PROTAC employs a warhead, a linker and an E3 ligase binding moiety to promote targeted proteolysis by hijacking the inherent ubiquitin-proteasome pathway of the cells. Research has shown that the PROTACs form a ternary complex with the POI and E3 ligase by being positioned between them, a concept that was first visualized in 2017 (Gadd et al., 2017). Gadd et al. (2017) used X-ray crystallography to confirm the formation of ternary structures between MZ1 (PROTAC), Brd4 (BET family proteins) and VHL (Von Hippel–Lindau protein). Two asymmetrical ternary complexes, with minor deviations, were observed in the following orientation: Brd4-MZ1-VHL (Gadd et al., 2017). This orientation allowed formation of novel interactions between the proteins, as well as between the proteins and their ligands, resulting in a more stable structure as well as specific folding of ligands to allow improved recruitment and productivity of targets (Gadd et al., 2017).

The ligands in the PROTACs bind to their respective targets, POI and E3 ligases, thereby bringing them into close proximity of each other (Figure 1). This results in the transfer of ubiquitin from E2 ligase onto the POI, a process which is catalyzed by the E3 ligases (Yin & Hu, 2020). Successful ubiquitination of the POI marks it for destruction, which is thus accomplished by the proteolytic activity of the 26S proteasome (Yin & Hu, 2020). Furthermore, PROTACs employ a catalytic mode of action wherein they disconnect from complex after ubiquitination, thus showing great potential for exhibiting significant activity at low doses (X. Sun et al., 2019). This also allows them to be less vulnerable towards surges in target expression and/or mutations (X. Sun et al., 2019).

Initially, emphasis was placed on selecting ligands possessing high affinity for their respective substrates, however, further research has found that greater prominence needs to be given to the association between the specific ligase and the POI chosen (Bondeson et al., 2018). The novel Protein-Protein Interactions (PPI) between them result in a more stable ternary complex that produces a more successful PROTACs, as noted in several studies (Lai et al., 2015; Chan et al., 2018; Smith et al., 2019).

According to the PROTACs database, PROTAC-DB (http://cadd.zju.edu.cn/protacdb/), over a hundred proteins have been targeted for degradation by PROTACs. Amongst the most popular include nuclear receptors, kinases, epigenetic proteins (such as BET proteins and histone deacetylases), STAT3 and even E3 ligases itself (Hu et al., 2020; X.; Zhou et al., 2020). Concurrently, the database also reveals 275 warheads that have up till now been employed in PROTACs. Currently, majority of them are established warheads i.e. actively inhibiting proteins by binding to their respective active sites (Yin & Hu, 2020). Only a handful of PROTACs have used warheads that bind allosterically to their respective target proteins (Burslem et al., 2019). Research has shown that warheads with limited or non-existent inhibiting activity can still produce significantly positive results (Paiva & Crews, 2019).

Furthermore, while more than 600 E3 ubiquitin ligases have been estimated to exist in the human genome, only a few of them have been used by PROTACs (Yin & Hu, 2020; Bricelj et al., 2021). The most popular of these are von Hippel-Lindau (VHL) and Cereblon (CRBN). VHL, together with Rbx-1, cullin 2, elongin B and elongin C, is a component of the CRL2VHL complex, a Cullin RING ligases (Smith et al., 2019; Bricelj et al., 2021). One of its two domains, which form after folding, behaves as a substrate binder, most particularly with HIF-1α (Hypoxia-inducible factor 1-alpha) (Smith et al., 2019). Crystal structures of VHL have proven their ability to bind with small molecule inhibitors in a method analogous to their binding with HIF-1α (Chen & Jin, 2020). This has made them ideal for use in PROTACs, one of the most popular being VH298 (Chen & Jin, 2020). Similarly, CRBN are a part of the Cullin-4-RING E3 ubiquitin ligase complex, where they act as specific substrate adapters (Bondeson et al., 2018; Bricelj et al., 2021). Generally, substrates for CRBN are immunomodulatory imide drugs or IMiDs (Bricelj et al., 2021), especially glutarimide compounds including thalidomide, pomalidomide and lenalidomide (Yin & Hu, 2020). Binding of the IMiDs to the CRBN results in modifications to their ligase activity, resulting in subsequent ubiquitination and proteolysis (Bricelj et al., 2021). Interestingly, E3 ligases c-Cbl (Casitas B-lineage lymphoma), C-terminus of Hsc70-interacting protein (CHIP) and chimeric ubiquitin ligase, SH2-U-box are involved to induce degradation of BCR-ABL (Tsukahara & Maru, 2010; Ru et al., 2016).

Although comparatively lesser importance has been given to linkers in the past, the significance of their composition and length on the stability and formation of the ternary complex, along with the proteolysis activity and target specificity, cannot be denied (Troup et al., 2020). However, as more focus has generally been on the warheads and E3 ligands selected, linkers have been optimized according to each individual PROTAC (Troup et al., 2020). Thus, there are no standard methods or rules for constructing linkers. Traditionally, linkers have been formed as a combination of several chemical motifs, particularly PEG and Alkyl motifs (Troup et al., 2020). According to PROTAC-DB, more than a thousand linkers have been developed to date.

The BCR-ABL oncoprotein is the product of reciprocal chromosomal translocation between the long arms of chromosome 9 and 22 result in the genesis of Chronic Myeloid Leukemia (CML) (Bracco et al., 2021). Its oncogenic properties are a result of its tyrosine kinase activity (Bracco et al., 2021). Generally, BCR-ABL + CML is treated with tyrosine kinase inhibitors (TKIs), usually requiring lifelong administration (T.-T. Huang et al., 2021). Several shortcomings have been associated with TKIs: First generation TKIs (Imatinib) show drug resistance and can result in oncoprotein overexpression; second generation TKIs (dasatinib, nilotinib, and bosutinib) show resistance against T3151 mutation; and third generation TKIs (Ponatinib) result in adverse cardiovascular side effects (T.-T. Huang et al., 2021; Pophali and Patnaik, 2016).

PROTACs against BCR-ABL were first developed in 2016, by linking imatinib, bosutinib or dasatinib to VHL or CRBN by any of 4 different linkers (Lai et al., 2015). Their activity was tested against K562 CML cells (Lai et al., 2015). Imatinib, although it bound to both ABL and BCR-ABL, showed no degradation (Lai et al., 2015). The boustinib-VHL PROTAC showed no degradation while the dasatinib-VHL PROTAC degraded ABL only (Lai et al., 2015). Both boustinib and dasatinib, when bound to CBRN, showed degradation of ABL and BCR-ABL (Lai et al., 2015). In the following 2 years, protein degraders using ABL kinase inhibitors and IAP ligands were used against BCR-ABL. SNIPER(ABL)2 (Table 2), imatinib connected to methyl bestatin by hexyl linker (Demizua et al., 2016), and SNIPER(ABL)-39 (Table 2), dasatinib and LCL161 derivative connected by polyethylene glycol (PEG) × 3 linker (Shibata et al., 2017), showed promising results. Consequently, researchers turned their attention towards allosteric sites on BCR-ABL as possible PROTACs targets. Shimokawa et al. were amongst the first to target allosteric site, thus developing SNIPER(ABL)-21 (Table 2), GNF-2/-5 (1–2) with LCL-161, and SNIPER(ABL)-62 (Table 2), ABL001 and LCL-161 (Shimokawa et al., 2017). Allosteric site targeting PROTAC GMB-475 (Table 2), developed using GNF-5 AND VHL, showed great potential, particularly against mutations that are clinically important (Burslem et al., 2019). The researchers also demonstrated the benefit of using protein degraders in unison with protein kinase inhibitors like imatinib (Burslem et al., 2019). The PROTAC that was established by linking dasatinib with VHL1, SIAIS178 (Table 2), also showed remarkable degradation and tumor regression (in K526 xenografts) capacity (Zhao et al., 2019). Furthermore, it degraded BCR-ABL containing clinically important mutations, which confer drug resistance, and, after treatment for a short period, exhibited a comparatively prolonged cellular response than inhibitors (Zhao et al., 2019). Other similar studies have been conducted against BCR-ABL proteins containing clinically important mutations, including one using the PROTAC SIAIS056, containing a sulfur-substituted carbon chain linker (H. Liu et al., 2021; Yang et al., 2020). A novel study involves the use of nimbolide, derived from Azadirachta indica, as a ligand for the E3 ligase RNF114, in combination with dasatinib to generate BT1 (Tong et al., 2020). Unlike previous studies involving VHL and CRBN, nimbolide showed greater degradation of BCR-ABL as compared to c-ABL (Tong et al., 2020).

Cyclin-dependent kinases (CDK) are a family of protein kinases that play an essential role in the regulation of cell cycle progression (Scheicher et al., 2015). The CDK4/6-cyclinD complex is of particular importance due to the crucial role of CDK6 in activating leukemic stem cells (LCSs), which are important for the development of AML and CML (Scheicher et al., 2015). CDK6 is also involved in hematopoietic malignancies such as Mixed-Lineage Leukemia (MLL) related AML (Scheicher et al., 2015) and acute lymphoblastic leukemia (ALL) (Linden et al., 2014). Owning to its critical role in development of hematopoietic malignancies, several small molecule inhibitors targeting the ATP-binding domain of CDK6 have been developed (He et al., 2020). However, the presence of an identical ATP-binding domain on CDK4 (He et al., 2020), thereby preventing CDK6 specific inhibition, along with the scaffolding activity and kinase-independent functions (that promote growth of hematopoietic tumors), make these inhibitors an insufficient treatment (Dominici et al., 2020).

YX-2-107 (Supplementary Table S1) is a PROTAC, designed using palbociclin and CBRN ligands, intended as a possible treatment for acute lymphoblastic leukemia (ALL), involving the Philadelphia chromosome positive (Ph+) ALL (Dominici et al., 2020). The PROTAC was designed to inhibit CDK6 activity as well as preferentially degrade CDK6 over CDK4, two proteins with almost identical amino acid sequences (Dominici et al., 2020). When tested in Ph + ALL cells and xenografts, and mice, YX-2-107 showed promising results, especially regarding selective CDK6 degradation (Dominici et al., 2020). However it requires more development before it can be considered as a possible treatment, especially to improve its half-life and assess its biological impacts (Dominici et al., 2020).

Other CDK6 PROTACs designed for selective degradation of CDK6 over CDK4 using CDK inhibitors and CRBN ligands include BSJ-03-123 (Supplementary Table S1) (against AML) (Brand et al., 2019) and CP-10 (Supplementary Table S1) (most potent degrader from a CDK6 degrader library) which not only degraded mutated CDK6 that underwent overexpression, but also inhibited hematopoietic malignant cell proliferation (Su et al., 2019). Selective CDK degradation was also achieved using VHL (Steinebach et al., 2020) and IAP (Anderson et al., 2020).

Bruton’s tyrosin kinase (BTK) is present in most hematopoietic cells, most notably in B cells (Woyach et al., 2014; Burger, 2019). BTK plays a key role in signal transduction of B cell receptors: the antigen-receptors activate BTK, which in turn transmits or amplifies the signal by stimulating multiple downstream signal cascades, for example nuclear factor-κB (NFκB) (Woyach et al., 2014; Burger, 2019). BTK is not only involved in intracellular signaling, it also involved in the microenvironment around the cell and assists in the growth and survival of tumor cells (Woyach et al., 2014; Burger, 2019). Furthermore, the poor maturation of B cells due to insufficient BCR response in the absence of BTK suggests BTK plays a key role in B cell survival and development as well (Woyach et al., 2014; Burger, 2019). This is further certified by mutations in BTK domains resulting in the development of X-linked agammaglobulinemia, which is characterized by obstructed B cell development (Woyach et al., 2014). BTK is also instrumental in the development of chronic lymphocytic leukemia (CLL), with the current effective treatments including BTK inhibitors such as Ibrutinib (Woyach et al., 2014; Burger et al., 2017; Burger, 2019). Similarly, BTK was also found to be crucial for the progression of AML (S. Huang et al., 2019; S. Li et al., 2021; Rushworth et al., 2014) and Hairy Cell Leukemia (HCL) (Sivina et al., 2012; Rogers et al., 2021).

Interestingly, PROTACs targeting BTK generally use CRBN instead of VHL, due to VHL exhibiting unsatisfactory degrading properties (Buhimschi et al., 2018; Jaime-Figueroa et al., 2020). DD-04-015 (Supplementary Table S2) (resulting from conjugating RN486 (warhead) to CRBN ligand) is a BTK specific PROTAC, designed by comparing its activity against TL12-186, a multiple kinase degrading PROTAC (H.-T. Huang et al., 2018). When MOLM-14 cells were treated with DD-04-015, effective BTK degradation could be observed after just 4 hours (H.-T. Huang et al., 2018). Furthermore, compared to RN486, the PROTAC showed more sustained pharmacodynamics effect (H.-T. Huang et al., 2018).

Certain BTK PROTACs were designed to target the specific C418S mutation in BTK. These included MT-802 (Supplementary Table S2) (Ibrutinib and pomalidomide) which showed full BTK degradation in CLL cells after about 4 hours at 250 nM concentration (Buhimschi et al., 2018). MT-802 also lacks an acrylamide moiety which results in fewer off-target kinase binding, as compared to the inhibitor ibrutinib (Buhimschi et al., 2018). Another such PROTACs is SJF620 (Supplementary Table S2), which showed pharmacokinetically superior results in vivo, as compared to MT-802, due to configurational adjustments in the linker and CRBN ligand (Jaime-Figueroa et al., 2020). P13I (Supplementary Table S2) (ibrutinib and pomalidomide) was successful in degrading both mutant BTK and a wild type that is resistant to ibrutinib (Y. Sun et al., 2018). DD-03-007 (Supplementary Table S2) and DD-03-17 (Supplementary Table S2), designed using CGI1746 and thalidomide, showed reduction in BTK levels within 4 hours, even at 100 nM (Dobrovolsky et al., 2019). Noncovalent PROTACs (Buhimschi et al., 2018; Dobrovolsky et al., 2019; Y.; Sun et al., 2018) and covalent PROTACs (irreversible (Xue et al., 2020) and reversible (Guo et al., 2020)) have also been developed against BTK. One such study compared the activity of reversible covalent BTK-degrading PROTACs against their irreversible covalent and noncovalent counterparts (Gabizon et al., 2020). Results showed that while the noncovalent PROTACs was the most effective, the reversible PROTAC (RC-3) still showed high potency and selectivity, thus resulting in fewer off-target reactions (Gabizon et al., 2020).

The Bromodomain (BRD) and extraterminal (BET) protein family, known as “epigenetic readers”, consist of BRD2, BRD3, BRD4 and BRDT (specific to germ cells) (Braun & Gardin, 2017; Reyes-Garau et al., 2019). BET proteins are involved in regulating RNA transcription and cell cycle progression by activating RNA polymerase II (Braun & Gardin, 2017; Reyes-Garau et al., 2019). This is accomplished by the binding of the BET proteins to the acetylated histone protein tails, by their two, conserved N-terminal bromodomains (Braun & Gardin, 2017; Reyes-Garau et al., 2019). BRD4 is better understood than the other BET proteins. It is involved in transcriptional regulation by interacting with cyclin T1 and CDK9, and Mediator complex (Reyes-Garau et al., 2019). BRD4 are involved in the progression of AML by activating the genes c-MYC and nucleophosmin, through transcription (Braun & Gardin, 2017; Reyes-Garau et al., 2019). Importance of BRD4 in AML can be inferred from the successful use of BET-inhibitors as treatments (Braun & Gardin, 2017; Reyes-Garau et al., 2019; Bill et al., 2021; Lee et al., 2021; Ramsey et al., 2021). In addition, BET-inhibitors have also been used to treat CLL (E. Kim et al., 2020; Ozer et al., 2018; Sundaram et al., 2020).

Remarkably, BET proteins have been a prime target for PROTACs research (Chan et al., 2018; Hines et al., 2019; S. A.; Kim et al., 2019; Ohoka et al., 2019; Zengerle et al., 2015). MZ1 (Supplementary Table S2) (using JQ1 and VHL ligand), has been used in several studies (Zengerle et al., 2015; Gadd et al., 2017; Roy et al., 2019). Zengerle et al. showed its potential for efficient and prolonged intracellular BRD4 degradation, as well as its preferential degradation of BRD4 as compared to BRD2/3 (Zengerle et al., 2015). Gadd et al. used MZ1 to confirm the formation of ternary complexes by PROTACs (Gadd et al., 2017), while Roy et al. used MZ1 to determine ternary complex stability (Roy et al., 2019). Some BET targeting PROTACs were designed by changing the E3 ligase binding moieties. A1874 (Supplementary Table S3), for example, utilized nutlin (for MDM2) and showed 98% degradation, even at nanomolar concentrations (Hines et al., 2019). MDM2 also provided an extra benefit of stabilizing the upregulation of p53 tumor-suppresser gene (Hines et al., 2019). Similarly, TD-428 (Supplementary Table S3) used TD-106 (unique IMiD analog) and displayed degradation of BET protein in 22Rv1 prostate cancer cells (S. A. Kim et al., 2019). Other such PROTACs include CCW 28-3 (Supplementary Table S3) (Ward et al., 2019), using RNF4 E3 ligase, and Kb02 (Supplementary Table S3) (Zhang et al., 2019), an electrophilic PROTAC that uses DCAF16 ligases. Additional BET targeting PROTACs include: macrocyclic PROTAC, a MZ1 with a cyclizing linker, (Testa et al., 2019); BEtd-246 (Supplementary Table S3) against triple-negative breast cancer (Bai et al., 2017); dBET1 (Supplementary Table S3), exhibiting antitumor activity against leukemia (Winter et al., 2015); ARV-825 (Supplementary Table S3) that induces degradation in BL cell lines (Lu et al., 2015); QCA570 (Supplementary Table S3) showing degradation in acute leukemia cell lines at picomolar concentrations (Qin et al., 2018); ARV-771 (Supplementary Table S3) against castration-resistant prostate cancer (CRPC) (Raina et al., 2016); and antibody-drug conjugates involving BRD4 targeting chimeric degraders (Dragovich et al., 2021).

FMS-like tyrosine kinase 3 are membrane bound receptors with two common mutated forms observed in various types of leukemia (Cheng et al., 2018). Internal Tandem Duplication (ITD), present within the juxtamembrane domain of FLT-3 proteins, is most frequently observed in AML (30%) (Cheng et al., 2018). On the other hand, Missense point mutations, found in the tyrosine kinase domain, are more common in ALL (Cheng et al., 2018). Mutated FLT-3 proteins are involved in activating several signaling pathways, for example MAPK/ERK and STAT5, and thus indicate a higher risk of developing leukemia as well as a worse prognosis (Cheng et al., 2018). High levels on FLT-3 can be found in leukemic cells, which in turn is responsible for overexpression of leukemic oncogenes (Cheng et al., 2018). Due to its prominent involvement in leukemia, FLT-3 specific inhibitors, such as quizartinib (Fathi & Chen, 2017), are being used to treat AML, either unaccompanied or in combination with other treatments (Oliva et al., 2021).

TL13-117 and TL13-149 (Supplementary Table S4) were PROTACs designed against AML that target FLT-3 proteins (H.-T. Huang et al., 2018). They were developed by combining quizartinib with a PEG linker and CRBN ligand (H.-T. Huang et al., 2018). However, the results were unexpected, with increased FLT-3 levels in MOLM-14 cells after treatment with PROTACs and reduced degradation by PROTACs as compared to quizartinib (H.-T. Huang et al., 2018). Conversely, PROTACs, which were designed to target ITD mutation in FLT-3 by coupling quizartinib and VHL ligand, showed enhanced selectivity and stimulation of apoptosis in MV4-11 and MOLM-14 cells, as well as in mice (Burslem et al., 2018). Another study supporting a positive outcome of PROTACs targeting FLT-3 proteins combined FF-10101, a novel FLT-3 inhibitor, with CRBN ligand. The study determined reversible covalent PROTACs displayed a much lower half maximal inhibitory concentration than the irreversible covalent (5 folds higher) and reversible noncovalent (34 fold higher) (Guo et al., 2020).

While the aforementioned oncoproteins are the most pertinent in various types of leukemia, there are many other oncoproteins that also play a role in the development of leukemia and have thus been targets for PROTACs. These include STAT3 (H. Zhou et al., 2019), Blc-2 (Bond et al., 2020), PLK1 (Mu et al., 2020) and SMARCA2 and SMARCA4 (Farnaby et al., 2019; Kargbo, 2020). As cancer research advances and the exact role of various oncogenes in the development of leukemia becomes more apparent, leukemic cells have become an ideal target for PROTACs. However, more research is still required to design efficient PROTACs that provide better outcomes with lower doses, before it can be used on a large scale.