Mechanisms of resistance to bruton’s tyrosine kinase inhibitors: synergistic effects of tumor microenvironment regulation and signaling pathways

Abstract

It is widely acknowledged that B-cell lymphoma represent a significant threat to human health, and Bruton Tyrosine Kinase inhibitors (BTKi) have been shown to exhibit superior clinical efficacy and safety in comparison to conventional chemotherapy and immunotherapy modalities. However, as patients continue to use BTKi over a time, they will inevitably encounter the drug resistance. This resistance renders the therapeutic efficacy of BTKi, thereby significantly constraining its clinical benefits. Drug resistance of tumor is a multifaceted process influenced by numerous factors, mainly including individual genetic variations, tumor stem cells, drug inactivation, reduced drug absorption, and altered metabolism of anti-tumor drugs. The tumor microenvironment (TME) has been demonstrated to exert an important influence on the process of therapy resistance. It is evident that non-cellular components (e.g. the extracellular matrix, hypoxia, an acidified microenvironment, exosome, and cytokines) modulate the drug resistance through different mechanisms. These mechanisms include physical barriers that impede drug delivery, the formation of an immunosuppressive microenvironment, metabolic reprogramming and the activation of bypass signal moueculars. Furthermore, the presence of mutations of moleculars involved in the BCR signaling pathways (e.g. BTK and PLCG2 mutations) and the aberrant activation of key pathways such as PI3K-AKT-mTOR, NF-κB, Wnt/β-catenin and MAPK/ERK signaling further weakened the efficacy of BTKi. This review focus on the mechanism of BTKi resistance, the role of the TME and its components in drug resistance. It emphasized that targeting TME remodeling and combined the inhibition of multiple pathways may provide a new strategy for overcoming drug resistance, optimizing the treatment paradigm of B-cell lymphoma.

Introduction

Bruton’s tyrosine kinase (BTK) is a pivotal enzyme in the B-cell receptor (BCR) signaling pathway, which regulates B-cell proliferation, differentiation, and survival. Abnormal activation of BTK has been identified as a critical driver in several B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL).The BTKi, such as ibrutinib, acalabrutinib, zanubrutinib, and orelabrutinib, has markedly improved the clinical outcomes of patients with these diseases, offering superior efficacy and tolerability compared with conventional chemotherapy and immunotherapy.

However, despite these therapeutic advances, the emergence of BTKi resistance has become a major clinical challenge. Many patients eventually experience disease progression, relapse, or transformation during long-term treatment. Genetic mutations—particularly BTK^C481S and PLCG2 gain-of-function mutations—have been recognized as the predominant mechanisms of acquired resistance, leading to reactivation of downstream BCR signaling and reduced drug sensitivity. Nonetheless, clinical and experimental evidence suggests that resistance cannot be fully explained by genetic alterations alone.

Despite several reviews describing genetic mechanisms of BTK inhibitor resistance, most have focused primarily on mutations within BTK or PLCG2. However, increasing evidence highlights that non-genetic factors—including metabolic reprogramming, tumor-microenvironmental signaling, and stromal protection—play essential complementary roles in determining therapeutic response. These multidimensional interactions between malignant B cells and their microenvironment have not yet been comprehensively reviewed.

The surrounding TME exerts profound influences on drug response through cell–cell interactions, cytokine secretion, and metabolic reprogramming, contributing to adaptive and reversible mechanisms of BTKi resistance. Previous reviews have primarily focused on genetic mutations and direct alterations in the BCR signaling cascade. In contrast, the role of the TME and its complex crosstalk with intracellular signaling pathways remains incompletely understood and under-reviewed. Accumulating studies have shown that components of the TME—such as tumor-associated macrophages, mesenchymal stem cells, endothelial cells, and extracellular matrix remodeling—can reshape signaling networks like PI3K-AKT-mTOR, NF-κB, and Wnt/β-catenin, thereby diminishing BTKi efficacy. In this review, we aim to comprehensively summarize the mechanisms by which the TME contributes to BTKi resistance, and to elucidate how these microenvironmental cues interact with intracellular signaling pathways. We also discuss the potential therapeutic implications of targeting TME remodeling and multi-pathway inhibition as synergistic strategies to overcome BTKi resistance in B-cell malignancies. This integrative perspective highlights an underexplored dimension of resistance biology and provides a conceptual framework for future translational research

The tumor microenvironment

TME is defined as the internal milieu in which neoplastic cells reside and evolve. It comprises a complex network of tumor cells, immune cells, and supporting cells, including but not limited to fibroblasts, stromal cells, and endothelial cells…. TME also encompasses the secreted products of these cells, such as cytokines and chemokines, in addition to non-cellular constituents found within the extracellular matrix (ECM). ECM is composed of non-cellular components…. The components of the specific TME are shown in Fig. 1. TME provides the space and conditions for tumor cells to survive and develop. In turn, these cells regulate and ‘transform’ the TME to ‘safeguard’ and ‘facilitate’ their malignant behaviors. In the subsequent section, an exploration will be conducted into the resistance mechanisms exhibited by the various components of TME in the context of lymphoma-based hematological diseases.

Fig. 1.

Components of the tumor microenvironment

The cellular components of the TME and its pro-resistance mechanism

Cancer-associated fibroblasts

Cancer-associated fibroblasts (CAFs) consist of multiple functionally distinct subtypes that exert pleiotropic and opposing functions in the TME. For instance, signals such as those involving the proteins CXCR4 and CXCR5 enable B cells to adhere to and persist within the bone marrow or lymph node stroma, thereby acquiring survival signals from the matrix [1]. This results in a reduction of exposure to BTK inhibitors, while concurrently providing survival signals. Consequently, this diminishes the efficacy of BTK inhibitors. Furthermore, upstream activation of CXCR4 and CXCR5 has been demonstrated to stimulate pathways such as PI3K–AKT and MAPK/ERK, or enhance integrin (VLA-4)-mediated adhesion [2]. This may result in the circumvention of BTK inhibition and the sustained proliferation/apoptosis resistance of the cells. Chemokines, such as CCL2, CCL3, CCL4, CCL5 and CXCR3, have been shown to recruit immunosuppressive cells, including TAMs, MDSCs and Tregs, or to modulate T-cell infiltration and function. This results in the creation of an immunosuppressive microenvironment, thereby diminishing the efficacy of immune-related therapies and promoting tumour survival [3]. This, in turn, indirectly facilitates the emergence of resistance. Chemokines have been demonstrated to enhance VLA-4 (CD49d) activity or its interaction with the matrix, thereby conferring ‘adhesion protection’ to tumour cells [4]. This results in the inhibition of BTKi-related cell migration/dissociation and a consequent diminution in drug efficacy. Finally, by recruiting or activating M2-TAMs, chemokines induce secretion of IL-6, CCL18 and others. These subsequently activate STAT3 and NF-κB, promoting anti-apoptotic gene expression (e.g., MCL-1) and enhancing tolerance to BTK inhibitors [5].

Tumor-associated macrophages (TAMs)

Tumour-associated macrophages (TAMs) represent a major subset of myeloid immune cells within the B-cell lymphoma microenvironment, exerting considerable influence on tumour growth, immune suppression, and drug response [6]. A plethora of experimental evidence has emerged that indicates the promotion of resistance to BTKIs by TAMs through multiple mechanisms, including the secretion of cytokines, the release of exosome, and immunomodulation.

TAMs have been observed to release cytokines, including IL-6, IL-10, and BAFF, which in turn activate STAT3 and NF-κB signalling within malignant B cells. The process in question has been shown to upregulate anti-apoptotic proteins, including BCL-2 and MCL-1, thereby counteracting BTKi-induced apoptosis [7]. The IL-6–STAT3 pathway has been identified as one of the most critical bypass survival signals following BTKi treatment.

Concurrently, BTKi therapy remodels macrophage polarisation states. Ibrutinib has been shown to inhibit BTK/ITK signalling, thereby altering the metabolic processes and chemotactic responses of macrophages. In addition, it has been demonstrated to promote the long-term conversion of these cells to the immunosuppressive M2 phenotype, thus creating an environment conducive to tumour survival [8]. M2-type TAMs have been shown to secrete transforming growth factor-β (TGF-β) and chemokine CCR18 in order to suppress T-cell activity. In addition, they have been observed to enhance B-cell adhesion-mediated resistance via the CXCR4-CXCL12 axis [9].

In addition, it has been demonstrated that TAM-derived exosome play a pivotal role in BTKi resistance development. The miR-155 they carry can be taken up by tumour cells, activating the PI3K-AKT pathway and promoting metabolic reprogramming, thereby enhancing drug resistance [10].

Collectively, TAMs and BTKi resistance exhibit bidirectional regulation: BTKi therapy has been demonstrated to influence macrophage polarisation and secretory profiles. In addition, TAMs have been shown to reinforce resistance through inflammatory signaling, metabolic support and immune suppression feedback. Interventions targeting TAM-associated signaling (e.g., IL-6/STAT3 inhibitors or CSF1R blockade) have been demonstrated to partially restore BTKi sensitivity in preclinical models [11].

T cell

Tumour-infiltrating T cells, most notably CD8 + cytotoxic T lymphocytes (CTLs) and CD4 + helper subsets, have been shown to play a pivotal regulatory role in determining therapeutic response and resistance to BTK inhibitors [12]. It is well-documented that persistent antigenic stimulation within the lymphoma microenvironment frequently induces T-cell exhaustion. This is manifested by increased expression of inhibitory receptors such as PD-1, TIM-3, and LAG-3, alongside diminished secretion of effector cytokines [13]. A number of studies have indicated that ibrutinib, by inhibiting BTK, partially reverses this exhaustion phenotype, thereby enhancing CD8 + T-cell proliferation and cytotoxic capacity in patients diagnosed with chronic lymphocytic leukaemia [14, 15]. The aforementioned effects may transiently enhance immune-mediated tumor control and delay disease progression. However, the TME may counteract these benefits through immunosuppressive remodeling [16].

The upregulation of PD-1 and PD-L1 expression, the recruitment of regulatory T cells, and the recruitment of inhibitory myeloid cells collectively diminish the capacity of effector T cells to eliminate malignant B cells. This, in turn, limits the durability of BTKi efficacy. Preliminary preclinical studies suggest that the combination of BTK inhibitors with PD-1 or PD-L1 blockade may restore T-cell function and achieve superior disease control. This observation lends support to the hypothesis that T-cell dysfunction contributes to adaptive resistance.

Furthermore, BTK inhibition has been demonstrated to alter cytokine secretion and intercellular communication within the lymphoid microenvironment. These alterations have been shown to impact the differentiation and persistence of both effector and memory T cell populations, thereby modulating the selective pressures exerted upon tumour clones [17]. Consequently, the composition and functional state of T cell populations constitute critical determinants of BTKi efficacy, and therapeutic strategies aimed at restoring T cell activity may help overcome or delay the development of resistance [18].

Mesenchymal stem cells

Mesenchymal stem cells (MSC) are a class of non-hematopoietic stem cells with multidirectional differentiation potential found in a wide range of tissues, with unique cytokine secretion that maintains the bone marrow microenvironment [19, 20].

MSC recruited into the lymphoma microenvironment promote BTK inhibitor resistance through multiple mechanisms, as supported by evidence from translational medicine. Cytokines secreted by MSCs, such as IL-6, activate the JAK/STAT signaling pathway (particularly STAT3) in malignant B cells. This promotes cell survival and reduces drug sensitivity, and the IL-6-mediated stromal protective effect has been validated in diffuse large B-cell lymphoma (DLBCL) models [21]. MSCs also produce chemokines such as CXCL12, which enhance cell adhesion by binding to the CXCR4 receptor on tumor cell surfaces. This enables retention within protective bone marrow or lymph node microenvironments. This adhesion-mediated resistance reduces effective BTKi exposure and functions in conjunction with matrix protection [21]. Several studies suggest that stromal/MSC support activates the PI3K-AKT-mTOR signaling pathway within tumors or stromal compartments, providing survival signals that bypass BTK inhibition. In preclinical models, inhibiting PI3K p110α/δ or downstream nodes overcomes stroma-mediated ibrutinib resistance [22]. Concurrently, matrix-driven focal adhesion kinase (FAK) signaling promotes NF-κB/AKT/MAPK activation and matrix remodeling. In MCL models, the combination of ibrutinib and FAK inhibitors exhibits synergistic effects, reversing matrix protection [23].

MSCs can also modulate the microenvironment via extracellular vesicles and angiogenesis/matrix factors (VEGF and MMPs). This reprograms tumor metabolism and impairs drug delivery, thereby diminishing BTKi efficacy [24].

Finally, clinical and translational analyses indicate that immune-exhausted or matrix-rich microenvironments are associated with a poor response to BTKIs, which supports the clinical relevance of MSC-mediated mechanisms and reveals therapeutic opportunities for combining BTKIs with drugs that disrupt matrix support [24].

Endothelial cells

Endothelial cells (ECs) form the inner layer of blood vessels, and tumor-derived endothelial cells (TECs) can arise directly from the differentiation of tumor cells.

TECs are key active determinants of drug response within the lymphoma microenvironment, directly or indirectly facilitating resistance to BTK inhibitors. Firstly, abnormalities in tumor vascular structure and function lead to uneven perfusion, elevated interstitial fluid pressure, and the formation of hypoxic zones. These factors both impair effective drug delivery and promote the initiation of adaptive resistance mechanisms in malignant cells; Currently, perfusion impairment and hypoxia-induced signaling are recognised as key mediators of therapeutic failure in tumors [25].

Secondly, TECs frequently acquire intrinsic stem-like properties (such as elevated aldehyde dehydrogenase activity and upregulation of MDR1/P-gp and other drug efflux transporters), conferring intrinsic tolerance to cytotoxic drugs upon the endothelial tissue itself while maintaining a microenvironment that shields tumor cells from therapeutic agents [26].

Moreover, TECs serve as major paracrine sources of pro-survival and vasoendocrine factors (VEGF, FGF, and other cytokines). These factors activate PI3K-AKT, MAPK, and NF-κB signaling pathways in adjacent tumor cells, providing alternative survival signals that reduce malignant B-cell dependence on BTK signaling [27].

Finally, TECs and associated stromal cells exchange extracellular vesicles and microRNAs with tumor cells; tumor-derived EVs can also reprogram ECs to develop drug-resistant phenotypes, establishing bidirectional cross-regulation that promotes drug tolerance [28].

BTK inhibitors themselves modulate endothelial biology; preclinical studies indicate that drugs such as ibrutinib can affect endothelial cell adhesion capacity, angiogenic potential, and blood-tumor barrier properties. These alterations may modify intratumoural pharmacokinetics and the immune microenvironment, thereby influencing the in vivo efficacy of BTK inhibitors [29].

Noncellular components of the TME and its pro-resistance mechanisms

Extracellular matrix (ECM)

In TME, the ECM is a non-cellular component that provides biochemical constituents and basic structural support for tumor cells.

ECM is a highly dynamic and biologically active component within the tumour microenvironment, exerting a critical influence on lymphoma progression and therapeutic response. Beyond functioning as a structural scaffold, the ECM delivers biochemical and mechanical signals that directly impact the efficacy of BTKi. Excessive ECM deposition and cross-linking by stromal fibroblasts and MSCs increase tissue stiffness and interstitial pressure, thereby impeding drug diffusion and reducing BTKi penetration within tumours [30]. Furthermore, the remodelling enzymes of the ECM, such as matrix metalloproteinases (MMP-2, MMP-9) and lysyl oxidases (LOX), promote fibrosis and hypoxic microenvironments, thereby activating adaptive signaling cascades within lymphoma cells [31].

The core mechanism by which ECM regulates BTK inhibitor resistance lies in cell adhesion-mediated resistance (CAM-DR). Integralins (particularly VLA-4/α4β1 and α5β1) bind to fibronectin, collagen, and laminin, activating downstream FAK, PI3K-AKT, and NF-κB signalling pathways. These pathways have been shown to sustain tumour cell survival and proliferation independently of BTK activity. In preclinical models of MCL, pharmacological inhibition of FAK or PI3K significantly restored ibrutinib sensitivity, thereby revealing that adhesion-triggered bypass pathways constitute a primary cause of microenvironment-driven resistance. Furthermore, ECM-derived stiffness has been shown to enhance the synergistic interaction between BCR and integrin signaling by activating Yap/Taz-mediated mechanotransduction, thereby promoting sustained B-cell survival under BTKi treatment [32].

Matrix components also influence angiogenesis and immune evasion. Hyaluronan, fibronectin and collagen fragments stimulate endothelial and myeloid cells to release IL-6, CXCL12 and VEGF, thereby intensifying matrix-vascular interactions and forming a protective microenvironment shield. Furthermore, matrix components have been demonstrated to influence angiogenesis and immune evasion. Hyaluronan, fibronectin, and collagen fragments have been shown to stimulate endothelial and myeloid cells to release IL-6, CXC chemokine ligand 12 (CXCL12), and vascular endothelial growth factor (VEGF). This, in turn, intensifies matrix-vascular interactions and establishes a protective microenvironment that shields lymphoma cells from BTK inhibition [33]. Concurrently, the presence of ECM degradation products has been observed to trigger the recruitment of macrophages and regulatory T cells. This process has been shown to perpetuate an immunosuppressive environment, thereby indirectly fostering the development of adaptive resistance [34]. These findings indicate that the composition, remodeling, and biomechanical properties of the ECM play a critical role in regulating BTKi efficacy by altering drug accessibility and activating integrin-dependent survival networks. Intervening in ECM-associated signaling through inhibitors targeting FAK, LOX, or ECM-derived growth factors represents a potential strategy to overcome microenvironment-mediated BTKi resistance in B-cell malignancies [35].

Laminin

The metabolic reprogramming of lipids has been identified as a significant hallmark of BTKi resistance in B-cell malignancies. Tumour cells within the lymphoma microenvironment exhibit enhanced cholesterol synthesis and uptake, increased fatty acid oxidation (FAO), and remodelled membrane lipid composition. These alterations collectively impact the efficacy of BTKi and tumour survival [36, 37].It has been demonstrated that dysregulated lipid metabolism supplies energy and biosynthetic precursors. In addition, it has been shown to compensate for BTK blockade under therapeutic pressure by modulating key survival pathways such as PI3K-AKT and NF-κB [38].

Firstly, it is imperative to acknowledge the pivotal function of cholesterol metabolism in the context of adaptive resistance. The investigation established that BTK-inhibited cells enhance endogenous cholesterol biosynthesis by upregulating sterol regulatory element-binding proteins (SREBPs) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). The validity of this process was confirmed in models of CLL and DLBCL. Even in the presence of BTK inhibition, this mechanism maintains the integrity of lipid rafts and facilitates sustained BCR signaling. Furthermore, BTKi-treated cells exhibit increased expression of scavenger receptor B1 (SR-B1) and low-density lipoprotein receptor (LDLR), promoting exogenous cholesterol uptake and reinforcing the lipid raft-dependent signalling platform. Inhibition of HMG-CoR or SREBP has been demonstrated to enhance the sensitivity of drug-resistant cells to ibrutinib, thereby underscoring the significance of cholesterol metabolism as a druggable determinant of BTKi efficacy.

Secondly, the process of FAO has been demonstrated to promote metabolic flexibility and drug tolerance. In the context of BTK blockade, lymphoma cells have been observed to engage in the sustained production of ATP and NADPH, a process that is instrumental in counteracting oxidative stress and preserving their proliferation. This augmentation in the production of ATP and NADPH is facilitated by the enhancement of FAO, a process that is mediated by carnitine palmitoyltransferase 1 A (CPT1A) and mitochondrial oxidative phosphorylation. The pharmacological or genetic inhibition of FAO has been shown to significantly restore BTKi sensitivity in preclinical models. This finding indicates the existence of metabolic bypass mechanisms downstream of BTK inhibition.

Thirdly, tumour-associated lipoproteins and matrix lipid exchange similarly influence BTKi resistance. The internalisation of lipid-rich apolipoproteins (ApoE, ApoC1) and extracellular vesicles secreted by stromal and ECs by lymphoma cells has been demonstrated to enhance membrane fluidity and activate pro-survival signaling. A study of a cohort of patients with chronic lymphocytic leukaemia revealed an elevated level of ApoE to be associated with a poor response to BTKi therapy and a reduced overall survival rate. This finding serves to confirm the existence of paracrine lipid transfer mechanisms.

Furthermore, the accumulation of lipid droplets within tumour cells forms a neutral lipid reservoir that buffers oxidative stress while stabilising BTK-independent signaling complexes. This process serves to exacerbate therapeutic resistance.

Finally, BTKi exposure itself remodels lipid metabolism: prolonged ibrutinib treatment alters SREBP2, AMPK, and PPARα activity in B cells, inducing adaptive reorganisation of lipid biosynthesis and FAO networks.The collective analysis of these data indicates that lipid metabolic plasticity is a pivotal factor in the determination of BTKi resistance. The targeting of cholesterol synthesis (statins), FAO (CPT1 inhibitors), or apolipoprotein-mediated lipid exchange has been identified as a promising strategy to overcome metabolic adaptation in BTKi-refractory B-cell lymphomas.

Hypoxia

Hypoxia is a prevalent feature of the microenvironments of solid tumors and haematological malignancies, and the adaptive programmes it instigates are closely associated with therapeutic resistance. Hypoxic stress has been demonstrated to stabilise hypoxia-inducible factors (HIFs), with a particular emphasis on HIF-1α. The role of HIF-1α in this process is significant, as it is known to promote angiogenesis, metabolic reprogramming (enhancing glycolysis and lactate production), induce autophagy, and upregulate pro-survival factors through the reorganisation of transcription networks. Collectively, these alterations enable tumour cells to withstand inhibitory stresses, including targeted therapeutics.

First, HIF-1α transcriptionally upregulates genes involved in drug efflux (multiple ABC transporters) and detoxification, reducing intracelular accumulation of therapeutics; hypoxia-driven ABC transporter expression has been linked to multidrug resistance across cancer types [38].

Secondly, the phenomenon of hypoxia-induced metabolic switching (Warburg-like glycolysis and enhanced FAO under specific conditions) has been shown to alleviate drug-induced oxidative stress by supplying ATP and reducing equivalents, whilst activating survival pathways (PI3K-AKT, NF-κB) that circumvent BTK inhibition [40].

Thirdly, it has been demonstrated that hypoxia induces autophagy and upregulates pro-survival BCL-2 family members (such as MCL-1). The correlation of both processes with targeted therapy resistance has been demonstrated, and it is hypothesised that they may attenuate the apoptotic effects downstream of BTK blockade [40].

Furthermore, it has been demonstrated that hypoxia can indirectly promote resistance by remodeling the microenvironment. The process of hypoxia-driven lactate accumulation has been shown to induce acidosis, which in turn has been demonstrated to reduce cellular uptake efficiency of weakly basic drugs and to alter drug distribution patterns. This physicochemical mechanism has been shown to diminish the effective concentration of BTKi at the tumor cell membrane [40].

Furthermore, the presence of hypoxia gives rise to an immunosuppressive microenvironment by means of the recruitment of regulatory myeloid/lymphoid cell populations and the upregulation of immune checkpoints. This, in turn, undermines immune-mediated tumour control mechanisms that could otherwise synergise with the action of BTKi [41].

Preclinical studies and translational analyses further demonstrate that the targeting of hypoxia-driven nodes can result in the restoration of drug sensitivity. A body of research has demonstrated the potential for HIF inhibitors, tumour perfusion normalisation approaches, blocking hypoxia-induced autophagy, and metabolic/efflux pathway inhibitors to demonstrate synergistic effects with targeted therapies across multiple tumour models [39].

Acidified microenvironments

The acidic tumour microenvironment is a hallmark of malignant B-cell lymphoma, exerting a pivotal influence on treatment response and drug tolerance. The process of acidification is primarily driven by the enhanced glycolysis and subsequent accumulation of lactic acid within the tumour cells. This process is subject to regulation by various oncogenic signalling pathways, including PI3K–AKT–mTOR and HIF-1α, which are influenced by hypoxia [42]. In the context of BTK inhibitor (BTKi) therapy, tumour cells undergo further activation of glycolysis, a process that serves to sustain their energy supply. This, in turn, has the effect of exacerbating tumour microenvironment acidification, thereby establishing a metabolic niche that is conducive to cell survival.

Firstly, the acidic microenvironment exerts a direct influence on the distribution and uptake of drugs. The majority of BTKIs (e.g., ibrutinib, acalabrutinib) are inherently weak basic small molecules that undergo facile protonation under low pH conditions, thereby reducing transmembrane permeability and diminishing intracelular drug concentrations [43]. This physicochemical mechanism directly impairs effective drug exposure within tumour regions.

Secondly, the acidified environment activates multiple pH-sensitive signalling pathways. In the presence of an acidic environment, the NF-κB and ERK1/2 pathways become persistently activated, inducing the expression of pro-survival cytokines such as IL-6 and IL-8. This maintains cellular growth signals despite BTK inhibition. Furthermore, acidification has been demonstrated to enhance integrin-FAK signalling and the expression of matrix metalloproteinases (MMP-2/9), thereby promoting CAM-DR.

Furthermore, the accumulation of lactic acid and the subsequent acidification of the environment have been demonstrated to exert a significant suppressive effect on the immune system. Elevated lactic acid concentrations have been demonstrated to inhibit the function of cytotoxic T cells and natural killer (NK) cells, whilst inducing TAMs to differentiate towards the M2 phenotype. This has the effect of impairing the synergistic antitumour effect between BTK inhibitors and the innate immune response.

Finally, the targeting of acidified metabolism has been demonstrated to be an effective strategy for reversing drug resistance. Inhibition of lactate transporters (MCT1/4) or lactate dehydrogenase A (LDHA) has been demonstrated to reduce lactate efflux, thereby restoring extracellular pH equilibrium and enhancing sensitivity to BTK inhibitors. In summary, the acidified microenvironment has been demonstrated to promote BTK inhibitor resistance through multiple mechanisms, including impaired drug penetration, activation of alternative signaling pathways, and induction of immune suppression. This finding suggests that metabolic pH regulation may represent a novel strategy for overcoming drug resistance.

Exosomes and cytokines

Exosomes and other extracellular vesicles (EVs) have been shown to play a pivotal signaling role within the tumour microenvironment, and are central to the development of BTKi resistance. These vesicles have been found to facilitate the transportation of proteins, lipids, mRNA, microRNA, and long non-coding RNA, thereby regulating signal transduction, metabolic states, and immune responses in recipient cells via intercellular transport [44].

Firstly, it is important to note that exosome derived from resistant cells have the capacity to transfer active kinase signals. Exosomes released by BTKi-resistant lymphoma cells have been found to be enriched with phosphorylated SYK, PI3K, and NF-κB signaling proteins. These molecules have the capacity to be absorbed by sensitive cells, which in turn activates downstream survival pathways. This process is known as ‘horizontal transmission’ and is a key mechanism through which resistance is propagated.

Secondly, extracellular miRNAs have been demonstrated to play a pivotal role in the regulation of resistance. Extracellular microRNAs (miRs) such as miR-155, miR-21 and miR-222 have been shown to be significantly increased following exposure to the BTK inhibitor. These microRNAs have been shown to enhance AKT pathway activity by suppressing negative regulators such as PTEN and SHIP1, thereby attenuating drug-induced apoptosis. Concurrently, matrix-derived exosome populations characterised by elevated levels of miR-146a and miR-451 have been observed to induce sustained activation of NF-κB and JAK/STAT signaling pathways, thereby further consolidating the drug-resistant microenvironment.

Thirdly, it has been demonstrated that exosome administration can promote drug efflux and metabolic tolerance. It has been demonstrated that these cells are capable of transporting ATP-binding cassette transporters (ABCG2, ABCC1) and metabolic enzymes, thereby enhancing the drug clearance capacity and antioxidant defence levels of recipient cells.

Fourthly, it has been demonstrated that the cells in question are capable of immune evasion. Exosomes containing PD-L1 or TGF-β have been shown to suppress effector T-cell activity, thereby diminishing the synergistic effect between the immune system and BTKi therapy [45].

Consequently, the inhibition of exosome generation or release has been shown to effectively restore drug sensitivity. Inhibition of sphingomyelinase 2 (nSMase2) via GW4869 or disruption of Rab27a-dependent secretory pathways has been demonstrated to reduce exosome release, thereby restoring BTKi efficacy in both in vitro and in vivo models. Consequently, it is evident that exosome function extends beyond their role as crucial mediators of drug resistance signaling, thus establishing them as a significant target for the development of future combination therapies.

Key signaling pathways and drug resistance mechanisms

Direct escape from the BCR signaling pathway

BTK is comprised of five structural domains, namely the BTK homology (PH) structural domain, the proline-rich TEC homology (TH) structural domain, the SRC homology (SH) structural domains (designated SH3 and SH2), and finally, the catalytic structural domain (illustrated). Its structure is shown in Fig. 2.

Fig. 2.

The structure of BTK

Acquired BTK mutations have been identified as a prevalent mechanism of resistance to BTKi in CLL patients. Ibrutinib exerts its inhibitory effect on BTK by means of an irreversible binding process to residue C481 within the ATP-binding pocket of BTK [46]. However, of the BTK mutations that have been acquired, BTK C481S is the most prevalent, accounting for over 90% of BTK mutations observed in patients treated with ibrutinib. This includes C481S, C481R, C481Y, C481F, T474I, T474S, L528W, and T316A, which disrupt ibrutinib binding and lead to ibrutinib resistance [47–49]. Furthermore, patients diagnosed with CLL who carry BTK mutations demonstrate resistance to second-generation BTK inhibitors through a mechanism analogous to that of ibrutinib. For instance, patients who experienced disease progression following acalabrutinib treatment exhibited BTK C481S, C481R, C481Y, and T474I mutations [50]. Woyach et al. ascertained that the prevalence of BTK mutations in patients with CLL who demonstrated disease progression during acalabrutinib or ibrutinib treatment was 66% and 37%, respectively, with the BTK C481S mutation being the most prevalent (93.5% and 90.9%, respectively) [51]. It is noteworthy that the T474I gate-keeping mutation was observed in 29% of patients who were resistant to Acalabrutinib, either in isolation or in conjunction with the C481S mutation. Patients demonstrating resistance to zanubrutinib, treatment exhibited BTK C481S, C481R and L528W mutations [52]. Conversely, patients treated with orelabrutinib exhibit BTK C481S, C481R, T474I, and L528W mutations. The presence of mutations within the BTK kinase domain, specifically at T474 and L528, has been demonstrated to result in resistance to non-covalent BTK inhibitors [47, 53]. Furthermore, other BTK mutations (V416L, A428D, M437R and L528W) have been shown to impair binding and BTK autophosphorylation of the third-generation BTK inhibitor pirtobrutinib in patients with CLL, thereby inducing drug resistance [47].

Mutations in PLCG2, a substrate of BTK in the BCR signaling pathway, represent the second most common mechanism of resistance to BTK inhibitors, occurring in 11% of ibrutinib-resistant cases, with specific mutations detected in R665W, L845F, S707Y, S707P, S707F, R742P, and D1140G. Furthermore, mutations in CLL patients resistant to acalabrutinib, zanubrutinib, and pirtobrutinib were also observed in patients with resistant CLL [47, 50, 54]. Mutations in BTK, PLCG2 and CARD11 have been identified as a factor in the development of BTKi resistance in patients diagnosed with MCL, through a mechanism analogous to that observed in patients with CLL [55, 56].

The PI3K-AKT-mTOR pathway

In ibrutinib-resistant CLL cells, the activation of VLA-4 via a BTK-independent, PI3K-mediated pathway impedes the capacity of ibrutinib to mobilize CLL cells from the protective lymphatic microenvironment to the peripheral blood. This enhanced VLA-4 activation strengthens the interaction of CLL cells with the microenvironment, allowing these cells to receive signals that promote survival against ibrutinib-induced apoptosis [57, 58].

In a manner analogous to that observed in CLL, stromal cell-mediated ibrutinib resistance has been demonstrated to arise from a direct interaction between stromal cells and MCL cells, involving the integrin VLA-4. Moreover, stromal cells have been demonstrated to promote MCL cell regrowth by attenuating the down-regulation of the PI3K-AKT pathway subsequent to ibrutinib withdrawal. The PI3K-α inhibitor BYL719 has been shown to potentiate ibrutinib-mediated down-regulation of pAKT levels, thereby overcoming stromal cell-mediated ibrutinib resistance [22].

ABC-DLBCL cells that carry a mutant form of MYD88 but retain a wild-type copy of CD79B are dependent on the MYD88-mediated NF-κB pathway for survival, rather than the BCR-mediated NF-κB pathway. This results in the ineffectiveness of ibrutinib [59, 60]. However, in ABC DLBCL cells carrying both MYD88 and CD79B mutations, a complex structure known as the My-T-BCR multiprotein supercomplex was assembled, consisting of MYD88, TLR9 and BCR. It was demonstrated that ibrutinib was capable of disrupting the formation of supercomplexes and blocking pro-survival signaling. Furthermore, a subset of ABC DLBCL patients who demonstrate unresponsiveness to ibrutinib have been identified as carrying activating mutations in CARD11 or inactivating mutations in tumor necrosis factor-α-inducible protein 3 (TNFAIP3), which functions as a classical inhibitor of the NF-κB pathway. The presence of mutations in either CARD11 or TNFAIP3 results in over-activation of NF-κB signaling, thereby facilitating ABC DLBCL cell survival and resistance to ibrutinib [59]. The Kelch-like protein 14 (KLHL14) gene is involved in endoplasmic reticulum (ER)-associated protein degradation and is considered to be a tumor suppressor.

NF-κB pathway

In the non-classical NF-κB signaling pathway, p100 is processed into p52, which forms a dimer with RelB and enters the nucleus to regulate gene transcription. Mutations in molecules associated with the NF-κB signaling pathway trigger the initiation of both the classical and the non-classical NF-κB pathways, which promotes cell survival and leads to resistance to BTK inhibitors.

Mutations in NFKBIE and BIRC3 were identified in patients with CLL who experienced disease relapse following treatment with ibrutinib [61]. NFKBIE mutations have been demonstrated to reduce the level of IκBε protein (encoded by the NFKBIE gene) and increase nuclear translocation of p50/p65, resulting in sustained activation of the classical NF-κB pathway and CLL cell survival [62]. BIRC3 has been shown to mediate the ubiquitin-dependent degradation of NIK, thereby inhibiting the non-classical NF-κB pathway [63]. Furthermore, mutations in BIRC3 have been observed to promote non-classical NF-κB pathway activation, which in turn enhances CLL cell survival [64]. CARD11 mutations have been identified in patients with CLL who are resistant to BTKi [65]. Mutations in the convoluted helical domain of the CARD11 gene have been demonstrated to induce sustained activation of NF-κB and to enhance NF-κB activity following antigen receptor stimulation [66]. CARD11 studies demonstrated that combination treatment with ibrutinib and MI2 rendered CLL mutant cell-sensitive MCL cells susceptible to ibrutinib, with BCR-induced activation of the classical NF-κB pathway, while non-classical NIK-NF-κB pathway-activated cells exhibited resistance to ibrutinib [67, 68]. Inactivating mutations in the TRAF2 and BIRC3 genes have been identified in MCL patients who have experienced relapse after treatment with ibrutinib, and these mutations activate NIK and promote MCL cell surviva [63]. Consequently, NIK inhibitors may offer a therapeutic alternative for MCL patients carrying TRAF2 and BIRC3 mutations and resistant to ibrutinib.

Analogous to the findings in CLL and MCL, mutations in BTK, PLCG2 and CARD11 were identified as a mechanism conferring resistance to BTK inhibitors in Waldenstrom’s macroglobulinemia (WM) patients [69]. It is noteworthy that the MYD88 L265P mutation was identified in 91% of WM patients, whereas it was present in only 14–29% of ABC DLBCL patients [70]. It is noteworthy that there was no statistically significant difference in response rates to ibrutinib between MYD88 mutant and wild-type ABC DLBCL patients [59]. However, WM cells carrying the MYD88 L 265P mutation exhibited increased sensitivity to ibrutinib in comparison to wild-type cells. Mechanistically, MYD88 L 265P mutated WM cells triggered NF-κB activation by activating BTK and IRAK1/4 [70]. However, in WM cells carrying both BTK C481S and MYD88 L 265P mutations, the BTK C481S mutation led to ibrutinib resistance by reactivating the BTK-PLCG2-ERK1/2 signaling pathway. Furthermore, the reactivation of ERK1/2 was accompanied by the secretion of multiple pro-survival and inflammatory cytokines, which further increased drug resistance. Treatment with the ERK1/2 inhibitors ulixertinib or GDC0994 resulted in the inhibition of the release of IL-6 and IL-10, and the induction of apoptosis in WM cells with the BTK C481S and MYD88 L265P double mutations. Furthermore, the combination of these inhibitors with ibrutinib exhibited a synergistic effect in terms of toxicity [70].

Inactivating mutations in the KLHL14 gene are frequently observed in mature B-cell malignancies, particularly in ABC DLBCL isoforms with double mutations in MYD88 and CD79b. Inactivation of KLHL14 promotes the assembly of the My-T-BCR supercomplex and the activation of NF-κB, sustaining the activation of NF-κB signaling even in the presence of ibrutinib. PIM1 mutations have been observed to occur with a higher frequency in patients diagnosed with ABC-DLBCL as opposed to GCB-DLBCL. These patients have been shown to demonstrate enhanced stability in comparison to the wild-type population, resulting in the consequent hyperactivation of the NF-κB pathway. The present study has demonstrated that combination therapy involving the pan-PIM inhibitor AZD1208 and ibrutinib has the capacity to inhibit the proliferation of ibrutinib-resistant ABC-DLBCL cells in a synergistic manner [71].

The Wnt/β-catenin pathway

The Wnt signaling pathway is a pivotal class of cell signaling pathways, whose functions involve cell proliferation, differentiation and migration.

The transfer of specific microRNAs (miRNAs) and proteins from CAF-derived EVs to tumor cells has been shown to activate the Wnt/β-catenin signaling pathway, thereby promoting an epithelial-mesenchymal transition (EMT). This, in turn, has been demonstrated to enhance metastasis and drug resistance in tumor cells, thus contributing to a more aggressive malignant phenotype. Furthermore, the Wnt/β-catenin signaling pathway has been demonstrated to exert influence on the tumor immune microenvironment, for instance, by modulating the recruitment and function of immune cells, thereby further promoting tumor immune escape. Consequently, the targeting of the CAF-EVs-mediated Wnt/β-catenin signaling pathway is regarded as a promising therapeutic strategy to impede tumor progression and metastasis [72, 73].

The Wnt/β-catenin pathway has been demonstrated to be instrumental in preserving the self-renewal capacity of tumor stem cells (CSCs). This is achieved through the activation of stem cell markers, such as LGR5. CSCs have been observed to exhibit a natural resistance to targeted therapies, including BTKi, and their survival is contingent on β-catenin-mediated metabolic reprogramming, which can be illustrated by enhanced glycolysis.

THE MAPK/ERK pathway

In a recent cohort study of disease progression, 58.3% (N = 21) of patients lacked BTK/PLCG2 mutations, which were predominantly characterised by alterations in TP53 (57.1%), del(17p) (47.6%), SF3B1 (28.6%), and NOTCH1 (28.6%), whereas the detection rate of these mutations was low in non-progressing patients [74]. The role of TP53 abnormalities and NOTCH1/SF3B1 mutations in the development of resistance to BTK inhibitors remains a subject of debate. However, the study revealed a significant enrichment of MAPK pathway mutations (BRAF, NRAS, KRAS, and MAP2K1) and elevated VAF in the progressed group [74]. This activation of the RAS/RAF/MAPK pathway may circumvent BTK inhibition, thereby sustaining CLL cell proliferation, a finding that aligns with prior observations in ABC-DLBCL and MCL, where CD79B-mediated MAPK activation led to ibrutinib resistance [75, 76]. In addition, in patients with CLL who experienced a relapse following treatment with ibrutinib, mutations in the transcription factor early growth response 2 (EGR2) were observed to be present in conjunction with BTK/PLCG2 mutations. BCR stimulation has been demonstrated to activate EGR2 through the process of ERK phosphorylation. The presence of mutations in EGR2 has been shown to result in the sustained dysregulation of BCR signaling, a condition that, when taken in conjunction with preexisting BTK/PLCG2 mutations that lead to ibrutinib-resistant cells, may contribute to the development of resistance to ibrutinib [61].

Stromal cells in lymph nodes and bone marrow secrete the chemokine, ‘CXCL12’, which binds to the surface of tumor cells to form a complex that is then able to activate the transmembrane chemokine receptor, CXCR4. This receptor is known to play a role in the processes of cell proliferation, survival and chemotaxis, and is able to do so through the activation of the AKT and ERK pathways [77]. Mutations in the CXCR4 gene have been identified in approximately 30% of patients diagnosed with WM, with the most prevalent mutation being at the S338X locus. The CXCR4 S338X mutation has been shown to induce resistance to ibrutinib, a drug used to treat WM, through enhanced activation of the AKT and ERK pathways. The utilization of CXCR4, AKT or MEK inhibitors has been demonstrated to restore sensitivity to ibrutinib in WM cells that carry the CXCR4 S338X mutation [78]. The specific mechanisms of the signaling pathway are summarized in Figs. 3.

Fig. 3.

Molecular Interplay Between BCR, PI3K/Akt/mTOR, Ras/Raf/MEK/ERK, NF-κB pathway, and Wnt/β-Catenin

Conclusion

BTKi resistance stems from the dual influence of intrinsic genetic mutations in tumor cells and adaptive mechanisms mediated by the TME. Within the TME, CAFs promote immune escape through the rejection of immune cells via chemokines and the recruitment of myeloid-derived suppressor cells. M2-type macrophages inhibit T-cell function and induce angiogenesis by secreting IL-10, VEGF and CCL18, while MSCs and ECs are associated with the formation of abnormal vascular structures through metabolic reprogramming and reduced drug penetration. Among the non-cellular components, physical barriers and mechanical signals in the ECM activate survival pathways; hypoxia and an acidified microenvironment enhance drug resistance through HIF-1α upregulation, lactate accumulation, and metabolic adaptations; and exosomes activate paracrine signaling by delivering resistance-associated molecules (e.g. microRNAs and mutant proteins).

At a molecular level, the BTK C481S mutation and the PLCG2 gain-of-function mutation lead directly to BCR signaling escape, while the PI3K-AKT-mTOR pathway enhances survival signaling through integrin VLA-4-mediated cell adhesion. Persistent activation of the NF-κB pathway (e.g. CARD11 and BIRC3 mutations) and abnormal activation of the Wnt/β-catenin pathway further maintain tumor stem cell properties and immunosuppression. Paracrine activation of the MAPK/ERK pathway drives proliferation via CXCR4 mutations or RAS/RAF signaling.

In summary, while genetic mutations such as BTK C481S remain the predominant cause of acquired resistance, our review emphasizes that functional resistance driven by the tumor microenvironment and signaling reprogramming is emerging as an equally important determinant of clinical outcomes. By integrating these aspects, this work provides that including microenvironment-targeted and metabolic-pathway-based strategies to restore BTKi sensitivity.

Author contributions

XY.D and SJ.T: Writing – original draft; Writing – review & editing.CL.W and L.Y: Conceptualization, Funding Acquisition.Resources, Supervision, Validation, Writing-Original Draft, Writing-Review &Editing;MH.C,QN.C,YF.W,YY.S,Y.D,Y.C and ZM.H, s:Data curation, Formal analysis; Writing – review & editing.All authors reviewed the manuscript.

Funding

The review was financially supported by the “533” talent project of Huaian city, the Huaian First People’s Hospital Innovation Team Project (YCT202306), the Huai’an First People’s Hospital Youth Innovative Talent Project (QC202218),the Huai’an First People’s Hospital High-level Talent Program Project (GQ202406), the Affiliated Huaian NO.1 People’s hospital of Nanjing Medical University, Northern Jiangsu Institute if Clinical Medical, Nanjing Medical University(SLKYMS20240114).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.