: aryl hydrocarbon receptor; AR: androgen receptor; ER: estrogen receptor; FKBP12: the 12-kDa FK506-binding protein; RAR: retinoic acid receptor; cIAP1: cellular inhibitor of apoptosis protein 1; SARM: non-steroidal androgen receptor ligand; MDM2: mouse double minute 2 homolog; BCR-ABL: TKI: tyrosine kinase inhibitor; BCR-ABL: breakpoint cluster region-abelson murine leukemia viral oncogene; BRD4: JQ1: a BET bromodomain inhibitor; BE04: a bestatin derivative that has affinity to cIAP1; CRPC: castration-resistant prostate cancer; CRABP: cellular retinoic acid-binding protein; ATRA: all-trans retinoic acid; MeBS: methyl bestatin; ERRα: estrogen-related receptor α; RIPK2: receptor interacting serine/threonine kinase 2; TACC3: transforming acidic coiled coil-containing protein 3; DHODH: dihydroorotate dehydrogenase; TBK1: TANK-binding kinase 1; MCL: mantle cell lymphoma; CDK9: cyclin-dependent kinase 9; SirReal: sirtuin rearranging ligand; SMC: small molecule compound; ERK: extracellular regulated protein kinase.

Thalidomide derivatives and CRL4 (CRBN)-based PROTAC

As mentioned above, thalidomide and its derivatives are able to directly target the substrate receptor CRBN of CRL4, promoting the degradation of multiple substrates for anti-tumor effects especially in multiple myeloma^162,208,209. Since these drugs feature non-ionic structures and are therefore suitable as small molecule ligands, many CRL4 (CRBN)-based PROTACs have been constructed via utilizing the moiety of thalidomide derivatives as the ligand for CRBN in conjugation with specific inhibitors as the substrate ligands.

In 2015, Winter and colleagues reported that by assembling phthalimide moiety and BET antagonist, a heterobifunctional PROTAC was formed which bridged CRBN and BRD4 (bromodomain-containing protein 4), leading to the highly selective degradation of BRD4 and consequently demonstrating anti-proliferative and tumor inhibitory effects in mouse models^210,211. Additionally, with a FKBP12 inhibitor as the substrate ligand, the phthalimide-equipped PROTAC could also connect CRBN with FKBP12 to facilitate its ubiquitin-dependent degradation²¹¹. Apart from the phthalimide-characterized PROTACs, thalidomide-equipped PROTACs have also emerged, which lead to the degradation of Sirtuin 2 (SIRT2)²¹² and CDK9 (cyclin-dependent kinase 9)²¹³, implicating their possible applications in regulating cell cycle and epigenetic stability (Table 10).

Other major E3 ligases for PROTAC technology

The MDM2-based PROTAC was the first reported example of small molecule PROTACs²¹⁴. It was constructed by a nonsteroidal AR ligand for AR recognition, while the MDM2 ligand was a polyethylene glycol (PEG)-contained nutlin, a pharmaceutical inhibitor of MDM2. Schneekloth and colleagues discovered that the expression of AR was effectively inhibited in HeLa cells after administration of MDM2-based PROTAC which could be reversed by the proteasome inhibitor epoxomicin, displaying therapeutic potential against AR-positive human malignancies^214,215.

Additionally, the cIAP1-based PROTAC has also been designed, featuring a bestatin-incorporated ligand for E3 ligase cIAP1²¹⁵. According to different target ligands for substrates, this set of PROTACs could successfully lead to the degradation of ERα^216,217, RAR (retinoic acid receptor)²¹⁶ and CRABP I/II (cellular retinoic acid binding protein I/II)^218,219, involved in multiple biological activities such as metastasis of neuroblastoma cells (Table 10).

PROTAC: Major biological impacts Peptide PROTAC

As the first-generation of PROTAC technology, peptide PROTACs had limited tissue distributions and membrane penetration. However, these PROTACs still exhibited various biological effects via degrading specific substrates.

CRL2 (VHL) is the major E3 ligase that is utilized for experimental design of peptide PROTACs. Targeted turnover of ER (estrogen receptor), FRS2α (fibroblast growth factor receptor substrate 2 α) and tau, VHL-based PROTACs lead to inhibition of endothelial cell differentiation^220–222, neuronal differentiation²²³ and reduced proteotoxic effects in AD (Alzheimer’s Disease) mouse models²²⁴, respectively, demonstrating vital physiological participation and therapeutic potential against pathological conditions. Additionally, the HBx-targeted PROTAC also displayed protective roles against HBV (hepatitis B virus) infection and liver carcinogenesis²²⁵ while the Smad3-targeted PROTAC prevented the progression of renal fibrosis²²⁶. Meanwhile, despite a lack of in vivo evidence, the remaining PROTACs are believed to have crucial roles in multiple biological events, especially for those targeting AKT²²⁷, AR²⁰² and MetAP-2²²⁰ for degradation.

Besides CRL2 (VHL), there are also two PROTACs, namely PROTAC-3 and PROTAC-2, that utilize CRL1 (β-TRCP) as E3 ligases to destabilize AR and ER, respectively²²⁸. Nonetheless, although the technology works well to degrade target proteins in vitro, more in vivo evidence are necessary to evaluate their potential efficacy.

Currently, although peptide PROTACs are no longer a primary focus in the development of PROTAC technology due to difficulties described above, there may still be instances where there is therapeutic value of peptide PROTACs under certain circumstances, which maybe act as alternative options to small molecule PROTACs in the future (Table 9).

Small molecule PROTAC

Due to its limited molecule weight and non-ionic features, small molecule PROTAC is thought to provide greater bioavailability and efficacy. CRL2 (VHL) and CRL4 (CRBN) are two key E3 ligases for small molecule PROTACs. Currently, owing to the incurability of malignant diseases, all evidence regarding VHL-based PROTACs is tumor associated, which serve as potential anti-cancer interventions (Table 10). Similarly, the pathological role of CRBN-based PROTACs is also centered on neoplastic relevant behaviors (Table 10). Besides this direct evidence, the biological functions of the remaining small molecule PROTACs still require in vivo verification.

In addition, other PROTAC members including cIAP1-based, APC/C (CDH1)-based and MDM2-based PROTACs also emerge as protein degraders for different targets, displaying potential therapeutic effects against RAR (retinoic acid receptor)-positive²¹⁶, TACC3 (transforming acidic coiled coil-containing protein 3)-positive²²⁹ and AR-positive²¹⁴ disorders respectively. Moreover, apart from these traditional small molecule PROTACs, two types of novel PROTACs have been reported. The first type, HaloTag7-fused PROTACs (HT7-PROTACs), where unlike other PROTACs the E3 ligases (β-TRCP) is engineered and fused to a HT7 tag. And then a chloroalkane ligand in the PROTAC is applied to attach the E3 ligase and leads to substrate degradation²³⁰. This suggests that if it is difficult to design a small molecule ligand for certain E3 ligases, engineered modifications of E3 ligases may be an alternative approach for cell and molecular based studies. Secondly, a homo-PROTAC has also been designed, which is a bivalent small-molecule dimerizer of VHL E3 ligase that induces its self-degradation²³¹. This likewise provides therapeutic implications against those disorders with an elevated expression of undruggable E3 ligases (Table 10).

PROTAC: anti-tumor functionality by degrading oncogenic proteins

Most of the PROTACS have thus far been designed with the purpose of treating cancers via degradation of specific oncogenic proteins, such as BRD4. Currently, investigations have confirmed that BRD4 acts as an oncoprotein in multiple malignancies such as prostate and hematological cancers, primarily by interacting with histones in order to trigger transcription of oncogenes²³². Nevertheless, due to toxicities and progressive inefficacies of BRD4 inhibitors including BET, more specific and tolerable targeted medications are required, such as BRD4-targeted PROTACs. So far, there are six PROTACs reported to effectively target and destabilize BRD4, either VHL-based or CRBN-based. Among them, four PROTACs have shown anti-tumor efficacy, including ARV-771, compound 23, dBET1 and ARV-825, which display great inhibitory effects against the malignant progression of castration-resistant prostate cancer (CRPC)²³³, xenograft leukemic models²³⁴, leukemic cells²¹¹ and Burkitt’s lymphoma²¹⁰ respectively. All these PROTACs, especially ARV-771 and ARV-825, are waiting for verification in animal studies before being assessed in the clinic. Furthermore, despite a lack of direct evidence, the other two PROTACs including compound MZ1^235,236 and CLIPTAC-1^236,237 also target BRD4, which are believed to have potential contributions against BRD4-positive neoplasms.

In addition to BRD4, other PROTACs have also been regarded as anti-tumor PROTACs. As the only peptide PROTAC, phosphoPROTAC-2 directly degrades PI3K via recruiting CRL2 (VHL), culminating in reduced viability of breast cancer cells²²³. BCR-ABL, is an oncogenic fusion protein for chronic myelogenous leukemia, can be targeted by TKI-PROTAC-1 which has shown in vitro effects on survival of chronic myelogenous leukemic cells²³⁸. CRABP I/II are cellular binding proteins of retinoic acid and mediate the biologic impact of retinoic acid under diverse circumstances. The oncogenic role of CRABP I/II in the pathogenesis of neuroblastoma has been widely shown, and correspondingly, compound 4, a CRABP I/II-targeted PROTAC, has also been shown to have a tumor suppressive role against the migration of neuroblastoma cells^218,219. Moreover, both brequinar-PROTAC and SNIPER (TACC3) are characterized by their tumor inhibitory effect among colon and breast cancer cells, respectively, via targeting DHODH (dihydroorotate dehydrogenase)²³⁹ and TACC3 (transforming acidic coiled-coil-containing protein 3), respectively²²⁹. These results have shown the therapeutic potential of PROTACs as a novel class of anti-tumor therapeutics.

Apart from those with direct evidence, the majority of current PROTACs are assumed to potentially play anti-neoplastic roles, which have not been shown yet in vivo despite their pro-degrading effects on certain oncogenic substrates in cells. Among those, AR and ER are the most popular targets for PROTACs. At present, there have been four PROTACs, either peptide or small molecule, believed to successfully and specifically destabilize AR in vitro and in vivo, including PROTAC-3^228,240, PROTAC-5^202,240, SNIPER-1^216,240 and SARM-nutlin PROTAC^214,240. The AR-targeted PROTACs are considered as potential therapeutic agents given the extensive oncogenic role of AR in a variety of cancers, especially prostate cancer. Moreover, ER, a notorious oncoprotein in gynecological malignancies²⁴¹, has been targeted by three PROTACs, namely PROTAC-2^228,242, E2-SMPI^{220–222,242} and SNIPER-2^216,217,243, all demonstrating degrading effects of the ER protein despite limited studies showing suppression of tumorigenesis.

Taken together, although currently no PROTAC has entered the clinic, the anti-tumor PROTACs are still expected to become important supplements to current targeted therapies. Compared to the direct evidence regarding the anti-tumor effects of small molecule PROTACs, only one peptide PROTAC has reported a direct effect on cancer cell viability, which highlights the advantage and therapeutic potential of small molecules PROTACs. Nevertheless, there are issues with the current status of PROTACs that should be addressed in future investigations. First, PROTAC related studies have lacked comparisons with standard of care agents, which may lower their credibility as an alternative to currently approved options. Second, although there has been direct evidence towards the anti-tumor impact of certain PROTACs, in vivo efficacy in animals has been limited, let alone clinical assessment, therefore more studies are necessary to demonstrate the clinical benefits of this promising technology (Figure 7 and Table 11).

Figure 7. Anti-tumor PROTACs and their working mechanisms.

Table 11.

Major anti-tumor PROTACs and their contributions

Name	Category	Target	E3 ligase	Evidence*
phosphoPROTAC-2	Peptide	PI3K	CRL2 (VHL)	Direct roles against viability of breast cancer cells223
TKI-PROTAC-1	Small molecule	BCR-ABL	CRL4 (CRB)	Direct roles against viability of chronic myelogenous leukemic cells238
ARV-771	Small molecule	BRD4	CRL2 (VHL)	Direct roles against the tumor growth in CRPC mouse models233 and MCL cells334
Compound 23	Small molecule	BRD4	CRL4 (CRB)	Direct roles against the growth of xenograft leukemic tumors234
dBET1	Small molecule	BRD4	CRL4 (CRB)	Direct roles against the progression of leukemia211
ARV-825	Small molecule	BRD4	CRL4 (CRB)	Direct roles against the proliferation of Burkitt’s lymphoma210 and MCL cells334
Compound 4	Small molecule	CRABP I/II	cIAP1	Direct roles against the migration of neuroblastoma cells218,219
brequinar-PROTAC	Small molecule	DHODH	CRL2 (VHL)	Direct roles against the viability of colon cancer cells239
SNIPER (TACC3)	Small molecule	TACC3	APC/C (CDH1)	Direct roles against the viability of breast cancer cells229
Api-PROTAC	Peptide	AHR	CRL2 (VHL)	Potential roles against multiple AHR positive cancers332,333,336
Tri_a-PROTAC	Peptide	Akt	CRL2 (VHL)	Potential roles against multiple Akt positive cancers227,337
PROTAC-3	Peptide	AR	CRL1 (β-TRCP)	Potential roles against multiple AR positive cancers228,240
PROTAC-5	Peptide	AR	CRL2 (VHL)	Potential roles against multiple AR positive cancers202,240
PROTAC-2	Peptide	ER	CRL1 (β-TRCP)	Potential roles against multiple ER positive cancers228,242
E2-SMPI	Peptide	ER	CRL2 (VHL)	Potential roles against multiple ER positive cancers220–222,242
phosphoPROTAC-1	Peptide	FRS2α	CRL2 (VHL)	Potential roles against multiple FRS2α positive cancers223,338
HBx-PROTAC	Peptide	HBx	CRL2 (VHL)	Potential roles against development of liver cancer225
Fu-SMPI	Peptide	MetAP-2	CRL2 (VHL)	Potential roles against multiple MetAP-2 positive cancers220,339
SNIPER-1	Small molecule	AR	cIAP1	Potential roles against multiple AR positive cancers216,240
SARM–nutlin PROTAC	Small molecule	AR	MDM2	Potential roles against multiple AR positive cancers214,240
Compound MZ1	Small molecule	BRD4	CRL2 (VHL)	Potential roles against multiple BRD4 positive cancers235,236
CLIPTAC-1	Small molecule	BRD4	CRL4 (CRB)	Potential roles against multiple BRD4 positive cancers236,237
TKI-PROTAC-2	Small molecule	c-ABL	CRL2 (VHL)	Potential roles against multiple c-ABL positive cancers238,340
TKI-PROTAC-3	Small molecule	c-ABL	CRL4 (CRB)	Potential roles against multiple c-ABL positive cancers238,340
CDK9-PROTAC	Small molecule	CDK9	CRL4 (CRB)	Potential roles against multiple CDK9 positive cancers213,341
SNIPER-2	Small molecule	ER	cIAP1	Potential roles against multiple ER positive cancers216,217,243
CLIPTAC-2	Small molecule	ERK1/2	CRL4 (CRB)	Potential roles against multiple ERK1/2 positive cancers237,342
PROTAC_ ERRα	Small molecule	ERRα	CRL2 (VHL)	Potential roles against multiple ERRα positive cancers203,343
SNIPER-3	Small molecule	RAR	cIAP1	Potential roles against multiple RAR positive cancers216,344
PROTAC_ RIPK2	Small molecule	RIPK2	CRL2 (VHL)	Potential roles against multiple RIPK2 positive cancers203,345
SirReal-PROTAC	Small molecule	Sirtuin 2	CRL4 (CRB)	Potential roles against multiple Sirtuin 2 positive cancers212,346
PROTAC 3i	Small molecule	TBK1	CRL2 (VHL)	Potential roles against multiple TBK1 positive cancers335,347

^*:If there are direct descriptions or results demonstrating the anti-tumor efficacy of specific PROTAC, then these kinds of evidences are defined as “Direct”, while if there are no direct descriptions, the anti-tumor effects can only be predicted from the role of substrates, then these kinds of evidences are defined as “Potential”.

Perspective and therapeutic implications

As we have described, many substrate receptors demonstrate tumor-inhibitory effects by degrading oncogenic substrates, serving as potential therapeutic targets for cancer treatment. Currently, there are two major types of medications that specially aim at the interactions between substrates and recognition receptors in order to exert anti-cancer impacts, namely sulfonamides and thalidomide derivatives, each characterized by specific mechanisms. Three members of anticancer sulfonamides E7820, CQS (chloroquinoxaline sulfonamide) and indisulam have displayed efficacy against various cancers, including colorectal cancer and melanoma^244–246. However, their mechanisms of action remain poorly defined. Investigations have found that their anti-cancer contributions in colorectal cancer cells may rely on the interaction between CRL4 (DCAF15) and the substrate CAPERα, promoting its degradation^173,247. Knockout of DCAF15 or overexpression of CAPERα confers resistance to the effects by sulfonamides, further proving DCAF15 as the mechanistic target of sulfonamides²⁴⁷. So far, all these drugs have undergone clinical trials and are waiting for approval by the FDA for clinical application against colorectal cancer.

Thalidomide and its derivatives, known as immunomodulatory drugs (IMiDs), have drawn much attention owing to their great anti-tumor effectiveness against hematological malignancies, especially multiple myeloma²⁴⁸. Current investigations have confirmed that IMiDs facilitates interaction between CRBN (cereblon) and its substrates. By binding to CRBN, IMiDs, including thalidomide, pomalidomide and lenalidomide, can trigger the degradation of substrates such as IKZF1/3²⁴⁸ and CK1α¹⁶⁴, thus halting tumor expansion of multiple myeloma and myelodysplastic syndrome. All these IMiDs have gained FDA approval against multiple myeloma. Besides IMiDs, there is another type of CRBN modulator CC-885 which promotes the interaction between CRBN and GSPT1 to trigger the degradation of GSPT1, showing tumor suppressive effects against leukemic cells¹⁶³. In addition, DCAF2 seems to function as a therapeutic target since its interaction with substrates SET8 and p21 could be specifically inhibited by Pevonedistat¹⁵⁰ (Table 8).

Table 8.

Major CRL4s targeted therapies

Drug	Mechanism	Function	Preclinical evidence	Clinical trial	FDA approval
E7820	Promoting DCAF15-CAPERα degradation	Anti-growth in colorectal cancer cells247	Yes	Completed	Not yet
CQS	Promoting DCAF15-CAPERα degradation	Anti-growth in colorectal cancer cells247	Yes	Completed	Not yet
Indisulam	Promoting DCAF15-CAPERα degradation	Anti-growth in colorectal cancer cells173,247	Yes	Completed	Not yet
CC-220	Promoting CRBN- IKZF1 and IKZF3 degradation	Anti-tumor effect against multiple myeloma cells331	Yes	Ongoing	Not yet
Lenalidomide	Promoting CRBN- CK1α degradation	Anti-tumor effect against myelodysplastic syndrome164	Yes	Completed	Approved
	Promoting CRBN- IKZF1 and IKZF3 degradation	Anti-tumor effect against multiple myeloma cells162,208,209	Yes	Completed	Approved
Thalidomide	Promoting CRBN- IKZF1 and IKZF3 degradation	Anti-tumor effect against multiple myeloma cells248	Yes	Completed	Approved
Pomalidomide	Promoting CRBN- IKZF1 and IKZF3 degradation	Anti-tumor effect against multiple myeloma cells248	Yes	Completed	Approved
CC-885	Promoting CRBN- GSPT1 degradation	Anti-tumor effect against leukemic cells163	Yes	Not yet	Not yet
Pevonedistat	Inactivation of DCAF2-SET8/p21 degradation	Anti-tumor effect against melanoma cells150	Yes	Completed	Not yet

Abbreviations: CQS: chloroquinoxaline sulfonamide; CC-220: alias Compound 6.

As a lesser understood member of the Cullin-RING ligase family, CRL4 has drawn much attention due to its extensive involvement in physiological and pathological conditions, especially in tumorigenesis. Based on the evidence described above from knockout and transgenic mouse models, human clinical data, and biochemical interactions, the scaffold protein CUL4A/B and RING finger protein RBX1 are believed to serve as oncoproteins in a variety of malignancies. Nevertheless, the adaptor DDB1 and substrate recognition receptors seem to play context dependent roles in tumorigenesis, adding diversity to the actual role of CRL4 in tumorigenesis.

Moreover, owing to its critical role in cancer regulation, many CRL4-targeted medications have been identified, including thalidomide and its derivatives that target CRBN and the sulfonamides that target DCAF15, which significantly suppress proliferation of multiple myeloma and colorectal cancer, respectively. PROTACs are a group of artificially assembled protein degraders that induce the ubiquitination-mediated degradation of substrates via recruiting endogenous E3 ligases, such as CRL4 (CRBN) and CRL2 (VHL). Due to their ability to increase the number of druggable targets and potential for high specificity, more than 30 PROTACs have been designed and display anti-tumor effects, especially BRD4-targeted PROTACs. Current and future studies in animal tumor models and clinical trials would lead to mechanistic and functional understanding of PROTACs as drug candidates, and set the stage for their clinical usage in cancer patients.