Resistance Mechanisms and Approach to Chronic Lymphocytic Leukemia after BTK Inhibitor Therapy

Abstract

Bruton tyrosine kinase (BTK), an essential component of the B-cell receptor (BCR)signaling pathway,is a validated target inchronic lymphocytic leukemia. Ibrutinib, acalabrutinib, and zanubrutinib are covalent BTK inhibitors (cBTKi) that bind toresidue C481, leading to sustained target inhibition. A significant proportion of patients develop resistancetocontinuous cBTKi therapy,predominantly viamutations in BTK and its immediate downstream effector,PLCG2.The noncovalent BTKi pirtobrutinib does not require binding to C481 and can restore BTK inhibition after progression on a cBTKi. However, non-C481 BTK mutations conferring resistance to pirtobrutinib have been identified. Furthermore, the scaffolding function of BTK, activation of bypass signaling pathways,and the tumor microenvironment maycontribute to BTKi resistance. Targeting BTK for degradation is an emerging strategy that appears effective against multipleBTK mutations,and inhibitors of downstream BCR signaling proteins are under development.This review addresses BTKi resistance mechanisms and therapeutic approachesafter cBTKi failure.

Introduction

Bruton tyrosine kinase (BTK) is a non-receptor tyrosine kinase that amplifiesupstream B-cell receptor (BCR) signaling and is essential for B-cell development and maturation(1, 2). In normal B cells, BTK activationleads to activation of nuclear factor kappa B (NF-κB) enabling increased B-cell survival and proliferation (Figure 1). BTK is also involved in toll-like receptor and integrin signaling, as well as chemokine-mediated cell migration(1). As a result, inhibition of BTK abrogatesBCR-driven survival and proliferation, and is an effective therapeutic strategyagainst several B-cell malignancies, particularly chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL)(3).

Figure 1:

Overview of the B-cell receptor (BCR) pathway with drug binding sites illustrated. Antigen binding by the BCR and/or ligand independent BCR signaling induces the formation of a signaling complex initiated by phosphorylation of tyrosine activation motif residues on the cytoplasmic tails of CD79A and CD79B. Subsequently, this recruits spleen tyrosine kinase (SYK), followed by activation of the B-cell linker protein (BLNK), followed by the activation of Bruton tyrosine kinase (BTK), phospholipase C gamma 2 (PLCγ2), and phosphoinositide 3’-kinase (PI3K).. Further downstream responses include activation of protein kinase C (PKC) as well as the nuclear factor-κB (NFκB) pro survival pathway. Ibrutinib, acalabrutinib, and zanubrutinib bind BTK covalently, whereas pirtobrutinib and nemtabrutinib bind to BTK non-covalently. There are several experimental inhibitors which target PKC as well as the MALT1-CARD11-BCL-10signalosome complex.

Ibrutinib is a first-in-class covalent BTK inhibitor (cBTKi) that received approval by the United States Food and Drug Administration (U.S. FDA) in 2014 for the treatment of CLL/SLL(4). Ibrutinib-based therapy results in durable remissions in most patients with CLL. Randomized phase 3 trials demonstrated that upfront treatment with ibrutinib ± rituximab prolonged progression-free survival (PFS) compared to chemoimmunotherapy(5, 6). Measurable residual disease(MRD)-guidedfixed-duration regimen of ibrutinib-venetoclax also improved PFS and overall survival (OS) when compared to chemoimmunotherapy(7). However, after 4 years of treatment with ibrutinib, up to 40% of patients eventually encounter treatment failure due to the development of resistance or intolerance (8). Several next-generation BTKis have been developed with the aim to reduce off-target toxicity and overcome acquired resistance. After ibrutinib, cBTKisacalabrutinib and zanubrutinib were approved by the U.S. FDA for CLL in 2019 and 2023 respectively, followed byaccelerated approval ofthe noncovalent (nc) BTKipirtobrutinib in 2023 for patients who have received ≥2 prior lines of therapy, including a BTKi and a B-cell lymphoma-2 inhibitor (BCL2i)(9–11).

Primary resistance,where a cBTKi fails to provide benefit, is uncommon and most often associated with Richter transformation(12, 13). Notably, some patients may take months before they meet iwCLL criteria for response, and this should not be confused with resistance(14).Secondary resistance is seen in patients who initially respond then progress on continued therapy. We distinguish between mechanisms of resistance that aretumor intrinsic due tomutations in signaling componentsor activation of bypass pathways, or extrinsic due to cytokine signaling or cell-cell interactionswithin the tumor microenvironment (TME)(15).

CLL cells have been shown to undergo significant evolution and genetic diversification after chemoimmunotherapy(16). Likewise, continuous BTKi therapy exerts a strong selective pressure, with resistant subclones often emerging after years of treatment(12, 17, 18). Ibrutinib-resistant CLL often harbors several subclones with BTK C481 and PLCG2mutations; different growth rates of these subclones aremost consistent with each mutation arising in an independent ancestor (12). Clonal evolution leading to BTKi resistance has implications for treatment of CLL. Duration of response with a BTKi is longer in settings where clonal complexity and evolutionary potential are lower, such as in treatment-naïve cases(12, 14, 17).

Genetic Mechanisms of BTKi Resistance in CLL

Ibrutinib, acalabrutinib, and zanubrutinib bind covalently to residue C481 of BTK, and thus are susceptible to mutations changing C481 to another amino acid, most commonly C481S(19–21).While mutations at C481 are the common mechanism of resistance to cBTKi, other mutations of BTK outside C481 may arise.There is a tendency for T747I mutations with acalabrutinib and L528W mutations with zanubrutinib among others (Figure 3)(20–22). Additional genetic mechanisms of resistance to BTKi in CLL include,PLCG2 mutations as well as chromosome 8p deletion (del [8p])(23). In published studies of disease progression during BTKi therapy,approximately 35% of patients were found to have aBTK mutation alone, an additional 10% had a coexisting mutations in PLCG2, 5% of cases had a PLCG2mutation alone, and the remaining 50% were wild-type (WT) for BTK and PLCG2 (Figure 2)(12, 19, 21–31). Because these mutations are not detected before treatment initiation,theyhave been labelled as acquired as a result of selective pressure during BTKi therapy.However, modeling based on the error rate of DNA replication, overall disease burden, and proliferative history of CLL suggests that these mutations are acquired during the natural history of the disease rather than after BTKi initiation(32). Further, there has been no indication that BTKi therapy, in contrast to PI3Kδ inhibition, increases genomic instability in B cells (33). Since the reported prevalence of mutations depends on assay sensitivity, it is likely that rare mutations at baseline will not be detected by most next-generation sequencing methods; consequently, mutations are likely under-reported in older studies utilizing less sensitive assays.Indeed, there has been a report ofa single patient in whom a PLCG2 M1141R mutation was detected in 0.0002% of pre-treatment cells using ultra-sensitive droplet-digital amplification technology(23). These observations are consistent with the hypothesis that these resistance mutations are pre-existingand expand under therapeutic pressure.

Figure 3:

Map of clinically documented BTK mutations associated with resistance to covalent Bruton tyrosine kinase inhibitor (cBTKi) (ibrutinib, acalabrutinib, zanubrutinib) and noncovalent Bruton tyrosine kinase inhibitor (ncBTKi) (pirtobrutinib). Size of circle corresponds to the number of patients identified with a specific variant. ‡: indicates 17 cases total for these 4 mutations, not further specified.

Figure 2:

Pie chart summary ofreported incidence of BTKand PLCG2 mutations in patients with progressive chronic lymphocytic leukemia (CLL) on ibrutinib.

BTK Mutations

Most BTK mutations are single base pair substitutions affecting C481(Figure 3) (12, 34).The first reported BTK mutation in CLL patients with progressive disease on ibrutinib (19, 35)was a missense mutation changing C481 to serine (C481S)(19).The C481S mutation disrupts the covalent binding of ibrutinib to BTK and results in a weak and reversible interaction (affinity [K_D], 0.2 nM vs. 10.4 nM) (19). Subsequently, additional missense mutations of C481 have been identified which make the BTK-ibrutinib bond reversible and unstable, including C481A, C481R, C481F, and C481Y, among others at C481 which are less common (12, 24, 36). In contrast to C481S, which retains the enzymatic activity of BTK, C481Y/R/F cause steric hindrance and structural clashing within the ATP binding pocket, preventing BTK Y223 autophosphorylation (34, 37).Mutations outside of C481 have been less commonly associated with treatment resistance in patients treated with a cBTKi (Figure 3) (38). BTKT474I/S have been termed “gatekeeper mutations” as they cause steric interference with binding for ibrutinib or acalabrutinib (39, 40). BTK T316A affect a binding residue for B-cell like linker protein, preventing activation of the downstream PLCγ2(28).In addition, several mutations in the BTK kinase domain such as L528W, M437R, and V416Ldemonstrate greatly reduced BTK enzymatic function(41).

BTKmutations may be classified as kinase proficient (e.g., T474I/S and C481S) or kinase impaired (e.g., M437R, V416L, C481Y/R/F, and L528W) (41–43). Downstream signaling is preserved in CLL cells with kinase impaired BTK mutations through novel protein interactions between mutant BTK and alternative kinases, as was demonstrated with BTK L528W (41).Invitro studies have shown thatcell lines transfected with both C481 and T474mutations are insensitive to exponentially higher pharmacological serum concentrations of ibrutinib, acalabrutinib, and zanubrutinib(40). However, co-occurrence of BTK mutations within the same cell has not been demonstrated in patient samples(12, 23).The variant allele frequency (VAF) of BTK mutations is highly variable between patients who relapse on a cBTKi.For example, in astudy of 15 patients treated with ibrutinib,the VAF of BTK C481S ranged from 0.02 to 0.78(12).Acquired ibrutinib resistance has also been observed in patients with Waldenstrom’s macroglobulinemia with BTK mutations at low VAFs. It was demonstrated that WM cells with BTK C481S confer protection against ibrutinib to neighboring BTK WT cells in vitro via a paracrine mechanism involving activation of extracellular signal–regulated kinase (ERK) 1/2(44).As progressive CLLhas been observed in patients withvery low VAF of BTK mutations, it raises the question of whether these mutations are underrepresented in peripheral blood testing, constitute actual drivers of the resistant phenotype or are just one ofcoexisting mechanisms of resistance(45).

PLCG2 Mutations

The second most common BTKi resistance mechanism described in patients with CLL is mutation ofPLCG2(26). PLCG2 is an effector phospholipase immediately downstream of BTK that connects BCR signaling to NF-kB activation. Mutations of PLCG have been observed to co-occur with BTK mutations in the same patient (12, 19, 24). PLCG2 S707Y, R665W, and L845F are gain-of-function missense mutations which prolong activation of BCR‐mediated downstream signaling, independent of BTK (19). Studies have shown that mutant PLCG2 protein can be activated via kinases LYN and SYK and also suggest functional dependency of PLCG2 on LYN and SYK (46). In this way, LYN and SYK, can contribute to activation of PLCG2 bypassing BTK.

Chromosome 8p Deletion

Del(8p) results in haploinsufficiency of tumor necrosis factor-related apoptosis-inducing ligand-R (TRAIL-R) protein, which binds to TRAIL and induces apoptosis, suggesting function as a tumor suppressor (47). CLL cells with del(8p) have been found to be refractory to TRAIL-induced apoptosis(47). In one longitudinal genomic study of 5 CLL patients who had disease progression on ibrutinib, del(8p) was observed in 3 patients withBTK and PLCG2 wild typeCLL(23). Since these patients achieved partial remission on ibrutinib before PD and del(8p) was present in pre-treatment samples, del(8p) alone is likely insufficient to cause resistance to ibrutinib therapy.

Tumor Microenvironment-Related Resistance to BTKi

Approximately 20% of CLL patients who develop ibrutinib resistance are not found to have any BTK or PLCG2 mutations, even when using highly sensitive testing, suggesting alternate mechanisms of resistance(38). The TME, comprised of various components such as stromal cells, cytokines, and growth factors, contributes to tumor cell survival and support.The non‐malignant cells and matrixcomponents which make up the TME have been shown to playa role in tumor cell survival, proliferation,andtreatment resistance (48). Integrins are adhesion molecules that function as anchors in retaining tumor cells in supportive tissues and facilitate tumor spread. Within the TME, the integrin very late antigen 4 (VLA-4) has a major role in B-cell development and function and isa master regulator of cell-cell and cell-matrix interactions via binding to vascular cell adhesion molecule-1 (VCAM-1) and fibronectin(49). VLA-4 is a heterodimer of CD49d (integrin α4) and CD29 (integrin β1).In normal B lymphocytes, inside-out signalingoriginating from the BCR activates VLA-4, an interplay which takes placein lymph nodes, spleen, and other secondary lymphoid organs during the process of antigen-specific B-cell differentiation(50). BCR stimulation may trigger a cascade of molecular events eventually leading to increased VLA-4 activity and prevents apoptosis of B-cells through tonic interactions with VCAM-1, the major ligand for VLA-4 (51).

CD49d is a strong prognostic factor in patients with CLL treated with ibrutinib or acalabrutinib(49).Positive or bimodal CD49d expression portends inferior PFS compared to cases entirely negative for CD49d(49, 52).Inibrutinib-treated patients, high CD49d expression (≥30% CD49+ cells) was correlated with reduced treatment-induced lymphocytosis and inferior nodal responses(53). Interestingly, BTK and/or PLCG2mutations are detected in most of these patients at time of progression(49). Bimodal CD49d cases, consisting of concurrent CD49d+ and CD49d− CLL subpopulations irrespective of the 30% cutoff value, have been found to demonstrate clinical behavior similar to CD49d+ CLL in the settings of treatment with chemoimmunotherapy and ibrutinib(52). Moreover, bimodal CD49d expression correlates with shorter PFS in ibrutinib and acalabrutinib-treated patients(49). In CD49d+ CLL cells, VLA-4 can be activated upon BCR engagement via a complex inside-out signaling mechanism, resulting in VLA-4 conformational change, enhancedCLL cell adhesion, and further interactions between the BCR and the TME(49). Treatment with BTKidoes not completely block this mechanism, leading to pro-survival and activation signals (49). Ongoing VLA-4 activation in CD49+ CLL has been proposed to decrease nodal response, redistribution lymphocytosis, and PFS compared to CD49− CLL treated with ibrutinib or acalabrutinib(49, 53).

Ways to Overcome BTKi Resistance

When patients treated with a BTKi develop progressive disease, a clinical trial should be considered as next line of treatment (Figure 4). Prior treatment status with venetoclax should also be factored into decisions. In patientswho have not previously received venetoclax, combination therapy with venetoclax and an anti-CD20 monoclonal antibody (mAb)can achieve durable remissions in most cases(54). Response to venetoclax re-treatment has been reported in patients who achieved a long treatment-free period after their initial venetoclax-based therapy, although shorter remissions are expected with re-exposure(55, 56). For double refractory patients, i.e. to BTKi and venetoclax, clinical trials are a good choice. Other available and FDA approved therapies could include ncBTKipirtobrutinib, CD19-directed, 4–1BB CAR-T product lisocabtagenemaraleucel (liso-cel),and PI3Kδ inhibitors duvelisib and idelalisib. The optimal sequencing of novel agents after BTKi failure is a matter of debate, and in need of further clinical investigation.

Figure 4:

Suggested management algorithm for patients with chronic lymphocytic leukemia (CLL) who develop resistance to a covalent Bruton tyrosine kinase inhibitor (BTKi).

BCL2 Inhibitors

Venetoclax is an oral, selective, small molecule inhibitor of the antiapoptotic protein BCL2. High levels of BCL2 protein are expressed in CLL, contributing to resistance to apoptosis; which is induced by venetoclax in CLL cells by a p53-independent mechanism(57). Venetoclax initiation and ramp-up require tumor lysis risk assessment and risk-stratified monitoring.Alone or in combination with anti-CD20 mAb or BTKi, venetoclax results in a high rate of durable response and undetectable minimal residual disease (uMRD)(58).

In a prospective multicenter phase 2 study, venetoclax monotherapy was administered to patients with CLL previously treated with cBTKi(59). The overall response rate (ORR) was 65% (complete response [CR] rate 9%) and similar among patients with and without high-risk genetic abnormalities (del[17p], del[11q], TP53 mutation, CD38, and ZAP-70; of note, there were incomplete data for IGHV mutational status and karyotype)(59). Of 21 patients evaluated for mutations, BTK or PLCG2mutations were present in 17 (81%), with VAF ranging widely from 1.2%to 98.8%(60). Twelve of 17 (71%) patients with BTK and/orPLCG2 mutations responded to venetoclax therapy, includingone CR.The median PFS of patients with known BTK and/orPLCG2mutations was 21.9 months (95% confidence interval [CI], 4.4 to not reached [NR])(59).

Refractoriness to cBTKi has been independently associated with a lower CR rate and shorter duration of response in multiple regression analyses of early-phase venetoclax studies (60). A retrospective, international, observational study assessed clinical outcomes of patients who received venetoclax after cBTKi therapy.A total of 184 patients discontinued cBTKi (83 [45.1%] due to intolerance and 78 [42.4%] due to progression)and initiated venetoclax-based therapy (115 venetoclax monotherapy and 69 venetoclax combined with rituximab or obinutuzumab)(61). Of 127 (69.0%)patients evaluable for response, the ORR was 78.0% (CR rate 43.3%) and the median PFS was 43.2 months (95% CI, 31.9 to NR).These results suggest that venetoclax is effective following cBTKi therapy particularly when used as second or third line treatment (61).

Venetoclax is the only BCL2 inhibitor currently approved by the U.S. FDA, but drug resistance driven, in part, byBCL2 mutations, has been reported(62).Sonrotoclaxis a second-generation BCL2 inhibitor under clinical investigation with activity againstBCL2 mutations, and superior potency and selectivity compared with venetoclax(63).

Noncovalent BTKi

One way of overcoming resistance mediated by BTK C481 mutations is the use of a reversible, ncBTKi, which does not require binding to the C481 site of BTK. Pirtobrutinib has been approved for CLL and several other ncBTKi have reached clinical development. Pirtobrutinib blocks the ATP binding interface of BTK, and is highly selective for BTK with >300 fold selectivity over 98% of other kinases (64). Consistent with its highly selective profile and reversible binding mechanism, pirtobrutinib has been observed to be well tolerated with lower rates of BTKi-associated toxicities such as atrial arrhythmias and hemorrhage(65). The BRUIN phase 1/2 study included 247 patients on pirtobrutinib who had previously been treated with a BTKi,84 (37.8%)of whom had a knownBTK C481mutation(66). The ORR was 73.3%and the median PFS was 19.6 months(95% CI, 16.9 to 22.1 months) (66).The median PFS was 17.5 months (95% CI, 13.8 to 19.6) among patients who discontinued cBTKi for disease progression compared to 28.4 months (95% CI, 22.1 to NR) among those who discontinued cBTKi for toxicity(66). A median PFS of 16.8 months (95% CI, 13.2 to 18.7) was observed in the cohort who had previously received both a BTKi and a BCL2i(66). The most common adverse events (AEs) were infections (71% of patients), bleeding (43%), and neutropenia (32%).Infections (28%) and neutropenia (27%) were the most common grade ≥3 AEs.(66).

Acquired resistance to pirtobrutinib has been observed. Several non-C481BTK mutations within the tyrosine kinase domain have been identified at relapse in patients treated with pirtobrutinib, includingBTKL528W, A428D, V416L, M437R, and T474I.Some mutations, such as A428D and L528W,conferred resistance to not only pirtobrutinib, but also cBTKi in in vitro assays(42).Proteomic studies of kinase-impaired BTK L528W have shown that BTK can act as a scaffold for other kinases to sustain downstream BCR signaling. Despite lacking BTK autophosphorylation, the downstream effects of BCR activation such as calcium release, ERK phosphorylation, and pro-survival signals were maintained in BTK L528W cells, suggesting compensation by other kinases such as hematopoietic cell kinase (HCK) and integrin-linked kinase (ILK)for the absence of BTK enzymatic activity(41, 67).

Another ncBTKi under clinical investigation, nemtabrutinib, has demonstrated clinical efficacy in relapsed CLL with and without C481S-mutant BTK, as well as inhibits multiple other kinases involved in BCR signaling, including LYN, SYK, and MEK1(68). A phase 1 first-in-human study of nemtabrutinib in patients with relapsed/refractory CLL reported an ORR of 75% at the recommended phase 2 dose(69).

Cellular Therapies

Allogeneic hematopoietic stem cell transplant (allo HSCT) is a potentially curative treatment for CLL. In a retrospective study of 65 patients who underwent alloHSCT following treatment with ≥1 novel agent such as BTKi, outcomes showed a 2-year PFS of 63%, a 2-year non-relapse mortality (NRM) of 13%, and an OS of 81% (70). Achievement of uMRD at 1 year after alloHSCT was associated with 10-year disease-free survival of 87% (71). However, 30–50% of patients experience serious complications such as graft-vs-host disease (GvHD), which is associated with mortality in 10–20% of patients(70, 71). AlloHSCT should be reserved for CLL patients who are medically fit, high-risk, heavily pre-treated, and lack other treatment options due to resistance or intolerance. The timing of allo HSCT should ideally be performed while CLL remains well-controlled. Pre-transplant disease status is a key determinant of outcomes after alloHSCT, with multiple studies consistently reporting improved PFS and lower relapse rates among patients in complete or partial response prior to transplant(72). Non-myeloablative or reduced-intensity conditioning is generally preferred over myeloablative conditioning to reduce the risk of GvHD and NRM after allo HSCT in CLL(72).

Chimeric antigen receptors (CARs) are engineered synthetic receptors that redirect lymphocytes (most commonly T cells) to recognize and eliminate cells expressing a specific target antigen. CARs binding to target antigens expressed on the cell surface, independent from the MHC receptor, resulting in vigorous T-cell activation and effective anti-tumor responses. Clinical trials suggest a role for CAR-T therapy in heavily pretreated CLL. In a phase 2 study, 24 patients with relapsed/refractory CLL previously treated with ibrutinib received anti-CD19 CART-cells (38). In 19 evaluable patients, the ORR was 74% (CR 21%), and 15 of 17 patients (88%) achieved uMRD in the bone marrow (73). There was significant treatment-related toxicity, with 20 patients (83%) developing cytokine release syndrome (CRS), and 8(33%) developing neurotoxicity, including a fatality(73). The results of this study suggest that CD19 CAR-T cells are effective with mostly manageable toxicity in patients with high-risk CLL, including those who are BTKi refractory. In along-term follow-up report of 2 CLL patients who remained in CR10 and 9 years after CAR-T infusion, flow cytometry detected CD4+ CAR T cellsexhibiting cytotoxic characteristics, ongoing functional activation, and proliferation (74).

Liso-cel is an autologous, CD19-directedCAR-Tcell product administered at equal target doses of CD8+ and CD4+ CAR+ T cells.In thephase 1/2 TRANSCEND CLL 004 study, 117 patients who had previous treatment failure on a BTKi received liso-cel(75). With a null hypothesis that the CR rate (including incomplete marrow recovery) was ≤5%, the study met its primary endpoint with a CR rate of 18% (p=0.0006).The secondary endpoint of ORR(43%) was not statistically significantcompared with the null hypothesis of ORR ≤40% (75). Median PFS was 11.9 months (95% CI, 5.7 to26.2), and the uMRD rate was 63% in blood and 59% in marrow(75).Remissions appear to be durable only among patients who achieve CR, and little is known about clonal stability and long-term persistence of the infused cells. One patient died from macrophage activation syndrome/hemophagocytic lymphohistiocytosis.Grade 3 CRS and neurological events were reported in 9% and 18% of patients,respectively, and were managed with tocilizumab and/or corticosteroid treatment (75). Based on theseresults, liso-cel was granted accelerated approval by the FDA for the treatment of CLL patients who have received ≥2 prior lines of treatment (76). Primary results from the phase 1/2 TRANSCEND CLL study for liso-cel combined with ibrutinib for relapsed/refractory disease demonstrated significant efficacy with deep remissions (ORR 86%, CR rate 45%, and blood uMRD rate 86%) and a manageable safety profile. While the authors urge interpretation should be made with caution due to differences in disease characteristics between cohorts, this represents a promising new therapeutic combination strategy.

PI3K Inhibitors

Idelalisib and duvelisib are oral phosphoinositide 3’-kinase inhibitors (PI3Ki) approved by the FDA for treatment of relapsed CLL. Among PI3K isoforms α, β, γ, and δ, PI3Kδ is the predominant isoform in BCR signal transduction,playing an important role in the survival and proliferation of CLL cells. Idelalisib, a specific PI3Kδ inhibitor, and duvelisib, aPI3Kγ and PI3Kδinhibitor, havedemonstrated activity in relapsed CLL (46, 77).

In a large multicenter, retrospective analysis of CLL patients treated with ibrutinib, idelalisib, or venetoclax, patients who discontinued ibrutinib due to progression or toxicity had improved outcomes if they received venetoclax (ORR 79%) versus idelalisib (ORR 46%) (78). Though idelalisib has shown promising initial clinical response, its therapeutic application has been limited by toxicities including enterocolitis, pneumonitis, and transaminitis; multiple lines of evidence indicate that the mechanism of idelalisib-induced hepatitis is autoimmune (79).

Safety concerns were raised by a U.S. FDA review of DUO, a global phase 3 randomized study of duvelisib versus ofatumumab monotherapy for patients with relapsed or refractory CLL/SLL who had received ≥2 prior lines of therapy excluding BTKi.There were more deaths in the duvelisib arm (50% [80/160])compared with the ofatumumab arm(44% [70/159]), and a higher rate of fatal adverse events in the duvelisib arm (15% [23/158])compared with the ofatumumab atm (3% [5/155]).Increased rates of serious AEs and grade ≥3 AEs were also reported with duvelisib (80). Prescribing information for duvelisib, which remains FDA-approved,carries a blackbox warning of treatment-related mortality and fatal and/or serious side effects.

Combination Therapies

Deepening response by achieving uMRD may help prevent the development of resistance. Studies demonstrate a consistent relationship between uMRD and PFS in patients treated with chemoimmunotherapy (81). Though BTKi monotherapy does not achieve high rates of uMRD, a promising option to overcome resistance to BTKi is a combination approach to deepen response. The phase 3 randomized trial ELEVATE-TN compared the efficacy of acalabrutinib with/without obinutuzumab to chlorambucil plus obinutuzumab in patients with treatment-naive CLL. At 6 years, the estimated PFS rates for acalabrutinib with/without obinutuzumab and chlorambucil plus obinutuzumab were 78%, 62%, and 17%, respectively. OS was significantly longer in patients treated with acalabrutinib-obinutuzumab versus chlorambucil-obinutuzumab (HR 0.62, p=0.0349), but was not significantly different between patients treated with acalabrutinib monotherapy and chlorambucil-obinutuzmab(82). Of note, the study was not powered to compare acalabrutinib versus acalabrutinib-obinutuzumab, and among patients receiving acalabrutinib-obinutuzumab, there were more frequent AEs of neutropenia, fatigue, and arthralgia compared to acalabrutinib alone.

The randomized phase 2 study CAPTIVATE evaluated combination therapy with fixed-duration ibrutinib plus venetoclax in previously untreated CLL patients (58). When venetoclax was combined with ibrutinib, the rate of uMRD improved from <10% to 55–75% in the frontline setting (58).Patients who achieved uMRD on this regimen showed a very favorable 30-month PFS of ≥95%.No mutations in BTK, PLCG2, or BCL2 have been detected so far, suggesting that fixed-duration combination therapy may mitigate the development of drug resistance (58, 83). In the GLOW study, fixed-duration ibrutinib plus venetoclax in previously untreated CLL patients with advanced age and/or comorbidities demonstrated superior PFS compared to chlorambucil plus obinutuzumab (74.6% [95% CI 65.0–82.0] vs. 24.8% [16.5–34.1], respectively, at 42 months) (84). The proportion of patients with sustained uMRD in peripheral blood from 3–12 months after end of treatment was 84.5% for the ibrutinib-venetoclax arm and 29.3% for the chlorambucil-obinutuzumab arm (85).

Two recent retrospective series reported the use of combined ibrutinib and venetoclax in patients refractory to BTKi and venetoclax (86, 87). In the first study, 22 patients had experienced disease progression on ibrutinib, but were venetoclax naïve, and 11 patients had received sequential ibrutinib and venetoclax, and were double-refractory. Treatment with combined ibrutinib and venetoclax was associated with median treatment-free and OS of 23.7 and 47.1 months respectively for venetoclax-naïve patients, and 11.2 and 27.0 months respectively for double-refractory patients (86). In the second study, 13 patients who previously received BTKi and venetoclax separately in any order were treated with cBTKi-venetoclax combination. Best response was partial response in 9 patients, stable disease in 2, and progressive disease in 2 (87). PFS at 1 year was 56.4% (95% CI, 24.4% to 79.3%) and OS was 70.0% (95% CI, 32.9% to 89.2%).

In the setting of BTKi resistance, even transient responses may be clinically relevant as a bridge to clinical trials or cellular therapies.

BTK Degrader

A targeted protein degrader is a chimeric small molecule that recruits target protein into the proximity of an E3 ubiquitin ligase, which then promotes polyubiquitylation of the target, triggering its degradation via the ubiquitin proteasome system (88, 89). In contrast to BTKi, BTK degraders eliminate both the enzymatic activity and scaffolding function of BTK (41). NX-2127 is an orally bioavailable, small molecule BTK degrader that is effective against both WT and mutant forms of BTK (41). Preliminary results of a phase 1, first-in-human clinical trial of NX-2127 in patients with CLL have demonstrated that >80% BTK degradation was achieved, and clinical responses were seen in 79% of evaluable patients (41, 90). Other BTK degraders in clinical development include NX-5948, BGB-16673 and ABBV-101, which have shown preliminary activity in ongoing studies(91–93).

Bispecific Antibodies

While several T-cell directed bispecific antibodies (bsAb) are FDA-approved for non-Hodgkin lymphoma, they are still under investigation for CLL. Preclinical work with anovel CD19/CD3 bsAbhas shown in vitro and in vivo(patient-derived xenograft) activity against CLL by recruiting autologous Tcells to form cytolytic synapses with the CD19+ CLL cells (94). Tcells obtained from patients on ibrutinib andtreated with CD19/CD3 bsAbin vitromounted faster cytotoxic responses than Tcells from ibrutinib-naïve patients (94). This differencewas also observed in T cells from acalabrutinib-treated CLL patients, suggesting that BTKi increases efficacy of CD19/CD3 bsAb irrespective of interleukin-2 inducible T-cell kinase (ITK) inhibition, an effect of ibrutinib but not acalabrutinib (95).EPCORE CLL-1 is an open-label, multicenter, phase 1b/2 trial evaluating the safety and efficacy of epcoritamab, a CD20/CD3 bsAb, in relapsed/refractory CLL(96). In a cohort of 23 patients, responses were seen early, with an ORR of 82%, CR of 33%, and 83% of responders remained in response at 9 months(96). Furthermore, with optimized step-up dosing of epcoritamab and corticosteroid prophylaxis,there were no CRS events greater than grade 2 in severity and no reports of immune effector cell-associated neurotoxicity syndrome (97).

Overall, bsAb represent a promising therapeutic strategy for relapsed/refractory CLL, and as a potential combination partner to deepen responses in patients on targeted therapy, especially in conjunction with BTKi and venetoclax (94, 95, 98).

Discussion

BTKi have dramatically changed the treatment landscape for CLL. However, a significant percentage of patients need to discontinue treatment with cBTKi due to toxicity or progression. Mutations in BTKand PLCG2 are the most common and best understood mechanism leading to BTKi resistance. Other mechanisms, including bypass mechanisms and clonal evolution, have been described, and may have implications for selection of subsequent treatment.

Therapeutic alternatives to overcome BTKi resistanceincludetargeted agents and cellular therapies. The introduction of venetoclax has improved PFS in cases of BTKi resistance, butremissions remain shorter compared to patients who are BTKi-naivePirtobrutinib has demonstrated clinical efficacy, and in clinical trialsappears to havelower rates of cardiac AEs,major bleeding episodes, bruising,and arthralgias compared to cBTKi.A head-to-head randomized trial is underway (NCT05254743). CAR-T may be an effective option for patients with high-risk CLL, but toxicity is considerable and durable remissions are only achieved in a small subset of patients. Novel drugs targeting BTK and immunotherapy are being developed for patients refractory to BTKi. BTK degraders utilizing the cellular proteasome to degrade BTK are currently under investigation and show early promise. Preliminary results from T-cell engaging bsAb suggest a role for harnessing the host immune system to overcome refractory CLL or possibly augment response with BTKi or venetoclax. The optimal sequencing of therapieshas not been systematically test, and should be tailored to the individual patient.