Acquired BTK mutations associated with resistance to noncovalent BTK inhibitors.
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ABSTRACT:
SUBMITTER: Qi J
PROVIDER: S-EPMC10539862 | biostudies-literature | 2023 Oct
REPOSITORIES: biostudies-literature
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Acquired BTK mutations associated with resistance to noncovalent BTK inhibitors.
Qi Jialei J Endres Sascha S Yosifov Deyan Yordanov DY Tausch Eugen E Dheenadayalan Rashmi Priyadharshini RP Gao Xiang X Müller Annika A Schneider Christof C Mertens Daniel D Gierschik Peter P Wist Martin M Jebaraj Billy Michael Chelliah BMC Stilgenbauer Stephan S
Blood advances 20231001 19
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Project description:The use of Bruton tyrosine kinase (BTK) inhibitors to block B-cell receptor (BCR)-dependent NF-κB activation in lymphoid malignancies has been a major clinical advance, yet acquired therapeutic resistance is a recurring problem. We modeled the development of resistance to the BTK inhibitor ibrutinib in the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma, which relies on chronic active BCR signaling for survival. The primary mode of resistance was epigenetic, driven in part by the transcription factor TCF4. The resultant phenotypic shift altered BCR signaling such that the GTPase RAC2 substituted for BTK in the activation of phospholipase Cγ2, thereby sustaining NF-κB activity. The interaction of RAC2 with phospholipase Cγ2 was also increased in chronic lymphocytic leukemia cells from patients with persistent or progressive disease on BTK inhibitor treatment. We identified clinically available drugs that can treat epigenetic ibrutinib resistance, suggesting combination therapeutic strategies.SignificanceIn diffuse large B-cell lymphoma, we show that primary resistance to BTK inhibitors is due to epigenetic rather than genetic changes that circumvent the BTK blockade. We also observed this resistance mechanism in chronic lymphocytic leukemia, suggesting that epigenetic alterations may contribute more to BTK inhibitor resistance than currently thought.See related commentary by Pasqualucci, p. 555. This article is highlighted in the In This Issue feature, p. 549.
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Project description:Inhibition of Bruton tyrosine kinase (BTK) is a breakthrough therapy for certain B cell lymphomas and B cell chronic lymphatic leukemia. Covalent BTK inhibitors (e.g., ibrutinib) bind to cysteine C481, and mutations of this residue confer clinical resistance. This has led to the development of noncovalent BTK inhibitors that do not require binding to cysteine C481. These new compounds are now entering clinical trials. In a systematic BTK mutagenesis screen, we identify residues that are critical for the activity of noncovalent inhibitors. These include a gatekeeper residue (T474) and mutations in the kinase domain. Strikingly, co-occurrence of gatekeeper and kinase domain lesions (L512M, E513G, F517L, L547P) in cis results in a 10- to 15-fold gain of BTK kinase activity and de novo transforming potential in vitro and in vivo. Computational BTK structure analyses reveal how these lesions disrupt an intramolecular mechanism that attenuates BTK activation. Our findings anticipate clinical resistance mechanisms to a new class of noncovalent BTK inhibitors and reveal intramolecular mechanisms that constrain BTK's transforming potential.
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Project description:Activated B-cell-like diffuse large B-cell lymphoma relies on B-cell receptor signaling to drive proliferation and survival. Downstream of the B-cell receptor, the key signaling kinases Bruton's tyrosine kinase and phosphoinositide 3-kinase δ offer opportunities for therapeutic intervention by agents such as ibrutinib, ONO/GS-4059, and idelalisib. Combination therapy with such targeted agents could provide enhanced efficacy due to complimentary mechanisms of action. In this study, we describe both the additive interaction of and resistance mechanisms to idelalisib and ONO/GS-4059 in a model of activated B-cell-like diffuse large B-cell lymphoma. Significant tumor regression was observed with a combination of PI3Kδ and Bruton's tyrosine kinase inhibitors in the mouse TMD8 xenograft. Acquired resistance to idelalisib in the TMD8 cell line occurred by loss of phosphatase and tensin homolog and phosphoinositide 3-kinase pathway upregulation, but not by mutation of PIK3CD. Sensitivity to idelalisib could be restored by combining idelalisib and ONO/GS-4059. Further evaluation of targeted inhibitors revealed that the combination of idelalisib and the phosphoinositide-dependent kinase-1 inhibitor GSK2334470 or the AKT inhibitor MK-2206 could partially overcome resistance. Characterization of acquired Bruton's tyrosine kinase inhibitor resistance revealed a novel tumor necrosis factor alpha induced protein 3 mutation (TNFAIP3 Q143*), which led to a loss of A20 protein, and increased p-IκBα. The combination of idelalisib and ONO/GS-4059 partially restored sensitivity in this resistant line. Additionally, a mutation in Bruton's tyrosine kinase at C481F was identified as a mechanism of resistance. The combination activity observed with idelalisib and ONO/GS-4059, taken together with the ability to overcome resistance, could lead to a new therapeutic option in activated B-cell-like diffuse large B-cell lymphoma. A clinical trial is currently underway to evaluate the combination of idelalisib and ONO/GS-4059 (NCT02457598).
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Project description:Inhibition of Bruton's tyrosine kinase (BTK) has proven to be highly effective in the treatment of B-cell malignancies such as chronic lymphocytic leukemia (CLL), autoimmune disorders, and multiple sclerosis. Since the approval of the first BTK inhibitor (BTKi), Ibrutinib, several other inhibitors including Acalabrutinib, Zanubrutinib, Tirabrutinib, and Pirtobrutinib have been clinically approved. All are covalent active site inhibitors, with the exception of the reversible active site inhibitor Pirtobrutinib. The large number of available inhibitors for the BTK target creates challenges in choosing the most appropriate BTKi for treatment. Side-by-side comparisons in CLL have shown that different inhibitors may differ in their treatment efficacy. Moreover, the nature of the resistance mutations that arise in patients appears to depend on the specific BTKi administered. We have previously shown that Ibrutinib binding to the kinase active site causes unanticipated long-range effects on the global conformation of BTK (Joseph et al., 2020). Here, we show that binding of each of the five approved BTKi to the kinase active site brings about distinct allosteric changes that alter the conformational equilibrium of full-length BTK. Additionally, we provide an explanation for the resistance mutation bias observed in CLL patients treated with different BTKi and characterize the mechanism of action of two common resistance mutations: BTK T474I and L528W.
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Project description:Ibrutinib produces high response rates and durable remissions in Waldenström macroglobulinemia (WM) that are impacted by MYD88 and CXCR4WHIM mutations. Disease progression can develop on ibrutinib, although the molecular basis remains to be clarified. We sequenced sorted CD19+ lymphoplasmacytic cells from 6 WM patients who progressed after achieving major responses on ibrutinib using Sanger, TA cloning and sequencing, and highly sensitive and allele-specific polymerase chain reaction (AS-PCR) assays that we developed for Bruton tyrosine kinase (BTK) mutations. AS-PCR assays were used to screen patients with and without progressive disease on ibrutinib, and ibrutinib-naïve disease. Targeted next-generation sequencing was used to validate AS-PCR findings, assess for other BTK mutations, and other targets in B-cell receptor and MYD88 signaling. Among the 6 progressing patients, 3 had BTKCys481 variants that included BTKCys481Ser(c.1635G>C and c.1634T>A) and BTKCys481Arg(c.1634T>C) Two of these patients had multiple BTK mutations. Screening of 38 additional patients on ibrutinib without clinical progression identified BTKCys481 mutations in 2 (5.1%) individuals, both of whom subsequently progressed. BTKCys481 mutations were not detected in baseline samples or in 100 ibrutinib-naive WM patients. Using mutated MYD88 as a tumor marker, BTKCys481 mutations were subclonal, with a highly variable clonal distribution. Targeted deep-sequencing confirmed AS-PCR findings, and identified an additional BTKCys481Tyr(c.1634G>A) mutation in the 2 patients with multiple other BTKCys481 mutations, as well as CARD11Leu878Phe(c.2632C>T) and PLCγ2Tyr495His(c.1483T>C) mutations. Four of the 5 patients with BTKC481 variants were CXCR4 mutated. BTKCys481 mutations are common in WM patients with clinical progression on ibrutinib, and are associated with mutated CXCR4.
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Project description:Bruton's tyrosine kinase (BTK) is a proven target in mantle cell lymphoma (MCL), an aggressive subtype of non-Hodgkin lymphoma. However, resistance to BTK inhibitors is a major clinical challenge. We here report that MALT1 is one of the top overexpressed genes in ibrutinib-resistant MCL cells, while expression of CARD11, which is upstream of MALT1, is decreased. MALT1 genetic knockout or inhibition produced dramatic defects in MCL cell growth regardless of ibrutinib sensitivity. Conversely, CARD11-knockout cells showed antitumor effects only in ibrutinib-sensitive cells, suggesting that MALT1 overexpression could drive ibrutinib resistance via bypassing BTK/CARD11 signaling. Additionally, BTK knockdown and MALT1 knockout markedly impaired MCL tumor migration and dissemination, and MALT1 pharmacological inhibition decreased MCL cell viability, adhesion, and migration by suppressing NF-κB, PI3K/AKT/mTOR, and integrin signaling. Importantly, cotargeting MALT1 with safimaltib and BTK with pirtobrutinib induced potent anti-MCL activity in ibrutinib-resistant MCL cell lines and patient-derived xenografts. Therefore, we conclude that MALT1 overexpression associates with resistance to BTK inhibitors in MCL, targeting abnormal MALT1 activity could be a promising therapeutic strategy to overcome BTK inhibitor resistance, and cotargeting of MALT1 and BTK should improve MCL treatment efficacy and durability as well as patient outcomes.
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Project description:BackgroundCovalent (irreversible) Bruton's tyrosine kinase (BTK) inhibitors have transformed the treatment of multiple B-cell cancers, especially chronic lymphocytic leukemia (CLL). However, resistance can arise through multiple mechanisms, including acquired mutations in BTK at residue C481, the binding site of covalent BTK inhibitors. Noncovalent (reversible) BTK inhibitors overcome this mechanism and other sources of resistance, but the mechanisms of resistance to these therapies are currently not well understood.MethodsWe performed genomic analyses of pretreatment specimens as well as specimens obtained at the time of disease progression from patients with CLL who had been treated with the noncovalent BTK inhibitor pirtobrutinib. Structural modeling, BTK-binding assays, and cell-based assays were conducted to study mutations that confer resistance to noncovalent BTK inhibitors.ResultsAmong 55 treated patients, we identified 9 patients with relapsed or refractory CLL and acquired mechanisms of genetic resistance to pirtobrutinib. We found mutations (V416L, A428D, M437R, T474I, and L528W) that were clustered in the kinase domain of BTK and that conferred resistance to both noncovalent BTK inhibitors and certain covalent BTK inhibitors. Mutations in BTK or phospholipase C gamma 2 (PLCγ2), a signaling molecule and downstream substrate of BTK, were found in all 9 patients. Transcriptional activation reflecting B-cell-receptor signaling persisted despite continued therapy with noncovalent BTK inhibitors.ConclusionsResistance to noncovalent BTK inhibitors arose through on-target BTK mutations and downstream PLCγ2 mutations that allowed escape from BTK inhibition. A proportion of these mutations also conferred resistance across clinically approved covalent BTK inhibitors. These data suggested new mechanisms of genomic escape from established covalent and novel noncovalent BTK inhibitors. (Funded by the American Society of Hematology and others.).
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Project description:BackgroundApproximately 75% of objective responses to anti-programmed death 1 (PD-1) therapy in patients with melanoma are durable, lasting for years, but delayed relapses have been noted long after initial objective tumor regression despite continuous therapy. Mechanisms of immune escape in this context are unknown.MethodsWe analyzed biopsy samples from paired baseline and relapsing lesions in four patients with metastatic melanoma who had had an initial objective tumor regression in response to anti-PD-1 therapy (pembrolizumab) followed by disease progression months to years later.ResultsWhole-exome sequencing detected clonal selection and outgrowth of the acquired resistant tumors and, in two of the four patients, revealed resistance-associated loss-of-function mutations in the genes encoding interferon-receptor-associated Janus kinase 1 (JAK1) or Janus kinase 2 (JAK2), concurrent with deletion of the wild-type allele. A truncating mutation in the gene encoding the antigen-presenting protein beta-2-microglobulin (B2M) was identified in a third patient. JAK1 and JAK2 truncating mutations resulted in a lack of response to interferon gamma, including insensitivity to its antiproliferative effects on cancer cells. The B2M truncating mutation led to loss of surface expression of major histocompatibility complex class I.ConclusionsIn this study, acquired resistance to PD-1 blockade immunotherapy in patients with melanoma was associated with defects in the pathways involved in interferon-receptor signaling and in antigen presentation. (Funded by the National Institutes of Health and others.).
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Project description:Covalent inhibitors of Bruton tyrosine kinase (BTK) have transformed the therapy of chronic lymphocytic leukemia (CLL), but continuous therapy has been complicated by the development of resistance. The most common resistance mechanism in patients whose disease progresses on covalent BTK inhibitors (BTKis) is a mutation in the BTK 481 cysteine residue to which the inhibitors bind covalently. Pirtobrutinib is a highly selective, noncovalent BTKi with substantial clinical activity in patients whose disease has progressed on covalent BTKi, regardless of BTK mutation status. Using in vitro ibrutinib-resistant models and cells from patients with CLL, we show that pirtobrutinib potently inhibits BTK-mediated functions including B-cell receptor (BCR) signaling, cell viability, and CCL3/CCL4 chemokine production in both BTK wild-type and C481S mutant CLL cells. We demonstrate that primary CLL cells from responding patients on the pirtobrutinib trial show reduced BCR signaling, cell survival, and CCL3/CCL4 chemokine secretion. At time of progression, these primary CLL cells show increasing resistance to pirtobrutinib in signaling inhibition, cell viability, and cytokine production. We employed longitudinal whole-exome sequencing on 2 patients whose disease progressed on pirtobrutinib and identified selection of alternative-site BTK mutations, providing clinical evidence that secondary BTK mutations lead to resistance to noncovalent BTKis.
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Project description:Bruton's tyrosine kinase (BTK) plays a key role in the B-cell receptor (BCR) signaling pathway and confers anti-apoptotic and proliferative properties to malignant B-cells in chronic lymphocytic leukemia (CLL). Small molecule BTK inhibitors were designed to bind BTK's active site and block downstream signaling. These drugs have now been used in the treatment of thousands of patients with CLL, the most common form of leukemia in the western hemisphere. However, adverse effects of early generations of BTK inhibitors and resistance to treatment have led to the development of newer, more selective and non-covalent BTK inhibitors. As the use of these newer generation BTK inhibitors has increased, novel BTK resistance mutations have come to light. This review aims to discuss previously known and novel BTK mutations, their mechanisms of resistance, and their relationship with patient treatment. Also discussed here are future studies that are needed to investigate the underlying cause allowing these mutations to occur and how they incite resistance. New treatments on the horizon that attempt to maneuver around these resistance mutations can be met with new resistance mutations, creating an unmet need for patients with CLL. Novel therapies and combinations that address all forms of resistance are discussed.
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