BTK Inhibitor Success Puts Focus on Resistance Mechanisms

Krithika Subramanian, PhD

Less than 5 years after Bruton tyrosine kinase (BTK) inhibition was introduced in hematologic malignancies, the need for strategies to address primary and secondary mechanisms of resistance has emerged.

The robust potency of ibrutinib (Imbruvica), which, in December 2013, became the first BTK inhibitor to gain FDA approval, has paved the way for second-generation agents with improved specificity profiles and expanded the potential for adding the drug to novel combination therapies. Ibrutinib use in a wider range of patients has also revealed the significance of resistance mechanisms and the need for options to manage ibrutinib-resistant cancers.

Targeting BTK in Cancer

Figure. BCR Signaling and Key Factors Involved in Ibrutinib Resistance

In October, the FDA approved acalabrutinib (Calquence), another BTK inhibitor, as second-line therapy in mantle cell lymphoma (MCL). Several other BTK-targeting drugs, including agents designed to address resistance mutations, are being investigated in clinical studies (Table).The identification of BTK as a target for anticancer therapy stems from its involvement in aberrant B-cell receptor (BCR) signaling, which plays a central role in B-cell malignancies. BCR signaling is initiated by antigen binding, resulting in receptor aggregation and subsequent phosphorylation of the cytoplasmic tyrosine-based activation motifs in BCR by the SRC family kinases SYK and LYN.This helps drive BTK to amplify and transmit BCR signaling, through phosphorylation of phospholipase C gamma 2 (PLC-gamma-2), mobilization of calcium secondary messenger, and activation of transcriptional programs driven by the nuclear factor к-B (NF—кB), AKT, RAS, mitogen-activated protein kinase, and nuclear factor of activated T cells pathways, ultimately promoting B-cell proliferation and survival (Figure).^1,2

BTK is a member of the highly conserved TEC kinase family.^3,4 BTK loss-of-function mutations result in X-linked agammaglobulinemia, characterized by absence of B cells, low serum immunoglobulin levels, and recurring infections, all consequences of impaired B-cell development. In addition to its role in BCR signaling, BTK is involved in chemokine-receptor, toll-like receptor, and Fc-receptor signaling in B cells.^1,5

Ibrutinib Enters the Picture

Table. The Landscape of BTK Inhibitors

The expression of BTK in B-cell malignancies and its pivotal role in the BCR signaling cascade, B-cell development, and lymphomagenesis mark BTK as a unique druggable target, providing a compelling rationale for use of BTK inhibitors in hematological malignancies.⁶Ibrutinib was initially approved for patients with MCL as a secondline monotherapy and has since gained indications in chronic lymphocytic leukemia (CLL)/small lymphocytic leukemia (SLL), and Waldenström macroglobulinemia (WM) as first-line therapy, as well as in marginal zone lymphoma (MZL) and chronic graft versus host disease after at least 1 prior therapy.⁷Ibrutinib inactivates BTK by binding covalently to cysteine 481 (C481) within the ATP-binding pocket in the kinase domain, acting as an irreversible inhibitor.⁸ Ibrutinib—BTK binding inhibits phosphorylation of BTK and its downstream targets and abrogates downstream BCR signaling. The potent activity of ibrutinib, first in MCL and subsequently in CLL, paired with the drugs relative tolerability underscores a paradigm shift in the treatment of aggressive B-cell malignancies.⁹

In CLL, ibrutinib has been a transformative therapy. The FDA approval of ibrutinib in CLL, for patients who had received at least 1 prior therapy and for previously untreated patients, was based on significantly improved overall survival (OS) compared with standard-of-care therapies.^10,11

In the first-line setting in CLL, the OS rate at 24 months among participants in the phase III RESONATE-2 study was 98% for patients treated with ibrutinib versus 85% among those treated with standard-of-care chlorambucil, which translated to an 84% reduction in the risk of death (HR, 0.16; 95% CI, 0.05-0.56; P = .001).¹⁰

In, improvements were noted in progression- free survival (PFS) and overall response rates (ORRs) in refractory/relapsed B-cell malignancies, including CLL and MCL.^12,13

In previously treated patients with WM, ibrutinib was found to be highly active (ORR, 90.5%) and was associated with durable responses.¹⁴ Ibrutinib induced durable responses (ORR, around 50%) with a favorable benefit—risk profile in patients with previously treated MZL.¹⁵

Adverse events (AEs) with ibrutinib in clinical trials have mostly been limited to grade 1 or 2 toxicities; however, ibrutinib is associated with high bleeding risk, with major bleeding events reported in around 3% of patients receiving the drug. Careful monitoring and concomitant anticoagulant and antiplatelet agents have been used to mitigate these AEs. Additionally, atrial fibrillation has been observed in up to 16% of patients.^16,17

Investigators hypothesize that ibrutinib’s toxicity profile stems from off-target inhibition of other kinases, including those of TEC, EGFR, and TXK.^18—20 Another notable characteristic in CLL with ibrutinib treatment is the development of lymphocytosis, because CLL cells mobilize from lymph nodes and the spleen. Although ibrutinib-associated lymphocytosis typically resolves within 8 months, it may persist past 12 months in some patients, concomitant with a continued response to ibrutinib.²¹

Two mechanisms account for ibrutinib’s clinical efficacy: inhibition of intrinsic B-cell signaling pathways that impede proliferation and survival and disruption of tumor—microenvironment interactions.

Mechanisms of Resistance

Primary Resistance

In CD40- or BCR-activated CLL cells, ibrutinib inhibits the ERK, PI3K, and NF-кB pathways, thereby suppressing cell survival.²² Ibrutinib also impedes migration of CLL cells toward chemokines and suppresses BCR-dependent chemokine secretion, suggesting that its clinical activity involves interfering with relocation and retention of malignant cells in their survival niches.^23,24In pivotal clinical trials across multiple indications, ibrutinib has demonstrated high response rates. These range from an ORR of 42.6% as a single agent in the phase III RESONATE study in patients with relapsed/refractory CLL/SLL to 82.7% in combination with bendamustine and rituximab (Rituxan) in the phase III HELIOS study in a similar patient population.²⁵

Despite the clinical efficacy of ibrutinib, primary and secondary resistance has been reported.^26,27 As a larger proportion of patients undergo treatment with this drug, the mechanisms of resistance and salvage or combination therapies for ibrutinib- resistant cancers have taken center stage in aggressive leukemia/lymphoma management. One-third of patients with MCL exhibit primary resistance to ibrutinib; moreover, further resistance develops with continued therapy, despite substantial response rates.¹²

Acquired Resistance

To date, bypass mutations in or sustained activation of downstream factors, rather than mutations in BTK, have been identified as drivers of primary resistance to ibrutinib.^28—30 For instance, sustained downstream PI3K/AKT activity, rather than BTK activity, correlated with resistance to ibrutinib in MCL.³¹ Gene-sequencing analysis of ibrutinibresistant MCL cells identified mutations in effectors of the BTK-independent nonclassical NF-кB pathway.³² A study evaluating ibrutinib resistance in ABC-subtype diffuse large B-cell lymphoma (DLBCL) found that aberrant activation of the NF-кB pathway, driven by mutations in myeloid differentiation primary response 88 (MYD88), was associated with primary ibrutinib resistance.³³Acquired mutations, arising after malignant clone persistence, have been described in secondary resistance to reversible kinase inhibitors such as BCR-ABL kinase—directed imatinib (Gleevec) and EGFR-directed erlotinib (Tarceva).^34,35 Recent studies highlight the importance of mutations in BTK and its downstream targets in secondary resistance to ibrutinib.

Two important classes of ibrutinib-resistance mutations have been characterized thus far: the BTK C481S mutation, which abolishes the covalent binding site for ibrutinib, and PLC-gamma-2 mutations.^36—39

Whole-exome sequencing of baseline and posttreatment relapse samples from 6 patients with CLL with acquired ibrutinib resistance identified the C481S mutation in BTK, and functional analysis showed that the mutant is reversibly inhibited by ibrutinib.³⁷ RNA sequencing analysis in a patient with relapsed/refractory CLL who failed previous lines of therapy and developed ibrutinib resistance identified the BTK C481S mutation as the driver of secondary ibrutinib resistance.³⁶

Three distinct PLC-gamma-2 mutations, S707Y, R665W, and L845F, were identified in 2 patients with CLL with acquired ibrutinib resistance. S707Y is a gain-of-function mutation that suppressed the auto-inhibitory effect of the SH2 domain in PLC-gamma-2, whereas R665W and L845F were shown to activate BTK-independent BCR-mediated signaling, bypassing ibrutinib’s inhibitory effect on BTK.³⁷ Interestingly, the BTK C481S and PLC-gamma-2 mutations were not detected prior to treatment or in patients with persistent lymphocytosis, although BTK and PLC-gamma-2 are inhibited in these patients.²¹ The first example of an activating mutation in BTK (L528W) that confers ibrutinib resistance in CLL and follicular lymphoma has also been described.^27,40

Clonal evolution is a central theme in B-cell neoplasms and studies have shown that relapses, particularly in aggressive B-cell lymphomas, are characterized by 2 patterns of clonal evolution: early divergence from a common progenitor and later divergence from a primary tumor.^41,42 A recent study provided evidence for clonal evolution during leukemia progression in ibrutinib-relapsed CLL, concomitant with a BTK mutation (T316A) in the SH2 domain, in addition to the BTK C481S mutation.^43,44 Notably, in patients with Richter transformation, the lymphoma cells were clonal descendants of circulating CLL cells that continued to undergo genetic drifts and evolution.⁴³

The frequency and significance of the mutations listed above have been described thus far in clinical trial settings and small studies, in real-world populations of ibrutinib-treated patients with relapsed disease or with continued/prolonged therapy, and have not been evaluated extensively.

Investigators at the San Raffaele Scientific Institute in Milan, Italy, are leading a multicenter international study that is addressing the prevalence of BTK/PLC-gamma-2 mutations in real-world populations of patients with CLL. Their preliminary analysis showed that about 50% of relapsing patients (10 of 22 evaluated so far) harbor the BTK C481S mutation, often associated with PLC-gamma-2 mutations. Notably, BTK mutations were also detected in 2 cases that were still responsive to ibrutinib. Longer follow-up would help elucidate whether the responsiveness will diminish or cease with continued therapy or will continue through unknown mechanisms.⁴⁵

In the Pipeline

High-sensitivity mutation testing in a recent phase II study of patients with CLL treated with single-agent ibrutinib showed that resistance mutations in BTK and PLC-gamma-2 predated disease progression by up to 15 months.⁴⁶ Although larger sample sizes and longer follow-ups will help illuminate resistance mechanisms, methods for tackling resistance can help improve ibrutinib use and patient outcomes.Clinical studies are actively exploring 2 immediate strategies to manage primary and secondary ibrutinib resistance: alternative BTK-independent targets within the BCR pathway and second-generation BTK inhibitors (Table).

Second-Generation BTK Inhibitors

The pathways that contribute to primary ibrutinib resistance, including the PI3K and alternative NF-кB (MCL), MYD88 (DLBCL), and CXCR (WM) pathways, can be targeted in ibrutinib-refractory patients to improve outcomes. In patients with secondary/acquired ibrutinib resistance, factors upstream of BTK and second-generation BTK inhibitors, including inhibitors of C481S-mutant BTK, may be useful targets.³⁹Acalabrutinib is a more potent and selective irreversible BTK inhibitor than ibrutinib, due to reduced off-target binding.⁴⁷ The FDA granted an accelerated approval for its use in adult patients with MCL who have received at least 1 prior therapy, not including a previous BTK inhibitor, based on the single-arm LY-004 trial, in which acalabrutinib monotherapy resulted in an 81% ORR by investigator assessment.⁴⁸

In the safety database of more than 600 patients that the FDA reviewed before approving the drug, the rate of grade 3 or higher bleeding events with acabrutinib monotherapy was 2% and the rate of atrial fibrillation and flutter of any grade was 3%.⁴⁸

Ongoing clinical studies are evaluating the safety and efficacy of acalabrutinib in hematological malignancies, including DLBCL, multiple myeloma, WM, and CLL.⁴⁷

Tirabrutinib (GS-4059), another potent and selective second-generation BTK inhibitor with lower affinity for other kinases, also inhibits autophosphorylation of BTK at Y223, thereby inhibiting downstream BCR signaling.⁶ Three clinical studies of tirabrutinib are underway in CLL and B-cell malignancies.

Zanubrutinib (BGB-3111) is a highly selective BTK inhibitor with higher bioavailability than ibrutinib; it effectively attenuates BCR signaling, resulting in growth inhibition and cell death in malignant B cells.^8,49 Ongoing clinical studies are evaluating zanubrutinib in B-cell malignancies, including phase III studies in CLL, SLL, and WM.

CG’806 is a noncovalent pan-FMS-like tyrosine kinase 3 /BTK multikinase inhibitor that impedes both wild-type (WT) and C481S-mutant BTK.^50,51 A recent study, presented at the 2018 European Hematology Association Congress, showed that CG’806 induced apoptosis in primary and cultured malignant B cells, inhibited both WT and mutant BTK with equivalent potency, and was well tolerated in murine xenograft models.⁵⁰

Alternative Targets in the BCR Pathway

For patients with BTK C481S mutation-induced ibrutinib resistance, agents that inhibit BTK via alternative mechanisms may also be options; 3 reversible BTK inhibitors—GDC-0853, ARQ 531, and vecabrutinib (SNS-062)—demonstrate preclinical efficacy and early-phase clinical studies are ongoing.^52—56Oncogenic pathways associated with BCR signaling, including PI3K-mTOR, NF-кB and alternative NF-кB pathways, are additional targets in ibrutinib-resistant disease. PI3K inhibitors such as idelalisib (Zydelig) and duvelisib, as well as mTOR inhibitors such as everolimus (Afinitor), have been proposed as rational choices for treating ibrutinib-resistant cancers. Multitargeting agents, such as heat shock protein 90 inhibitors and selinexor, an inhibitor of nuclear exportin, also are being explored. Some agents have been evaluated in clinical studies of ibrutinib-treated patients.^57—60

Findings for the BCL2 inhibitor venetoclax (Venclexta) have provided the most promising results to date, although data in an ibrutinib-treated population are limited. In June, the FDA granted a standard approval to venetoclax for patients with CLL or SLL, with or without 17p deletion, following at least 1 prior therapy. Most patients in the pivotal MURANO trial had received chemotherapy and/or anti-CD20 antibodies in previous lines of therapy; only about 2% of the nearly 400 participants in the study had taken unspecified BCR inhibitors.⁶¹

In a phase II study, single-agent venetoclax demonstrated an ORR of 65% in 91 patients with ibrutinib-refractory disease.⁵⁷ A real-world study of Ibrutinib-resistant CLL patients treated with idelalisib reported an ORR of 28%, with a median PFS of 8 months.⁵⁸

Future Considerations

Although preliminary data for duvelisib did not demonstrate significant efficacy in 1 study of ibrutinib-resistant CLL, the study did not include stratification based on BTK mutation status.⁶⁰The potential of this class of agents is a new and exciting area of research, especially in the treatment of aggressive blood cancers of B-cell origin. Although the BCR signaling pathway is the focus of BTK-targeted therapies, emerging data for the role of B cells in solid malignancies and the off-target effects of BTK inhibitors, especially ibrutinib, may help expand their use in hematological and solid malignancies.⁶² Clinical trials assessing antitumor activity in solid neoplasms are underway in ibrutinib and acalabrutinib.

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