New means and challenges in the targeting of BTK

Abstract

Bruton’s tyrosine kinase (BTK) is central to the survival of malignant and normal B-lymphocytes and has been a crucial therapeutic target of several generations of kinase inhibitors and newly developed degraders. These new means for targeting BTK have added additional agents to the armamentarium for battling cancers dependent on B-cell receptor (BCR) signaling, including chronic lymphocytic leukemia and other non-Hodgkin lymphomas. However, the development of acquired resistance mutations to each of these classes of BTK inhibitors has led to new challenges in targeting BTK as well as novel insights into BCR signaling. The first generation covalent BTK inhibitor ibrutinib is susceptible to mutations affecting the covalent binding site, Cysteine 481 (C481). Newer noncovalent BTK inhibitors, such as pirtobrutinib, overcome C481 mutation-mediated resistance but are susceptible to other kinase domain mutations, particularly at residues Threonine 474 and Leucine 528. Additionally, these novel BTK inhibitor resistance mutations have been shown biochemically and in patients to cause cross-resistance to some covalent BTK inhibitors. Importantly, newer generation covalent BTK inhibitors zanubrutinib and acalabrutinib are susceptible to the same mutations which confer resistance to non-covalent inhibitors. The BTK L528W mutation is of particular interest as it disrupts the kinase activity of BTK, rendering it kinase dead. This observation suggests that BTK may act independently of its kinase activity as a scaffold. Thus, the timely development of BTK degrading proteolysis targeting drugs has allowed for degradation, rather than just enzymatic inhibition, of BTK in B-cell lymphomas and early clinical trials to evaluate BTK degraders are underway.

INTRODUCTION

Bruton’s tyrosine kinase (BTK) inhibitors have revolutionized the treatment of chronic lymphocytic leukemia (CLL) and other B-cell non-Hodgkin lymphomas (NHL). Mutations in the BTK binding site for all covalent BTK inhibitors (Cysteine residue 481 (C481)) were recognized early in the use of ibrutinib as a major cause of clinical resistance (1). With widespread use of BTK inhibitors, including next generation covalent BTK inhibitors and the recently approved non-covalent BTK inhibitor pirtobrutinib, non-C481 BTK mutations have come to light as novel mediators of resistance (2,3). In this review, we provide background on the role of BTK signaling in normal and malignant B-cells, the targeting of BTK in B-cell NHLs, the studies that lead to the approval of BTK inhibitors in NHLs, and emerging data on resistance mechanisms arising to the different classes of these targeted therapies. Finally, we will discuss potential ways to overcome this resistance with novel therapeutic approaches.

BTK as a target for kinase inhibition

BTK is a 77kDa cytoplasmic, nonreceptor tyrosine kinase expressed primarily in B-cells, and can be activated by many surface receptors including B-cell receptors (BCRs), Toll like receptors (TLRs), chemokine receptors, and Fc receptors (Figure 1A) (4). As a kinase of the TEC family, BTK contains several domains (pleckstrin homology, Tec homology, Src-homology 3, Src-homology 2, and tyrosine kinase domain) required for the formation of signalosomes, as well as phosphorylation sites for activation (Y551, Y223) and inactivation (S21, S115, and S180) (4). BTK activation leads to ATP and substrate binding in the kinase domain, and subsequently to substrate phosphorylation and downstream signaling. BTK plays a key role in the development, activation, and maturation of normal B-cells. Therefore, knockdown of BTK induces cell death of malignant B-cells, and consequently BTK inhibitors have been developed to target B-cell malignancies (5).

Figure 1. Cell surface receptors that stimulate BTK activation in B-cells.

(A) BTK can be activated by many surface receptors including BCRs, TLRs, chemokine receptors and Fc receptors. Canonically BTK kinase activity leads to phosphorylation of PLCγ2, and subsequent activation of calcium flux and PKCβ signaling. This in turn leads to the activation of transcription regulators including NFAT, NF-kB and ERK1/2.

(B) BTK mutations can allow additional scaffolding functions, such as with HCK and ILK, that leads to alternative signaling.

BCAP, B-cell adaptor protein; BCL10, B-cell lymphoma/leukemia 10; BCR, B-cell receptor; BLNK, B-cell linker; BTK, Bruton’s tyrosine kinase; CaM, Calmodulin; CARD11, Caspase recruitment domain-containing protein 11; DAG, Diacylglycerol; ERK1/2, Extracellular signal-regulated kinase 1/2; HCK hematopoietic cell kinase; ILK, integrin-linked kinase; IP3, Inositol 1,4,5-trisphosphate; MALT1, Mucosa-associated lymphoid tissue lymphoma translocation protein 1; mTOR, mammalian target of rapamycin; NFAT, Nuclear factor of activated T-cell;

NF-kB, Nuclear Factor Kappa B; PI3K, Phosphoinositide 3-kinase; PIP3, Phosphatidylinositol (3,4,5)-trisphosphate; PKC, Protein kinase C; PLCy2, Phospholipase C gamma 2; RAC2, Ras-related C3 botulinum toxin substrate 2; SHIP1, SH-2 containing inositol 5’ polyphosphatase 1; SYK, spleen tyrosine kinase; TLR, Toll-like receptor. (Image created with BioRender.com.)

Currently, all clinical BTK inhibitors target BTK’s kinase domain either through covalent binding or non-covalent binding. Covalent BTK inhibitors bind irreversibly to residue C481 in the ATP binding pocket and prevent BTK phosphorylation at Y223 (but not Y551) (3,6–8). On the other hand, non-covalent BTK inhibitors can bind to the ATP binding pocket or deeper in the H3 pocket in the kinase domain to inhibit Y223 and Y551 phosphorylation (6,8). Both types of inhibitors prevent the binding of ATP to the kinase domain and in turn prevent substrate phosphorylation and downstream signaling (3,6–8)

BTK signaling

While multiple B-cell surface receptors work in concert through BTK to regulate B-cell signaling and B-cell function (Figure 1A), in mature B-cells, BCRs are the primary activators of BTK. Antigen binding and phosphorylation of BCR by Lyn and Syk kinases lead to recruitment of numerous kinases and adaptor proteins to form a signalosome (4). BCR aggregation and microcluster formation are facilitated by CD19 and BTK activation. Protein-protein interactions with BTK enable the transphosphorylation of Y551 and autophosphorylation of Y223 leading to the stabilization of the BTK active conformation (4). This allows ATP and substrate binding, enabling substrate phosphorylation. Phospholipase C gamma 2 (PLCγ2) phosphorylation leads to calcium flux and PKCβ signaling, and in turn to the activation of transcription regulators including NFAT, NF-kB and ERK1/2 (4). BCR stimulation and signaling through BTK is crucial regulator of B-cell development, differentiation, maturation, migration, and apoptosis.

BCR and TLR signaling synergy has been found to be significant in B-cell activation, differentiation, and cancer development. MyD88-mediated TLR signaling results in production of cytokines (including TNF-α, IL-6, and IL-10), inhibition of antigen processing, and decrease in the affinity maturation of antigen-specific B-cells (9). BTK is capable of directly binding with MyD88 affecting cytokine production, B-cell maturation, and differentiation.

B-cells express two chemokine G-protein coupled receptors, CXCR4 and CXCR5, at various stages of their development, which are crucial for the regulation of B-cell migration, localization, and homeostasis (4). BTK directly interacts with CXCR4 by binding to the heterotrimeric G protein subunits Gα and Gβγ, thereby influencing CXCR4 signaling through the PI3K, AKT and MAPK pathways (10). BCR stimulation in B-cells also leads to the internalization of CXCR4 and involves several proteins including Syk, BTK, PLCγ2, PKC, and PIM-1, as well as the activation of the ERK/MAPK cascade (11).

Both activating and inhibitory Fc-receptors regulate the immune response in B-cells (12,13). Activating Fc-receptors such as FcμR can directly interact with BCR regulating B-cell activation and survival (13). Inhibitory FcγRIIB (exclusively expressed in B cells) can recruit phosphatases that subsequently inhibit BTK activation and dampen immune responses (12). The coordination between Fc receptors and BCR signaling pathways allows B-cells to mount effective immune responses and generate tailored antibody-mediated immune defenses (13).

CLINICAL IMPORTANCE OF BTK AND BTK INHIBITORS

Before the discovery of BTK inhibitors, numerous genetic findings in patients highlighted the importance of BTK in B-cell disorders. Loss of function of BTK activity is responsible for X-linked agammaglobulinemia (XLA), while reliance on BTK is implicated in the hematologic malignancies, CLL, mantle cell lymphoma (MCL), Waldenström macroglobulinemia (WM), activated B-cell subtype diffuse large B-cell lymphoma, and marginal zone lymphoma (MZL). Consequently, the covalent BTK inhibitors ibrutinib, acalabrutinib, and zanubrutinib were approved by the U.S. FDA, and tirabrutinib and orelabrutinib are clinically used for the treatment of CLL/SLL, MCL, WM, and/or MZL outside of the U.S. (Figure 2, Table 1) (14–23).

Figure 2. Clinical approvals of BTK inhibitors for B-cell non-Hodgkin lymphomas.

R/R, relapsed refractory; TN, treatment naïve; del(17p), deletion of chromosome 17p13.1; CLL, chronic lymphocytic leukemia; SLL, small lymphocytic lymphoma; MCL, mantle cell lymphoma; WM, Waldenström macroglobulinemia; MZL, marginal zone lymphoma

Table 1.

Published clinical trials of covalent BTK inhibitors for CLL/SLL, MCL, WM and/or MZL.

Drug tested	Control	Cancer	Phase	National Clinical Trial number	Number of patients in test arm	Median follow up (months)	Overall response rate %	Median progression-free survival (months)	Discontinuations (for any reason)
Ibrutinib	Ofatumumab	R/R CLL/SLL	3	NCT01578707 (33,65)	195	65.3	91%	44.1	88%
	Chlorambucil	TN CLL/SLL	3	NCT01722487 (66,67)	136	82.7	92%	NR 59% at 7 years	58%
	Temsirolimus	R/R MCL	3	NCT01646021 (68)	139	20	72%	14∙6	53%*
	Zanubrutinib	WM	3	NCT03053440 (41)	99	19.4	93%	NR 84% at 18 months	21%*
	N/A	R/R MZL	2	NCT01980628 (69)	63	33.1	58%	15.7	71%*
Acalabrutinib	Ibrutinib	R/R CLL/SLL	3	NCT02477696 (39)	268	40.9	81.0%	38.4	54%
	Obinutuzumab+Chlorambucil	TN CLL/SLL	3	NCT02475681 (70)	179	46.9	89.9%	NR 77.9% at 48 months	30.7%*
	N/A	R/R MCL	2	NCT02213926 (71)	124	15·2	81%	NR 67% at 12-months	44%
	N/A	WM	2	NCT02180724 (72)	106	27·4	93·4%	NR 90% at 24 months	28%
	N/A	MZL	2	NCT02180711 (73)	43	13.3	53%	27.4	42%
Zanubrutinib	Ibrutinib	R/R CLL/SLL	3	NCT03734016 (42)	324	29.6	83.5	NR 78.4% at 24 months	27.2%
	Bendamustine+Rituximab	TN CLL/SLL del(17p)+	3	NCT03336333 (74)	109	18.2	94.5%	NR 89% at 18 months	11%%
	N/A	R/R MCL	2	NCT03206970 (75)	86	35.3	83.7%	33.0	54.7%
	Ibrutinib	WM	3	NCT03053440 (41)	102	19.4	94%	NR 85% at 18 months	20%*
	N/A	MZL	2	NCT03846427 (76)	68	15.7	68.2%	NR 82.5% at 15 months	41.2%
Orelabrutinib	N/A	CLL/SLL	2	NCT03493217 (48)	80	32.3	92.5%	NR 70.9 % at 30 months	33%*
	N/A	R/R MCL	1/2	NCT03494179 (49)	106	23.8	81.1%	22	51%*
	N/A	R/R WM	2	NCT04440059 (77)	47	16.4	89.4%	NR 89.4% at 12 months	13%*
Tirabrutinib	N/A	R/R CLL/SLL	1	NCT01659255 (44,47)	28	32.5	96%	38.5	39.3%
	N/A	MCL	1	NCT02457559 (45)	16	22.3	68.8%	25.8	69%*
	N/A	WM	2	ONO-4059-05 (46)	27	24.8	96.3%	NR	18.5*

⁺deletion of chromosome 17p13.1;

^*calculated from remained on treatment;

R/R, relapsed/refractory; TN, treatment naïve; CLL, chronic lymphocytic leukemia; SLL, small lymphocytic lymphoma; MCL, mantle cell lymphoma; WM, Waldenström macroglobulinemia; MZL, marginal zone lymphoma; NR, not reached; N/A, not applicable

XLA is characterized by lack of immunoglobulins in the blood due to the absence of mature B-cells, leading to frequent severe infections (24). XLA is caused by loss-of-function mutations in BTK (which is located on the X chromosome). Physiologically, BTK in pre-B-cells is constitutively phosphorylated leading to stimulatory signals for cell growth and proliferation (25). Consequently, the loss of function of BTK leads to disruption of this signal and hindered B-cell development and immune response.

CLL is the most common adult leukemia and is characterized by the presence of CD5+ CD19+ B-cells (5). Cells from patients with CLL have high BTK mRNA and constitutive BCR signaling which is mediated by BTK (5,26,27). The importance of BTK in CLL has been shown clinically by the success of U.S. FDA approved BTK inhibitors (ibrutinib, acalabrutinib, zanubrutinib, and pirtobrutinib) in treating patients with CLL(17,18,27).

MCL is a mature B-cell non-Hodgkin lymphoma caused by constitutive overexpression of Cyclin D1 (due to chromosomal translocation) leading to malignant transformation of the B-cells in the mantle zone (4). BTK was also found to be commonly overexpressed in MCL, and consequently, clinical trials of BTK inhibitors in MCL led to the FDA approval of four BTK inhibitors (ibrutinib, acalabrutinib, zanubrutinib, and pirtobrutinib) for MCL treatment (14,20,21).

Other non-Hodgkin B-cell malignancies can be treated with BTK inhibitors due to its role as a signaling mediator between multiple receptors and transcription regulators. WM is caused by somatic MYD88 mutation and BTK mediated NF-κB signaling, which leads to elevated monoclonal IgM levels and IgM-producing lymphoplasmacytic cells in bone marrow (28). Ibrutinib and zanubrutinib are approved for the treatment of WM (15,22). MZL is caused by dysregulation in antigen-mediated BCR activation leading to heterogenous B-cell lymphoma (29). Ibrutinib and zanubrutinib were approved as treatments of MZL(16,23).

First and second generation covalent BTK inhibitors

Ibrutinib represents the first generation of BTK inhibitors and inhibits BTK activity with high potency (7). Covalent inhibition of BTK by ibrutinib results in downregulation of B-cell activation genes (BCL2A1, Cyclin D2, c-FLIP), CCL3/CCL4 secretion (markers of B-cell activation), and CXCL12/CXCL13 induced CLL cell migration (27,30–32). In a phase 3 trial of relapsed, refractory patients with CLL/SLL receiving ibrutinib or ofatumumab (a CD20 antibody), the overall response rate was 91% and median progression-free survival was 44.1 months for ibrutinib, and ibrutinib-treated patients had a higher overall survival (33). Ibrutinib was subsequently approved for the treatment of CLL, MCL, WM, and MZL (14–17). The exact indications for drug approval for each drug noted here is provided in Table 1. However, the accelerated approval of ibrutinib was recently withdrawn for MCL and MZL due to the lack of evidence for clinical benefit for these indications. Ibrutinib can also modestly inhibit other protein kinase families containing a cysteine analogous to C481 such as epidermal growth factor receptor (EGFR) kinases, SRC kinases (BLK), TEC kinases (BMX, ITK and TEC) leading to side effects (7,33,34). Between 16–50% of patients with CLL/SLL discontinued ibrutinib due to adverse effects, which motivated development of additional BTK inhibitors (33,35). Currently, due to these novel BTK inhibitors and possible fatal cardiac arrythmias caused by ibrutinib, it’s use in the clinic is declining.

The second generation of BTK inhibitors, acalabrutinib, zanubrutinib, tirabrutinib, and orelabrutinib, also function as covalent inhibitors (6,8,36,37). Acalabrutinib also potently inhibits BTK but is more selective for BTK than ibrutinib in that it does not inhibit ITK or EGFR signaling and exhibits more than 10x lower potency against BMX and TEC (38). In a phase 3 trial comparing acalabrutinib with ibrutinib in previously treated patients with CLL, median progression-free survival was similar between the treatments (39). From a clinical perspective, there was less diarrhea, infection, and cardiovascular incidents (atrial fibrillation/atrial flutter incidence, 9% vs 16%; hypertension, 9% vs 23%) with acalabrutinib compared to ibrutinib, but greater headache. Discontinuations due to adverse effects were lower with acalabrutinib (14.7%) compared with ibrutinib (21.3%).

Zanubrutinib has an IC₅₀ of 1nM but binds to EGFR kinases, SRC kinases (BLK), and TEC kinases (BMX, TXK and TEC) similar to ibrutinib (40). In contrast to ibrutinib, zanubrutinib is selective over ITK, JAK3, and LCK. In a phase 3 trial comparing zanubrutinib to ibrutinib in patients with WM (MYD88L265P positive), progression-free survival and overall response rate was similar, however zanubrutinib had less adverse effects (41). Discontinuation rates due to adverse events were lower with zanubrutinib (4%) compared to ibrutinib (9%). In another phase 3 trial comparing zanubrutinib versus ibrutinib in patients with R/R CLL/SLL, 24-month progression-free survival and overall response rate was higher with zanubrutinib (78.4% and 83.5%, respectively) versus ibrutinib (65.9% and 74.2%, respectively) (42). Again, zanubrutinib had less adverse effects and discontinuation (16.2%) compared to ibrutinib (22.8

Orelabrutinib and tirabrutinib are the most recent clinically approved (although not approved in the Unites States) covalent BTK inhibitors. Orelabrutinib has an IC₅₀ of 1.8nM and is reported to be selective for BTK over EGFR, TEC, and BMX (36). Tirabrutinib has an IC₅₀ of 6.8nM and selective for BTK and TEC over ITK and EGFR (37). Owing to their good efficacy and low side effect profile, both orelabrutinib and tirabrutinib have been approved for MCL and CLL/SLL in China, and for primary central nervous system lymphoma (PCNSL) and WM in Japan, respectively (19,43–49).

Rationale for non-covalent BTK inhibition

Despite the success of covalent BTK inhibitors, approximately 60% of patients acquire resistance to covalent inhibitors due to BTK mutations, although typically occurring after years of response to covalent BTK inhibitors (1,50). The most common mutations causing resistance to ibrutinib occur at the BTK C481 residue, which is required for irreversible inhibitor binding. It is most frequently mutated predominantly to a serine residue (C481S) and is a predictor of CLL relapse (3,50). Second most common after BTK C481 mutations are activating mutations in the downstream substrate of BTK, Phospholipase C gamma 2 (PLCγ2). To overcome C481S resistant mutations, non-covalent ATP-competitive BTK inhibitors have been developed which maintain an ability to inhibit BTK signaling by the BTK C481S mutant protein. It is not known if the non-covalent inhibitors also overcome resistance due to other substitutions besides serine at the C481 residue (such as R/F/Y) or whether they overcome resistance caused by PLCγ2 mutations. In the study by Wang et al, 2022, of the nine patients with ibrutinib and pirtobrutinib resistant CLL, 2 patients had PLCγ2 mutations. The cancer cell fraction with PLCγ2 S707F and PLCγ2 D1140E were decreased or eliminated while PLCγ2 E1139del cell fraction increased after pirtobrutinib treatment suggesting context dependency (2). Nonetheless, BTK inhibitors that are not susceptible to BTK C481S mutations are attractive for drug development.

Pirtobrutinib is the first and only non-covalent BTK inhibitor which currently has U.S. FDA-approval. Pirtobrutinib has an IC₅₀ of 1–3nM at BTK WT and the C418S mutant, and is at least 10x selective for BTK over most EGFR, SRC, and TEC kinases (2,8). Pirtobrutinib binds at the ATP binding pocket of BTK, without interacting with C481, to prevent BTK Y223 and Y551 phosphorylation and activation of PLCγ2, ERK1/2, and NF-kB (8). In a phase 1/2 clinical trial with patients with B-cell malignancies, the overall response rate for patients with CLL/SLL was 63% and for patients with MCL was 52% (51). 1% discontinued treatment due to adverse effects. Pirtobrutinib has been approved for patient with R/R MCL after at least two lines of therapy, including a BTKi. It is also approved for patients with R/R CLL who have received at least two prior lines of therapy, including a BTKi and a BCL-2 inhibitor. Ongoing clinical trials are evaluating pirtobrutinib in patients with WM and in combination with venetoclax in CLL (51) (Table 2).

Table 2.

Clinical trials of non-covalent BTK inhibitors and degraders for CLL/SLL, MCL, WM and/or MZL.

Drug tested	Control	Class of drug	Cancer	Phase	National Clinical Trial number	Number of patients	Median follow up (months)	Overall response rate %	Median progression-free survival (months)	Discontinuations (for any reason)
Pirtobrutinib	N/A	non-covalent	R/R CLL/SLL	1/2	NCT03740529 (51)	170	6	63%	NR	14%*
			MCL			61	6	52%	NR	46%*
			WM			26	5	68%	NR	42%*
Pirtobrutinib	Idelalisib+Rituximab or Bendamustine+Rituximab	non-covalent	CLL/SLL	3 recruiting	NCT04666038	250	NA	NA	NA	NA
Pirtobrutinib	Ibrutinib	non-covalent	CLL/SLL	3 recruiting	NCT05254743	650	NA	NA	NA	NA
Pirtobrutinib	Bendamustine+Rituximab	non-covalent	TN CLL/SLL	3 recruiting	NCT05023980	250	NA	NA	NA	NA
Pirtobrutinib + Venetoclax	N/A	non-covalent	WM	2 recruiting	NCT05734495	42	NA	NA	NA	NA
Pirtobrutinib + Venetoclax	N/A	non-covalent	R/R MCL	2 recruiting	NCT05529069	30	NA	NA	NA	NA
Pirtobrutinib	Ibrutinib, Acalabrutinib or Zanubrutinib	non-covalent	MCL	3 recruiting	NCT04662255	500	NA	NA	NA	NA
Nemtabrutinib	N/A	non-covalent	R/R CLL/SLL	1/2	NCT03162536 (53)	57	8.1	56%	26.3	68%
			Other B-cell NHL (including WM)			55	NA	NA	NR	13%
Nemtabrutinib	N/A	non-covalent	Hematologic Malignancies	2 recruiting	NCT04728893	450	NA	NA	NA	NA
Nemtabrutinib	Ibrutinib or Acalabrutinib	non-covalent	TN CLL/SLL	3 recruiting	NCT06136559	1200	NA	NA	NA	NA
Nemtabrutinib + Venetoclax	Venetoclax+Rituximab	non-covalent	R/R CLL/SLL	3 recruiting	NCT05947851	720	NA	NA	NA	NA
NX-2127	N/A	degrader of BTK, IKZF1, IKZF3	R/R B-cell malignancies	1a/b	NCT04830137 (60)	160	NA	NA	NA	NA
			R/R CLL/SLL			23	5.6	33%	NR	39%*
NX-5948	N/A	degrader of BTK	R/R B-cell malignancies	1 recruiting	NCT05131022 (59,78)	130	NA	NA	NA	NA
BGB-16673	N/A	degrader of BTK	R/R B-cell malignancies	1/2 recruiting	NCT05006716	291	NA	NA	NA	NA
AC676	N/A	degrader of BTK	R/R B-cell malignancies	1 recruiting	NCT05780034	60	NA	NA	NA	NA
ABBV-101	N/A	degrader of BTK	R/R B-cell malignancies	1 recruiting	NCT05753501	188	NA	NA	NA	NA
HSK-29116	N/A	degrader of BTK	R/R B-cell malignancies	Ia/b recruiting	NCT04861779	156	NA	NA	NA	NA

^*calculated from remained on treatment;

R/R, relapsed/refractory; TN, treatment naïve; CLL, chronic lymphocytic leukemia; SLL, small lymphocytic lymphoma; MCL, mantle cell lymphoma; WM, Waldenström macroglobulinemia; MZL, marginal zone lymphoma; NHL, non-Hodgkin lymphoma; RT, Richter Transformation; NR, not reached; NA, not available; N/A, not applicable

Nemtabrutinib (formerly ARQ 531/MK-1026) has a potency of 0.4–0.9nM at the WT and the C418S mutant, and also binds to the ATP binding pocket of BTK, without interacting with C481, to inhibit Y223 and Y551 phosphorylation, and hence ERK1/2 and NF-κB signaling (52). It is not selective for BTK over some SRC kinases (BLK), TEC kinases (BMX, ITK, TEC), or Trk kinases (52). In a Phase 1/2 study of nemtabrutinibin patients with B-cell malignancies, patients with CLL/SLL had an overall response rate of 58%, progress free survival of 10.1 months, and discontinuation due to adverse events of 14% (53).

The case for targeted BTK degradation

Despite the success of the non-covalent inhibitor pirtobrutinib in R/R MCL and CLL, a recent study showed that novel non-C481 mutations in the BTK kinase domain can cause resistance to pirtobrutinib as well as other non-covalent and covalent BTK inhibitors (2,54). These BTK mutations most commonly involve Threonine T474 (T474I) and Leucine 528 (L528W) (Figure 3). They can occur with covalent inhibitors prior to noncovalent inhibitors although with a lower frequency depending on the covalent BTKi used (55). Furthermore, the manuscript describing these mutations showed that the L528W, as well as other mutations A428D, V416L and M437R, were kinase dead but could still signal through BCR pathways that bypass BTK and suggest a newly described scaffolding function for BTK (2,54,56). Further work by this same group showed that the kinase dead BTK L528W mutation confers a neofunction of BTK to bind to other SRC kinases such as hematopoietic cell kinase (HCK) and others (Figure 1B) (57). Therefore, they investigated a novel strategy using PROteolysis TArgeting Chimera (PROTAC) degraders of BTK. PROTAC degraders are heterobifunctional molecules that link a ligand of a target protein to a ligand of an E3-ligase (58). The proximity allows the target protein to be ubiquitinated by the E3-ligase, leading to proteasomal degradation. Additionally, in contrast to covalent inhibitors, the stoichiometry between drug and target binding is not 1:1. One degrader can degrade multiple targets, therefore less drug needs to be administered. Unlike other classes of BTK inhibitors, the dose-response relationship for PROTAC degraders is not sigmoidal but is bell shaped. After the ubiquitination and degradation of the target protein, target protein activity is not recovered until its resynthesis. NX-2127 and NX-5948 exemplify BTK PROTAC degraders and are currently in clinical trials for patients with B-cell malignancies (Table 2) (59,60).

Figure 3. Mutations in BTK that cause resistance to BTK inhibitors.

Highlighted in red are kinase impaired BTK mutants while in black are kinase proficient. PH, pleckstrin homology; TH, Tec homology; SH3, Src-homology 3; SH2, Src-homology 2 domain. (Image created with BioRender.com.)

NX-2127 links a BTK ligand to a ligand of the E3-ligase, cereblon so the proximity allows BTK to be ubiquitinated and degraded by cereblon (61). NX-2127 has a DC₅₀ of 4–6nM (concentration to achieve half maximum degradation) for BTK WT and 13nM for BTK C481S in cancer cell lines and inhibits BTK WT and BTK C481S tumor growth in mouse xenografts (61,62). It also degrades additional kinase dead BTK mutants (C481R and L528W) that confer resistance to BTK enzymatic inhibitors (57). As a potential added benefit, NX-2127also degrades IKZF1 and IKZF3 (25nM and 54nM, respectively), hypothetically leading to T cell activation and IL-2 secretion (61,62). In an ongoing phase I trial, NX-2127 has been orally administrated to 36 patients with R/R B-cell malignancies (23 had CLL) with prior BTK inhibitor therapies (60). 14 of the 23 patients with R/R CLL remained on treatment (at median follow-up of 5.6 months) and had a response rate of 33%. In vivo BTK degradation was 86% as measured directly from peripheral blood B-cells using intracellular flow cytometry and B-cell activation indirectly by measuring plasma CCL4 concentrations (57).

NX-5948 is a novel additional BTK degrader which does not degrade IKZF1 or IKZF3. NX-5948 has a DC₅₀ of 0.32nM and 1.0nM for BTK WT and C481S, respectively, in cell lines (63). In addition to BTK peripheral inhibition, importantly NX-5948 is blood–brain barrier permeable and inhibits tumor growth when administered systematically to mice with intracranial tumors. A phase I clinical trial of NX-5948 is under way with patients with R/R B-cell malignancies (59). Additional BTK degraders that are in trials include AC676 (NCT05780034), BGB-16673 (NCT05294731) and ABBV-101 (NCT05753501) (Table 2).

CONCLUSION AND KEY QUESTIONS

Although BTK inhibitors are a remarkable demonstration of successful targeted drug development, the first-generation drug ibrutinib is associated with a range of side effects and all covalent BTK inhibitors are susceptible to resistance mutations at C481 covalent binding site. Non-covalent BTK inhibitors overcome BTK C481S mutation-mediated resistance; however, non-C481 mutations can also limit the efficacy of these drugs. Elucidation of on-target resistance BTK mutations in response to BTK inhibitors led to the discovery of novel kinase dead BTK mutations, with BTK L528W being the most common and causing resistance to pirtobrutinib and cross-resistance to certain covalent inhibitors such as zanubrutinib. Recent mechanistic studies have revealed that kinase dead BTK mutations act through a novel scaffolding function of BTK that involves binding of other non-receptor kinases. Several BTK degraders have now been generated and appear to downregulate most recurrent mutant BTK proteoforms and effectively kill cells bearing these mutations. As such, BTK degraders may represent a novel “fourth generation” of BTK inhibitors and are currently in clinical trials for CLL and other non-Hodgkin lymphomas.

Despite the wave of mechanisms and modalities to inhibit BTK, some key questions remain unanswered. For example, the success of pirtobrutinib in the relapsed/refractory setting following covalent BTK inhibitors has led to a question of the optimal order of use of BTK inhibitors. Should covalent inhibitors always be used before non-covalent inhibitors? Currently, there are no data on the use of non-covalent BTK inhibitors in treatment of patients with treatment naïve CLL, although clinical trials are underway (NCT05254743 BRUIN CLL-314 for BTKi naïve CLL and NCT04662255 BRUIN MCL-321 for BTKi naïve MCL). Assuming non-covalent BTK inhibitors would be as effective as 2^nd generation covalent BTK inhibitors and given the lower frequency of side effects due to their selectivity, it might become preferable to start with non-covalent BTK inhibitors such as pirtobrutinib for frontline CLL. However, we also do not know the true frequency of BTK inhibitor resistance to the 2^nd generation covalent BTKi or to non-covalent BTK inhibitors. Thus, a randomized trial would be the best way to answer this question. However, until results from such a randomized trial are generated, there are practical questions about whether mutational data should be used to guide BTK inhibitor usage in patients already exposed to one inhibitor. For example, it seems obvious that if a patient is progressing on a covalent BTK inhibitor with a BTK C481S mutation, pirtobrutinib could be a good next treatment option for that patient. However, if a patient is progressing on zanubrutinib with a BTK L528W mutation, should they avoid pirtobrutinib and perhaps be trialed on ibrutinib? In the absence of clinical trial data designed to answer the question of optimal BTKi sequencing, current clinical data suggests that patients with L528W do exhibit some clinical responses to pirtobrutinib (64). Furthermore, patients progressing on BTK inhibitors who are BCL2-inhibitor naïve still demonstrate excellent responses to venetoclax therapy, and patients with relapsed/refractory disease exposed to currently approved agents have promising alternative options including BTK degraders.

It is important to also acknowledge that there are BTK independent mechanisms of BTK inhibitor resistance such as PLCγ2 mutants. These mutations activate PLCγ2, which is downstream of BTK and existence of PLCγ2 mutants begs the question of whether any BTK inhibitor strategy would work in PLCG2 mutants. Combining BTK inhibitors with other targeted agents may be able to avert or overcome PLCG2 mutations but the frequency and mechanisms of BTK inhibitor resistance when used in combinations with other agents has not been thoroughly described. There could be a role for heterobifunctional BTK degraders that also degrade IKZF1/3 in addition to BTK such as NX-2127 in the setting of PLCG2 mutations. NX-2127 has shown to be able to overcome resistance caused by all the currently described mutations in BTK and its IKZF1/3 targeting ability may be of utility in non-BTK mutation mediated resistance mechanisms. While much more research needs to be done on the efficacy and safety of BTK degraders, whether there will there be resistance to degraders remains unanswered. Given their requirement for engaging E3 ligases, there could be greater potential for more means of drug resistance not mediated through mutations in BCR signaling components.

The discovery of a scaffold function for BTK provides a rationale for using BTK degrader therapies but also prompts the question of where there could also be utility for allosteric BTK inhibitors. Allosteric BTK inhibitors do not bind the active site of BTK and therefore are theoretically impervious to mutations occurring in the kinase domain that have been shown to cause resistance to enzymatic inhibitors of BTK. Allosteric inhibitors might potentially block BTKs interaction with other proteins that enable signaling downstream or by harnessing the power of targeted protein degradation could also be used as novel BTK degraders.

Overall, recent developments in BTK inhibitor therapy have led to new means for targeting BTK but also new challenges in various cancer types in which these treatments are used in the form of acquired resistance mutations. Untangling these challenges and finding answers to some of the remaining key unanswered question is a task that will require innovations in drug development, preclinical and translational research, and additional clinical trials. Nevertheless, the future of targeting BTK inhibitors remains promising.