Mechanisms Of Ibrutinib Resistance Research Articles

Epigenetic Rewiring of BCR Signaling as a Novel Mechanism of Ibrutinib Resistance in ABC-DLBCL.

In this issue of Blood Cancer Discovery, Schaffer and colleagues uncover a novel epigenetic mechanism of resistance to the Bruton tyrosine kinase inhibitor ibrutinib in activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), whereby tumor cells rewire the B-cell receptor (BCR)-driven NF-κB signaling cascade through the small GTPase RAC2. This circuit can be efficiently targeted by RAC1/2 small-molecule inhibitors, suggesting a promising new therapeutic approach to overcome acquired ibrutinib resistance in ABC-DLBCL and possibly other B-cell malignancies relying on active BCR signaling.See related article by Shaffer et al., p. 630.

Dissection of Clonal Evolution By Temporal Mutation Profiling in Chronic Lymphocytic Leukemia Patients Treated with Ibrutinib

Introduction: The irreversible Bruton tyrosine kinase (BTK) inhibitor ibrutinib is changing the paradigm of the treatment of chronic lymphocytic leukemia (CLL) with remarkable outcomes in first line as well as in relapsed CLL, including high risk patients with TP53 aberration. While the majority of patients demonstrate durable responses, with a median follow up of 3 years, approximately 18% of them develop resistance and relapse. Mutations in the BTK and PLCG2 genes emerged as predominant mechanisms conferring secondary ibrutinib resistance with BTK and/or PLCG2 mutations detected in the majority of resistant patients, often even 10 months before the clinical relapse. Given the very poor outcome of patients discontinuing ibrutinib, comprehensive understanding of the mechanisms of ibrutinib resistance and changes in the clonal architecture induced by the selective pressure of the drug are of major clinical importance. To dissect the clonal evolution in the context of all relevant mutation targets in CLL, we performed a temporal mutation profiling of 31 genes in paired pre- and post-treatment samples of patients receiving ibrutinib therapy.Methods: Sequential samples from 18 patients treated with single-agent ibrutinib were included in this study. Pre-treatment peripheral blood samples were available in all patients, with post-treatment samples at 18 months for 6 patients, at 12 months for 7 patients, at 8 months for 3 patients and at 3 months for 2 patients, with a median follow up of 12 months. Targeted deep NGS analysis of 31 recurrently mutated target genes, including ATM, BCOR, BIRC3, BRAF, BTK, CHD2, DDX3X, EGR2, EIF2A, EP300, FBXW7, HIST1H1E, IGLL5, KLHL6, KMT2D, LRP1B, MAPK1, MED12, MGA, MYD88, NFKBIE, NOTCH1, PLCG2, POT1, RIPK1, RPS15, SAMHD1, SF3B1, TP53, XPO1 and ZMYM3 was performed using the TruSeq Custom Amplicon approach (Illumina) on genomic DNA specimens extracted from peripheral blood mononuclear cells. Variant calling was performed using the VariantStudio3.0 application (Illumina) and only variants supported with more than 100 mutant reads were considered. Our cohort represented a heavily pretreated patient group with a median of 2.5 (range: 1-5) lines of prior therapies.Results: The ultra-deep NGS analysis revealed a total of 473 somatic mutations in the 18 paired samples with a median allelic depth of 25.550x across the 31 genes analyzed. The post-treatment samples carried a considerably higher number of mutations compared to the pre-treatment samples (114 vs 359 mutations) with more than half of the variants representing subclonal alterations with variant allele frequencies (VAF) of <10%. The most frequently mutated genes at baseline included TP53 (50%) , NOTCH1 (50%) and POT1 (28%) with similarly high mutation frequencies in the post-treatment samples. MYD88, BIRC3 and SF3B1 mutations were enriched in the post-ibrutinib samples with mutation frequencies of 11% vs 33%, 11% vs 39% and 11% vs 50%, respectively. Interestingly, XPO1 mutations emerged only in the post-treatment samples of five (28%) patients. As for the BTK and PLCG2 mutations, none of the pre-treatment samples carried a mutation in either of these genes, however, we detected emerging subclonal BTK and PLCG2 mutations (with VAFs between 1.5% and 11%) in 28% (5/18) and 33% (6/18) of the post-treatment samples, respectively. A subset of cases harbored multiple mutations in both genes with some of these representing novel BTK and PLCG2 mutations affecting previously unreported amino acid residues. Scrutiny of the clonal evolution revealed that all individual cases were characterized by almost unique combinations of mutations as well as patterns of emerging and disappearing mutant subclones upon the selective pressure of ibrutinib.Conclusions: The ultra-deep NGS analysis performed on paired samples of CLL patients treated with ibrutinib provided a significant insight into the mutational repertoire as well as subclonal dynamics of the leukemic compartment. Furthermore, it has revealed a profound subclonal heterogeneity that seems to be further enhanced by ibrutinib therapy, as demonstrated by a higher frequency of subclonal variants in the post-treatment samples. The clinical significance and the potential role of the novel BTK and PLCG2 variants identified in our study will require a longer follow up time, as currently all patients harboring these subclonal variants are still on ibrutinib therapy. DisclosuresNo relevant conflicts of interest to declare.

Phase II/1b Trial of Ibrutinib Plus Prednisone and Rituximab Induction Followed By Venetoclax Plus Ibrutinib Maintenance in Newly Diagnosed High-Risk Chronic Lymphocytic Leukemia

Introduction: Therapy options for chronic lymphocytic leukemia (CLL) are rapidly changing with the approval of ibrutinib, an irreversible Bruton's tyrosine kinase inhibitor, and venetoclax, a Bcl2 inhibitor. In the RESONATE studies, ibrutinib showed significant improvements in overall response rates (ORR), progression-free-survival (PFS), and overall survival (OS). Nonetheless, complete response (CR) rates were only about 4%.During the first few weeks of treatment, ibrutinib will mobilize CLL cells from tissue sites into peripheral blood. Rituximab, a chimeric monoclonal CD20 antibody, when used with ibrutinib, has shown to immediately clear CLL from the blood with durable responses but with CR's of only 8%.Venetoclax is approved for relapsed or refractory CLL with 17p deletion. In a phase 2 study, at a median follow-up of 12.1 months, the ORR was ~79%, but with only 16% CR's. Amongst the 45 patients of whom minimal residual disease (MRD) was assessed, 24 were negative for MRD.In vitro studies have shown that nurse-like cells (NLCs) will protect CLL cells against spontaneous apoptosis by producing chemokines and interleukins such as SDF1, suggestive of a mechanism of ibrutinib resistance. SDF1 was shown to bind to its receptor CXCR4 located on CLL cells and activate a signal transduction pathway that would reduce spontaneous apoptosis. Dexamethasone was found to decrease NLC viability. This suggests that a combined therapy of ibrutinib plus steroids along with rituximab and venetoclax may be necessary to achieve deeper CR's in high-risk CLL.In addition, CLL with overexpression of exosome miR-155 is associated with a poor response to chemoimmunotherapy. In prior preclinical studies, exosome miR-150 and miR-155 have been shown to increase with BCR activation via α-IgM stimulation. We hope that via next generation microRNA sequencing from our patients' CLL plasma cells we will observe a decrease in the levels of exosome miR-150 and miR-155 in response to inhibition of BCR-pathway by ibrutinib as seen in preclinical studies.Methods: Patients with newly diagnosed high-risk CLL per FISH studies will be enrolled. Ibrutinib will be administered orally 420 mg daily, rituximab will be dosed at 375 mg/m2 intravenously on days 1, 8, 15, 22 of cycle 1 and on day 1 for cycle 2-6. Prednisone will be administered as 10 mg one week on, one week off per cycle. Each cycle will last 28 days. After 6 cycles, if patients have MRD, venetoclax will be added in on a weekly schedule with weekly ramp up from 20mg to 50mg, 100mg, and 200mg for a total duration of 6 months plus ibrutinib at 420mg as maintenance until disease progression or drug intolerance. There is the option to reintroduce venetoclax, prednisone, or rituximab if patients progress during maintenance.Results: The primary objectives of this study are to evaluate safety and MRD negative CR rates based on IWG-CLL criteria. For the evaluation of safety, the proportions of the 29 subjects with any adverse events, each specific adverse event, and study-related adverse events will be reported along with the 95% confidence intervals. The proportion of the 29 subjects who demonstrate a MRD negative CR will be reported along with the 95% confidence interval. A sample size of 29 will produce a two-sided 95% confidence interval with a width≤ 0.380.In addition, Kaplan-Meier method will be performed to evaluate time to first MRD negativity (TTMN), time to first relapse (TTFR), PFS and OS, respectively. Based on the Kaplan-Meier curve, the median time to TTMN, TTFR, PFS and OS, respectively, will be reported along with the 95% confidence interval if it exists. Correlation coefficients will be derived to describe the relationship between continuous endpoints and Kendall's tau will be derived to describe the relationship between categorical endpoints.There is no planned interim analysis for the CR rate. However, at any time point if an excessive number of adverse events are observed, the study will be terminated.Conclusion: In this Phase IB/II clinical trial, we seek to observe prolonged response rates of ibrutinib via inhibiting NLC's by adding prednisone. Also, we anticipate that rituximab will further amplify the CR's. For maintenance therapy, we hope that by combining venetoclax with ibrutinib in high-risk CLL patients who were unable to achieve negative MRD's, we will be able to induce deeper remissions. [Display omitted] DisclosuresPersky:Genentech: Consultancy; MorphoSys: Other: Independent Data Monitoring Committee member ; Verastem: Consultancy; Spectrum Pharmaceuticals: Research Funding.

Predicting Mechanisms of Ibrutinib-Resistance in Chronic Lymphocytic Leukemia (CLL) Using Computational-Based Modeling for Personalized Therapy with Clinical Validation

Background: Bruton's tyrosine kinase (BTK), a mediator of B cell receptor signaling, has been effectively exploited as a therapeutic target in Chronic lymphocytic leukemia (CLL). Ibrutinib is a BTK inhibitor that has shown excellent efficacy in treatment-naïve, relapsed/refractory, and high-risk CLL. However, concerns regarding ibrutinib resistance have emerged and characterizing the underlying mechanisms may aid in identifying patients with increased risk of resistance/disease progression. Moreover, understanding the interplay between determinants of ibrutinib resistance may reveal alternate therapeutic targets. Using a computational biology modeling (CBM) simulation-based method, we reverse-engineered the salient molecular architecture of a CLL patient progressing on ibrutinib to identify potential drug combinations to overcome drug resistance and induce remission.Methods: For validation of the computational biological modeling (CBM) software (Cellworks group), we selected a cohort of 15 ibrutinib resistant CLL patients from previously published studies (Table 1). Genomic data derived from whole exome sequencing (WES), targeted next-generation sequencing (NGS), copy number variation (CNV), and/or karyotype data were used in this study. All available genomic data for each profile was entered into the CBM, which generates a disease-specific protein network map using PubMed and other online resources. Disease-biomarkers that are unique to each patient were identified within the protein network maps by using CBM technology. Ibrutinib was selected from a digital drug library and simulated on every relapsed CLL patient case. Drug impact was assessed by quantitatively measuring the drug's effect on a cell growth score, which is a composite of cell proliferation, viability, and apoptosis, along with the simulated impact on each disease-specific biomarker score. Each patient-specific map was digitally screened to identify any Ibrutinib resistance mechanism(s).Results: In our modeled patient set, 4 different resistance mechanisms were identified across 14/15 ibrutinib- refractory CLL cases. 1) BTK C481S mutation (n=4) impacts ibrutinib binding affinity; 2) PLCG2 mutations (n=4) lead to activation of PRKCB and AKT1 signaling; 3) CARD11 mutation (n=2) results in constitutive activation of BTK downstream effectors; and 4) decreased G-protein coupled receptor (GPCR) pathway signaling, an upstream activator of BTK (n=5), was found to be a novel resistance mechanism identified by CBM. Knockdown (KD) of ALOX5AP, ARRB1 and CYSLTR2 reduced GPCR pathway signaling, resulting in reduced BTK activity (Table 1). After validating the CBM algorithm, we rendered a digital avatar for a relapsed-refractory CLL patient (Del17p+); progressing on ibrutinib. The patient had rapidly escalating ALC and lymphadenopathy; with the largest node measuring ~16cm. In addition to p53 loss, simulation modeling accounted for BTK C481S mutation (BAF 0.69) expressed in the patients tumor cells. CBM predicted venetoclax would be the most effective therapeutic for the patient due to several upstream signaling pathways converging on Bcl-2. Indeed, addition of venetoclax to the ongoing ibrutinib monotherapy dramatically reduced the patients ALC (from 113 to 3.3 x 109/L) by day 4 post-treatment, accompanied by reduction in lymphadenopathy (left axillary node decreased to ~3cm). Ibrutinib was discontinued within 1 month of starting the combination, with venetoclax monotherapy continued per dosing guidelines thereafter. Notably, the patient maintained remission status on venetoclax monotherapy alone.Conclusions: Computational modeling was used to identify ibrutinib resistance mechanisms in CLL patients. Using this novel approach, we identified an effective therapy (venetoclax) for a high-risk CLL patient who was progressing on ibrutinib. CBM-based approaches can be used to predict alternative therapeutic regimens for drug-refractory patients, targeting the disease-specific biomarkers unique to each patient. Altogether, our study demonstrates proof-of-principle on the use of computational predictive algorithms to simulate multiple protein-interaction networks operational in a tumor cell to exploit individualized therapeutic vulnerabilities. [Display omitted] DisclosuresYsebaert:Janssen: Consultancy, Research Funding, Speakers Bureau. Kumar:Cellworks: Employment. Singh:Cellworks Research India Pvt. Ltd: Employment. Ullal:Cellworks Research India: Employment. Bhargav:Cellworks: Employment. Azam:cellworks: Employment. Abbasi:Cellworks Group Inc.: Employment. Vali:Cellworks Group Inc.: Employment. Cogle:Celgene: Other: Membership on Steering Committee for Connect MDS/AML Registry.

Oncogenic MALT1 Promotes Cell Survival and Mediates Ibrutinib Resistance and Ibrutinib-Venetoclax Resistance in Mantle Cell Lymphoma

Both as monotherapies and in combination, the Bruton's tyrosine kinase inhibitor ibrutinib and the BH3 mimetic BCL2 inhibitor venetoclax have proven to be efficacious and are now widely used treatment options for mantle cell lymphoma (MCL) patients. However, mono- and dual- resistance frequently develops, necessitating investigation into the mechanisms mediating resistance to these therapies. To investigate the mechanism of ibrutinib resistance, we generated two ibrutinib-resistant cells due to marked BTK knockdown via CRISPR/CAS9 from JeKo-1, which is ibrutinib-sensitive and venetoclax-resistant. To understand the mechanism of venetoclax resistance, we generated three venetoclax-resistant cell lines with acquired resistance via chronic exposure to increasing doses of venetoclax from ibrutinib/venetoclax double sensitive Mino and Rec-1 cells, and ibrutinib-resistant but venetoclax-sensitive Granta519 cells. All these paired cell lines with various resistance to ibrutinib and venetoclax were subject to whole transcriptome sequencing of these paired MCL cell lines. We discovered that mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is significantly overexpressed in ibrutinib-resistant and ibrutinib-venetoclax dual-resistant MCL cells, especially in cells with BTK knockdown. This was further validated in primary MCL patient cells (n=24). Interestingly, MALT1 overexpression inversely correlates with CARD11 expression and enhances non-canonical NF-κB signaling, suggesting a switch from a highly-dependent BTK-CARD11 mechanism to an independent mechanism in both ibrutinib-resistant and ibrutinib-venetoclax dual-resistant MCL cells. Chromosomal translocations of MALT1 are the hallmarks of MALT lymphoma, which result in oncogenic fusion of MALT1 products. MALT1 is constitutively active in driving aggressive ABC-type DLBCL. This indicates MALT1 can be oncogenic when its activity is dysregulated. Therefore, we hypothesized that constitutive MALT1 activity may be responsible for the resistance to ibrutinib and venetoclax in MCL cells. To demonstrate this relationship, MALT1 ablation using genetic manipulation resulted in significant growth defects both in vitro and in vivo. Pharmaceutical approaches using MALT1 inhibitor MI-2 resulted in similar effects on cell survival and using cell viability assays. Whole transcriptome sequencing analysis revealed that MYC, NF-kB, ROS, cell cycle and mTOR signaling are the most significantly downregulated pathways upon MI-2 treatment. Intriguingly, MYC, NF-kB, PI3K-AKT-mTOR and mTOR signaling pathways were reported to be upregulated in ibrutinib-resistant MCL cells compared to sensitive MCL cells. To address this further, proteomics analysis by reverse phase protein array (RPPA) using more than 400 antibodies confirmed that MI-2 significantly downregulated AKT-mTOR signaling. NF-kB modulation, ROS production, AKT-mTOR, and metabolism changes were further confirmed through multiple biochemical approaches. In addition, MI-2 treatment resulted in a dramatic reduction of MALT1 expression, suggesting that MI-2 treatment affected both its scaffold and paracaspase activities. Furthermore, MI-2 treatment resulted in significant inhibition of in vivo tumor growth of ibrutinib-venetoclax dual-resistant MCL subcutaneous xenografts and tumor homing to the spleen and bone marrow in an ibrutinib-venetoclax dual-resistant MCL patient-derived xenograft (PDX) mouse model. In conclusion, we discovered that MALT1, an essential regulator of NF-κB signaling, is hyperactive in ibrutinib-resistant cells and ibrutinib-venetoclax dual-resistant MCL cells, which puts MALT1 forward as a potentially new therapeutic target in ibrutinib and venetoclax-resistant MCL tumors. Genetic depletion or pharmaceutical inhibition of MALT1 resulted in remarkable defects in cell survival and cell proliferation. The MALT1 inhibitor MI-2 proved its in vivo potency by its pro-apoptotic effect and its significant tumor growth inhibition. In conclusion, targeting a hyperactive MALT1 is a promising therapeutic strategy that could lead to clinical implementation of a new treatment strategy meant to overcome ibrutinib and ibrutinib-venetoclax dual resistance in MCL patients by reversing the NF-kB and ROS/mTOR- mediated resistance in these tumors. Disclosures Wang: Targeted Oncology: Honoraria; Loxo Oncology: Consultancy, Research Funding; Pulse Biosciences: Consultancy; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding; InnoCare: Consultancy; Oncternal: Consultancy, Research Funding; Nobel Insights: Consultancy; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; OncLive: Honoraria; Beijing Medical Award Foundation: Honoraria; Lu Daopei Medical Group: Honoraria; MoreHealth: Consultancy; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; OMI: Honoraria, Other: Travel, accommodation, expenses.

Ibrutinib in Chronic Lymphocytic Leukemia: Clinical Applications, Drug Resistance, and Prospects.

Bruton’s tyrosine kinase (BTK), a pivotal component of B-cell receptor (BCR) signaling, has been recognized as an important driver of the pathogenesis of chronic lymphocytic leukemia. Ibrutinib is a highly active and selectively irreversible inhibitor of BTK, which has been approved to be effective in both frontline and recurrent therapy of CLL. Acquired resistance has become a greater problem than initially anticipated with the widespread use of ibrutinib. An ongoing exploration of the mechanisms of ibrutinib resistance (IR) in CLL has revealed potentially useful therapeutic targets. New drugs expected to overcome IR in CLL are in the early stages of clinical development. This study aimed to summarize the possible mechanisms of IR and retrospectively analyze promising therapies that might have superior efficacy in overcoming IR.

Dissection of Subclonal Evolution by Temporal Mutation Profiling in Chronic Lymphocytic Leukemia Patients Treated with Ibrutinib

Abstract Introduction: The irreversible Bruton tyrosine kinase (BTK) inhibitor ibrutinib is changing the paradigm of the treatment of chronic lymphocytic leukemia (CLL) with remarkable outcomes in first line as well as in relapsed CLL, including high risk patients with TP53 aberration. While the majority of patients demonstrate durable responses, with a median follow up of 3 years, approximately 18% of them develop resistance and relapse. Mutations in the BTK and PLCG2 genes emerged as predominant mechanisms conferring secondary ibrutinib resistance with BTK and/or PLCG2 mutations detected in the majority of resistant patients, often even 10 months before the clinical relapse. Given the very poor outcome of patients discontinuing ibrutinib, comprehensive understanding of the mechanisms of ibrutinib resistance and changes in the clonal architecture induced by the selective pressure of the drug are of major clinical importance. To dissect the clonal evolution in the context of all relevant mutation targets in CLL, we performed a temporal mutation profiling of 31 genes in paired pre- and post-treatment samples of patients receiving ibrutinib therapy. Methods: Sequential samples from 18 patients treated with single-agent ibrutinib were included in this study. Pre-treatment peripheral blood samples were available in all patients, with post-treatment samples at 18 months for 6 patients, at 12 months for 7 patients, at 8 months for 3 patients and at 3 months for 2 patients, with a median follow up of 12 months. Targeted deep NGS analysis of 31 recurrently mutated target genes, including ATM, BCOR, BIRC3, BRAF, BTK, CHD2, DDX3X, EGR2, EIF2A, EP300, FBXW7, HIST1H1E, IGLL5, KLHL6, KMT2D, LRP1B, MAPK1, MED12, MGA, MYD88, NFKBIE, NOTCH1, PLCG2, POT1, RIPK1, RPS15, SAMHD1, SF3B1, TP53, XPO1 and ZMYM3 was performed using the TruSeq Custom Amplicon approach (Illumina) on genomic DNA specimens extracted from peripheral blood mononuclear cells. Variant calling was performed using the VariantStudio3.0 application (Illumina) and only variants supported with more than 100 mutant reads were considered. Our cohort represented a heavily pretreated patient group with a median of 2.5 (range: 1-5) lines of prior therapies. Results: The ultra-deep NGS analysis revealed a total of 473 somatic mutations in the 18 paired samples with a median allelic depth of 25.550x across the 31 genes analyzed. The post-treatment samples carried a considerably higher number of mutations compared to the pre-treatment samples (114 vs 359 mutations) with more than half of the variants representing subclonal alterations with variant allele frequencies (VAF) of Conclusions: The ultra-deep NGS analysis performed on paired samples of CLL patients treated with ibrutinib provided a significant insight into the mutational repertoire as well as subclonal dynamics of the leukemic compartment. Furthermore, it has revealed a profound subclonal heterogeneity that seems to be further enhanced by ibrutinib therapy, as demonstrated by a higher frequency of subclonal variants in the post-treatment samples. The clinical significance and the potential role of the novel BTK and PLCG2 variants identified in our study will require a longer follow up time, as currently all patients harboring these subclonal variants are still on ibrutinib therapy. Disclosures No relevant conflicts of interest to declare.

Glutamine-fuelled OXPHOS - a new target in MCL.

In a study published in Science Translational Medicine, Zhang et al. report a mechanism of ibrutinib resistance in mantle cell lymphoma that involves metabolic reprogramming towards glutamine-fuelled oxidative phosphorylation, revealing a novel therapeutic opportunity.

Targeting BTK in CLL: Beyond Ibrutinib.

While the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib has revolutionized the treatment of chronic lymphocytic leukemia (CLL), current limitations include off-target toxicities and the development of resistance. In this review, we summarize the emerging data for alternative BTKi. Second-generation BTKi include acalabrutinib, zanubrutinib, and tirabrutinib which offer greater BTK selectivity. While these agents may limit off-target toxicity, they do not overcome common mechanisms of ibrutinib resistance. Reversible BTKi including vecabrutinib and LOXO-305 inhibit BTK in the presence of C481S mutation, and non-selective reversible BTKi, including ARQ-531, may retain activity despite mutations within PLCG2. Early-phase studies are underway to establish the clinical efficacy and toxicity of these agents. A randomized trial of ibrutinib versus acalabrutinib is ongoing, and acalabrutinib may be an option for ibrutinib-intolerant patients. Results from ongoing trials of alternate BTKi will help to define their role in CLL therapy as single agents or in combination therapy.

Targeting Kinome Reprogramming for Overcoming Ibrutinib-Resistance (IR) in Mantle Cell Lymphoma (MCL)

Mantle cell lymphoma (MCL) is an aggressive and incurable B-cell malignancy. Prognosis remains poor due to emergence of drug resistance and MCL progression. Ibrutinib is a novel B-cell receptor (BCR) downstream bruton's tyrosine kinase (BTK) inhibitor with high response rates reported in MCL patients. The initial success of ibrutinib is likely attributed to attenuation of BCR-dependent lymphoma-tumor microenvironment (TME) interactions. Moreover, despite the dramatic responses to ibrutinib, ibrutinib resistance (IR) inevitably develops. Remarkably, once patients relapse after ibrutinib treatment, MCL patients experience disease progression and die within 12 months. Thus, there is an urgent need to define mechanisms of ibrutinib resistance (IR) and to identify novel targets to bring forward novel treatment options with real curative potential for this fatal complication. Currently the mechanisms driving IR are poorly understood and no recurrent driver mutations have been identified in MCL. We have modeled acquired resistance to ibrutinib and implemented chemical proteomics and a cell-based drug screen approaches in IR MCL lines and primary samples, we have shown that MCL cells become resistant to ibrutinib through a kinome-adaptive reprogramming mechanism that lead to constitutive activation of the PI3K/AKT/mTOR pathway with increased levels of Myc and sustained transcription activation. Significantly, the acquired IR MCL cells have increased rates of growth and augmented adhesion to stromal cells. Collectively, our published and preliminary studies indicate that IR MCL relies on global transcriptome remodeling and subsequent kinome reprogramming, leading to molecularly and clinically aggressive phenotype and resistance to ibrutinib therapy. Rather than there being a single mechanism of acquired IR, kinase networks are rewired in a plethora of ways in MCL cells as they become resistant to ibrutinib treatment. Adaptive remodeling of the kinome creates therapeutic challenges, where even combination targeted kinase inhibitor treatments are unlikely to be successful. We reasoned instead that global adaptive kinome as a whole must be blocked for overcoming IR. Sustained transcriptional activation and associated adaptive signaling changes drive the malignant phenotype of IR and, accordingly, that targeting the transcriptional machinery responsible for conferring IR triggers synthetic lethality in MCL. We leveraged powerful genomic and pharmacoproteomic approaches available to our laboratory and IR model to identify the key upstream genetic and epigenetic driver for IR. Ultimately, we designed transcription activation based combination therapy to overcome drug resistance and hinder MCL progression. In addition, performing cell-based drug screen assay on primary MCL cells of MCL patients allowed us to predict drug response and tailor therapeutic drugs for individual patients, thus, creating a personalized therapeutic strategy for MCL patients DisclosuresNo relevant conflicts of interest to declare.

Abstract 2857: PI3K/AKT activation in de novo and acquired resistance to ibrutinib in lymphoid malignancies

Abstract B-cell lymphoid malignancies, including CLL and DLBCL, are characterized by abnormal activation of Bruton's tyrosine kinase (BTK)-mediated B-cell receptor signaling that contributes to malignant transformation. Ibrutinib (IB) is an oral covalent BTK inhibitor that has shown impressive clinical activity. However, many IB-treated patients relapse over time with fulminant progression. We sought to define the molecular pathways that mediate Ib resistance by studying changes that occurred in vitro in cell lines and primary patient samples with prolonged exposure to IB. We found IB-increased apoptosis in a dose- and time-dependent manner, with upregulated levels of PTEN, BIM, and FOXO3a in Mec-1 and RIVA cells; followed by decreased phosphorylation (p) of Ser473-AKT and Ser253-FOXO3a. mRNA expression levels of Foxo3a and its targets Bim and Pten were significantly upregulated in a dose- and time-dependent manner. Interestingly, a subset of miRNAs, including miRNA-494 were significantly downregulated with upregulation of target transcripts Pten and Bim. Similar results were observed after IB treatment of primary CLL cells of naïve patients and those on new drug targets at high risk for relapse. To examine if miRNA-494-dependent PTEN regulation and activation of PI3K/AKT/FOXO3a signaling has a role in de novo and acquired resistance to IB in B-cell malignancies, we investigated the resistance mechanism that develops to IB therapy by generating IB-resistant (Ib-R) cells. Chronic exposure of RIVA cells to IB demonstrated AKT activation and increased pSer253-FOXO3a, in turn, leading to decreased PTEN and BIM levels; attributed to its decreased transcription as assessed by qRT-PCR in Ib-R RIVA cells. Interestingly, Ib-R RIVA cells had strikingly higher miRNA-494 expression and decreased Pten mRNA levels, indicating further regulation of PTEN/AKT/FOXO3a signaling. Moreover, unlike IB-sensitive CLL samples, patients with partial remission and Ib-R, before and after in vivo IB treatment, demonstrated significant upregulation of miRNA-494 and decreased Pten mRNA expression. The Ib-R cells were sensitized to treatment with IB by PI3K (idelalisib) and AKT (MK-2206) inhibition. MK-2206 downregulated pAKT and sensitized Ib-R cells through AKT-mediated PTEN activation. siRNA-mediated downregulation of AKT and FOXO3a significantly modulated cell survival, demonstrating the importance of these cell survival factors for Ib-R. Importantly, overexpression of miRNA-494 inhibitor increased Pten mRNA levels, further indicating regulation of apoptosis by AKT/FOXO3a signaling. Collectively, these findings reveal a novel mechanism of ibrutinib resistance in B-cell malignancies that modulates the expression and activity of tumor suppressor proteins and pro-apoptotic factors to confer drug resistance that could be effectively inhibited by a rational combination of therapeutic regimens. Citation Format: Yue Li, Isha Kapoor, Huayuan Zhu, Brian T. Hill, Wei Xu, Alex Almasan. PI3K/AKT activation in de novo and acquired resistance to ibrutinib in lymphoid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2857.

Ibrutinib.

Abnormal B-cell receptor (BCR) signalling is a key mechanism of disease progression in B-cell malignancy. Bruton's tyrosine kinase (BTK) has a pivotal role in BCR signalling. Ibrutinib (PCI-32765) is a small molecule which serves as a covalent irreversible inhibitor of BTK. It is characterized by high selectivity for BTK and high potency. Ibrutinib is currently approved by the FDA and EMA for use in chronic lymphocytic leukaemia in any line of treatment, for treatment of Waldenstrom macroglobulinemia in patients who have received previous treatments or are not suitable to receive immunochemotherapy as well as for second line treatment of mantle cell lymphoma and for patients with marginal zone lymphoma who have received at least one prior anti-CD20-based therapy. In addition, there is emerging clinical data on its efficacy in ABC subtype diffuse large B-cell lymphoma, multiple myeloma and primary central nervous system lymphoma. Ibrutinib has opened new options for treatment of those patients that have relapsed or have been refractory to more classical modes of treatment. Moreover, Ibrutinib has been shown to be effective in patients that have been known to have little sensitivity to classical immunochemotherapy. Having a favourable risk profile, the substance is, unlike conventional immunochemotherapy, also suitable for the less physical fit patients. Cases of primary and secondary resistance to Ibrutinib have emerged and there is an ongoing effort to identify their mechanism and develop strategies to overcome them. Beyond its direct effects on survival and apoptosis of malignant B-cells, there is increasing evidence that Ibrutinib is able to modulate the tumour microenvironment to overcome mechanisms of immune evasion. This has sparked interest in use of the substance beyond lymphoid malignancy. This chapter discusses structure, mechanism of action and toxicities of Ibrutinib and also presents important preclinical and clinical data as well as mechanisms of Ibrutinib resistance. Combination strategies with immunotherapeutic strategies such as immune checkpoint blockade and CAR T-cell therapy may be synergistic and are currently under investigation.

Waldenstrom macroglobulinemia cells devoid of BTKC481S or CXCR4WHIM-like mutations acquire resistance to ibrutinib through upregulation of Bcl-2 and AKT resulting in vulnerability towards venetoclax or MK2206 treatment

Although ibrutinib is highly effective in Waldenstrom macroglobulinemia (WM), no complete remissions in WM patients treated with ibrutinib have been reported to date. Moreover, ibrutinib-resistant disease is being steadily reported and is associated with dismal clinical outcome (overall survival of 2.9–3.1 months). To understand mechanisms of ibrutinib resistance in WM, we established ibrutinib-resistant in vitro models using validated WM cell lines. Characterization of these models revealed the absence of BTKC481S and CXCR4WHIM-like mutations. BTK-mediated signaling was found to be highly attenuated accompanied by a shift in PI3K/AKT and apoptosis regulation-associated genes/proteins. Cytotoxicity studies using the AKT inhibitor, MK2206±ibrutinib, and the Bcl-2-specific inhibitor, venetoclax±ibrutinib, demonstrated synergistic loss of cell viability when either MK22016 or venetoclax were used in combination with ibrutinib. Our findings demonstrate that induction of ibrutinib resistance in WM cells can arise independent of BTKC481S and CXCR4WHIM-like mutations and sustained pressure from ibrutinib appears to activate compensatory AKT signaling as well as reshuffling of Bcl-2 family proteins for maintenance of cell survival. Combination treatment demonstrated greater (and synergistic) antitumor effect and provides rationale for development of therapeutic strategies encompassing venetoclax+ibrutinib or PI3K/AKT inhibitors+ibrutinib in ibrutinib-resistant WM.

Ibrutinib in CLL: a focus on adverse events, resistance, and novel approaches beyond ibrutinib.

Bruton's tyrosine kinase (BTK), a mediator in B cell receptor signaling has been successfully exploited as a therapeutic target in treatment of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Ibrutinib is a BTK inhibitor that has shown excellent efficacy in treatment-naïve, heavily pre-treated, and high-risk CLL/SLL. With remarkable efficacy, good oral bioavailability, and modest adverse events profile, ibrutinib use is likely to continue to increase. As data with ibrutinib use in CLL matures, concerns regarding adverse events and drug resistance have emerged. New insights into mechanisms of ibrutinib resistance in CLL have uncovered potential therapeutic targets. Several promising novel agents are currently in early phases of development for overcoming ibrutinib resistance in CLL/SLL. We provide a comprehensive analysis of emerging adverse events profile of ibrutinib, summarize our current understanding of ibrutinib resistance in CLL, and review promising novel therapeutic tools to overcome this challenge.

Progress in Waldenstrom macroglobulinemia

Waldenstrom macroglobulinemia (WM) is a malignant B-cell lymphoproliferative disorder, which still remains incurable. Reports on the latest research progress of WM were presented in the 58th American Society of Hematology (ASH) Annual Meeting, covering the theoretical and clinical researches of this disease. In basic research, mechanism of ibrutinib resistance has been further explored. The application of modern technologies, such as next-generation sequencing, has promoted the pathogenesis and prognosis of WM. In clinical research, retrospective analyses of traditional treatment provide new theoretical foundation in the choice of regimen, while clinical trials on new drugs including BGB-3111 and oprozomib may improve the therapy. The research advances in WM will be summarized in this paper. Key words: Waldenstrom macroglobulinemia; American Society of Hematology Annual Meeting

Molecular Mechanisms of Ibrutinib Resistance: Defining a Logical Approach to Improving Targeted Therapy in CLL

IntroductionChronic lymphocytic leukemia (CLL) is one of the most common B cell malignancies in the western world. The importance of B cell receptor (BCR) signaling in CLL pathology has led to the emergence of a number of targeted therapies specific to kinases in this pathway. Ibrutinib, a potent irreversible Bruton's Tyrosine Kinase (BTK) inhibitor, is highly effective in CLL with minimal side effects. However, resistance to Ibrutinib is now emerging among patients in several clinical trials.Aims/MethodsTo understand the molecular mechanisms underlying ibrutinib resistance to aid in early detection of resistant clones and to inform further treatment choices, we have developed an in vitro ibrutinib resistant model using BCR signalling dependent B cell lymphoma lines (Ramos and Namalwa). These cells have been conditioned by exposure to escalating doses of ibrutinib - starting from 0.001μM and increased over time to a lethal dose of 0.5μM in order to select drug-resistant cells. Single cell clones have been isolated from both the naïve and the resistant cells. The growth kinetics of 7 Ramos and 5 Namalwa resistant clones has reached the same pattern as the parental cells. Additionally, 3 naïve clones were isolated from each sensitive parental cell line.Dysregulation of the BCR signaling pathway was characterized by both flow cytometry, using specific anti-phosphokinase antibodies, and by Western blotting. In addition, RNA from these clones was isolated and analyzed on an Affymetrix HTA2.0 gene expression array (n=6 from two cell lines paralleled to one naïve clone from each cell line).ResultsIn Ramos (n=3), gene expression profiling revealed significant upregulation of BCL2 and interferon response genes including interferon gamma (IFNG). However, in Namalwa (n=3), in addition to BCL2 upregulation, the expression arrays showed the overexpression of BCR signaling pathway genes including BTK itself, CD79B, LCK and GAB2 (Growth factor receptor-bound protein 2- associated-binder 2), suggesting hyper-activation of BCR pathway and constitutive activation of BTK and downstream kinases.Moreover, initial screening showed that not all of the resistant clones behave similarly. In Ramos, one clone apparently escapes ibrutinib inhibition by modifying different pathways compared to the other analysed clones, suggesting the potential for multiple mechanisms by which the malignant B cell clones can evade the toxic activity of targeted therapy.Results of this analysis were validated by qPCR and additional resistant clones were tested for BCL2 and IFNG expression. In Ramos, 7 resistant clones showed significant upregulation of BCL2 (mean 9.7-fold increase, range 2.1- 31.8, SEM 4.5) and IFNG(mean increase 8.3, range 2.6-19.1, SEM 2.2). IFNγ, is a known immune modulator linked to various stress signals indicating one possible resistance mechanism of malignant cells to anti-cancer therapies.In Namalwa, qPCR of 5 resistant clones showed significant upregulation of BTK (mean increase 10.3, range 2- 31: SEM: 5.4) and confirmed at the protein expression level by Western blotting as well; where the resistant cells over-express BTK protein compared to their respective naïve cells (n=6, mean increase 1.6, range 1.1- 1.9, SEM 0.16). Moreover, CD79B was over-expressed (mean over-expression 12.4, range 1.5- 22.2, SEM: 4.3) and 6-fold overexpression of LCK (mean increase 6, range 3.1- 9.5, SEM: 1.4) compared to the analysed naïve clones. Furthermore, validation with qPCR showed 12-fold upregulation of the GAB2gene (mean upregulation 12.7, SEM: 4.8), an adaptor protein which transmits crucial signals to activate downstream PI3K signaling with links to leukemogenesis.ConclusionOur results demonstrate that common mechanisms of resistance to BCR inhibition include both the up-regulation of B-cell receptor signaling activity and the up-regulation of anti-apoptotic molecules, particularly BCL2. More potent and more target-specific BTK inhibitors may overcome the hyperactivity of the BCR pathway. In contrast, inhibition of BCL2 with venetoclax may be beneficial in overcoming the emergent resistance to ibrutinib as these cells become more sensitive to BCL2 inhibition. These initial results support the combination of ibrutinib with the pro-apoptotic BCL2 inhibitor, venetoclax, which we are currently studying in the Bloodwise TAP CLARITY study. DisclosuresHillmen:Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Research Funding.

Prognostic Models Predictive of Disease Progression in CLL Patients Treated with Ibrutinib

Introduction: Progressive disease on ibrutinib is an emerging area of an unmet need. The key mechanism of ibrutinib resistance is driven by the growth of subclones with BTK and/or PLCG2 mutations under selective pressure from therapy [Woyach JA et al, N Engl J Med 2014; Burger et al, Nat Commun 2016]. It is unclear, however, how to effectively discriminate patients who are at risk of progression during ibrutinib. Established prognostic factors in CLL were developed in the context of chemoimmunnotherapy, and only limited data are available on predictive values of complex karyotype [Thompson et al, Cancer 2015] and deletion 17p [Byrd et al, Blood 2015] in patients receiving ibrutinib. To determine predictive values of conventional prognostic factors in CLL, we performed multivariate analyses of factors relevant to progression-free survival (PFS) on ibrutinib.Methods and Patients: 84 evaluable CLL patients received single-agent ibrutinib 420mg once daily under a phase II investigator-initiated trial (NCT01500733). 3-year safety and efficacy results from this trial were previously reported [Farooqui et al, 2015 ASH Annual Meeting Abstract 2937]. Briefly, eligible patients had either TP53 aberration (deletion 17p by FISH or TP53 mutation by targeted sequencing) or were 65 years or older. The study included both treatment-naive and relapsed/refractory patients. Metaphase karyotype was not routinely performed. The study was approved by the institutional review board, and conducted in accordance with the Declaration of Helsinki. Cox regression models were used to identify independent predictors of progression. Probability of PFS was estimated by the Kaplan-Meier method. Log-rank test was used to compare PFS probabilities between subgroups. Statistical analyses were performed using R statistical software version 3.2.3.Result: At the median follow up of 34.3 months, 19 (22.6%) of 84 evaluable patients progressed or died on study; 15 (17.9%) patients progressed and 4 (4.8%) additional patients died due to reasons unrelated to progression. Independent factors of PFS identified from univariate analysis were; TP53 aberration, prior treatment, advanced Rai stage (III/IV) and high beta-2 microgloblin (B2M) (>4mg/L or =<4mg/L) (P<0.05 for all tests). Patients with unmutated IGHV had a trend towards inferior outcome compared to mutated IGHV, but the difference between two groups did not reach statistical significance (P=0.088). Six variables were considered for multivariate models; age (continuous variable), IGHV mutation, TP53 aberration, Rai stage, prior treatment and B2M. B2M was significantly correlated with previously treated disease (median B2M 5.2 mg/L in relapsed/refractory CLL vs. 3.3 mg/L in previously untreated CLL, P=0.0006). To avoid multicollinearity, we performed two multivariate Cox regression models separately including B2M (model 1) or prior treatment status (model 2) (Table). Two models showed similar predictive values for PFS (c-statistic: 0.80 for model 1, 0.79 for model 2). Multivariate analysis identified the presence of TP53 aberration, advanced Rai stage, and high B2M or prior treatment were independently associated with PFS. PFS of patients who had all three factors on either model 1 or model 2 were significantly inferior than those who had two or less factors (P=5.49x10-5 for model1, P=5.95x10-6 for model 2; Figure). The median PFS was 38.8 moths for those who were high-risk by model 1 and 38.4 months for those who were high risk by model 2, and was significantly shorter than non-high-risk patients (median PFS: not reached in both models).Conclusion: This is the first integrated risk scoring system developed from patients receiving ibrutinib If confirmed in independent cohorts this model could enable early risk stratification for treatment trials with combination regimens or with alternative targeted agents. Conversely, the absence of adverse prognostic factors can be used to identify a subgroup who may benefit from single-agent ibrutinib and can potentially discontinue therapy if deep responses are achieved after long-term ibrutinib. [Display omitted] [Display omitted] DisclosuresFarooqui:Merck: Employment. Wiestner:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding.

Postibrutinib outcomes in patients with mantle cell lymphoma

AbstractDespite unprecedented clinical activity in mantle cell lymphoma (MCL), primary and acquired resistance to ibrutinib is common. The outcomes and ideal management of patients who experience ibrutinib failure are unclear. We performed a retrospective cohort study of all patients with MCL who experienced disease progression while receiving ibrutinib across 15 international sites. Medical records were evaluated for clinical characteristics, pathological and radiological data, and therapies used pre- and postibrutinib. A total of 114 subjects met eligibility criteria. The median number of prior therapies was 3 (range, 0-10). The Mantle Cell Lymphoma International Prognostic Index (MIPI) scores at the start of ibrutinib were low, intermediate, and high in 46%, 31%, and 23% of patients, respectively. Of patients with available data prior to ibrutinib and postibrutinib, 34 of 47 and 11 of 12 had a Ki67 >30%. The median time on ibrutinib was 4.7 months (range 0.7-43.6). The median overall survival (OS) following cessation of ibrutinib was 2.9 months (95% confidence interval [CI], 1.6-4.9). Of the 104 patients with data available, 73 underwent subsequent treatment an average of 0.3 months after stopping ibrutinib with a median OS of 5.8 months (95% CI, 3.7-10.4). Multivariate Cox regression analysis of MIPI before postibrutinib treatment, and subsequent treatment with bendamustine, cytarabine, or lenalidomide failed to reveal any association with OS. Poor clinical outcomes were noted in the majority of patients with primary or secondary ibrutinib resistance. We could not identify treatments that clearly improved outcomes. Future trials should focus on understanding the mechanisms of ibrutinib resistance and on treatment after ibrutinib.

Developing Therapeutic Strategies for CLL with Mutant p53

Chronic lymphocytic leukemia (CLL) is a clonal disease marked by genetic heterogeneity, often resulting in varied therapeutic responses. As a consequence of these genetic variations, cytogenetic analyses are routinely employed to select the most efficacious treatment strategies. One of the most critical genetic variants used for CLL risk-stratification is deletion of the short arm of chromosome 17 (17p-). The tumor suppressor TP53 maps to this region, and its loss correlates with dismal outcomes. While FISH analyses for 17p- are routinely performed, the mutational status of TP53 is typically unknown. The importance of p53 mutations in leukemic progression have recently become apparent, as clinical studies have identified subsets of CLL patients that harbor TP53 mutations without an accompanying 17p- (Zenz et. al., JCO 2010); while other studies have suggest that p53 mutations are present in leukemic clones that expand following cytotoxic treatment (Wong et. al., Nature 2015). Thus, it is critical that we identify frontline treatment modalities that do not directly place undo selective pressure on the p53 pathway in CLL patients harboring a single TP53 mutation.In recent clinical trials, the Bruton's tyrosine kinase (BTK) inhibitor, Ibrutinib, has been shown effective in improving progression free survival in patients with CLL regardless of 17p- status (O'Brien et. al., Lancet Oncology 2014). However, little is known regarding how p53 mutations impact therapeutic responses given their known dominant-negative and gain-of-function effects. To this end, we have utilized a preclinical in vivo mouse model of B-CLL (Eµ-TCL1) in the presence or absence of a TP53 hot-spot mutation (p53R172H, corresponding to p53R175H in humans) to study its impact on therapeutic response, survival, and dynamic loss of the remaining wild-type Trp53 allele during the natural course of B-CLL following ibrutinib-based therapy. Cohorts of Eµ-TCL1; p53R172H/+ and Eµ-TCL1; p53WT mice were treated with ibrutinib 25mg/kg/day by oral gavage starting at 8 months of age. Ibrutinib significantly extended survival in both the Eµ-TCL1; p53R172H/+ and Eµ-TCL1; p53WT cohorts (Fig. 1A) and resulted in a reduction in CD5+CD19+ cells (Fig. 1B). Together, these data indicate ibrutinib's therapeutic efficacy even in the presence of mutated p53. To investigate the molecular pathways altered by ibrutinib in both the wild type and p53 mutant setting, we performed RNA-Seq analyses using malignant B-cells from treated and untreated Eµ-TCL1; p53R172H and Eµ-TCL1; p53WT mice. Critically, these analyses revealed that ibrutinib did not impact genes or cellular programs governed by p53 (Fig. 1C). Furthermore, RNA and protein analyses of tumor samples revealed that ibrutinib was directly impacted the BTK-, PLC-, MAPK-, and ERK- pathways regardless of p53 mutational status. Perhaps most important, we did not observe a significant loss of heterozygosity of the remaining wild type Trp53 allele in the ibrutinib treated Eµ-TCL1; p53R172H/+ lymphomas compared to untreated Eµ-TCL1; p53R172H/+. Together, these results directly indicate that ibrutinib does not place undo selective pressure on the remaining wild type Trp53 allele (Fig. 1D) and demonstrates that ibrutinib based treatment modalities may be effective treatment regimens in CLL patients harboring mutant p53.Even though ibrutinib effectively improved overall survival compared to vehicle treated animals, these mice eventually succumbed to the disease. Thus, it will be critical to examine mechanisms of ibrutinib-resistance in the setting of Trp53 mutations. To this end, expression analysis of ibrutinib treated Eµ-TCL1; p53R172H/+ mice revealed an interesting increase in T-cell immune regulatory pathways compared to Eµ-TCL1; p53WT treated cohorts. Currently, we are exploring the mechanism by which mutant p53 expression, exposure to ibrutinib, and regulations of immune-checkpoint genes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mechanisms of ibrutinib resistance in chronic lymphocytic leukaemia and non-Hodgkin lymphoma.

Bruton tyrosine kinase (BTK), a mediator of B-cell receptor (BCR) signalling, has been implicated in the pathogenesis of chronic lymphocytic leukaemia (CLL) and other B-cell malignancies. Ibrutinib is an orally bioavailable and highly specific BTK inhibitor that was recently approved for treatment of patients with recurrent CLL and mantle cell lymphoma (MCL). In addition, ibrutinib has shown efficacy in subsets of patients with diffuse large B cell lymphoma (DLBCL) and Waldenstrom macroglobulinaemia (WM). However, despite ibrutinib's activity in multiple B-cell malignancies, cases of primary and secondary resistance have emerged. The overall reported frequency of resistance is low, but follow-up in many trials was short, and we predict that the incidence of observed resistance will increase as clinical use outside clinical trials expands over time. Mutations within BTK have been described and clearly interfere with drug binding; however, there are also emerging alternative mechanisms that bypass BTK entirely and offer new opportunities for other targeted agents. Improved understanding of mechanisms of primary and secondary resistance is essential to developing appropriate therapeutic strategies to both prevent and address resistance. This review provides a comprehensive analysis of ibrutinib resistance in CLL, MCL, DLBCL and WM and considers potential strategies for further study.