BTK Inhibitors: present and future

Abstract

Bruton tyrosine kinase (BTK) is a nonreceptor tyrosine kinase which plays a central role in the signal transduction of the B cell antigen receptor (BCR) and other cell surface receptors, both in normal and malignant B lymphocytes. BCR signaling is activated in secondary lymphatic organs and drives the proliferation of malignant B cells, including chronic lymphocytic leukemia (CLL) cells. During the last ten years, BTK inhibitors (BTKi) are increasingly replacing chemotherapy-based regimen, especially in patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). BTKi are particularly active in patients with CLL and MCL, but also received approval for Waldenström’s macroglobulinemia, small lymphocytic lymphoma, marginal zone lymphoma, and chronic graft versus host disease. Current clinical practice is continuous long-term administration of BTKi, which can be complicated by side effects or the development of drug resistance. Alternatives to long-term use of BTKi are being developed, such as combination therapies, permitting for limited duration therapy. Second generation BTKi are under development, which differ from ibrutinib, the first in class BTKi, in their specificity for BTK, and therefore may differentiate themselves from ibrutinib in terms of side effects or efficacy.

Bruton’s tyrosine kinase (BTK)

The BTK gene encodes a cytoplasmic non-receptor tyrosine kinase which belongs to the Tec (tyrosine kinase expressed in hepatocellular carcinoma) kinase family. In humans, members of this protein family are primarily expressed in hematopoietic cells, and their activation is one of the first steps in antigen receptor signaling¹. BTK is expressed in most hematopoietic cells, especially in B cells, myeloid cells, and platelets, whereas T lymphocytes and plasma cells have low or undetectable levels of BTK. BTK is a 659 amino acid protein that contains five signaling domains, which is characteristic for members of the Tec family, and BTK has diverse partner molecules. This allows BTK to transmit and amplify signals from a variety of surface molecules though which cells communicate with other cells within the tissue microenvironment. Receptors that can activate BTK include antigen-receptors, especially the B cell receptor (BCR), growth factor and cytokine receptors, G-protein coupled receptors (GPCRs), such as chemokine receptors, and integrins. Upon activation, BTK triggers several downstream signaling cascades, including the phosphoinositide 3-kinase (PI3K)-AKT pathway, PLC, PKC, and nuclear factor-κB (NFκB). The role of BTK in BCR signaling and in cell migration appear to be the primary targets of BTKi.

BTK activation following antigen engagement with the BCR triggers a downstream signaling cascade which results in B cell survival, proliferation, and differentiation. After BCR engagement, first, the signal transduction molecules Igα and Igβ (CD79a/CD79b) cluster and become and phosphorylated within the cytoplasmic tails of their immune-receptor tyrosine-based activation motifs (ITAMs). Subsequently, spleen tyrosine kinase (SYK) binds to the ITAM motifs, which, in turn, activates the B cell linker scaffold protein (BLNK, also known as SLP65 or BASH). Subsequently, BTK and CD19 are activated, which activates PI3K and consequently increases cytoplasmic PIP₃ levels. Downstream, phospholipase Cγ2 (PLCγ2) is activated, which results in calcium and PKC signaling and transcriptional activation though nuclear factor κB (NF-κB) and ERK. In the absence of BTK, BCR signaling is insufficient to induce B cell differentiation into mature peripheral B cells. This leads to altered B cell development and defects in functional responses, including cellular proliferation, expression of activation markers, cytokine and antibody production and responses to infectious diseases. Interestingly, BTK overexpressing in B cells results in the spontaneous formation of germinal centers, antinuclear autoantibody production, and a systemic lupus erythematosus (SLE)–like autoimmune disease, caused by hyper-responsive BCR signaling and increased NFκB activation, which was reversible with the BTKi ibrutinib.

Besides its role in BCR signaling, BTK also plays an role in signaling of cytokine receptors, CD19, CD38, CD40, chemokine receptors, such as CXCR4², tumor necrosis family receptors (TNFR), toll-like receptors (TLRs), and integrins. Of particular interest are effects of BTK on cell motility and tissue homing, given that the BTKi cause redistribution of tissue-resident (CLL) B cells into the peripheral blood, causing lymphocytosis that depends on the continuous presence of the BTK inhibitor^{3, 4}. The role of BTK in chemokine receptor- and integrin-signaling in B cells^{2, 5} is considered to be the basis for this clinical phenomenon.

Ibrutinib

Ibrutinib, previously called PCI-32765, is a potent (IC₅₀, 0.5 nM) and selective BTK inhibitor⁶ that entered clinical development in late 2009. BTK and ibrutinib were named after Dr. Ogden Bruton, a pediatrician who described a primary immunodeficiency syndrome, now termed Bruton’s agammaglobulinemia or X-linked agammaglobulinemia (XLA) in the 1950s⁷. Mutations in a kinase encoded on the X chromosome, now termed BTK, were found to be responsible for XLA. XLA patients, as a consequence of defective BCR signaling, lack mature B cells and immunoglobulins⁸. The initial clinical evaluation of ibrutinib in CLL demonstrated a distinct response pattern with rapid shrinkage of enlarged lymph nodes during the first days of treatment, along with a transient lymphocytosis in the peripheral blood³. This clinical phenomenon called “redistribution lymphocytosis”⁴ is due to CLL cell redistribution from lymphoid tissues into the peripheral blood, a class effect that is shared between BTK³, SYK⁹, and PI3K inhibitors¹⁰. Redistribution lymphocytosis is caused by inhibition of signaling and function of chemokine receptors (CXCR4, CXCR5) and adhesion molecules, such as integrins^{4, 11–14}.

The efficacy and safety of ibrutinib in patients with relapsed CLL was evaluated in a phase 1b/2 study. This population included patients with relapsed or refractory CLL or small lymphocytic leukemia (SLL) who had received a median of 4 prior therapies as well as a small cohort of elderly, previously untreated patients. In the primary analysis, ibrutinib was associated with an objective overall response rate (ORR) of 71% (primarily PRs) in both cohorts¹⁵. An additional 20% of patients achieved a PR with lymphocytosis at 12 months, but this rate decreased to 5% after 36 months, as the lymphocytosis resolved in practically all patients. Consequently, over time, the ORR increased accordingly, reaching 89%, with complete response rates increasing over time to 29% in untreated patients and 10% in relapsed or refractory patients. ¹⁶. In this trial, ibrutinib was associated with an improved PFS when compared with historic data using more traditional salvage options. For relapsed or refractory patients with del(17p) the median duration of remission (DOR) was 31 months, those with del(11q) or complex karyotype (CK) also tended to experience shorter DOR than patients without these abnormalities, with a median DOR of 39 months in patients with del(11q) and 31 months in patients with CK. Overall, PFS rates remained high in treatment-naive patients and declined over time in patients with relapsed/refractory CLL¹⁶, primarily due to progression in patients with high-risk features, such as del17p and/or complex karyotype abnormalities, or intolerance and other complications^{17, 18}. However, the majority of patients have durable remissions, even in the relapsed/refractory setting.

Ibrutinib generally is not myelosuppressive, and therefore patients can remain on continuous therapy for an extended time. In anemic and/or thrombocytopenic patients, hemoglobin and platelet levels generally improve and then normalize, as patients achieve remissions. In the phase 1/2 trial, the most common toxicities were grade 1/2 and did not require any treatment adjustments or interruptions. Such common adverse events (AEs) were diarrhea (49%), upper respiratory tract infections (33%), and fatigue (32%)¹⁵. Low-grade musculoskeletal side effects (eg, myalgias or arthralgias) and minor bruising also are relatively common side effects. Bleeding complications have been recognized as a drug-related AE due to effects of ibrutinib on BTK-mediated platelet adhesion^19–21. In randomized trials, the incidence of severe bleeding events was not significantly higher in ibrutinib-treated patients, and many patients concomitantly receive anticoagulants or anti-platelet agents together with ibrutinib, without any major complications^22–24. Infections are common in patients with CLL, and consequently also were noted in patients on these clinical trials. However, these events were generally attributed more to the disease activity than the treatment with ibrutinib. Infectious complications tend to occur more frequently in relaped/refractory CLL during the first year on ibrutinib therapy, and seem to decline rather than increase in frequency over time, after patients achieve a durable remission, based on the currently available data^{16, 25, 26}.

Randomized ibrutinib trials in patients with CLL

The randomized, open-label, phase 3 RESONATE trial compared ibrutinib with the anti-CD20 monoclonal antibody ofatumumab in patients with relapsed/refractory CLL or SLL^{22, 27}. A total of 391 patients were randomly assigned to ibrutinib or ofatumumab. Patients in the ofatumumab arm could cross over to receive ibrutinib after disease progression. Ofatumumab, which is FDA-approved in the salvage setting, is associated with a median PFS of approximately 6 to 12 months^{28, 29}. After a median follow-up of 9.4 months, ibrutinib was significantly more effective than ofatumumab, with a median PFS not reached in the ibrutinib arm, compared to 8.1 months in the ofatumumab arm. The 12-month OS rate was 90% with ibrutinib and 81% with ofatumumab, and the ORR was 42.6% vs 4.1%, respectively; an additional 20% of patients treated with ibrutinib had a partial response with lymphocytosis. With longer follow-up, overall response to ibrutinib increased, with 91% of patients attaining a response. The PFS benefit with ibrutinib was independent of baseline risk factors, although patients with ≥2 prior therapies had shorter PFS than those with <2 prior therapies, and the presence of TP53 or SF3B1 mutations showed a trend toward shorter PFS vs without these factors. A long-term follow-up analysis of the RESONATE trial with median follow-up of 65.3 months recently was reported²⁷. This analysis confirmed a major survival benefit (PFS and OS) for patients receiving ibrutinib versus ofatumumab (median PFS 44.1 vs 8.1 months; hazard ratio [HR]: 0.148; 95% confidence interval [CI]: 0.113–0.196; P˂0.0001), irrespective of high-risk clinical or genomic features. The ibrutinib safety profile remained consistent with prior reports; cumulatively, grade ≥3 hypertension and atrial fibrillation occurred in 9% and 6% of patients, and only 16% discontinued ibrutinib because of adverse events (AEs).

The efficacy and safety of ibrutinib in the frontline setting were established in the randomized, open-label, phase 3 RESONATE-2 trial, which compared ibrutinib with chlorambucil in treatment-naive, older patients with previously untreated CLL or SLL²³. A total of 296 patients at least 65 years of age (median age: 73 years) were randomly assigned to ibrutinib continuously daily, or chlorambucil, administered once every 2 weeks. After a median follow-up of 18.4 months, ibrutinib was significantly more effective than chlorambucil, with the median PFS not reached vs 18.9 months. The estimated 2-year OS rates were 98% and 85%, respectively. Subgroup analyses identified no significant difference in outcomes based on CLL risk factors in the ibrutinib arm, a finding consistent throughout several studies. In contrast, in the chlorambucil arm, higher-risk patients (eg, del[11q], or IGHV-unmutated CLL) had worse outcomes than other patients. Finally, the ORR was also significantly higher with ibrutinib vs chlorambucil. Ibrutinib was also associated with greater sustained improvements in hemoglobin and platelet levels. The AE profile was similar to that observed in the phase 1/2 study, aside from additional safety signals, including arterial hypertension (reported in 14% of ibrutinib-treated patients), atrial fibrillation (6%) and hemorrhage, reported in 4% of patients receiving ibrutinib vs 2% of patients receiving chlorambucil²³. The incidence of major bleeding events was lower than 5% and did not significantly differ between the treatment arms. Overall, the data from the RESONATE-2 trial indicate that ibrutinib is a valuable option for elderly or high-risk patients who are not candidates for chemo-immunotherapy.

A randomized three arm trial for elderly (>65) CLL patients with untreated CLL (Alliance trial) compared chemo-immunotherapy (CIT) with bendamustine plus rituximab (BR) given for 6 cycles, with continuous ibrutinib alone or in combination with rituximab³⁰. The estimated PFS at 2 years was 74% with BR, which was significantly lower than the PFS with ibrutinib (87%) or ibrutinib combined with rituximab (88%). This difference in survival outcome was driven by patients with high-risk disease features who are know to have shorter survival with CIT, whereas the difference between BR and ibrutinib did not appear to be significant in low-risk patients. Regarding the potential benefit from adding rituximab to ibrutinib, there was no significant PFS difference between ibrutinib monotherapy versus ibrutinib combined with rituximab. Similarly, a randomized trial comparing ibrutinib monotherapy with ibrutinib plus rituximab in relapsed/refractory and untreated high-risk CLL patients failed to show any PFS benefit from the addition of rituximab to ibrutinib³¹. The addition of rituximab (and other anti-CD20 mAbs) appears to accelerate responses to BTKi therapy, and result in deeper responses, but does not prolong survival when compared with ibrutinib alone.

Another randomized phase 3 trial (iLLUMINATE) compared ibrutinib plus obinutuzumab with chlorambucil plus obinutuzumab CIT for treatment of elderly CLL patients (>65) or patients under 65 with comorbidities³². After a median follow-up of 31 months, median PFS was significantly longer in the ibrutinib plus obinutuzumab group (median not reached [95% CI 33·6–non-estimable]) than in the chlorambucil plus obinutuzumab group (19·0 months [15·1–22·1]; hazard ratio 0·23; 95% CI 0·15–0·37; p<0·0001). Estimated 30-month PFS was 79% in the ibrutinib plus obinutuzumab group and 31% in the chlorambucil plus obinutuzumab group.

The randomized ECOG Cancer Research Group (E1912) trial compared CIT with fludarabine, cyclophosphamide, and rituximab (FCR) with ibrutinib plus rituximab in previously untreated younger CLL patients (<70, patients with del17p were excluded)³³. After a median follow-up of 34 months, PFS was significantly longer in patients that received ibrutinib-rituximab versus FCR (89.4% vs. 72.9% at 3 years; p<0.001). Overall survival also favored ibrutinib-rituximab (98.8% vs. 91.5% at 3 years; P<0.001). However, in subgroup analysis, low risk-patients with IGHV mutation had similar PFS outcomes with ibrutinib plus rituximab compared to FCR (3 year PFS 87.7% vs. 88.0%)³³.

A phase 2 study in previously untreated high-risk and older CLL patients combined ibrutinib with the BCL2 antagonist venetoclax (ibrutinib monotherapy for 3 months, followed by the addition of venetoclax) for a duration of 24 months³⁴. After 12 months of combined treatment, 88% of the patients had CR or CR with incomplete count recovery, and 61% had remission with undetectable minimal residual disease (MRD). Responses were noted in older adults and across all high-risk subgroups. This trial is of importance, demonstrating that limited-duration combination therapy with ibrutinib plus venetoclax can induce deep remissions that permit for therapy discontinuation. Long follow-up is needed to determine whether the early success of this combination approach translates into long-term disease-free survival.

Ibrutinib in other B cell malignancies

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor prognosis, characterized by the overexpression of cyclin D1. MCL initially is treated with intensive chemo-immunotherapy (CIT), oftentimes followed by autologous stem-cell transplantation in younger patients, whereas older patients or those with coexisting conditions are treated with CIT and rituximab maintenance. A phase 2 study of ibrutinib in patients with relapsed or refractory MCL showed an ORR of 68% (CR:21%), and a median PFS of 14 months³⁵. A phase 3 trial of ibrutinib versus temsirolimus, an inhibitor of the mTOR pathway, showed a best overall response rate of 72% with ibrutinib, a rate of complete response of 19%, and a median progression-free survival of 14.6 months³⁶, and ibrutinib was superior to temsirolimus with terms of ORR, safety, and PFS. The side effect profile of ibrutinib in MCL was similar to that in CLL, with bleeding, diarrhea, rash, and atrial fibrillation as the most common related AEs. In contrast to CLL, where BTK and PLCG2 mutations are frequent among resistant patients, ibrutinib resistance in MCL appears to activate multiple possible bypass pathways including the nuclear factor κB (NF-κB) pathway³⁷, and PI3K-AKT-mTOR, and integrin-β1 signalling³⁸. A phase 2 trial of ibrutinib plus venetoclax in relapsed/refractory MCL (n=23) demonstrated feasibility and safety of this combination, which resulted in a relatively high CR rate of 42%, with MRD negativity in 67% of the patients. 78% of patients maintained a response at 15 months on therapy³⁹.

For patients with Waldenström macroglobulinemia (WM), a rare mature B cell malignancy characterized by elevated serum levels of IgM and infiltration of the bone marrow and other organs by IgM-producing clonal lymphoplasmacytic cells, ibrutinib has been approved in combination with rituximab for frontline and salvage therapy. In a randomized trial of ibrutinib plus rituximab versus placebo plus rituximab in patients with treatment-naïve or relapsed WM, patients on the ibrutinib arm had a significantly longer PFS (82% versus 28% at 30 months)⁴⁰. Accordingly, the rate of major response was higher with ibrutinib–rituximab (72% vs. 32%). AE of grade 3 or higher that occurred more frequently with ibrutinib–rituximab than with placebo–rituximab included atrial fibrillation (12% vs. 1%) and hypertension (13% vs. 4%).

In patients with marginal zone lymphoma (MCL), ibrutinib was approved as salvage therapy following at least one prior anti-CD20-based treatment, based on findings from a single-arm phase II study, which showed an ORR of 48%⁴¹. With median follow-up of 19.4 months, median duration of response was not reached and median PFS was 14.2 months.

Second generation BTK inhibitors

Several second generation BTK inhibitors are in clinical development. The majority of these agents covalently bind with the C481 residue in BTK, similar to ibrutinib, but exhibit higher target selectivity.

Acalabrutinib (Calquence), originally referred to as ACP-196, is a covalent, potent inhibitor of BTK, targeting the C481 BTK residue, is given at a dose of 100mg twice daily. Acalabrutinib has a higher selectivity for BTK when compared to ibrutinib, and therefore may have fewer adverse events⁴². For example, acalabrutinib does not inhibit EGFR signaling, ITK, or TEC kinases. Acalabrutinib currently is approved for patients with relapsed or refractory MCL, and received breakthrough therapy designation as monotherapy for CLL.

Whether acalabrutinib has clinically relevant differences when compared to ibrutinib ultimately will be determined in a randomized trial comparing ibrutinib with acalabrutinib ( NCT02477696). A phase I/II multi-center study demonstrated high response rates in patients with relapsed/refractory CLL that was comparable to that seen with ibrutinib, with an overall response rate of 85% (95% when including PRL)⁴³. As in MCL, acalabrutinib side effects in CLL patients include headache (46%), diarrhea (43%), upper respiratory tract infection (28%), fatigue (27%), nausea (27%), arthralgia and pyrexia (each 23%), contusion (22%), petechiae and weight increased (each 21%), hypertension (11%) and atrial fibrillation (3%). The approval of acalabrutinib in CLL will depend on the outcome of three pivotal trials, a randomized study comparing acalabrutinib with acalabrutinib plus obinutuzumab or chlorambucil plus obinutuzumab in treatment-naïve older CLL patients ( NCT02475681), a randomized study comparing ibrutinib with acalabrutinib in patients with relapsed/refractory CLL with high-risk features ( NCT02477696), and a randomized trial of acalabrutinib versus either idelalisib plus rituximab or bendamustine plus rituximab in relapsed/refractory CLL ( NCT02970318). In a Phase 2 trial in relapsed or refractory MCL patients, after a median follow-up of 15 months, ORR was 81%, with 40% of patients achieving a CR. 12-month PFS was 67% and 12-month OS 87%. The most common AE were headache (38%), diarrhea (31%), fatigue (27%), and myalgia (21%), neutropenia (10%), anemia (9%), and pneumonia (5%). There were no cases of atrial fibrillation and one case of grade 3 or worse hemorrhage. The median duration of treatment was 13·8 months⁴⁴.

Zanubrutinib (BGB-3111) is another covalent BTK inhibitor which binds to C481 and is more selective than ibrutinib⁴⁵. Zanubrutinib received breakthrough therapy designation for the treatment of adult patients with mantle cell lymphoma (MCL). In the initial Phase 1/2 trial, after median follow-up of 13.7 months, 95% of CLL/SLL patients remain on study. Among 78 evaluable CLL/SLL patients, the ORR was 96%, and the estimated PFS at 12 months was 100%⁴⁶. Most toxicities were grade 1/2; neutropenia was the only grade 3/4 toxicity observed in >2 patients. One patient experienced a grade 3 subcutaneous hemorrhage. Registration trials are ongoing.

Evobrutinib, another covalent oral BTK inhibitor with greater BTK selectivity than first generation inhibitors, inhibits B-cell activation and cytokine release, as well as activation, differentiation, and polarization of M1 macrophages. In a randomized trial in patients with relapsing multiple sclerosis, those treated with evobrutinib had significantly fewer enhancing CNS lesions than patients who received placebo⁴⁷.

Tirabrutinib (ONO/GS-4059) is another covalent inhibitor of BTK, targeting BTK C481 that is more selective than ibrutinib. GS-4059 demonstrated potent activity in patients with CLL/SLL; among the 25 evaluable patients, ORR was 96%, with a typical BTK inhibitor-induced rapid reduction in lymphadenopathy, redistribution lymphocytosis and improvement in hematologic parameters⁴⁸. Ongoing trials with GS-4059 in CLL combine this BTK inhibitor with the SYK inhibitor entospletinib (GS-9973) or the PI3Kδ inhibitor idelalisib, with or without the CD20 antibody obinutuzumab ( NCT02983617, NCT02968563).

SNS-062 is a potent, non-covalent BTK inhibitor, and thus differs from the previous BTK inhibitors⁴⁹. This agent is being developed to potentially overcome ibrutinib-resistance related to BTK Cys481Ser mutation; in vitro studies have demonstrated that SNS-062 can have antitumor activity even in cells that carry this mutation. It is in early clinical development in patients with different B cell malignancies ( NCT03037645) and has not yet proceeded towards registration trials.

Fungal infection in patients treated with BTKi

As larger patient populations are treated with BTKi, especially with ibrutinib, for longer periods of time, additional safety concerns have emerged. Whether these effects are class effects of BTKi or more specific for ibrutinib is not clear at this time. These include invasive fungal infections, especially with aspergillus species^50–52. In a small trial for central nervous system (CNS) lymphoma, 39% of patients treated with ibrutinib plus corticosteroids developed aspergillosis⁵³. A potential mechanism to explain these infections could be inhibitory BTK-related effects of ibrutinib on macrophages, suppressing the phagocytosis of aspergillus⁵⁴. In CLL or MCL patients treated with ibrutinib, however, the incidence of Aspergilloses appears to be much lower than in CNS lymphoma⁵⁰. Predominant sites of infection (pulmonary, CNS) and early onset fungal infection after start of ibrutinib therapy seem to be characteristic⁵². While anti-fungal prophylaxis is not warranted for the general population of ibrutinib-treated patients, particular attention and close follow-up, especially during the first months of therapy, is recommended for patients with high-risk features, such as concomitant corticosteroid use, higher number of prior therapies, diabetes, or liver disease.

Cardiac arrhythmias in patients treated with BTKi

Ibrutinib appears to have arrhythmogenic properties, although the underlying mechanism is not well established. In cultured cardiomyocytes, ibrutinib can trigger abnormal action potentials and increases late sodium current, leading to enhanced automaticity. In cultured cardiomyocytes, ibrutinib triggers abnormal action potentials and increases late sodium current, ultimately leading to enhanced automaticity⁵⁵. The kinase responsible for these changes is unknown and may not be BTK, given that ibrutinib inhibits several other off-target kinases. Several randomized studies of ibrutinib versus control therapy consistently demonstrated a higher incidence of atrial fibrillation (AF) in ibrutinib-treated patients^{22, 23, 36}. In a pooled analysis of 1505 CLL and MCL patients enrolled in four large, randomized trials AF incidence was 6.5% for ibrutinib at 16.6-months versus 1.6% for comparator therapy, and 10.4% after 36-months. Ibrutinib treatment, prior history of AF and age over 65 years were independent risk factors for AF. Most patients with AF (86%) did not discontinue ibrutinib, and more than half received anticoagulant/antiplatelet medications. Low-grade bleeds were more frequent with ibrutinib, but serious bleeds were uncommon (ibrutinib, 2.9%; comparator, 2.0%). The authors concluded that AF among ibrutinib-treated patients is generally manageable without permanent ibrutinib discontinuation⁵⁶. Another meta-analysis estimated the increase in AF risk among ibrutinib-treated patients when compared with other treatments to be approximately 3.9 fold⁵⁷. Over a median follow-ups of up to 26 months, the pooled rate of AF among ibrutinib recipients was 3.3 per 100 person-years, compared to 0.84 per 100 person-years for comparator-treated patients.

Besides the risk for developing atrial fibrillation, ventricular arrhythmias (VA) and sudden cardiac death have been reported among patients treated with ibrutinib. Analyzing published clinical trial data with ibrutinib that enrolled approximately 1000 total patients, 10 cases of sudden death or cardiac arrest were noted⁵⁸. The weighted average of the incidence rates was 788 events per 100 000 person-years for ibrutinib-treated patients, compared to rates of sudden cardiac death for normal 65-year-olds are in the range of 200 to 400 events per 100 000 person-years. In a larger observational study in 582 patients treated with ibrutinib for hematologic malignancies (median follow-up: 32 months)11 patients developed symptomatic VA, of which 7 (1 sudden cardiac death/ventricular fibrillation, and 2 recurrent sustained ventricular tachycardia) had a probable association with ibrutinib. Compared with a reported idiopathic VA incidence among similar non–ibrutinib-treated subjects, the authors estimated that ibrutinib increases the relative risk of VA approximately 12 fold⁵⁹.

Second generation BTKi generally have a higher selectivity and therefore may have a lower incidence for arrhythmias, if those are off-target effects. Given the limited experience with these agents and relatively short follow-up, only randomized studies, especially those comparing newer BTKi with ibrutinib, will determine the potential benefit from these newer agents when it comes to cardiac arrhythmias.

Resistance to BTK inhibitors

Primary sensitivity or resistance to BTK mirrors the importance of BCR signaling and the respective kinases for growth and survival of the respective malignant B cells. For example, CLL cells are exquisitely sensitive to ibrutinib, and therefore primary resistance to ibrutinib only occurs in patients with CLL who have disease transformation, namely Richter transformation. On the other hand, patients with GCB-DLBCL almost uniformly lack responsiveness to ibrutinib⁶⁰, in line with this lymphoma subtype not being dependent on active BCR signaling. Secondary resistance to BTK inhibitors is best characterized in CLL, where it can manifest as Richter transformation during the first year of therapy^{18, 61}, or as progressive CLL, characteristically occurring at later stages in a relatively small fraction of high-risk patients with del(17p) and/or complex cytogenetic abnormalities^{15, 62}. CLL progression often coincides with an expansion of clones carrying BTK mutations at the ibrutinib binding site (C481S) or mutations in PLCG2 (R665W, L845F, S707Y), the gene encoding this BCR signaling-related molecule^{18, 61, 63}. While BTK mutations generally reduce binding and thereby the efficacy of the kinase inhibitor, activating PLCG2 mutations result in pathway activation that is independent from BTK⁶³. In addition, ibrutinib therapy can also promote the expansion of CLL sub-clones carrying del(8p), with additional driver mutations⁶⁴. Based on mathematical models⁶⁵ and verified with a highly sensitive droplet method for detection of single cells with somatic gene mutations⁶⁴, it is apparent that in some patients miniscule populations of resistant cells can be already present before therapy initiation, which then become selected and expand under therapeutic pressure.

Patients with WM generally have durable remissions on ibrutinib therapy, and, as in CLL, BTK C481S mutations emerge in those patients developing resistance⁶⁶. In contrast, development of resistance during therapy is more common in patients with MCL, where C481S BTK mutations can be associated with resistance, along with additional PI3K–AKT and CDK4 resistance pathway activity⁶⁷. Besides infrequent C481S BTK mutations, resistance to ibrutinib in MCL has been shown to arise from adaptive changes in the kinome usage in tumor cells; in particular enhanced PI3K–AKT signaling^{38, 67}. In part, adaptive changes appear to be facilitated by integrin β1 signaling and tumor microenvironment interactions³⁸. Another alternative mechanism of BCR-independent growth of malignant B cells was recently described in MYC-driven lymphoma cells, where ablation of tonic BCR signaling, in vitro and in vivo, resulted in adaptive changes in carbon metabolism, along with activating RAS mutations that restored the growth fitness of BCR-deleted cells⁶⁸. Similar activating RAS mutations also represent a survival mechanism in BL, where a subset of the tumor bulk lacks BCR expression⁶⁸.

Conclusions

BTK inhibitors, especially ibrutinib, have revolutionized the therapy of CLL, MCL and other B cell malignancies over the past few years, which is based on higher efficacy in patients with high-risk disease features, and better tolerability in elderly and frail patients when compared to CIT. BTK inhibitors target the B cell receptor (BCR) and its downstream signaling cascade, which are critical for normal and malignant B cell survival and proliferation. The approved BTK inhibitors (i.e. ibrutinib, acalabrutinib) generally lack myelotoxicity and induces long-lasting remissions in the majority of CLL patients, while remissions in MCL patients are less durable. Long-term treatment is warranted when used as a single agent, because remissions normally are not deep. Long-term use of BTKi, however, is expensive and can be complicated by the emergence of long-term toxicities and/or resistance. Second generation kinase inhibitors may have an improved side-effect profile that may facilitate long-term use, while combination therapy approaches induce deeper remissions which opens the door to limited-duration therapy. Clearly, the BTKi have already changed the life and outlook for many patients with CLL and MCL, especially those with high-risk CLL and relapsed/refractory MCL, for whom prior treatment options were particularly unsatisfactory. The challenge for the next years will be to transition this early success into practical and tolerable regimens that reduce the duration of exposure to BTKi and the associated risk of toxicity and emergence of resistance.

Figure 1: Tissue microenvironment activates signaling pathways that are targeted by BTK inhibitors.

CLL cells and other malignant B cells proliferate in the microenvironment of secondary lymphatic organs (SLO, i.e. lymph nodes, spleen). CLL cells (and presumably other malignant B cells, such as MCL cells) engage in a complex cross talk with non-malignant cells which constitute the microenvironment⁶⁹. Monocyte-derived nurselike cells (NLC), called lymphoma-associated macrophages (LAM), and T lymphocytes are important elements of the microenvironment. B cell receptor (BCR) activation and BCR downstream signaling signaling promote the proliferation and survival of CLL and other malignant B cells. BCR signaling is activated in SLO after engagement of the BCR with soluble or surface bound antigens (Ag). An alternative mechanism of BCR activation is through homotypic interactions between two BCR molecules (red star, left upper corner). Other molecules that contribute to survival and proliferation are B cell-activating factor (BAFF) and APRIL (also known as also known as TNFSF13), which are expressed by NLC and activate corresponding receptors on CLL cells (BAFF-receptor [BAFF-R], B cell maturation antigen [BCMA] and TACI, also known as TNFRSF13B). Activated T helper express CD154 (CD 40 ligand/CD40L) which can promote the growth of malignant B cells via CD40 engagement. NLC and other stromal cells secrete chemotactic factors (chemokines, such as CXCL12 and CXCL13) which attract and retain CLL cells within the tissue compartment, by engaging corresponding chemokine receptors (CXCR4, CXCR5) on CLL cells. Primary target of the BTKi is BCR signaling cascade. However, BTK also plays a role in signaling of other surface receptors, such as chemokine receptors (CXCR4, CXCR5) and adhesion molecules (integrins). Furthermore, BTK is not only expressed by B cells, but also by bystander cells in the microenvironment, such as in NLC and LAM. As such, BTKi induce complex changes, disrupting B cell-microenvironment interactions. Disrupted signaling and function of chemokine receptors and adhesion molecules explains the redistribution lymphocytosis that is characteristically seen in CLL patients treated with BTK inhibitors.

Figure 2: Improtance of BTK in the signaling of the BCR, chemokine receptors, and adhesion molecules (integrins).

Antigen binding by the B cell receptor (BCR) induces the formation of a signaling complex that is initiated by phosphorylation of immunoreceptor tyrosine-based activation motif (ITAM) residues on cytoplasmic tails of CD79A (Igα) and CD79B (Igβ). In turn, spleen tyrosine kinase (SYK) is activated, which is followed by Bruton tyrosine kinase (BTK), PI3K, and phospholipase Cγ2 (PLCγ2) activation. Further downstream responses include calcium (Ca^{2 +}) mobilization, activation of protein kinase C (PKC), and the ERK (MAPK) and nuclear factor-κB (NFκB) pathways. The signaling response, including downregulation of the transcriptional repressor BCL6, promotes activation of transcription in the nucleus. This signaling cascade is effectively disrupted by BTKi

Table 1:

Key clinical trial data for BTK inhibitors.

BTK inhibitor	Disease state	ORR	PFS	Survival	Side effects (any grade unless specified)
Ibrutinib (Imbruvica)	CLL, r/r	71% after 20.9 months15 94% after 44 months26	88% after 6 months22, 76% after 30 months26, del17p: 60%, del11q:82%26;	90% after 12 months22, 87% after 30 months26, for del17p: 81%, del11q: 88%26	Diarrhea (55%)25, Hypertension (22%)26, Pneumonia (19%)26, Neutropenia (16%)26, Grade 3 or 4 infections (11%)26, atrial fibrillation (3%)22, diarrhea (48%)22, any grade bleeding events (44%)22, major hemorrhage (1%)22
	CLL, treatment-naive	85% with 26% CR after 44 months follow-up26 86% with 4% CR at 18.4 months23	90% after 18 months23, 96% after 30 months26	98% at 24 months23, 96% at 30 months26	Diarrhea (68%)25, Hypertension (22%)26 Pneumonia (4%)26, Neutropenia (4%)26, Grade 3 or 4 infections (48%)26, atrial fibrillation (6%)23, major hemorrhage (4%)23
	MCL, r/r	ORR 68% (21% CR)35	Median PFS 13.9 months	58% at 18 months	Mild or moderate diarrhea, fatigue, and nausea. Infrequent cytopenias.
	Relapsed WM	ORR 90.5%, major response 73%70	69.1% after 2 years	95.2% after 2 years	Neutropenia, thrombocytopenia, bleeding atrial fibrillation
Ibrutinib+rituximab	WM	Major response in 72% with sustained Hb increases in 73%40	82% at 30 months	94% at 30 months	diarrhea, arthralgia, nausea, atrial fibrillation and hypertension
Ibrutinib plus venetoclax	CLL, treatment-naïve	96% CR/Cri after 18 cycles, 61% MRD-negative34	Not reported	Not reported	Easy bruising, arthralgia, and diarrhea, grade 3 or 4 neutropenia in 48%
Acalabrutinib (ACP-196)	CLL, r/r	95% after 14 months43	90% at 16 months43	Over 90% at 16 months43	Headache, diarrhea, arthralgia and pyrexia, atrial fibrillation (3%). Grade ¾ neutropenia (11%) and pneumonia (10%)71
	MCL, r/r	ORR 81% (40% CR)44	67% at 12 months	87% at 12 months	Headache, diarrhea, fatigue, myalgia, infrequent neutropenia, anemia, and pneumonia
GS-4059 (ONO-4059)	Mature B cell malignancies	CLL: 96% (LN response), MCL: 92%, DLBCL: 35% in non-GCB DLBCL48	CLL: 874 days, MCL: 341 days, DLBCL: 54 days48	Not reported	Anemia (32%), thrombocytopenia (18%), diarrhea (18%), petechiae (14%), bruising (12%)48
Zanubrutinib (BGB-3111)	Relapsed/refractory B-cell malignancies	96.2% ORR in CLL46	100% at 12 months	Not reported	Grade 3/4 AEs: neutropenia, anemia, pneumonia, hypertension, febrile neutropenia. Infrequent atrial fibrillation and major hemorrhage.