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  • Waste disposal An attractive strategy for cancer …...REVIEW Waste disposal—An attractive strategy for cancer therapy Jemilat Salami1 and Craig M. Crews1,2,3* Targeted therapies
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REVIEW Waste disposal— An attractive strategy for cancer therapy Jemilat Salami 1 and Craig M. Crews 1,2,3 * Targeted therapies for cancer are typically small molecules or monoclonal antibodies that act by inhibiting the activity of specific proteins that drive tumor growth. Although many of these drugs are effective in cancer patients, the response is often not durable because tumor cells develop resistance to the drugs. Another limitation of this strategy is that not all oncogenic driver proteins are “druggable” enzymes or receptors with activities that can be inhibited. Here we describe an alternative approach to targeted therapy that is based on co-opting the cellular quality-control machinery—the ubiquitin-proteasome system—to remove specific cancer-causing proteins from the cell.We first discuss examples of existing cancer drugs that work by degrading specific proteins and then review recent progress in the rational design and preclinical testing of small molecules that induce selective degradation of specific target proteins. C ancer continues to be a leading global health problem; it has been estimated that by 2025 there will be nearly 20 million new cancer cases diagnosed each year (1). Notable progress is being made in cancer drug development, particularly in the areas of im- munotherapy and targeted therapy, but the enor- mity of the cancer problem requires a variety of therapeutic strategies. Many targeted therapies for cancer are small molecules or monoclonal antibodies that inhibit the activity of proteins driving tumor growth. Tumor cells often develop resistance to these drugs through overexpression of the target protein and/or through the acquisi- tion of new mutations in the target protein that allow it to escape the inhibitory effect of the drug. Over the past 15 years, researchers have be- gun to explore an alternative therapeutic ap- proach that aims to control protein function by regulating protein expression levels rather than activities. These efforts to harness controlled proteostasis as a therapeutic strategy evolved from the discovery that proteasome inhibitors that block protein degradation have anticancer activity. Carfilzomib and bortezomib are two ex- amples of such proteasome inhibitors approved by the U.S. Food and Drug Administration (FDA) for the treatment of multiple myeloma (MM) (2). Investigators have explored other ways in which the ubiquitin-proteasome system (UPS) can be manipulated to stabilize or promote the degradation of disease-causing proteins (Table 1). For example, efforts have been made to disrupt the interaction between proteins and the ubi- quitin E3 ligases responsible for their degradation (3). The interaction between the tumor suppres- sor p53 and its ubiquitin E3 ligase, MDM2 (mouse double minute 2 homolog), has been an attract- ive oncology target: A potent inhibitor of this in- teraction (RG7112) was found to kill wild-type p53- expressing cancer cells and to inhibit tumor growth in preclinical models of cancer. A reduction in the levels of oncoproteins can also be achieved by inhibiting enzymes that function to stabilize these proteins (3). For example, inhibition of USP7 (the ubiquitin-specific protease 7), a deubiquitinating enzyme (DUB) that deubiquitinates and stabilizes MDM2, reduces MDM2 levels and consequently increases p53 levels. To that end, the USP7 in- hibitor P5091 has shown promising antitumor activity in MM xenograft models (4). More recently, interest has focused on di- rectly using the UPS to induce degradation of specific target proteins, especially proteins for which there are no established DUBs or E3 ligases. These newer strategies involve the phar- macological hijacking of the cellular quality- control system to posttranslationally eliminate disease-causing proteins (Table 1). In this Re- view, we discuss the initial applications of this concept of targeted protein degradation to achieve controlled proteostasis and the strategies em- ployed to generate protein degraders. We high- light the progress achieved to date, as well as the some of the challenges inherent in this approach. The limitations of “occupancy-driven” pharmacology In this postgenomic era, a better understanding of the molecular drivers of cancer has led to the development of several successful cancer ther- apies that inhibit the activity of enzymes such as protein kinases (e.g., imatinib, erlotinib, and palbociclib), histone deacetylases (e.g., belinostat), and poly(ADP-ribose) polymerase (e.g., olaparib). These small-molecule inhibitors generally work by occupying a binding pocket or active site, resulting in the loss of protein function. However, given that most enzyme inhibitors bind noncova- lently (and thus reversibly), high drug concentra- tions must be maintained to ensure active-site occupancy and to sustain the clinical benefit (5). This is understood as an “ occupancy-driven” phar- macological paradigm: one that necessitates that the binding pocket remain occupied to maintain effectiveness of the small molecule (Fig. 1). Unfor- tunately, achieving and maintaining high systemic drug levels is one of the major challenges in drug development. Moreover, these high dosages can lead to undesirable off-target effects. In the case of kinases [which all bind ATP (adenosine triphos- phate)], active-site inhibition by ATP analogs is challenging because many kinases show structu- ral similarity. However, this promiscuity can some- times be advantageous, especially when targeting cancers that rely on the activation of multiple cellular signaling pathways. Creating a new paradigm: Induced protein degradation Targeted protein degradation has emerged as an alternative paradigm that requires only brief inter- action between a small molecule and its target protein to elicit the desired loss of protein func- tion. Under this “event-driven” paradigm, loss of function is due to the removal of the target pro- tein as a result of the transient binding event (Fig. 2). The strategy is to engage the cellular quality-control machinery (the UPS) and thereby tag unwanted proteins for destruction via ubi- quitination. Because the small-molecule drug sur- vives the target-protein tagging and destruction steps, it is free to engage in multiple cycles of target-protein degradation, resulting in substoi- chiometric activity. This event-driven approach is catalytic in nature, eliminating the need to main- tain high levels of drug. For the inhibitory effect of degradation to be reversed, the protein must be resynthesized. Protein degradation by the UPS involves a se- ries of steps culminating in the conjugation of the 8.5-kDa protein ubiquitin to the targeted protein by an E3 ligase (6–8). This monoubiquitinated protein then undergoes multiple cycles of ubi- quitination to become polyubiquitinated, which allows it to be recognized and then degraded by the 26S proteasome. Small molecules that both inhibit protein activity and induce protein degradation Early literature reports suggested that certain small molecules developed to inhibit protein activity could also induce the degradation of their targets, although the mechanism was often unknown. Canertinib (CI-1033) is one example of an irreversible tyrosine kinase inhibitor that also induces the polyubiquitination and degra- dation of ErbB-2 (a receptor tyrosine-protein kinase also known as HER2/neu), an important therapeu- tic target in several human cancers ( 9). Fulvestrant is another example of a small molecule that in- hibits the signaling function of its target, ERa (estrogen receptor alpha), and induces its degra- dation. Fulvestrant produced better outcomes in patients with ERa-positive breast cancers in com- parison with tamoxifen, another ERa antagonist that acts primarily by inhibiting ERa signaling (10). Arsenic trioxide is a highly effective therapy for acute promyelocytic leukemia (APL), and it works in part by degrading PML-RARa (promye- locytic leukemia–retinoic acid receptor alpha), the Salami et al., Science 355, 1163–1167 (2017) 17 March 2017 1 of 5 1 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA. 2 Department of Chemistry, Yale University, New Haven, CT 06520, USA. 3 Department of Pharmacology, Yale University, New Haven, CT 06520, USA. *Corresponding author. Email: [email protected] on August 13, 2020 http://science.sciencemag.org/ Downloaded from

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Page 1: Waste disposal An attractive strategy for cancer …...REVIEW Waste disposal—An attractive strategy for cancer therapy Jemilat Salami1 and Craig M. Crews1,2,3* Targeted therapies

REVIEW

Waste disposal—An attractivestrategy for cancer therapyJemilat Salami1 and Craig M. Crews1,2,3*

Targeted therapies for cancer are typically small molecules or monoclonal antibodiesthat act by inhibiting the activity of specific proteins that drive tumor growth.Although many of these drugs are effective in cancer patients, the response is oftennot durable because tumor cells develop resistance to the drugs. Another limitation ofthis strategy is that not all oncogenic driver proteins are “druggable”enzymes or receptors withactivities that can be inhibited. Here we describe an alternative approach to targeted therapythat is based on co-opting the cellular quality-control machinery—the ubiquitin-proteasomesystem—to remove specific cancer-causing proteins from the cell.We first discuss examples ofexisting cancer drugs that work by degrading specific proteins and then review recentprogress in the rational design and preclinical testing of small molecules that induceselective degradation of specific target proteins.

Cancer continues to be a leading globalhealth problem; it has been estimatedthat by 2025 there will be nearly 20millionnew cancer cases diagnosed each year (1).Notable progress is being made in cancer

drug development, particularly in the areas of im-munotherapy and targeted therapy, but the enor-mity of the cancer problem requires a variety oftherapeutic strategies. Many targeted therapiesfor cancer are small molecules or monoclonalantibodies that inhibit the activity of proteinsdriving tumor growth. Tumor cells often developresistance to these drugs through overexpressionof the target protein and/or through the acquisi-tion of new mutations in the target protein thatallow it to escape the inhibitory effect of the drug.Over the past 15 years, researchers have be-

gun to explore an alternative therapeutic ap-proach that aims to control protein function byregulating protein expression levels rather thanactivities. These efforts to harness controlledproteostasis as a therapeutic strategy evolvedfrom the discovery that proteasome inhibitorsthat block protein degradation have anticanceractivity. Carfilzomib and bortezomib are two ex-amples of such proteasome inhibitors approvedby the U.S. Food and Drug Administration (FDA)for the treatment of multiple myeloma (MM)(2). Investigators have explored other ways inwhich the ubiquitin-proteasome system (UPS)can be manipulated to stabilize or promote thedegradation of disease-causing proteins (Table 1).For example, efforts have been made to disruptthe interaction between proteins and the ubi-quitin E3 ligases responsible for their degradation(3). The interaction between the tumor suppres-sor p53 and its ubiquitin E3 ligase, MDM2 (mousedouble minute 2 homolog), has been an attract-ive oncology target: A potent inhibitor of this in-

teraction (RG7112) was found to kill wild-type p53-expressing cancer cells and to inhibit tumor growthin preclinical models of cancer. A reduction in thelevels of oncoproteins can also be achieved byinhibiting enzymes that function to stabilize theseproteins (3). For example, inhibition of USP7 (theubiquitin-specific protease 7), a deubiquitinatingenzyme (DUB) that deubiquitinates and stabilizesMDM2, reduces MDM2 levels and consequentlyincreases p53 levels. To that end, the USP7 in-hibitor P5091 has shown promising antitumoractivity in MM xenograft models (4).More recently, interest has focused on di-

rectly using the UPS to induce degradation ofspecific target proteins, especially proteins forwhich there are no established DUBs or E3ligases. These newer strategies involve the phar-macological hijacking of the cellular quality-control system to posttranslationally eliminatedisease-causing proteins (Table 1). In this Re-view, we discuss the initial applications of thisconcept of targeted protein degradation to achievecontrolled proteostasis and the strategies em-ployed to generate protein degraders. We high-light the progress achieved to date, as well as thesome of the challenges inherent in this approach.

The limitations of“occupancy-driven” pharmacology

In this postgenomic era, a better understandingof the molecular drivers of cancer has led to thedevelopment of several successful cancer ther-apies that inhibit the activity of enzymes suchas protein kinases (e.g., imatinib, erlotinib, andpalbociclib), histone deacetylases (e.g., belinostat),and poly(ADP-ribose) polymerase (e.g., olaparib).These small-molecule inhibitors generally workby occupying a binding pocket or active site,resulting in the loss of protein function. However,given that most enzyme inhibitors bind noncova-lently (and thus reversibly), high drug concentra-tions must be maintained to ensure active-siteoccupancy and to sustain the clinical benefit (5).This is understood as an “occupancy-driven” phar-macological paradigm: one that necessitates that

the binding pocket remain occupied to maintaineffectiveness of the small molecule (Fig. 1). Unfor-tunately, achieving and maintaining high systemicdrug levels is one of the major challenges in drugdevelopment. Moreover, these high dosages canlead to undesirable off-target effects. In the caseof kinases [which all bind ATP (adenosine triphos-phate)], active-site inhibition by ATP analogs ischallenging because many kinases show structu-ral similarity. However, this promiscuity can some-times be advantageous, especially when targetingcancers that rely on the activation of multiplecellular signaling pathways.

Creating a new paradigm:Induced protein degradation

Targeted protein degradation has emerged as analternative paradigm that requires only brief inter-action between a small molecule and its targetprotein to elicit the desired loss of protein func-tion. Under this “event-driven” paradigm, loss offunction is due to the removal of the target pro-tein as a result of the transient binding event(Fig. 2). The strategy is to engage the cellularquality-control machinery (the UPS) and therebytag unwanted proteins for destruction via ubi-quitination. Because the small-molecule drug sur-vives the target-protein tagging and destructionsteps, it is free to engage in multiple cycles oftarget-protein degradation, resulting in substoi-chiometric activity. This event-driven approach iscatalytic in nature, eliminating the need to main-tain high levels of drug. For the inhibitory effectof degradation to be reversed, the protein mustbe resynthesized.Protein degradation by the UPS involves a se-

ries of steps culminating in the conjugation of the8.5-kDa protein ubiquitin to the targeted proteinby an E3 ligase (6–8). This monoubiquitinatedprotein then undergoes multiple cycles of ubi-quitination to become polyubiquitinated, whichallows it to be recognized and then degraded bythe 26S proteasome.

Small molecules that both inhibit proteinactivity and induce protein degradation

Early literature reports suggested that certainsmall molecules developed to inhibit proteinactivity could also induce the degradation oftheir targets, although the mechanism was oftenunknown. Canertinib (CI-1033) is one exampleof an irreversible tyrosine kinase inhibitor thatalso induces the polyubiquitination and degra-dation of ErbB-2 (a receptor tyrosine-protein kinasealso known as HER2/neu), an important therapeu-tic target in several human cancers (9). Fulvestrantis another example of a small molecule that in-hibits the signaling function of its target, ERa(estrogen receptor alpha), and induces its degra-dation. Fulvestrant produced better outcomes inpatients with ERa-positive breast cancers in com-parison with tamoxifen, another ERa antagonistthat acts primarily by inhibiting ERa signaling(10). Arsenic trioxide is a highly effective therapyfor acute promyelocytic leukemia (APL), and itworks in part by degrading PML-RARa (promye-locytic leukemia–retinoic acid receptor alpha), the

Salami et al., Science 355, 1163–1167 (2017) 17 March 2017 1 of 5

1Department of Molecular, Cellular and DevelopmentalBiology, Yale University, New Haven, CT 06520, USA.2Department of Chemistry, Yale University, New Haven, CT06520, USA. 3Department of Pharmacology, Yale University,New Haven, CT 06520, USA.*Corresponding author. Email: [email protected]

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fusion protein that is characteristic of this diseaseand whose activity disrupts normal myeloid dif-ferentiation (11). More recently, lenalidomide, aneffective immunomodulatory drug (IMiD) usedin the treatment of MM, was found to causethe selective ubiquitination and degradation ofCK1a (casein kinase 1a) (12) and two importanttranscription factors inMM, Ikaros and Aiolos (13).Other examples of small molecules that promotethe degradation of specific proteins include thecancer-preventive isothiocyanates, which selec-tively bind and induce the degradation of a- andb-tubulins in human cancer cells (14), and CaCCinh-AO1, which causes the degradation of the calcium-activated chloride channel ANO1. CaCCinh-AO1reduces the proliferation of ANO1-dependent can-cer cells by inducing an endoplasmic reticulum–associated proteasomal degradation of its target(15). The success of these drugs has spurred in-creased interest in the development of additionaldegrader compounds. However, because the abi-lity of these small molecules to induce target-protein degradation was discovered fortuitously,

the challenge has been to develop a strategy thatwould allow for the rational design of degradersthat can target any specific protein of choice.

Proteolysis targeting chimeras (PROTACs)

Typically, an E3 ubiquitin ligase requires a spe-cific recognition signal to recruit and ubiquitinateits natural substrate (8). However, the proteol-ysis targeting chimera (PROTAC) technology isone approach that can achieve the same resultfor any protein target, even those not naturallyubiquitinated. By using a heterobifunctional mol-ecule to form a complex between the target pro-tein of interest and a recruited E3 ligase, PROTACsinduce the ubiquitination and subsequent pro-teasomal degradation of a target protein. ThePROTAC concept was introduced in a study inwhich an aliphatic linker was used to connect twoprotein ligands: ovalicin, which covalently bindsthe enzyme methionine aminopeptidase 2, anda phosphopeptide that associates with the ubi-quitin ligase complex SCFb-TrCP (Skp1/Cullin/Fboxb-TrCP) (16). Incubation of this PROTAC with

Xenopus egg extract resulted in ubiquitinationof methionine aminopeptidase 2 and its degra-dation by the proteasome (16). The same approachwas subsequently used to ubiquitinate and de-grade the estrogen and androgen receptors (17),protein targets that play major roles in the path-ogenesis of breast and prostate cancers (18, 19),respectively. These first-generation PROTACs hadlow activity in cells, presumably because of thepoor cell permeability of the peptide employed.The next generation of PROTACs moved away

from SCFb-TrCP and instead engaged the vonHippel–Lindau (VHL) tumor suppressor pro-tein to ubiquitinate recruited proteins. The E3ligase binding moiety of these PROTACs con-sisted of a short, hydroxyproline-containing pep-tide sequence derived from the VHL protein’snatural substrate, the transcription factor HIF1a(hypoxia-inducible factor 1a). When coupled to atargeting ligand and with incorporation of a cell-penetrating peptide, the resulting PROTACsproved selective for degradation of their targetproteins in intact cells (20).

Salami et al., Science 355, 1163–1167 (2017) 17 March 2017 2 of 5

Table 1. Representative cancer drugs and drug candidates that work bya controlled proteostasis mechanism. This list is not intended to be com-

prehensive but rather is an illustrative selection of the many compounds thatwork by this mechanism and are currently in the cancer drug development

pipeline. ERa, estrogen receptor alpha; PML-RARa, promyelocytic leukemia–

retinoic acid receptor alpha; CRBN, cereblon; USP7, ubiquitin-specific protease 7;

MDM2, mouse double minute 2 homolog; HER3, human epidermal growth

factor receptor 3; BET, bromodomain and extra-terminal; BRD4, bromodomain-

containing protein 4; APL, acute promyelocytic leukemia; AML, acute myeloid

leukemia; CK1a, casein kinase 1a; SNIPER, specific and nongenetic IAP-dependentprotein eraser; PROTAC, proteolysis targeting chimera; IMiD, immunomodula-

tory drug; MM, multiple myeloma; DUB, deubiquitinating enzyme.

Molecule/drug name Target Disease Mode of action Development stage Refs.

Bortezomib 20S proteasome MM Proteasome inhibition Clinically approved (2).. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .

Carlfizomib 20S proteasome MM Proteasome inhibition Clinically approved (2).. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .

Lenalidomide CRBN MM CK1a, Ikaros, and

Aiolos degradation

Clinically approved (12, 13)

.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .

P5091 USP7 MM DUB inhibition Preclinical/research (4).. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .

Fulvestrant ERa Breast cancer Selective estrogen

receptor degradation

Clinically approved (10)

.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .

RAD1901 ERa Breast cancer Selective estrogen

receptor degradation

Phase 1

clinical trial

(47)

.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .

SNIPER (ER) ERa Breast cancer SNIPER PROTAC–mediated

target degradation

Cultured cancer cells (28)

.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .

SARD279 AR Prostate cancer Hydrophobic

tagging–mediated

target degradation

Cultured cancer cells (32)

.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .

ARV-771 BETproteins Prostate cancer PROTAC-mediated

target degradation

Preclinical/xenograft (33)

.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .

dBET1 BETproteins AML IMiD-based

PROTAC-mediated

target degradation

Preclinical/xenograft (43)

.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .

Arsenic PML-RARa APL Target degradation Clinically approved (11).. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .

RG7112 MDM2 Hematologic cancers Ubiquitin E3

ligase inhibition

Phase 1

clinical trial

(48)

.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .

ARV825 BRD4 Burkitt’s lymphoma PROTAC-mediated

target degradation

Preclinical/xenograft (42)

.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .

TX2-121-1 HER3 Multiple cancers Hydrophobic

tagging–mediated

target degradation

Cultured cancer cells (31)

.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .

MZ1 BRD4 Multiple cancers PROTAC-mediated

target degradation

In vitro (44)

.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .

FRONTIERS IN CANCER THERAPY on A

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Though these early PROTACs were cell-permeable, they were far from drug-like be-cause they still required peptide sequences forE3 ligase recognition and cell penetration. Thus,the challenge was to design a nonpeptidic E3ligase ligand to allow for the creation of “all-small-molecule” PROTACs. This was achievedwith the development of a compound consist-ing of a selective androgen receptor modulatortethered to nutlin. Nutlin is a ligand for the E3ubiquitin ligase MDM2; it disrupts MDM2 bind-ing to its natural substrate, p53. This PROTACwas shown to recruit androgen receptors (ARs)to MDM2, leading to the ubiquitination anddegradation of ARs in HeLa cells (21). A keystep in the evolution of the PROTAC technologywas the development of hydroxyproline-basedsmall-molecule VHL ligands that could replacethe HIF1a peptide used in earlier studies (22).Bondeson et al. used these ligands to designtwo all-small-molecule PROTACS that targetedthe nuclear hormone receptor, ERRa (estrogen-related receptor alpha), and the serine-threoninekinase RIPK2. At a concentration 1000 times lowerthan that of previously reported compounds,PROTAC_ERRa and PROTAC_RIPK2 induceddegradation of their target proteins to below10% of levels present in untreated cells (23). Theactivity of these PROTAC molecules was cata-lytic and specific for the intended protein tar-gets. Several research groups have developedadditional PROTAC molecules that are like-wise showing promise in preclinical studies.Some of these examples will be discussed laterin the Review.

Further PROTACtechnology development

As discussed above, there are three basic com-ponents of a small-molecule PROTAC: the target-binding ligand, the E3 ligase-binding ligand,

and the linker that holds these two moietiestogether. A successful PROTAC molecule willrequire optimization of each of these compo-nents. Fortunately, a plethora of high-affinitysmall-molecule ligands and drugs have beendeveloped to target the many proteins of in-terest in the cancer research field. However,despite an estimated >700 endogenous humanE3 ligases (8), the use of PROTAC technologyhas largely been limited to the few E3 ligases

for which researchers have successfully de-veloped selective small-molecule ligands: VHL,CRBN (cereblon), MDM2, and cIAP1 (cellularinhibitor of apoptosis protein 1). The VHL lig-and was described above (22). Three small mol-ecules have been identified as CRBN ligands;these include thalidomide, lenalidomide, andpomalidomide (a class of immunomodulatory anti-tumor compounds in myeloma cells) (24). Methylbestatin (MeBS) and nutlin are small-molecule lig-ands for cIAP1 (25) and MDM2 (21), respectively.In addition to target-protein and E3 ligase moi-eties, the connecting linker is an important com-ponent of successful PROTACs. For each PROTAC,the length, hydrophilicity, and rigidity of thelinker must be optimized for improved cell per-

meability and optimal presentation of a target tothe E3 ligase for ubiquitination.Recent work highlights the importance of

each of these components within the contextof a PROTAC molecule and illustrates how smallchanges can substantially affect outcomes (26).The study focused on PROTACs targeting BCR-ABL, an oncogenic fusion tyrosine kinase thatdrives the development of chronic myelogenousleukemia. Small-molecule inhibitors of the tyro-sine kinase activity are highly effective thera-pies for this disease and are now the clinicalstandard of care, although the development ofdrug resistance can limit their efficacy in a subsetof patients. The PROTACs were designed usinga variety of linkers to attach imatinib, bosutinib, ordasatinib (structurally dissimilar small moleculesthat inhibit the c-ABL kinase domain) to the VHLsmall-molecule ligand (22) or to pomalidomide, athalidomide derivative (24). Of the four linkerstested, only one showed a considerable loss ofPROTAC affinity for the target, suggesting a widelatitude of flexibility in linker design for thesePROTACs. Whereas the imatinib PROTACs failedto degrade BCR-ABL or c-ABL despite verifiabletarget binding and inhibition in cells, the dasatinib-CRBNandbosutinib-CRBNPROTACs successfullydegraded both BCR-ABL and c-ABL. This impliesthat, beyond affinity considerations, target engage-ment alone is not sufficient for degradation andthat the identity of the target ligand is importantin determining the efficacy of PROTAC mole-cules in cells. The observation that the bosutinib-VHL compound does not degrade BCR-ABL orc-ABL and that the dasatinib-VHL compounddegrades only c-ABL suggests that changing theE3 ligase recruited for ubiquitination and subse-quent degradation can considerably alter the abil-ity to degrade a target, as well as the specificity ofthis degradation. In essence, it is possible to tunedegradation specificity—from no degradation to

Salami et al., Science 355, 1163–1167 (2017) 17 March 2017 3 of 5

A Active-state protein B Low inhibitor concentration/partially-active protein

C High inhibitor concentration/inactive-state protein

D Mutated protein can be activated upon inhibitor binding

Active site

Inactive protein

Inhibitor

Mutated activatedprotein

Fig. 1. Occupancy-driven pharmacological paradigm. (A) In the absenceof inhibitor, the target-protein active site is unoccupied and the protein re-mains active. (B) At low concentrations of inhibitor, the activity of the proteinis only partially inhibited because the active site is not maximally occupied.(C) At high concentrations of inhibitor, excess compound ensures that theactive site is occupied and protein enzymatic activity is disrupted, yielding

maximum efficacy. Some proteins have additional functions that are inde-pendent of their active-site enzymatic activities (e.g., scaffolding). These func-tions will remain unaffected despite high inhibitor concentrations. (D) Incertain mutated proteins in cancer [e.g., the Phe876→Leu876 mutation in theandrogen receptor (49)], the inhibitor compound can behave as an agonistand have the adverse effect of further activating the target protein.

“…achieving and maintaininghigh systemicdrug levels isone of themajor challenges indrugdevelopment. Moreover,these high dosages can leadto undesirable off-targeteffects.”

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selective degradation (only c-ABL) to promiscuous-degradation (BCR-ABL and c-ABL)—by testing dif-ferent target-ligand and E3 ligase combinations.Conceivably, this type of “specificity dial” wouldallow researchers to use promiscuous inhib-itors as a starting point for the design of more-selective degraders.

Other strategies for targetedprotein degradation

A number of other promising approaches havebeen developed that, like the PROTAC strategy,use small molecules to induce targeted proteinknockdown. For example, Itoh et al. developed ahybrid compound that fuses MeBS, a ligand forcIAP1, to all-trans retinoic acid (ATRA), a lig-and for retinoic acid receptors and an effec-tive drug for APL (27). Through recruitment ofcIAP1, the compound degraded cellular retinoicacid–binding proteins (CRABPs), based on theirability to bind ATRA (27). Furthermore, thiscompound inhibited migration of IMR-32 neuro-blastoma cells, a process that requires CRABP-II.This class of PROTACs is known as SNIPER(specific and nongenetic IAP-dependent pro-tein eraser) PROTACS (28) and consists of MeBSlinked to ligands for other protein targets, thusrecruiting cIAP1 to induce ubiquitination andproteasomal degradation of the target protein.Using a SNIPER with 4-hydroxy tamoxifen asthe ERa ligand, Okuhira et al. showed success-ful degradation of ERa and resultant necroticdeath of ERa-expressing MCF-7 breast cancercells (28). Another strategy involves treatingcells with two halves of a PROTAC molecule (asopposed to a larger single compound with lessoptimal physiochemical properties) that then self-assembles intracellularly (29). This approach wasused to recruit the E3 ligase cereblon to success-fully degrade BRD4 (bromodomain-containingprotein 4) and ERK1/2 (extracellular signal–regulated kinases 1 and 2), both important tar-gets for cancer therapy.Similar to the PROTAC technology, hydro-

phobic tags have been used to hijack the UPSto degrade proteins of interest. The underly-ing concept is that partially unfolded or mis-folded proteins expose hydrophobic patchesthat are otherwise buried, serving as a recruit-ing signal for E3 ubiquitin ligases that thencatalyze ubiquitination and subsequent degra-dation. Researchers have successfully mimickedthis protein unfolding for specific targets byappending a low–molecular weight hydropho-bic tag to the target’s small-molecule ligand;this results in recruitment of the UPS to degradethe target protein (30). This strategy has beenapplied to degrade the HER3 (human epidermalgrowth factor receptor 3) pseudokinase, a cur-rently “undruggable” cancer target, by append-ing a “greasy tag” to the potent and selectiveHER3ligand TX1-85-1 to generate the bifunctional TX2-121-1 compound (31). The resulting TX2-121-1–induced HER3 knockdown led to inhibition ofdownstream signaling and reduced proliferationof HER3-dependent cell lines. Gustafson et al.generated selective androgen receptor degraders

(SARDs) that degrade the AR via hydrophobic tag-ging (32). SARD279 was shown to be as effectiveas enzalutamide (an FDA-approved inhibitor ofAR signaling) in suppressing proliferation of hu-man prostate cancer cells; this drug also sup-pressed proliferation of enzalutamide-resistantprostate cancer cells (32).

Protein degradation strategies inpreclinical models of cancer

Within the past 2 years, considerable progresshas been made in the advancement of protein

degradation technology to selectively and ef-fectively degrade key cancer targets. The BET(bromodomain and extra-terminal) proteins, suchas BRD4, play important roles in the progres-sion of various cancers, including acute myeloidleukemia (AML), MM, Burkitt’s lymphoma (BL),ovarian cancer, and prostate cancer (33, 34–39).To that end, potent small-molecule inhibitorsof BET proteins have been developed and are inclinical trials (40, 41). The experience to date sug-gests that the effectiveness of these inhibitorsmay be limited by incomplete suppression of the

Salami et al., Science 355, 1163–1167 (2017) 17 March 2017 4 of 5

Target protein Target engagementand recruitment ofE3 ubiquitin ligase

Polyubiquitinationof target protein

Degradation by proteasome and re-engagement ofremaining protein pool

A

B

Active-site

No active-site

E3 ligase ligand E3 ligaseInhibitor E2 Ubiquitination Binding ligand

Fig. 2. Event-driven pharmacological paradigm. (A) Active-site binding and/or enzymatic proteomeengagement. A chimeric molecule binds to the active site of the target protein and inhibits itsactivity. This molecule also recruits the cellular protein degradation machinery to tag (ubiquitinate)the target for proteasomal degradation. This molecule displays catalytic (i.e., processive) activityover repeated cycles of induced degradation, thus eliminating the need for high concentrations toachieve efficacy. It also disrupts nonenzymatic functions of the target protein. Restoration of target-protein function requires resynthesis of the protein. Mutated proteins that retain the ability to bindthe ligand and would otherwise become activated are now vulnerable. (B) Non–active-site binding and/ornonenzymatic proteome engagement. A chimericmolecule functions in the samemanner as in (A), exceptthat the molecule binds to any crevice (as opposed to an active site) on the surface of the protein. Thisenables targeting of proteins that lack an active site (e.g., transcription factors and scaffolding proteins).G

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downstream oncogene c-MYC (39) and a compen-satory increase in BRD4 protein levels as a wayto circumvent inhibition (42).Several labs have reported PROTACs that

degrade the BRD4 protein in cells (33, 42–44).For example, Lu et al. designed ARV-825, whichdegrades BRD4 by engaging the CRBN E3 li-gase and incorporates the potent BET inhibitorOTX015 as a recruiting moiety (42). At subnano-molar concentrations, ARV-825 induced near-complete BRD4 degradation in BL cell lines. Itsuppressed c-MYC expression and was a more po-tent inducer of apoptosis than conventional BETinhibitors such as JQ1 and OTX015. Winter et al.designed a PROTAC called dBET1 by append-ing JQ1 to a phthalimide moiety, which hijacksthe cereblon E3 ubiquitin ligase complex (43).dBET1 showed greater antiproliferative effectsin AML and lymphoma cells when comparedwith JQ1 inhibition. Furthermore, dBET1 inhib-ited leukemia progression in a mouse xenograftmodel of AML. Raina et al.’s ARV-771 compoundrecruits BET proteins to the VHL E3 ligase fordegradation at subnanomolar concentrations inseveral prostate cancer cell lines. ARV-771 inhib-ited the proliferation of enzalutamide-resistantprostate cancer cells and inhibited tumor growthin a castration-resistant prostate cancer mousexenograft model (33). Zengerle et al. designeda BRD4 PROTAC in which JQ1 is tethered to aligand for the VHL E3 ligase and showed thatthis compound selectively induces degradationof BRD4 in cultured cells (44).

Conclusion

The most promising aspect of targeted degra-dation as a therapeutic strategy may be its po-tential for targeting proteins for which therecurrently is no drug. Undruggable proteins suchas scaffolding proteins, pseudokinases, and tran-scription factors make up ~80% of the humanproteome; these proteins are neither enzymes norreceptors and lack an enzymatic activity or func-tional interaction that can be compromised byan inhibitor (45). The ability to target any of thesewould require the identification of a specific lig-and. However, because event-driven protein degra-

dation can be mediated via any binding site on thesurface of the target protein rather than restrictedto a single, identifiable active site (Fig. 2), thedevelopment of simple but potent and selectiveligands may be easier. The targeted degradationapproach to eliminate such proteins has thepotential to render these otherwise-undruggableproteins pharmaceutically vulnerable.Targeted degradation may also prove useful

in drug-resistance mechanisms that involve acompensatory increase in the expression ofinhibited proteins or mutations that result inthe loss of inhibition despite maintained targetengagement. Given the encouraging preclinicalstudies on targeted degradation of BET proteinsand the AR, it appears that tools to win thispharmacological “arms race” may be available.One possible application is the subgroup ofcetuximab-resistant non–small cell lung cancersthat show increased expression of the epidermalgrowth factor receptor, the protein targeted bycetuximab (46). In this case, substantial loss oftarget-protein levels and activity would still beachieved because of the catalytic nature of thePROTAC mechanism of action. Finally, althoughthis Review has focused specifically on appli-cations in cancer therapy, other disease con-texts may also benefit from this emerging drugparadigm.

REFERENCES AND NOTES

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16669–16674 (2011).40. Z. Cheng et al., Clin. Cancer Res. 19, 1748–1759 (2013).41. R. K. Prinjha, J. Witherington, K. Lee, Trends Pharmacol. Sci.

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ACKNOWLEDGMENTS

C.M.C. is the chief scientific adviser of and is a shareholder inArvinas, a biotechnology company focused on developingprotein degradation therapeutics for cancer and other diseases.C.M.C. is an inventor on patent US7041298 B2 and patentapplications PCT/US2013/040551, PCT/US2013/021136,EP20150180508, and PCT/US2011/063401 (submitted by YaleUniversity) and PCT/US2015/025813 (submitted by Arvinas),which cover targeted protein degradation. C.M.C. acknowledgessupport from the Leukemia and Lymphoma Society and the NIH (grantR35CA197589). C.M.C. also receives research funding from Arvinas.

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An attractive strategy for cancer therapy−−Waste disposalJemilat Salami and Craig M. Crews

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