Bruton Tyrosine Kinase Degraders in B-Cell Malignancies

Bruton tyrosine kinase (BTK) belongs to the TEC family of nonreceptor kinases and is expressed in various hematopoietic cells, including B cells, myeloid cells, and platelets (1). Within B cells, BTK plays a critical role in the B cell receptor (BCR) signaling pathway, where it contributes to the differentiation, proliferation, and survival of normal B cells (2).BCR pathway activation begins when antigen stimulation induces phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAM) by SRC family protein tyrosine kinases (e.g., LYN; ref. 2). Consequently, spleen tyrosine kinase (SYK) becomes activated, facilitating recruitment of BTK to the cell membrane and subsequent phosphorylation of tyrosine 551 (Y551) within BTK's kinase domain (2). This activation of BTK prompts autophosphorylation at a second site, tyrosine 223 (Y223), located within BTK's SH3 domain (3). With complete activation achieved, BTK can then phosphorylate its immediate downstream target, phospholipase-Cγ2 (PLCγ; refs. 2, 3). In addition to its kinase activity, BTK also serves as a scaffold, shuttling phosphatidylinositol-4-phosphate 5-kinase (PIP5KI) from the cytosol to the plasma membrane to produce phosphatidylinositol (4, 5)-bisphosphate (PIP2; ref. 4). PIP2 is a substrate for both PLCγ and PIP3 (4). Consequently, these events trigger a cascade of signaling pathways important for B cell homeostasis, including AKT, MAPK (including ERK and JNK), and NF-κB (2, 3).However, in the context of B-cell malignancies, chronic antigen stimulation of the BCR pathway leads to the survival and proliferation of malignant B cells, with BTK playing a pivotal role in disease pathogenesis (2, 3). BTK is overexpressed and constitutively phosphorylated in patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL; refs. 3, 5, 6). In addition, diffuse large B-cell lymphoma of the activated B-cell type (ABC-DLBCL) relies on the NF-κB pathway, in part driven by BTK, with a majority of ABC-DLBCL patients harboring mutations within this pathway (3, 7). In multiple myeloma (MM), high expression of BTK results in increased AKT signaling associated with the upregulation of stemness and MM cell self-renewal (2, 8). Collectively, these observations highlight BTK's oncogenic role in B-cell malignancies.BTK has become a well-validated target with multiple covalent BTK inhibitors (BTKi's, e.g., ibrutinib, acalabrutinib, and zanubrutinib) receiving regulatory approvals in CLL, MCL, lymphoplasmacytic lymphoma, and marginal zone lymphoma. A recent FDA approval of the noncovalent BTKi pirtobrutinib further contributed to field evolution (9). However, resistance to BTKi's is inevitable, and is in part driven by mutations in BTK's kinase domain (10). Mutations in BTK are not the sole explanation of resistance to BTK inhibitors, particularly in patients with non-Hodgkin lymphoma (NHL), where activation of alternative prosurvival signaling pathways, such as PI3K pathway, underlies drug resistance (11, 12). Nevertheless, BTKC481S mutation is commonly observed among patients with CLL and a subset of patients with MCL treated with ibrutinib and results in constitutive BCR signaling, whereas additional mutations have been described among patients treated with both covalent and noncovalent BTKi's (13–16). Currently, no therapeutic agent has regulatory approval in BTKi-resistant B-cell malignancies, thereby representing a strong unmet medical need.Proteolysis-targeting chimeras (PROTAC) represent a promising class of small heterobifunctional molecules, which are designed to selectively degrade specific proteins of interest (POI). A PROTAC consists of three components: a ligand that binds to the POI ("hook"), a linker, and a ligand that binds to an E3 ubiquitin ligase ("harness"; ref. 17; Fig. 1). Through these components, the PROTAC can bind to the POI and E3 ligase to form a POI–PROTAC–E3 ternary complex, which leads to the ubiquitination and subsequent degradation of the POI through the ubiquitin proteasome system (UPS; ref. 17).When a PROTAC molecule links the POI to the E3 ligase, it stimulates a polyubiquitination process of the former (18). Specifically, it causes the repeated transfer of ubiquitin from the ubiquitin-activating enzyme (E1) to the ubiquitin-conjugating enzyme (E2), which then allows the E3 ligase to ultimately attach the ubiquitin to the POI, marking it for degradation (18). Currently, more than 600 E3 ligases have been identified in humans, and under physiologic conditions, each E3 ligase has relative specificity towards its own set of proteins (19). Of these, von-Hippel-Lindau (VHL) and cereblon (CRBN) are most frequently used in PROTAC development due to their ubiquitous expression in human tissue (19, 20). Others include murine double minute 2 (MDM2) and inhibitor of apoptosis protein (IAP; refs. 18–22).CRBN is the direct molecular target of immunomodulatory drugs (IMiD), some of which are used in the clinic and include thalidomide, lenalidomide, and pomalidomide (23, 24). Although CRBN targets various endogenous substrates, additional neosubstrates (i.e., substrates recognized by CRBN only in the presence of IMiDs) have also been identified, such as Ikaros Family Zinc Finger 1 (IKZF1) and 3 (IKZF3; refs. 23, 25, 26). In B-cell malignancies, such redirection of CRBN's substrate specificity to degrade IKZF1 and IKZF3 elicits both antitumor and immunomodulatory effects (23, 27–35). Briefly, IMiDs have been shown to inhibit proliferation and induce apoptosis of malignant B cells, favorably modulate T cells, and repair the immunologic synapse both in vitro and in vivo (27–35). These results suggest that the pharmacological effects of IMiDs are due, in part, to the degradation of these neosubstrates, and hence such effect may contribute to the activity of CRBN-based PROTACs (23).The unique mechanism of action employed by PROTACs sets them apart from conventional small molecule inhibitors. In a recent review by Madan and colleagues, PROTACs are described to operate in a catalytic manner, meaning a single PROTAC molecule can degrade multiple target proteins, potentially allowing for lower concentrations and less off-target effects (19, 36). In contrast to inhibitors, PROTACs can target POIs without having to bind to the active site (i.e., ATP-binding), substantially broadening the scope of targetable proteins (19). As we mentioned above, BTK mutations have been identified amongst patients treated with BTKi's (13,14). Some of these mutations, such as BTKV416L and BTKL528W, are known to lack kinase function (i.e., BTK phosphorylation) but still facilitate downstream BCR signaling (37). This suggests that these "kinase dead" BTK mutants confer resistance to BTKi's by maintaining BTK's scaffolding function (37). Thus, PROTACs have the potential to target both the kinase activity as well as the scaffolding function of BTK, underscoring their applicability to BTKi-resistant B-cell malignancies.Despite these advantages, the effective design of PROTACs warrants careful consideration. Although PROTACs operate in a catalytic manner, the POI degradation efficiency is not solely determined by the quantity of targets that are engaged and degraded (i.e., it is not dependent on the affinities of the ligands for either the POI or E3 ligase; refs. 19, 38). Instead, the degradation efficiency relies on a multitude of factors, including the efficiency of ubiquitin transfer from the E3 ligase to the POI, the rate at which the POI is degraded by the proteasome, the basal expression level of the POI, and the rate at which the POI is replenished (19, 38–41).One design aspect that should be considered to ensure efficient degradation lies in the strategic selection of the employed linker (19, 38). This selection is important as both the length and composition of the linker play a major role in ternary complex formation (19, 38). This is because the linker not only contributes to the PROTAC's target specificity but also to the POI's susceptibility to ubiquitination (38). Furthermore, the linker's length and composition also affect the PROTACs' overall conformation and the orientation through which they bind to their ligands (19).Another intrinsic challenge faced by PROTACs is known as the "hook effect," characterized by a concentration-dependent activity curve that follows a bell-shaped pattern (19, 42). To elaborate, at higher concentrations, the effectiveness of PROTACs diminishes due to the saturation of binding sites to favor the formation of binary, rather than ternary, complexes with either the POI or E3 ligase (19, 42). This, in turn, renders the POI resistant to degradation, representing an obstacle in PROTAC development and optimization, especially in the context of dosing strategies (19, 42).With these aspects in mind, PROTACs present an exciting new therapeutic strategy to overcome limitations associated with traditional small molecule cancer therapies, including BTK inhibitors. This review will now highlight the various groups focused on developing BTK-targeted PROTACs for the treatment of lymphoid malignancies. A comprehensive list of the PROTACs discussed in this review can be found in Table 1.In 2019, Tinworth and colleagues were the first to evaluate the differences between covalent (irreversible) and covalent-reversible BTK-targeted PROTACs (43). To generate covalent-reversible PROTACs, the authors selected covalent BTK binding moieties that could be altered to maintain reversible BTK binding. In this study, IAP-based PROTACs containing covalent-irreversible- and covalent-reversible-based ligands derived from ibrutinib were compared. Although both covalent-irreversible (PROTAC 2) and covalent-reversible (PROTAC 3) PROTACs inhibited BTK in THP1 cells, only PROTAC 3 resulted in BTK degradation. It was suggested that covalent-irreversible PROTACs lost degradation capabilities due to the transfer of ubiquitin onto the POI or access to the proteasome being blocked (43).Similarly, Guo and colleagues concluded that the covalent-reversible PROTAC, RC-1, which contained ibrutinib- and pomalidomide (CRBN)-based ligands, exhibited potent BTKWT degradation in MOLM-14 cells at 8 and 40 nmol/L concentrations, compared with their covalent-irreversible or noncovalent PROTACs (41).Xue and colleagues also showed that their covalent-reversible PROTAC, "compound 7," which contained an ibrutinib-based hook and a VHL-based harness, was able to significantly reduce BTK in K562 cells 18 hours post-treatment (44). When compared against their noncovalent PROTACs, the authors found that their covalent PROTAC was a more potent inhibitor and better degrader of BTK (44).In addition, Yu and colleagues developed another covalent-reversible BTK-targeted PROTAC, PS-2, using poseltinib-based (an irreversible BTKi) and pomalidomide-based ligands, which potently degraded BTK, IKZF1, and IKZF3 in Mino, RAMOS, A20 and HEK-293 cells (45). Specifically, PS-2 degraded IKZF1 and IKZF3 in Mino cells with a half-maximal degradation concentration (D50) of 27.8 and 2.5 nmol/L, respectively, and reduced Mino cell growth at a half-maximal inhibitory concentration (IC50) of 77 nmol/L in vitro (45).In contrast to these findings, Gabizon and colleagues also explored noncovalent, covalent-irreversible, and covalent-reversible BTK-targeted PROTACs that contained ibrutinib-based and thalidomide (CRBN)-based ligands (46). The noncovalent PROTAC, NC-1, demonstrated the highest BTK degradation potency in Mino cells (DC50 = 2.2 nmol/L), with maximum degradation occurring at 2 to 4 hours. Similar trends were observed in primary CLL patient samples (46). Treatment with NC-1 abolished BTK phosphorylation as well as partially inhibited phosphorylation of AKT, ERK, and PLCγ2 and restricted CLL cell activation, viability, and migration regardless of the presence or absence of BTK mutation (47). Although NC-1 was the most potent PROTAC, the covalent PROTACs IR-2 (irreversible) and RC-3 (reversible) also induced BTK degradation with DC50's <10 nmol/L (46).In 2018, Huang and colleagues were the first group to develop noncovalent BTK-targeted PROTACs (48). CJH-005–067 consisted of a bosutinib-based hook (targets BCR-ABL and BTK), whereas DD-04–015 consisted of an RN486-based hook (a selective BTKi), with both integrating a pomalidomide-based harness. Both PROTACs effectively degraded BTK in MOLM-14 cells within 4 hours, with peak efficiency observed at 100 nmol/L concentration. In addition, treatment of TMD8 cells with DD-04–015 resulted in decreased cell viability when compared to treatment with RN486 (control; ref. 48).Building upon this momentum, Buhimschi and colleagues generated an array of BTK-targeted PROTACs, among which MT-802 was the most potent, and contained an ibrutinib-based hook and a pomalidomide-based harness (49). Specifically, MT-802 fully degraded BTK at a concentration as low as 250 nmol/L 4 hours post-treatment in Namalwa cells. In addition, a "hook effect" was not observed at compound concentrations ≤2.5 μmol/L. Because ibrutinib has low selectivity towards BTK, the authors used a KINOMEscan assay and determined that although both MT-802 and ibrutinib strongly bound BTK and TEC, MT-802 displayed weaker binding to other kinases, including IL-2-inducible T-cell kinase (ITK), a well-recognized ibrutinib target (49, 50). This highlights a feature where BTK degraders may have altered and improved POI specificity compared with BTKi's, and MT-802 may have fewer side effects mediated by ibrutinib's off-target activity in the clinic (10, 49). Importantly, although both MT-802 and ibrutinib targeted BTKWT, only MT-802 maintained potency against BTKC481S and exhibited similar degradation kinetics for both BTK variants (49). Furthermore, MT-802, but not ibrutinib, effectively reduced BTK levels in primary CLL samples from patients who had relapsed on ibrutinib and harbored the BTKC481S mutation. Of note, the group also generated VHL-based PROTACs, which were found to be less potent than their pomalidomide-based agents (49).Concurrently, Sun and colleagues generated a panel of BTK-targeted PROTACs active against both BTKWT and BTKC481S (22). Their PROTACs contained ibrutinib- or spebrutinib-based hooks and pomalidomide- or RG-7112-based (MDM2) harnesses. Interestingly, CRBN-recruiting PROTACs showed the highest potency, with ibrutinib- and pomalidomide-based PROTAC, P13I, demonstrating the highest levels of BTK degradation in Ramos cells (73% at 10 nmol/L). P13I, but not ibrutinib, degraded BTKC481S in both HeLa cells and DLBCL cell lines while not targeting ITK. A recent PROTAC created by the same group, L18I, contained a lenalidomide (CRBN)-based harness and induced degradation of BTKC481S at low nanomolar concentrations (51). Moreover, the authors demonstrated L18I's ability to target other clinically relevant mutations at the C481 locus, including C481T/G/W/A. Treatment with L18I induced rapid tumor regression in a BTKC481S HBL-1 xenograft mouse model with no signs of toxicity (51).Exploring design optimization, Zorba and colleagues created an 11-compound library of BTK-targeted PROTACs containing a PF-06250112 (BTKi)-based hook, pomalidomide-based harness, and various linker lengths (52). From their library, they discerned that longer linker length directly correlated with ternary complex formation, with this effect not being observed with shorter linkers. As a result, "compound 10," which possessed a longer linker (20 atoms), was selected as the most potent PROTAC and was found to selectively degrade BTK and the CRBN-substrates, ZFP91, IKZF1, and IKZF3 in vitro. Treatment with increasing concentrations of compound 10 resulted in dose-dependent BTK degradation in the spleen of healthy rats, with BTK not being degraded in lung tissues, thereby indicating in vivo tissue dependency (52).Dobrovolsky and colleagues created a series of BTK-targeted PROTACs encompassing a CGI1746/vecabrutinib-based hook as well as a thalidomide-based harness (53). From this library, DD-03–171 selectively bound to BTK at 1 μmol/L concentration and reduced BTK levels in Ramos cells at concentrations as low as 100 nmol/L following 4 hours of treatment. Both BTKWT and BTKC481S were degraded when overexpressed in TMD8 cells. Extending investigations into MCL models, DD-03–171 potently degraded BTK and blocked proliferation of Mino and Maver-1 cells. Furthermore, BTK degradation and a reduction in tumor burden was observed following treatment with DD-03–171 in both DLBCL and MCL patient-derived xenograft mouse models (53).In an attempt to improve the degradation of BTKWT and BTKC481S variants as well as increase the selectivity of BTK, Zhao and colleagues created a series of pomalidomide-based PROTACs encompassing ARQ531/nemtabrutinib (a reversible noncovalent BTK inhibitor) as a hook (54). Following optimization, "compound 3e" inhibited growth of cells with BTKWT and BTKC481S with a DC50 of 7 nmol/L (55). In addition, compound 3e possessed increased metabolic stability with a T1/2 >145 minutes. Similar metabolic stability was observed when mice were given a single dose of 2 mg/kg of compound 3e (55).To rationally design orally bioavailable PROTACs, Zhang and colleagues used dimensionality reduction analysis and model molecule validation (56). Among a series of BTK-targeted PROTACs with ibrutinib-based and thalidomide-based ligands, C13 was identified as a highly efficient and selective PROTAC. 50 nmol/L of C13 induced 91% BTK degradation in Mino cells after 8 hours. C13 degraded both C418- and T316-mutated BTK in a dose-dependent manner with a DC50 of 5.8 nmol/L. C13 was well absorbed in mice via oral gavage and exhibited wide tissue distribution and moderate clearance, suggesting that it was suitable for in vivo investigation. These results were further bolstered by the observation that a single oral dose of C13 at 100 mg/kg effectively reduced BTK protein levels in mouse monocytes after 6 hours, which was further sustained for 24 hours. Finally, treatment of OCI-LY10 xenografted mice with C13 (30 mg/kg twice a day for 17 days) inhibited tumor growth by 97%, which was accompanied by a decrease in BTK levels in tumors (56).Adding to the pool of orally bioavailable BTK-targeted PROTACs, Lim and colleagues generated UBX-382, which possessed a novel ligand to target BTK and a thalidomide-based harness (57). UBX-382 had a DC50 of 4 nmol/L and possessed greater antiproliferative activity against TMD8, OCI-LY3, and U2932 cells as well as greater inhibition of BCR signaling than either ibrutinib or ARQ531. In an in vivo TMD8 xenograft mouse model, oral treatment with 30 mg/kg UBX-382 rapidly (3 hours) degraded BTK within spleens. Daily treatment with 10 or 30 mg/kg UBX-382 resulted in complete tumor regression within 15 days. Furthermore. UBX-382 elicited degradation of C481X, L528W, and E41K BTK variants, but not BTKT474I (57).Huang and colleagues generated a series of PROTACs containing ibrutinib-based and pomalidomide-based ligands with an aim of exploring BTK-targeted PROTACs in inflammatory conditions (58). After creating 23 different variations, the authors found that "compound 15" was the most potent BTK degrader with a DC50 of 3.18 and 7.07 nmol/L in Ramos and Mino cells, respectively, which was much better than their MT-802 control. Furthermore, the authors found that treatment of Ramos cells with 100 nmol/L of compound 15 resulted in BTK degradation as early as 4 hours, with near-complete BTK degradation occurring at 8 hours. They also noted that these effects could be maintained for at least 48 hours following wash out. To explore the anti-inflammatory effects of compound 15, the authors treated LPS-induced RAW264.7 cells with compound 15 and found that it suppressed gene expression and secretion of proinflammatory cytokines, like IL-6 and IL-1β, by inhibiting NF-κB. Furthermore, compound 15 suppressed inflammatory responses in a zymosan-induced peritonitis (ZIP) mouse model (58).Zhang and colleagues investigated NRX-0492, a tool compound and a preclinical congener of NX-2127 and NX-5948 described below. NX-0492 contains a noncovalent BTK-based hook and a thalidomide-based harness (59). In BTKWT and BTKC481S-expressing TMD8 cells, NRX-0492 degraded BTK at DC50's of 0.1 and 0.2 nmol/L, respectively. Similarly, in primary CLL cells, NRX-0492 induced potent eradication of both BTKWT and BTKC481S at DC50's of ≤0.2 and ≤0.5 nmol/L, respectively, and downregulated BCR-mediated signaling. NRX-0492 promoted minimal degradation of ITK (at concentrations in which BTK degradation was induced) and downregulated IKZF1 and IKZF3 in a dose-dependent manner. In a CLL patient-derived xenograft mouse model, NRX-0492 induced BTK degradation and inhibited activation and proliferation of CLL cells in both the blood and spleen while simultaneously reducing tumor burden. Similar observations were seen in their BTKC481S CLL patient-derived xenograft mouse model (59).Thus, several groups demonstrated that BTK-targeted PROTACs could be developed and successfully applied in preclinical models. these for the of these PROTACs in the clinic as as a have novel BTK-targeted PROTACs that have patients with CLL and other lymphoid malignancies. A in the was shown to degrade both BTKWT and BTKC481S protein in vitro and in and mouse xenograft tumor models either or In addition, NX-2127 has shown preclinical activity similar to IMiDs by the ubiquitination of IKZF1 and in increased T cell activation is a and the and of NX-2127 in patients with CLL and B-cell ref. NX-2127 orally in at 100 and to The results a of patients patients with 3 patients with with a of Treatment with NX-2127 to complete BTK degradation by 1 in decreased BCR signaling as by reduction of plasma activity was by IKZF1 degradation by to in a dose-dependent manner. The events observed with NX-2127 were with seen with BTK events were and 3 events were and was observed in of patients with CLL were with a of after a of lines of The majority of patients with CLL exhibited a reduction in and among the the was of patients had disease and responses to Importantly, responses were noted in patients and who on a noncovalent BTKi is a BTK degrader which not immunomodulatory activity, and not degrade with this agent were patients with and CLL NX-5948 50 to orally in a BTK degradation was complete within 8 of treatment. Treatment was well with most events of and was the most 3 but were responses were observed in patients with CLL, diffuse large B-cell lymphoma, and MCL is a novel BTK degrader without immunomodulatory activity that is being investigated in a in patients with B-cell ref. on 50 patients with and CLL demonstrated and and were the most events and of whereas 3 events were and Treatment due to events were of Among the was but were ref. is another selective BTK degrader which However, with this agent are not the BTK degraders discussed in this suggests that the of E3 ligase may and CRBN may confer higher potency in some models (22). However, and VHL-based PROTACs may have target effects due to the ubiquitous expression of both E3 thereby potentially As a result, additional E3 ligases that are specific are being explored further discussed in ref. the and may be by the of E3 ligase will be a of BTK degraders and will to be explored in the given the effect of targeting of BTK and in CLL, should explore BTK degraders in with inhibitors (e.g., the other immunomodulatory of PROTACs such as NX-2127 may to with and therapies, such as or antigen receptor T of BTK degrader be by immunomodulatory function or will function be The to this will on multiple factors, including the disease and the potential for additional side effects which due to IKZF1 and IKZF3 degradation. this is no preclinical or that is able to on this BTK degraders be in patients who had been treated with an (e.g., we can be by a which explored of resistance to lenalidomide and pomalidomide in a large of patients with multiple myeloma The of mutations in the CRBN of the mutations within the Thus, it is that PROTACs may have in this BTK degraders BTK inhibitors in of CLL and it is to on this given the potential of BTK degraders to resistance to therapies, the on is to the In BTK degraders to and they should be investigated in lines of in CLL and the has been in the field of BTK-targeted PROTACs, with several groups demonstrating their in preclinical in vitro and in vivo models. These PROTACs induce the formation of a ligase ternary complex, allowing for the catalytic degradation of BTK through the PROTACs not to bind to the active site of BTK to induce degradation (i.e., binding is not by mutations in BTK), a highly relevant to the context of BTKi BTK-targeted PROTACs a novel in the treatment of B-cell malignancies, with early demonstrating and in CLL and other B-cell malignancies. Thus, is a strong for the development of BTK-targeted and from from and and from the were by the other is a in