Development of a Potent Protein Degrader against Oncogenic BCR-ABL Protein

Abstract

Chromosomal translocation occurs in some cancer cells, resulting in the expression of aberrant oncogenic fusion proteins that include BCR-ABL in chronic myelogenous leukemia (CML). Inhibitors of ABL tyrosine kinase, such as imatinib and dasatinib, exhibit remarkable therapeutic effects, although emergence of drug resistance hampers the therapy during long-term treatment. An alternative approach to treat CML is to downregulate expression of the BCR-ABL protein. Recently, we have devised a protein knockdown system by hybrid molecules named Specific and Nongenetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers (SNIPER). This system is designed to induce IAP-mediated ubiquitylation and proteasomal degradation of target proteins. In this review, we describe the development of SNIPER against BCR-ABL, and discuss the features and prospect for treatment of CML.

1. Introduction

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized by the Philadelphia (Ph) chromosome in cancer cells.¹⁾ The Ph chromosome results from a translocation between the long arms of chromosomes 9 and 22, t(9;22) (q34;q11),²⁾ and a fusion gene, Bcr-Abl, which encodes a constitutively active protein tyrosine kinase was generated by this translocation.^3–5) Several small molecule tyrosine kinase inhibitors (TKIs) have been developed for CML treatment. Imatinib is the first-generation TKI against BCR-ABL; it competitively binds to the ATP-binding site resulting in the inhibition of cell proliferation.^6,7) Currently, imatinib is used as first-line therapy for CML patients. In the International Randomized Study of Interferon and STI571 (IRIS) study, patients receiving imatinib 400 mg/daily achieved 83.3% 10 years survival.⁸⁾ However, approximately 15–20% CML patients treated with imatinib fail to achieve a satisfactory therapeutic effect,⁸⁾ mainly because of the emergence of imatinib resistance, which is commonly attributable to point mutations in the tyrosine kinase domain of BCR-ABL protein. To overcome this imatinib resistance, second-generation (i.e., dasatinib,⁹⁾ nilotinib,¹⁰⁾ and bosutinib¹¹⁾) and third-generation (i.e., ponatinib¹²⁾) TKIs have been developed and approved for the treatment of CML. Such TKIs are capable of saving most CML patients, however, drug resistance also occurs against them. Therefore, novel drug candidates with different mechanisms of action are required.

An alternative to the inhibition of BCR-ABL kinase activity is the downregulation of BCR-ABL protein, which may have a potential therapeutic effect. To achieve downregulation of BCR-ABL protein, there are two strategies: inhibition of the protein biosynthesis and induction of its degradation (Fig. 1). To inhibit BCR-ABL biosynthesis, omacetaxine was developed. This compound binds the ribosome aminoacyl-tRNA acceptor site, and thereby inhibits the synthesis of several oncoproteins including BCR-ABL.¹³⁾ In 2012, U.S. Food and Drug Administration approved omacetaxine for the treatment of CML with resistance and/or intolerance to two or more TKIs.¹⁴⁾ However, clinical study revealed that 18% of patients with chronic phase CML and 33% of those with accelerated phase CML had adverse reactions leading to withdrawal from omacetaxine therapy, probably because of the inhibition of total protein synthesis.¹⁴⁾

Fig. 1. Scheme of Therapeutic Strategies against CML

TKIs (a) inhibit ABL tyrosine kinase activity. Omacetaxine (b) suppresses the synthesis of BCR-ABL protein expression. HSP90 inhibitors (c) reduce BCR-ABL half-life inducing its degradation. SNIPERs or PROTACs (d) induce the ubiquitylation and subsequent degradation of BCR-ABL via the UPS. (Color figure can be accessed in the online version.)

As for degradation of BCR-ABL, there are some reports that heat shock protein 90 (HSP90) inhibitors induce degradation of BCR-ABL protein. HSP90 is a major mammalian molecular chaperone, which facilitates proper folding of many client proteins and inhibits their proteasomal degradation. Since HSP90 induces stabilization and maturation of BCR-ABL protein, HSP90 inhibitors reduce BCR-ABL protein expression, leading to CML cell death.¹⁵⁾ However, the inhibitors show low selectivity to BCR-ABL, which may increase their toxicity.^16,17) Therefore, novel compounds that induce the degradation of BCR-ABL protein in a highly specific manner are still needed.

2. Protein Knockdown Technology

Recently, we and others have developed protein knockdown technologies, which induce degradation of target proteins using hybrid small molecules named Specific and Non-genetic inhibitor of apoptosis protein (IAP)-dependent Protein Erasers (SNIPERs)^18–34) and Proteolysis Targeting Chimeras (PROTACs).^35–54) SNIPERs and PROTACs are chimeric molecules composed of two different ligands connected by a linker; one ligand is for the target protein and the other is for E3 ubiquitin ligases. Accordingly, these molecules are expected to crosslink the target protein and E3 ubiquitin ligases in cells, resulting in ubiquitylation and subsequent degradation of the target protein via the ubiquitin–proteasome system (UPS) (Fig. 2). Currently, several proteins including oncogenic ones have been degraded effectively via the UPS using the protein knockdown technology.

Fig. 2. Scheme of the Mechanism of a Degrader Based on Protein Knockdown Technology

(Color figure can be accessed in the online version.)

In 2008, we reported that (−)-N-[(2S,3R)-3-amino-2-hydroxy-4-phenyl-butyryl]-L-leucine methyl ester (MeBS) destabilizes cellular inhibitor of apoptosis protein 1 (cIAP1), an E3 ubiquitin ligase belonging to IAP family.⁵⁵⁾ MeBS directly interacts with cIAP1 at the third baculoviral IAP repeat domain and induces auto-ubiquitylation of cIAP1 depending on its RING domain, resulting in the proteasomal degradation of cIAP1.⁵⁵⁾ Structure–activity relationship study indicated that analogs with a carboxyl-ester reduce the amount of cIAP1 even though the methyl group is substituted to other residues, while modifications of bestatin backbone seriously affected the activity. Based on these observations, we hypothesized that the methyl group can be substituted to a ligand for a target protein without ablating the ubiquitin ligase activity of cIAP1, and we designed hybrid molecules composed of MeBS and the ligands for target protein connected by a linker. The molecules are expected to crosslink the target proteins and cIAP1 in the cells, resulting in the ubiquitylation and subsequent degradation of the target proteins via the UPS. As a proof-of-concept study, we designed and synthesized a series of hybrid molecules consisting of MeBS and all-trans retinoic acid, and found that they induced the proteasomal degradation of cellular retinoic acid binding protein-II.^18,19,21,29) Then, we developed various hybrid molecules by connecting tamoxifen, androgen receptor (AR) antagonists, and KHS-108 to MeBS, which induce the degradation of estrogen receptor (ER),^20,22) AR,³⁴⁾ and transforming acidic coiled-coil containing protein 3,^23,27) respectively. In addition to these targets, HaloTag-fused proteins^24,26) and huntingtin³²⁾ were successfully degraded by bestatin-based SNIPERs. Thus, selective degradation of target proteins can be attained by hybrid molecules that crosslink target protein and cIAP1.

IAPs are family of anti-apoptotic proteins and regarded as attractive targets for tumor therapy because IAPs are overexpressed in multiple human malignancies and implicated in promoting tumor progression, treatment failure, and poor prognosis. Thus, many potent and cell-permeable IAP antagonists have been developed, some of which are under evaluation in clinical studies as anti-tumor drugs.^56,57) Because these IAP antagonists show higher affinity to IAPs than MeBS, we reasoned that novel SNIPERs with potent protein degradation activity could be developed by incorporating IAP antagonist into SNIPERs. Then, to develop a potent protein degrader against ERα (SNIPER(ER)), we tested various combinations of ERα ligands and IAP antagonist for protein degradation activity of SNIER(ER).²⁸⁾ The resulting SNIPER(ER)-87, in which an ERα ligand 4-hydroxytamoxifen is conjugated to an IAP antagonist LCL161 derivative, shows potent activity to degrade the ERα protein. The effective protein degradation of ERα by SNIPER(ER)-87 was observed at a concentration ≥3 nM in an in vitro cell culture system. Furthermore, the SNIPER(ER)-87 reduced ERα levels in tumor xenografts and suppressed the growth of ERα-positive breast tumors in mice.^28,33) In addition, by derivatization of the IAP ligand molecule, we have developed novel SNIPER(ER)s whose protein degradation and anti-tumor activities are more potent than SNIPER(ER)-87.³³⁾

PROTACs^58–61) are hybrid molecules that recruit different E3 ubiquitin ligases, such as CRL2^VHL,^35,41,46,47) CRL4^CRBN,^{38,39,42,43,45,48,50–52)} Mdm2,³⁶⁾ and CRL3^KEAP1.⁴⁹⁾ Currently, several oncogenic proteins, such as AR,³⁵⁾ ER,^35,36) bromodomain containing 4 (BRD4),^{38,39,41,43,53)} TRIM24,⁴⁷⁾ receptor tyrosine kinases (epidermal growth factor receptor, HER2, and c-MET),⁴⁶⁾ BRD9⁴²⁾ EML4-ALK,^51,52) fms related tyrosine kinase 3,⁴⁸⁾ Bruton’s tyrosine kinase (BTK),^45,54) and CDK9⁵⁰⁾ have been degraded effectively via the UPS by PROTACs. For example, BETd-260, a PROTAC against BRD4 induced degradation of BRD4 at a concentration ≥0.03 nM in RS4;11 cells.⁵³⁾ In addition, the PROTACs against estrogen related receptor α,³⁷⁾ BRD4,^39,43,53) and BTK⁵⁴⁾ provided knockdown of the target proteins in a tumor xenograft mouse model and a rat model.

Compared with PROTACs, SNIPERs recruit IAPs and induce degradation of both target proteins and IAPs, which may limit the full potential and duration of knockdown activity by SNIPERs. However, IAPs are frequently overexpressed in human cancer cells and implicated in promoting tumor progression and resistance to anticancer drugs. Therefore, SNIPERs are suitable for development of degraders against oncogenic proteins, because degradation of IAPs simultaneously with target oncogenic proteins by SNIPERs could be beneficial to kill cancer cells.

3. Development of a Potent Degrader against BCR-ABL, SNIPER(ABL)

To apply the protein knockdown technology to BCR-ABL degradation, we designed and developed a hybrid molecule, SNIPER(ABL)-2 (Table 1) by conjugating MeBS to imatinib as a ligand for BCR-ABL.²⁵⁾ As expected, SNIPER(ABL)-2 induced degradation of BCR-ABL protein at 30–100 µM. BCR-ABL is a constitutively active tyrosine kinase and drives uncontrolled cellular proliferation through signaling pathways that involve the STAT5 and CrkL.^62,63) In accordance with the degradation of BCR-ABL protein, SNIPER(ABL)-2 suppressed signaling pathways downstream of BCR-ABL and inhibited CML cell growth. These data suggest that the protein knockdown technology can be applied for the degradation of BCR-ABL protein.

Table 1. List of Degraders against BCR-ABL

Symbols in the “Degradation activity” cell represent the DC₅₀ values: ++++ ≤ 100 nM; 100 nM< +++ ≤ 1 µM; 1 µM< ++ ≤ 10 µM; 10 µM<+ ≤ 100 µM. n.d., not determined.

To improve the protein knockdown activity, we tested various combinations of ABL inhibitors and IAP ligands, and the linker was optimized for protein knockdown activity of SNIPER(ABL).³⁰⁾ The resulting SNIPER(ABL)-39 (Table 1), in which dasatinib is conjugated to LCL161 derivative by polyethylene glycol ×3 linker, shows potent activity. At 10 nM, SNIPER(ABL)-39 induced dramatic reduction of BCR-ABL protein in CML cell lines such as K562 and KU812. Consistent with the degradation of BCR-ABL protein, SNIPER(ABL)-39 inhibited the signaling pathways initiated by BCR-ABL, and suppressed the growth of BCR-ABL-positive CML cells.³⁰⁾ These results suggest that SNIPER(ABL)-39 could be a lead as a degradation-based novel anti-cancer drug against BCR-ABL-positive CML.

In 2016, Dr. Crews’ group developed PROTACs against BCR-ABL by conjugation of ABL inhibitors (imatinib, bosutinib, and dasatinib) to a von Hippel–Lindau (VHL) E3 ligase ligand or pomalidomide that is a ligand for Cereblon (CRBN) E3 ligase.⁴⁰⁾ Interestingly, only bosutinib-CRBN and dasatinib-CRBN PROTAC showed degradation activity for BCR-ABL (Table 1), suggesting that finding a good combination of an E3 ligase and a target protein is important to induce the target degradation efficiently.

4. Features of SNIPER(ABL)

Identification and understanding of the oncogenic driver proteins have led to the development of rational cancer therapies. One of the most successful examples is the development of small molecule inhibitors against protein kinases such as imatinib, crizotinib, gefitinib, and sorafenib. Compared with TKIs, SNIPERs and PROTACs have distinguishing features.

The small molecule inhibitors are based on an “occupancy strategy”; enzyme inhibitors must bind to a target enzyme to inhibit their activity^59,61) (Fig. 3). Therefore, high concentrations of the inhibitors should be maintained to ensure active-site occupancy and to sustain the inhibitory effect. Maintaining high drug levels in vivo are one of the challenging issues, since high concentrations often cause undesirable off-target effects. In contrast, degraders, such as SNIPERs and PROTACs are based on “event-driven strategy”; these degraders suppress protein function by reducing the target protein level^59,61) (Fig. 3). Therefore, once oncogenic proteins are degraded, tumor cells can not drive cell proliferation until sufficient amounts of oncogenic protein accumulate in the cells by de novo synthesis of the protein, which occasionally results in apoptosis of the cells. Consistently, short-term treatment with SNIPER(ABL) showed long-lasting suppression of the proliferation of CML cells, whereas a TKI dasatinib slightly retarded the growth of the cells after drug removal.⁶⁴⁾ In addition, transient treatment with SNIPER(ABL) markedly reduced the level of BCR-ABL protein, which remained low level thereafter. Currently, imatinib is a frontline therapy for CML and dasatinib is effective for treating CML patients who failed imatinib therapy. These TKIs are usually dosed once or twice daily. Although the efficacy and the potential benefit of BCR-ABL degraders should be carefully evaluated in clinical settings, we speculate the sustained suppression of cell growth by BCR-ABL degraders, such as SNIPER(ABL), might benefit CML patients.

Fig. 3. Pharmacological Difference between Inhibitors (a) and Degraders (b)

(Color figure can be accessed in the online version.)

In contrast to enzyme inhibition, the protein knockdown activity was rather attenuated at higher concentrations of SNIPERs and PROTACs. Indeed, SNIPER(ABL)-39 showed effective protein knockdown activity at a concentration ≥10 nM and the maximum activity was observed at 100 nM, whereas the activity diminished at 1 µM.³⁰⁾ This is known as a high-dose hook effect, where a certain pharmaceutical activity is interfered with higher concentration of drugs. This is explained by the formation of ternary complex consisting of target protein-SNIPERs or PROTACs-E3 ubiquitin ligase required for the protein knockdown activity, which would be suppressed by excess amount of the hybrid molecules, and therefore, the protein knockdown activity is attenuated.

5. Prospect of SNIPER(ABL)

There are many small molecule inhibitors that are clinically successful and some of them are used as the first-line therapy for patients. However, significant number of patients develop resistance. Therefore, novel enzyme inhibitors should be constantly developed in an effort to overcome drug resistance. We think the protein knockdown technology would provide an additional strategy to overcome the problem.

SNIPERs (and PROTACs) require a ligand for target protein, which can interact with the target protein. Since BCR-ABL protein has multiple domains besides the tyrosine kinase domain, it is possible to develop novel ligands that bind to such domains. Incorporation of such ligands into SNIPERs (or PROTACs) would allow us to induce the degradation of BCR-ABL proteins resistant to TKIs.

Currently, we and Dr. Crews’ group successfully developed degraders against the BCR-ABL protein.^30,40) When dasatinib was incorporated as a ligand for BCR-ABL protein, degraders recruiting IAP and CRBN showed protein knockdown activity against the BCR-ABL protein, whereas VHL-recruiting degrader showed negligible activity (Table 1). However, when HG-7-85-01⁶⁵⁾ (another inhibitor of BCR-ABL kinase) was incorporated as a target ligand, VHL-recruiting degrader showed effective protein knockdown activity, whereas IAP- and CRBN-recruiting degraders showed weak and no activity, respectively⁶⁴⁾ (Table 1). Probably, effective degraders can recruit E3 ligases to an appropriate position so that the lysine residues on the surface of BCR-ABL can be ubiquitylated. Accordingly, development of VHL- and CRBN-recruiting PROTACs based on a promiscuous kinase inhibitor ferotinib demonstrated that the degradation profiles of the two PROTACs were not identical, and that there was no correlation between the binding affinity of either PROTAC to the kinases and the extent of PROTAC-induced degradation of the kinases.⁴⁴⁾ Thus, the suitable combination of an E3 ligase ligand and a target ligand is critically important to develop a potent degrader. In this point of view, it is important to identify a novel small molecule that recruits another E3 ubiquitin ligase to the target protein appropriately.

After the preclinical studies including optimization of the degraders by combination of the ligands for BCR-ABL and E3 ligases, we need to investigate the absorption, distribution, metabolism, excretion, and toxicity aspects of the BCR-ABL degraders, and to evaluate the efficacy and the potential benefit of BCR-ABL degraders in clinical settings. Through the clinical studies, the potential utility of BCR-ABL degraders for CML treatment may be demonstrated.

6. Conclusion

To overcome the imatinib resistance, second- and third-generation TKIs and omacetaxine have been developed and approved for the treatment of CML. However, further drug resistance or adverse reactions also occur by the treatment. Multiple treatment options are helpful for patients with imatinib-resistant CML, because treatment selection is based on factors such as the patient’s disease state, prior therapies, comorbidities, treatment toxicity, and goals of therapy. Here, we show that BCR-ABL degraders based on the protein knockdown technology could be a novel option for the treatment of CML. Further studies are required to develop clinically useful degraders against BCR-ABL protein.

Acknowledgments

This study was supported, in part, by Japan Society for the Promotion of Science (KAKENHI Grant Numbers 26860050 and 18K07311 to N.S., 26860049 to N.O., 16H05090 to T.H. and M.N., and 16K15121 to N.O. and M.N.), by the Project for Cancer Research And Therapeutic Evolution (P-CREATE) (JP17cm0106522j0002 to N.S. and JP17cm0106124j0002 to N.O.) and the Research on Development of New Drugs (JP16ak0101029j1403 to M.N.) from the Japan Agency for Medical Research and Development (AMED), by Takeda Science Foundation (to N.O.) and by Takeda Pharmaceutical Co., Ltd. (to M.N.). The authors thank M. Seki for measurement of the protein knockdown activities.

Conflict of Interest

M. N. received research funds from Takeda Pharmaceutical Co., Ltd. and Daiichi-Sankyo Co., Ltd. The other authors declare no potential conflict of interest.

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