Bruton's Tyrosine Kinase Gene Research Articles

The Evolving Role of Bruton's Tyrosine Kinase Inhibitors in B Cell Lymphomas.

Bruton's tyrosine kinase (BTK), a non-receptor tyrosine kinase crucial for B cell development and function, acts downstream of the B cell receptor (BCR) in the BCR pathway. Other kinases involved downstream of the BCR besides BTK such as Syk, Lyn, PI3K, and Mitogen-activated protein (MAP) kinases also play roles in relaying signals from the BCR to provide pro-survival, activation, and proliferation cues. BTK signaling is implicated in various B-cell lymphomas such as mantle cell lymphoma, Waldenström Macroglobulinemia, follicular lymphoma, and diffuse large B cell lymphoma, leading to the development of transformative treatments like ibrutinib, the first-in-class covalent BTK inhibitor, and pirtobrutinib, the first-in-class noncovalent BTK inhibitor. However, kinase-deficient mutations C481F, C481Y, C481R, and L528W in the BTK gene confer resistance to both covalent and non-covalent BTK inhibitors, facilitating B cell survival and lymphomagenesis despite kinase inactivation. Further studies have revealed BTK's non-catalytic scaffolding function, mediating the assembly and activation of proteins including Toll-like receptor 9 (TLR9), vascular cell adhesion protein 1 (VCAM-1), hematopoietic cell kinase (HCK), and integrin-linked kinase (ILK). This non-enzymatic role promotes cell survival and proliferation independently of kinase activity. Understanding BTK's dual roles unveils opportunities for therapeutics targeting its scaffolding function, promising advancements in disrupting lymphomagenesis and refining B cell lymphoma treatments.

Screening and Characterization of Allosteric Small Molecules Targeting Bruton's Tyrosine Kinase.

Bruton's Tyrosine Kinase (BTK) is a nonreceptor tyrosine kinase that belongs to the TEC family. Mutations in the BTK gene cause X-linked agammaglobulinemia (XLA) leading to an arrest in B-cell development. BTK is also a drug target for B-cell lymphomas that rely on an intact B-cell receptor signaling cascade for survival. All FDA approved drugs for BTK target the ATP binding site of the catalytic kinase domain, leading to potential adverse events due to off-target inhibition. In addition, acquired resistance mutations occur in a subset of patients, rendering available BTK inhibitors ineffective. Therefore, allosteric sites on BTK should be explored for drug development to target BTK more specifically and in combination with active site inhibitors. Virtual screening against nonactive site pockets and in vitro experiments resulted in a series of small molecules that bind to BTK outside of the active site. We characterized these compounds using biochemical and biophysical techniques and narrowed our focus to compound "C2". C2 activates full-length BTK and smaller multidomain BTK fragments but not the isolated kinase domain, consistent with an allosteric mode of action. Kinetic experiments reveal a C2-mediated decrease in Km and an increase in kcat leading to an overall increase in the catalytic efficiency of BTK. C2 is also capable of activating the BTK XLA mutants. These proof-of-principle data reveal that BTK can be targeted allosterically with small molecules, providing an alternative to active site BTK inhibitors.

Trial in Progress: A Phase 1b Study of AS-1763, an Oral, Potent and Selective Noncovalent BTK Inhibitor, in Patients with Previously Treated Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma or Non-Hodgkin Lymphoma

Background: Covalent Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib are approved for treating patients with B-cell malignancies, but their long-term efficacy is limited by toxicity related to off-target kinase inhibition and acquired resistance due to BTK C481 mutation. AS-1763 is a potent, highly selective, orally available, and non-covalent BTK inhibitor, equipotent against both wild-type and C481S-mutated BTK with sub-nanomolar IC50 values (Kawahata et al. J Med Chem 2021; 64,14129-14141). In a Phase 1 single-ascending dose study in healthy volunteers, AS-1763 was well tolerated and safe at doses of 5-600 mg and achieved maximum inhibition of B cell activation at doses of 100 mg and above at 1-2 h post-dose and the duration of inhibitory effect increased with dose [Arimura et al. AACR 2022; Cancer Res 2022;82(12_Suppl)]. In preclinical studies, AS-1763 demonstrated a broad spectrum of inhibition against BTK mutations that confer resistance to other non-covalent BTK inhibitors in addition to the C481 mutation. Study Design and Methods: This study is an open-label, multi-center, Phase 1b study of oral AS-1763 in patients with advanced chronic lymphocytic leukemia (CLL)/small lymphocytic leukemia (SLL) and other B-cell non-Hodgkin lymphomas (NHLs) who have failed or are intolerant to at least two prior lines of systemic therapy. Prior therapy with a covalent BTK inhibitor is permitted; prior use of a noncovalent BTK inhibitor is excluded. This study consists of two parts, i.e., dose escalation and expansion parts. The dose escalation part will follow a 3+3 design with a starting dose of 100 mg BID. Each cycle will be 28 days. Three dose levels will be selected for dose expansion part based on a comprehensive review of data from dose escalation. The dose expansion part consists of 2 cohorts, i.e., Cohort 1 for CLL/SLL and Cohort 2 for B-cell NHL. In each cohort, the first 30 patients will be allocated to the 3 dose levels (10 patients for each), then the same provisional recommended Phase 2 Dose (RP2D) will be selected for Cohort 1 and Cohort 2 after 30 patients have enrolled in either cohort. After the provisional RP2D is selected, up to 28 patients in Cohort 1 and up to 15 patients in Cohort 2 may be treated at the provisional RP2D. In this study, up to 110 patients will be enrolled. Treatment will continue for 24 cycles or until disease progression, occurrence of unacceptable toxicity or discontinuation because of other reasons. Eligible patients include CLL/SLL, Waldenstrӧm macroglobulinemia (WM), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), and follicular lymphoma (FL). Patients with transformed disease (e.g., Richter's transformation) prior to or during screening are not eligible. Key exclusion criteria include CNS involvement by systemic lymphoma, stem cell transplant or CAR-T therapy <30 days, and clinically significant cardiovascular disease. The primary objective of the dose escalation part is to determine the maximum tolerated dose/dose-limiting toxicity. Key secondary endpoints include the evaluation of safety profile and tolerability, pharmacokinetics properties, and preliminary anti-tumor activity based on overall response rate (ORR) by investigator according to iwCLL 2018 for CLL, IWW6 for WM, and Lugano Treatment Response Criteria for MCL, MZL, SLL and FL. The primary objective of the dose expansion part is to assess the preliminary anti-tumor activity based on ORR by the Safety Monitoring Committee (SMC). Key secondary objectives are to investigate safety, tolerability, and pharmacokinetic profiles and to assess the preliminary antitumor activity based on ORR by investigator and best overall response, duration of response, progression-free survival and overall survival by investigator and the SMC. This trial will evaluate several key exploratory objectives including evaluation of minimal residual disease negativity, characterization of patient subsets defined by mutation status of BTK and phospholipase C gamma 2 (PLCG2), and characterize BTK and PLGC2 gene mutation before and after disease progression. Changes in the level of biomarkers such as CCL3 and CCL4 chemokines and B-cell receptor pathway signaling will be evaluated prior to, during therapy, and at time of disease progression. The RP2D will be determined based on all the data generated in the study. The study has been registered on ClinicalTrials.gov (NCT05602363).

Clinical features and mutational analysis of X-linked agammaglobulinemia patients in Malaysia.

Bruton's tyrosine kinase (BTK) is a cytoplasmic protein involved in the B cell development. X-linked agammaglobulinemia (XLA) is caused by mutation in the BTK gene, which results in very low or absent B cells. Affected males have markedly reduced immunoglobulin levels, which render them susceptible to recurrent and severe bacterial infections. Methods: Patients suspected with X-linked agammaglobulinemia were enrolled during the period of 2010-2018. Clinical summary, and immunological profiles of these patients were recorded. Peripheral blood samples were collected for monocyte BTK protein expression detection and BTK genetic analysis. The medical records between January 2020 and June 2023 were reviewed to investigate COVID-19 in XLA. Twenty-two patients (from 16 unrelated families) were molecularly diagnosed as XLA. Genetic testing revealed fifteen distinct mutations, including four splicing mutations, four missense mutations, three nonsense mutations, three short deletions, and one large indel mutation. These mutations scattered throughout the BTK gene and mostly affected the kinase domain. All mutations including five novel mutations were predicted to be pathogenic or deleterious by in silico prediction tools. Genetic testing confirmed that eleven mothers and seven sisters were carriers for the disease, while three mutations were de novo. Flow cytometric analysis showed that thirteen patients had minimal BTK expression (0-15%) while eight patients had reduced BTK expression (16-64%). One patient was not tested for monocyte BTK expression due to insufficient sample. Pneumonia (n=13) was the most common manifestation, while Pseudomonas aeruginosa was the most frequently isolated pathogen from the patients (n=4). Mild or asymptomatic COVID-19 was reported in four patients. This report provides the first overview of demographic, clinical, immunological and genetic data of XLA in Malaysia. The combination of flow cytometric assessment and BTK genetic analysis provides a definitive diagnosis for XLA patients, especially with atypical clinical presentation. In addition, it may also allow carrier detection and assist in genetic counselling and prenatal diagnosis.

Bruton's Tyrosine Kinase in Ankylosing Spondylitis.

Prospective case-control study. To explore the role of Bruton's tyrosine kinase (BTK) in ankylosing spondylitis (AS). AS substantially affects patients, impairing the range of motion in the whole spine and peripheral joints, as well as overall quality of life. However, surveillance for this condition is limited, and biomarkers that can predict disease activity are not well documented. The expression of the BTK gene in peripheral blood mononuclear cells (PBMCs) was measured using flow cytometry and real-time quantitative polymerase chain reaction in 36 patients with AS and 30 healthy controls. Demographic features, Ankylosing Spondylitis Disease Activity Score-C-reactive protein (CRP) based, Bath Ankylosing Spondylitis Disease Activity Index, Bath Ankylosing Spondylitis Functional Index, HLA-B27, erythrocyte sedimentation rate (ESR), and CRP were evaluated to identify factors associated with BTK expression. Analyses were performed using the Spearman rank correlation test for continuous data, the χ 2 test for categorical data, and that between continuous and dichotomous variables was measured using a point-biserial correlation test. The area under the curve of the receiver operating characteristic curve was used to assess the performance of each candidate biomarker. BTK gene expression was significantly higher in patients with AS than in controls ( P = 0.026) according to quantitative polymerase chain reaction results. BTK Y223 was also high in CD19 + PBMCs from patients with AS, with CD 19+ BTK Y223+high cells being significantly positively correlated to ESR, CRP, and Ankylosing Spondylitis Disease Activity Score. A negative association was observed between BTK expression and the chest expansion distance. The area under the curve for CD 19+ BTK Y223+ was larger than that for ESR, but CRP still had the largest area. BTK expression was higher in PBMCs from patients with AS when compared with controls, and was associated with a higher disease activity index, inflammatory reactants, and arthritis and extra-articular manifestations. These findings suggest that BTK expression may play a crucial role in the inflammatory process in individuals with AS.

Overexpression of Bruton Tyrosine Kinase Inhibits the Proliferation, Migration, and Invasion of Non-Small Cell Lung Cancer Cells.

Lung cancer is one of the most lethal malignant tumors in the world. Non-small cell lung cancer (NSCLC) is the most common pathological subtype. However, the molecular mechanism of NSCLC progress is still unclear. We extracted the expression data of the Bruton's tyrosine kinase (BTK) gene in NSCLC tissues from the TCGA database. The results of paired t-test showed that the BTK gene was significantly underexpressed in NSCLC tissues. To further verify the above results, we detected the expression of the BTK gene in NSCLC cell lines A549, H1299, and H1650 at the RNA and protein levels by real-time fluorescent quantitative polymerase chain reaction and Western Blot analysis, respectively. The results showed that BTK was low expressed in NSCLC tissues and cells. More importantly, the expression of the BTK gene is also significantly related to the patient's age, gender, tumor range (T), lymph node invasion (N), tumor stage, and prognosis, and its expression level gradually decreases with the progress of the disease. It is speculated that BTK may be an independent prognostic factor of NSCLC. Our experimental results are consistent with the above clinical correlation analysis results. Overexpression of BTK can significantly inhibit the proliferation, migration, and invasion of NSCLC cells and can block the G0/G1 tumor cell cycle, indicating that overexpression of BTK can inhibit the growth, migration, and invasion of NSCLC cells.

Association Between Bruton's Tyrosine Kinase Gene Overexpression and Risk of Lymphoma in Primary Sjögren's Syndrome.

We undertook this study to analyze whole blood gene expression and to investigate the role of B cell genes in primary Sjögren's syndrome-related non-Hodgkin lymphoma (primary SS-NHL). Peripheral whole blood samples were collected from 345 well-phenotyped patients with primary SS enrolled in the prospective Assessment of Systemic Signs and Evolution in Sjögren's Syndrome (ASSESS) cohort. Transcriptomic analysis was performed using human Clariom S Arrays (Affymetrix). In our primary analysis, we considered patients with incident lymphoma (i-primary SS-NHL) as the case group and all patients without lymphoma as the comparison group. In our sensitivity analyses, we considered all patients with primary SS-NHL, including those with a history of lymphoma (h-primary SS-NHL), as the case group and primary SS patients without lymphoma, stratified on their risk factors of lymphoma, as the comparison group. Twenty-one patients with primary SS-NHL (including 8 with i-primary SS-NHL and 13 h-primary SS-NHL) were eligible for transcriptomic analysis; we compared these patients to 324 primary SS controls without lymphoma, including 110 with moderate to severe disease activity and 61 with no risk factor of lymphoma. Functional clustering analyses revealed an enrichment of genes related to innate and adaptive immunity, including B cell-related genes. Bruton's tyrosine kinase (BTK) and a proliferation-inducing ligand (APRIL) genes were overexpressed before the occurrence of lymphoma in patients with incident lymphoma compared with patients without lymphoma. In sensitivity analyses, BTK was consistently up-regulated across all comparisons performed. BTK expression was associated with risk of lymphoma on multivariate analyses, which considered 9 validated predictors of lymphoma in primary SS. BTK and APRIL were overexpressed in the peripheral blood of primary SS patients prior to lymphoma. The association between BTK, APRIL, and primary SS-NHL requires confirmation in other prospective cohorts.

Decreased or Absent B Cells in patients with Severe Reduction in All Serum Immunoglobulin Isotypes, a registry based study

BackgroundSevere Reduction in All Serum Immunoglobulin Isotypes with Profoundly Decreased or Absent B Cells is a subgroup of inborn error of immunities (IEI). 80 to 90% carry mutations in X-chromosomal Bruton tyrosine kinase (BTK) and around 5–10% of the cases are autosomal recessive Agammaglobulinemia (ARA). Very few of Hypogammaglobulinemia with no B cells classified in other subgroups. All of these patients benefit from a genetic evaluation, especially in highly consanguine populations. Materials and Methods27 patients from 11 families with hypogamma-globinemia and no B-cells registered in Immunodeficiency Diseases Research Center registry, Isfahan University of Medical Sciences were phenotypically and genetically evaluated. ResultsThe most prevalent genetic cause was mutations in BTK. The novel mutations in BTK gene includes c.176 G>A (p. Glu89Lys); c.115T>C (p. Tyr39His); c.685-686insTTAC (p. Asn229llefs5), in ARA includes a homozygous stop mutation in Immunoglobulin Heavy Constant Mu Chain (IGHM) gene, a frameshift mutation of B-Cell Antigen Receptor Complex-Associated Protein (CD79A), a bi-allelic stop-gain mutation in Transcription Factor 3 (TCF3), and in PIK3CD also in others IEI includes :novel homozygous silent mutations in Phosphoinositide-3-Kinase Regulatory Subunit (RASGRP1), homozygote SAMD9 mutation. DiscussionWe did not observe any phenotypically significant differences in XLA patients.The main clinical manifestation of ARA cases is, recurrent infections. However, patient with IGHM deficiency had earlier onset of the disease and more severe complications. In CD79A deficient case with 5 affected members and four deaths in her family, the symptoms started very early but managed properly according to the family history. Due to the late diagnosis of PIK3CD cases, more severe complications like bronchiectasis, neurologic sequels after poliomyelitis, and SLE nephropathy has been developed. Our novel SAMD9 had quite different phenotype and the novel RASPGR2 case with no B cells unfortunately managed as ARA before genetic diagnosis and has died of lymphoma as her 3 siblings. However, we observed that in families with more than one affected family member, the disease was better controlled in the second patient. ConclusionOur study broadens the genetic spectrum of hypogammaglobulinemia with no B cells in Iran. This helps differentiate phenotypes and genotypes and manage patients appropriately.

Abstract CT128: Phase 1 study of HSK29116, a Bruton tyrosine kinase (BTK) proteolysis-targeting chimera (PROTAC) agent, in patients with relapsed or refractory B-cell malignancies

Abstract BACKGROUND: BTK, a non-receptor kinase in the B-cell receptor signaling pathway, plays an essential role in B-cell development and differentiation. BTK is critical for malignant B-cell survival. Oral BTK inhibitors (BTKis), standard treatments for patients (pts) with B-cell malignancies, prevent proliferation and induce apoptosis of B cells. Currently approved BTKis covalently bind to BTK residue C481, irreversibly inhibiting phosphorylation of downstream kinases and blocking B-cell activation. Because these agents are often given continuously, their use may lead to clinical resistance and unacceptable toxicities. Indeed, acquired resistance to covalent BTKis is common and caused by the expansion of clones with a cysteine-to-serine mutation at residue 481. A novel strategy being developed to overcome BTKi resistance, is PROTAC-induced degradation of BTK. PROTACs are small molecules with one domain that binds the target protein linked to another domain that binds to ubiquitin E3 ligase, resulting in ubiquitin-dependent proteasome degradation of the target protein. HSK29116 is a small BTK PROTAC molecule that is currently undergoing clinical investigation for the treatment of B-cell malignancies. In preclinical studies, HSK29116 overcame drug resistance caused by the C481S BTK mutation. Also, HSK29116 has demonstrated inhibition of B-cell proliferation without measurable loss of other BTK-family kinase activities. This kinase selectivity may limit some of the off-target toxicities seen with currently available BTKis. METHODS: This first-in-human, multicenter, open-label, phase 1 study (NCT04861779) began recruiting in 2021 to evaluate the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics of oral HSK29116. Eligible pts are ≥18 years old with diagnosis of a B-cell malignancy that has relapsed or become refractory to standard therapy (≥2 prior systemic therapies, which may have included a BTKi) and for which no other treatments known to provide clinical benefit exist. Pts must have an Eastern Cooperative Oncology Group performance status ≤2 and life expectancy >3 months. Phase 1a includes dose escalation to identify dose-limiting toxicities and establish the maximum tolerated dose and/or recommended dose of HSK29116 for a phase 1b dose expansion, initiating in the US. Approximately 36 pts will be enrolled in phase 1a and 90 pts in phase 1b (≥40 pts must have previously received covalent BTKis). The primary endpoint is safety. Secondary endpoints include PK and investigator-assessed overall response rate, duration of response, time to response, and progression-free survival. Exploratory analyses will evaluate the relationship between HSK29116 anti-tumor activity and BTK gene mutations, as well as BTK protein degradation effects of HSK29116. Descriptive statistics will be used to summarize all data. Citation Format: Jianyong Li, Wei Xu, Pangke Yan, Yong Cao, Mengyue Hu, William Daley. Phase 1 study of HSK29116, a Bruton tyrosine kinase (BTK) proteolysis-targeting chimera (PROTAC) agent, in patients with relapsed or refractory B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT128.

Polatuzumab-bendamustine-rituximab as bridge to CD19-directed CAR T cells in mantle cell lymphoma refractory to ibrutinib and venetoclax.

Polatuzumab-bendamustine-rituximab as bridge to CD19-directed CAR T cells in mantle cell lymphoma refractory to ibrutinib and venetoclax.

P606: META-ANALYSIS OF LOW ALLELIC BURDEN C481 BTK-MUTATIONS CALLS FOR CAUTION IN IBRUTINIB RESISTANCE EVALUATION

Background: Introduction of the orally administered tyrosine kinase inhibitor ibrutinib, a drug approved for relapsed or refractory mantle cell lymphoma in late 2013 and chronic lymphocytic leukemia in 2014, has changed treatment algoritms for B-cell malignancies and improved progression-free survival. Ibrutinib irreversibly targets Bruton’s tyrosine kinase (BTK), thus interrupting B-cell receptor signaling. However, it is known that mutations in the BTK gene, located on the X chromosome, can mediate acquired resistance, specifically modifying cysteine residue 481 to serine (C481S, X:101356176, GRCh38). Aims: Despite this knowledge, it is still unknown how low mutational burden can contribute to general resistance towards ibrutinib treatment. Thus, we performed a meta-analysis based on previous cardinal studies to investigate the collective and gender-specific distribution of variant allele frequencies. Methods: We implemented meta-analysis of single-nucleotide variant calls from seven studies involving whole-exome sequencing (Woyach et al. 2014 and 2017, Maddocks et al. 2015, Ahn et al. 2017, Kanagal-Shamanna et al. 2019, Gángó et al. 2020, Bödör et al. 2021) on mutational profiling concerning ibrutinib resistance and CLL. Four of these studies contained gender specific data. Computational analyses, statistics, and visualization were performed in Wolfram Language, R, and GraphPad Prism. Results: From the available data included in these studies, we were able to base our analyses on 226 non-BTK mutations and 170 BTK mutations within a variant allele frequency (VAF) range of 0.0001–0.9, of which 93 mutation candidates from males (VAF < 0.9) and 77 from females (VAF < 0.44) were observed. Extrapolative analysis of allele frequency distribution from all other variants (excl. BTK) in the studies revealed an exponential increase in the number of low burden variants below 10% VAF (R2>0.99). These variants were not significantly different between genders (PMann-Whitney=0.24, nmale=130, nfemale=73). In contrast, a significant difference was observed between male variant allele frequencies (PMann-Whitney=0.005) and females owing to BTK hemizygosity in males. However, this significance persisted when female BTK mutations were transformed to reflect a single copy (VAF/2, PMann-Whitney=0.015, ΔVAF=0.05) for gender comparison and even when observations for males were alternatively transformed to reflect two copies (2*VAF, PMann-Whitney<0.001, ΔVAF=0.19). The previously discovered threshold of 10% was set as the detection limit. Summary/Conclusion: Defining specific molecular contributions in drug resistance is imperative for the correct use of targeted therapy. It has been questioned how the low allelic burden of BTK mutations plays a role. Therefore, it is crucial to investigate the lower limit of detection. The meta-analysis suggests that variants below a certain threshold, here empirically 10%, may in many cases be false-positive variants, in agreement with other reports involving WES in general, introduced by erroneous base calling, PCR errors, etc. Although intriguing, it remains to elaborate whether the haploinsufficiency of BTK in males skew in the degree of ibrutinib resistance.

A comparison of DNA repair pathways to achieve a site-specific gene modification of the Bruton's tyrosine kinase gene

Gene editing utilizing homology-directed repair has advanced significantly for many monogenic diseases of the hematopoietic system in recent years but has also been hindered by decreases between in vitro and in vivo gene integration rates. Homology-directed repair occurs primarily in the S/G2 phases of the cell cycle, whereas long-term engrafting hematopoietic stem cells are typically quiescent. Alternative methods for a targeted integration have been proposed including homology-independent targeted integration and precise integration into target chromosome, which utilize non-homologous end joining and microhomology-mediated end joining, respectively. Non-homologous end joining occurs throughout the cell cycle, while microhomology-mediated end joining occurs predominantly in the S phase. We compared these pathways for the integration of a corrective DNA cassette at the Bruton’s tyrosine kinase gene for the treatment of X-linked agammaglobulinemia. Homology-directed repair generated the most integration in K562 cells; however, synchronizing cells into G1 resulted in the highest integration rates with homology-independent targeted integration. Only homology-directed repair produced seamless junctions, making it optimal for targets where insertions and deletions are impermissible. Bulk CD34+ cells were best edited by homology-directed repair and precise integration into the target chromosome, while sorted hematopoietic stem cells contained similar integration rates using all corrective donors.

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.

Optimizing Integration and Expression of Transgenic Bruton's Tyrosine Kinase for CRISPR-Cas9-Mediated Gene Editing of X-Linked Agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a monogenic primary immune deficiency characterized by very low levels of immunoglobulins and greatly increased risks for recurrent and severe infections. Patients with XLA have a loss-of-function mutation in the Bruton's tyrosine kinase (BTK) gene and fail to produce mature B lymphocytes. Gene editing in the hematopoietic stem cells of XLA patients to correct or replace the defective gene should restore B cell development and the humoral immune response. We used the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform to precisely target integration of a corrective, codon-optimized BTK complementary DNA (cDNA) cassette into its endogenous locus. This process is driven by homologous recombination and should place the transgenic BTK under transcriptional control of its endogenous regulatory elements. Each integrated copy of this cDNA in BTK-deficient K562 cells produced only 11% as much BTK protein as the wild-type gene. The donor cDNA was modified to include the terminal intron of the BTK gene. Successful integration of the intron-containing BTK donor led to a nearly twofold increase in BTK expression per cell over the base donor. However, this donor variant was too large to package into an adeno-associated viral vector for delivery into primary cells. Donors containing truncated variants of the terminal intron also produced elevated expression, although to a lesser degree than the full intron. Addition of the Woodchuck hepatitis virus posttranscriptional regulatory element led to a large boost in BTK transgene expression. Combining these modifications led to a BTK donor template that generated nearly physiological levels of BTK expression in cell lines. These reagents were then optimized to maximize integration rates into human hematopoietic stem and progenitor cells, which have reached potentially therapeutic levels in vitro. The novel donor modifications support effective gene therapy for XLA and will likely assist in the development of other gene editing-based therapies for genetic disorders.

X-Linked Agammaglobulinemia With Chronic Meningoencephalitis: A Diagnostic Challenge

X-linked agammaglobulinemia (XLA) is a primary disorder of humoral immunity characterized by Bruton tyrosine kinase gene mutations resulting in a primary antibody deficiency. While an intact T-cell function largely protects against majority of viral infections, enteroviruses are notorious for infecting these patients due to impaired mucosal immunity. Although the incidence of enteroviral meningoencephalitis in XLA is only 1-5%, yet the mortality is quite high. A typical presentation of enteroviral encephalitis in XLA is a subacute to chronic nervous system infection. A progressive loss of motor and cognitive milestones, spastic quadriplegia, coma, and death are common presentations. The histopathological features reflect gliosis, gradual neuronal loss, neuronophagia, and microglial proliferation. We describe the clinical and brain histopathological findings in a 2-year-old boy with XLA and progressive encephalitis, possibly due to an enteroviral infection.

X-Linked Immunodeficient Mice With No Functional Bruton's Tyrosine Kinase Are Protected From Sepsis-Induced Multiple Organ Failure.

We previously reported the Bruton's tyrosine kinase (BTK) inhibitors ibrutinib and acalabrutinib improve outcomes in a mouse model of polymicrobial sepsis. Now we show that genetic deficiency of the BTK gene alone in Xid mice confers protection against cardiac, renal, and liver injury in polymicrobial sepsis and reduces hyperimmune stimulation (“cytokine storm”) induced by an overwhelming bacterial infection. Protection is due in part to enhanced bacterial phagocytosis in vivo, changes in lipid metabolism and decreased activation of NF-κB and the NLRP3 inflammasome. The inactivation of BTK leads to reduced innate immune cell recruitment and a phenotypic switch from M1 to M2 macrophages, aiding in the resolution of sepsis. We have also found that BTK expression in humans is increased in the blood of septic non-survivors, while lower expression is associated with survival from sepsis. Importantly no further reduction in organ damage, cytokine production, or changes in plasma metabolites is seen in Xid mice treated with the BTK inhibitor ibrutinib, demonstrating that the protective effects of BTK inhibitors in polymicrobial sepsis are mediated solely by inhibition of BTK and not by off-target effects of this class of drugs.

Bruton's Tyrosine Kinase (BTK) Is a Novel KMT2A Partner Gene

Histone-lysine N-methyltransferase 2A gene (KMT2A) rearrangements are common genetic events in acute leukemia. They more frequent in infants and are found in 30-45% of acute myeloid leukemias (AML). They demonstrate great molecular heterogeneity with more than 90 partner genes and multiple KMT2A breakpoints involved (Meyer et al., 2018). These characteristics challenge both initial molecular diagnostics and MRD monitoring in ALs with KMT2A rearranged. As the accurate detection of all KMT2A-r types is crucial in order to correct patients' therapy and risk groups' definition, we aim to fully characterize KMT2A rearrangements within our cohort of patients using various techniques. Our recent study demonstrates a novel KMT2A partner gene - Bruton's tyrosine kinase (BTK), in a case of childhood AML. The patient is 9 m.o. girl who presented with WBC 69,3*109/L and hepatosplenomegaly. Morphological, immunochemical and immunological examination revealed acute monocytic leukemia. Bone marrow aspirates were analyzed by G-banded karyotyping, FISH with KMT2A breakapart probe. The karyotype was 45,X,der(X?)t(X;11)(q22.1;q23.3),del(11)(q14),-20[7] with 95% KMT2A-rearranged nuclei. Real-time RT-PCR for 8 most common KMT2A rearrangements screening was negative. Targeted RNA-seq with FusionPlex Myeloid kit (ArcherDX, CO, USA) followed by high-throughput sequencing identified novel fusion transcript KMT2A-BTK with exon 9-exon 2 breakpoint junction. Sanger sequencing was used for validation. The achieved data on KMT2A translocation partner and breakpoint location was used for subsequent patient-specific MRD monitoring. The patient was treated according to AML-MRD-2018 local protocol and achieved complete MRD-negative remission after induction course. Then, a severe myelodepression developed after consolidation therapy, and the patient died due to infectious complications. BTK gene is located at Xq22.1 and encodes for Bruton's tyrosine kinase protein (Hashimoto et al., 1996), which is a cytoplasmic non-receptor tyrosine kinase essential for B-cell maturation and development (Shinners et al., 2007). BTK deficiency is strongly associated with X-linked agammaglobulinemia, where 80-90% of patients express either truncated or misfolded or mutated BTK protein (Valiaho, Smith, &amp; Vihinen, 2006). BTK is also implemented in B-cell acute lymphoblastic leukemia, where it often has a deleted or a truncated kinase domain (Feldhahn et al., 2005). The opposite situation was reported for AML in several studies showing increased BTK activity (Gu et al., 2011; Tomasson et al., 2008). The presented t(X;11)(q22.1;q23.3) childhood AML is to our knowledge the first case of BTK translocations in hematologic malignancies, and its value for leukemogenesis is to be further investigated. However, as the breakpoint junction is located in BTK intron 1, upstream of its coding region (the translation starts in exon 2), we can speculate that BTK's structure itself is not committed and its activity is increased due to upregulated expression together with KMT2A. Therefore, the novel fusion KMT2A-BTK was for the first time revealed during the molecular profiling of KMT2A-rearranged AML in Russian Federation. The work was supported by RFBR grant №17-29-06052. Disclosures No relevant conflicts of interest to declare.

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.

A Novel BTK Gene Mutation in a Child With Atypical X-Linked Agammaglobulinemia and Recurrent Hemophagocytosis: A Case Report

X-linked agammaglobulinemia (XLA), caused by a mutation in the Bruton's tyrosine kinase (BTK) gene, is rarely reported in patients with recurrent hemophagocytic lymphohistiocytosis (HLH). This mutation leads to significantly reduced numbers of circulatory B cells and serum immunoglobulins in patients. Therefore, they exhibit repetitive bacterial infections since infancy, and immunoglobulin (Ig) replacement therapy is the primary treatment. HLH is a life-threatening condition with manifestations of non-remitting fever, hepatosplenomegaly, cytopenias, coagulopathy, lipid disorder, and multiple organ failure. It is caused by the immune dysregulation between cytotoxic T cells, NK cells, and histiocytes. The treatment is based on HLH-2004 protocol including immunotherapy, chemotherapy, supportive therapy, and stem cell transplantation. However, as we know more about the classification and pathophysiology of HLH, the treatment is modified. T-cell-directed immunotherapy is effective in patients with primary HLH, and strong immunosuppression is contraindicated in patients with severe ongoing infections or some primary immunodeficiency diseases (PIDs). Here, we report the case of a 7-year-old boy who presented with ecthyma gangrenosum and several episodes of pyogenic infections during childhood. At the age of 5 years, he exhibited cyclic HLH every 2–3 months. The remission of HLH episodes finally achieved after he received monthly Ig replacement therapy (400 mg/kg) at the 4th HLH. However, transient elevation of IgM was incidentally discovered after 6 cycles of monthly Ig replacement therapy. IgM-secreting multiple myeloma, Waldenström's macroglobulinemia, and lymphoma were excluded. The IgM levels then declined and returned to the normal range within a year. The patient and his parents received whole-genome sequencing analysis. It revealed a novel hemizygous c.1632-1G>A mutation in the BTK gene and XLA was diagnosed. XLA exhibits a spectrum of clinical and immunological presentations in patients. The identification of the mutation in the BTK gene contribute to an accurate diagnosis. Ig replacement therapy is the primary treatment for HLH in patients with XLA.

Dissection of subclonal evolution by temporal mutation profiling in chronic lymphocytic leukemia patients treated with ibrutinib.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is inducing durable responses in chronic lymphocytic leukemia (CLL) patients with refractory/relapsed disease or with TP53 defect, with BTK and phospholipase C gamma 2 (PLCG2) mutations representing the predominant mechanisms conferring secondary ibrutinib resistance. To understand the landscape of genomic changes and the dynamics of subclonal architecture associated with ibrutinib treatment, an ultra-deep next-generation sequencing analysis of 30 recurrently mutated genes was performed on sequential samples of 20 patients, collected before and during single-agent ibrutinib treatment. Mutations in the SF3B1, MGAand BIRC3 genes were enriched during ibrutinib treatment, while aberrations in the BTK, PLCG2, RIPK1, NFKBIE and XPO1 genes were exclusively detected in posttreatment samples. Besides the canonical mutations, four novel BTK mutations and three previously unreported PLCG2 variants were identified. BTK and PLCG2 mutations were backtracked in five patients using digital droplet PCR and were detectable on average 10.5 months before clinical relapse. With a median follow-up time of 36.5 months, 7/9 patients harboring BTK mutations showed disease progression based on clinical and/or laboratory features. In conclusion, subclonal heterogeneity, dynamic clonal selection and various patterns of clonal variegation were identified with novel resistance-associated BTK mutations in individual patients treated with ibrutinib.