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 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. Together, these studies provided compelling justification for the continued exploration of these PROTACs in the clinic as single-agents and/or as a combination strategy.Several biotech companies have engineered novel BTK-targeted PROTACs that have entered clinical trials enrolling patients with CLL and other lymphoid malignancies. A pioneer in the field, NX-2127, was shown to degrade both BTKWT and BTKC481S protein in vitro and in non-human primate studies and mouse xenograft tumor models expressing either WT or C481S-mutant protein. In addition, NX-2127 has shown preclinical activity similar to IMiDs by catalyzing the ubiquitination of IKZF1 and IKZF3, resulting in increased T cell activation (60).NX-2127–001 is a first-in-human, multicenter, open-label, phase I dose-escalation (Phase Ia) and dose-expansion (Phase Ib) trial, evaluating the safety, tolerability, and preliminary efficacy of NX-2127 in adult patients with relapsed/refractory (R/R) CLL and B-cell malignancies (NCT04830137; ref. 61). Patients received NX-2127 orally once daily in 28-day cycles starting at 100 mg, and up to 300 mg. The recently updated results included a total of 54 patients (21 patients with NHL, 3 patients with CLL) with a median follow-up of 9.7 months. Treatment with NX-2127 led to near complete BTK degradation by Cycle 1 Day 8, resulting in decreased BCR signaling as measured by reduction of plasma CCL4. Immunomodulatory activity was evidenced by IKZF1 degradation by 25% to 50% in a dose-dependent manner. The adverse events observed with NX-2127 therapy were consistent with those seen with BTK inhibition. Most common adverse events were fatigue (46%), neutropenia (46%), hypertension (33%), contusion (30%), and diarrhea (30%). Most common grade 3 adverse events were neutropenia (43%), hypertension (15%), and anemia (15%). Atrial fibrillation was observed in 11% of patients. Thirty-three patients with R/R CLL were enrolled, with a median age of 74 years, after a median of 5 prior lines of therapy (range 2–11). The majority of patients with CLL exhibited a reduction in lymphadenopathy, and among the 27 response-evaluable patients, the best ORR was 40.7%. Meanwhile, 44.4% of patients had stable disease and responses continue to deepen over time. Importantly, responses were noted in BTKi/BLC2i double-refractory patients and those who progressed on a noncovalent BTKi (61).NX-5948 is a BTK degrader which does not possess immunomodulatory activity, and thus does not degrade IKZF1. Initial data with this agent were presented recently (62). Twenty-six patients with NHL and CLL received NX-5948 50 to 200 mg orally daily in a first-in-human phase I trial (NCT05131022). BTK degradation was near complete within 8 days of treatment. Treatment was well tolerated with most common adverse events of purpura (46%), thrombocytopenia (38%), and neutropenia (31%). Neutropenia was the most common grade 3 adverse event (19%), but infections were uncommon. Early responses were observed in patients with CLL, diffuse large B-cell lymphoma, and MCL (62).BGB-16673 is a novel BTK degrader without immunomodulatory activity that is being investigated in a first-in-human study in patients with R/R B-cell malignancies (NCT05294731; ref. 63). An initial report on 50 patients with NHL and CLL demonstrated acceptable safety and preliminary efficacy. Contusion, diarrhea, and fatigue were the most common adverse events (30%, 24%, and 20% of patients, respectively), whereas common grade 3 events were neutropenia (12%) and pneumonia (6%). Treatment discontinuations due to adverse events were rare (6% of patients). Among the 28 response-evaluable patients, ORR was 57% (all but one were partial responses; ref. 63).Finally, ABBV-101 is another selective BTK degrader which entered clinical trials (NCT05753501). However, clinical data with this agent are not yet publicly available (64).Interestingly, the BTK degraders discussed in this section employ CRBN. Preclinical evidence suggests that the choice of E3 ligase may impact efficacy and CRBN may confer higher potency in some models (22). However, CRBN- and VHL-based PROTACs may have off target effects due to the ubiquitous expression of both E3 ligases, thereby potentially impacting safety. As a result, additional E3 ligases that are tissue/target specific are being explored e.g., brain (FBXL16, KCTD8), pancreas (ASB9), skeletal muscle (KLHL40, KLHL41), etc.; further discussed in ref. 20. How exactly the clinical efficacy and safety may be affected by the choice of E3 ligase will be a subject of future studies.As BTK degraders demonstrate acceptable safety and tolerability, combination therapies will need to be explored in the future. For example, given the synergistic effect of concurrent targeting of BTK and BCL-2 in CLL, future studies should explore BTK degraders in combination with BCL-2 inhibitors (e.g., venetoclax). On the other hand, immunomodulatory activities of PROTACs such as NX-2127 may lend themselves to successful combinations with immune and cellular therapies, such as bispecific antibodies or chimeric antigen receptor T cells.A number of provocative questions remain:1) Will BTK degrader efficacy be aided by immunomodulatory function (CRBN), or will BTK-degrading function be sufficient? The answer to this question will depend on multiple factors, including the disease setting (NHL vs. CLL) and the potential for additional side effects which might occur due to IKZF1 and IKZF3 degradation. At this time, there is no preclinical or clinical data that is able to shed light on this issue.2) Will BTK degraders be efficacious in patients who had been treated with an IMiD (e.g., lenalidomide)? Here, we can be guided by a study which explored mechanisms of resistance to lenalidomide and pomalidomide in a large cohort of patients with multiple myeloma (65). The study documented emergence of mutations in the CRBN gene, however none of the mutations occurred within the drug-binding region. Thus, it is possible that PROTACs may have efficacy in this setting.3) Will BTK degraders eventually displace BTK inhibitors in therapy of CLL and NHL? While it is tempting to speculate on this issue given the theoretical potential of BTK degraders to avoid resistance to BTK-targeting therapies, the current follow-up on clinical trials is too short to know the answer. In our opinion, if BTK degraders continue to demonstrate favorable safety and long-term efficacy, they should be investigated in earlier lines of therapy in CLL and NHL.Within the past five years, great progress has been achieved in the field of BTK-targeted PROTACs, with several groups demonstrating their efficacy in preclinical in vitro and in vivo models. These PROTACs induce the formation of a BTK–PROTAC–E3 ligase ternary complex, allowing for the catalytic degradation of BTK through the UPS. Notably, PROTACs do not need to bind to the active site of BTK to induce degradation (i.e., PROTAC-mediated binding is not affected by acquired mutations in BTK), a distinct characteristic highly relevant to the context of BTKi resistance. BTK-targeted PROTACs offer a novel avenue in the treatment of B-cell malignancies, with early clinical data demonstrating preliminary safety and efficacy in CLL and other B-cell malignancies. Thus, there is a strong rationale for the continued development of BTK-targeted PROTACs.A.V. Danilov reports grants and personal fees from Nurix, Beigene, Abbvie, MEI Pharma, Astra Zeneca, Lilly Oncology, Presage; grants from Incyte and Bayer; and personal fees from Regeneron outside the submitted work. No disclosures were reported by the other authors.A.V. Danilov is a Leukemia & Lymphoma Society Scholar in Clinical Research (No. 2319–19).