The aim of this study was to characterize clinical effects and biomarkers in three patients with chronic mucocutaneous candidiasis (CMC) caused by gain-of-function (GOF) mutations in the STAT1 gene during treatment with Janus kinase (JAK) inhibitors. Mass cytometry (CyTOF) was used to characterize mononuclear leukocyte populations and Olink assay to quantify 265 plasma proteins. Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA) was used to quantify the reactivity against Candida albicans. Overall, JAK inhibitors improved clinical symptoms of CMC, but caused side effects in two patients. Absolute numbers of neutrophils, T cells, B cells, and NK cells were sustained during baricitinib treatment. Detailed analysis of cellular subsets, using CyTOF, revealed increased expression of CD45, CD52, and CD99 in NK cells, reflecting a more functional phenotype. Conversely, monocytes and eosinophils downregulated CD16, consistent with reduced inflammation. Moreover, T and B cells showed increased expression of activation markers during treatment. In one patient with a remarkable clinical effect of baricitinib treatment, the immune response to C. albicans increased after 7 weeks of treatment. Alterations in plasma biomarkers involved downregulation of cellular markers CXCL10, annexin A1, granzyme B, granzyme H, and oncostatin M, whereas FGF21 was the only upregulated marker after 7 weeks. After 3 months, IFN-ɣ and CXCL10 were downregulated. The clinical effect of JAK inhibitor treatment of CMC is promising. Several biological variables were altered during baricitinib treatment demonstrating that lymphocytes, NK cells, monocytes, and eosinophils were affected. In parallel, cellular reactivity against C. albicans was enhanced.

BTKbase is an international database for disease-causing variants in Bruton tyrosine kinase (BTK) leading to X-linked agammaglobulinemia (XLA), a rare primary immunodeficiency of antibody production. BTKbase was established in 1994 as one of the first publicly available variation databases. The number of cases has more than doubled since the last update; it now contains information for 2310 DNA variants in 2291 individuals. 1025 of the DNA variants are unique. The human genome contains more than 500 protein kinases, among which BTK has the largest number of unique disease-causing variants. The current version of BTKbase has numerous novel features: the database has been reformatted, it has moved to LOVD database management system, it has been internally harmonized, etc. Systematics and standardization have been increased, including Variation Ontology annotations for variation types. There are some regions with lower than expected variation frequency and some hotspots for variations. BTKbase contains, in addition to variant descriptions at DNA, RNA and protein levels, also laboratory parameters and clinical features for many patients. BTKbase has served clinical and research communities in the diagnosis of XLA cases and provides general insight into effects of variations, especially in signalling pathways. Amino acid substitutions and their effects were investigated, predicted, and visualized at 3D level in the protein domains. BTKbase is freely available.

Pirtobrutinib is a non-covalent BTK inhibitor (BTKi) designed to maintain activity despite the most common resistance mutation to covalent inhibitors, at BTK C481. To investigate mechanisms of disease progression on pirtobrutinib, we evaluated the effect of pirtobrutinib in vitro on the BCR pathway in pre- and post-treatment patient samples (n=5), and on 2 of these patients, we performed whole exome sequencing longitudinally including prior to and at relapse on pirtobrutinib. Phylogenetics and subclonal dynamics associated with resistance were evaluated using the PhylogicNDT and Concerti tools. To investigate the impact of identified BTK mutations on BCR activation, we generated 6 single and 3 double mutants using site directed mutagenesis and expressed them in the BTK null DT40 B cell line. We demonstrate that primary CLL cells from responding patients on the pirtobrutinib trial show reduced BCR signaling, reduced CCL3/CCL4 chemokine secretion, as well as effective induction of apoptosis and inhibition of proliferation, in response to pirtobrutinib. At time of progression, these primary CLL cells show increasing resistance to pirtobrutinib in signaling inhibition and cytokine production, with reduced inhibition of proliferation and induction of apoptosis. In WES analysis, patient #1 had 16 samples evaluated prior to, during, and at relapse on acalabrutinib, vecabrutinib and pirtobrutinib. Clonal analysis of samples collected during acalabrutinib shows steady selection of a clone harboring BTK p.C481S mutation with CCF 92% at relapse but then steadily decreases during pirtobrutinib treatment. Concerti's time-scaled phylogenetic tree shows the birth of a new clone containing the BTK gatekeeper mutation, p.T474I, during acalabrutinib treatment, which then grows rapidly under pirtobrutinib treatment, taking over nearly the entire cancer cell population and replacing the prior p.C481S clone. This complete clonal shift during pirtobrutinib treatment suggests that pirtobrutinib effectively inhibits the p.C481S clone, while the p.T474I gatekeeper clone is likely driving resistance in this patient. We also observed an additional gatekeeper clone BTK p.T474L develop at low levels, as well as another previously undescribed BTK mutation at p.M477I. Manual inspection showed that BTK mutations p.M477I and p.T474I are in cis and therefore in the same clone. Patient #2 had 10 samples evaluated prior to, during and at relapse on ibrutinib and pirtobrutinib. During ibrutinib therapy, we observed a steady increase in a clone with TP53 p.S240G and SF3B1 p.K666N mutations, reaching CCFs >40% at relapse on ibrutinib. We also noted a significant increase in BTK p.C481R (CCF 33%), BTK p.C481S (CCF 11%) and TP53 p.R196* (CCF 5%) at progression on ibrutinib. Under pirtobrutinib treatment the clone carrying BTK p.C481R decreased to CCF 20%, while BTK p.C481S (28%) and TP53 p.R197* (35%) both increased. Concerti's phylogenetic tree captures the birth of a resistant clone, harboring BTK p.L528W which significantly increases to CCF 30% at progression on pirtobrutinib. Functional characterization of the identified BTK mutations demonstrated that only T474I, T474L and C481S mutants showed adequate kinase activity, while all the other mutants including M477I, C48IR and L528W essentially lacked kinase activity as judged by phosphorylation at BTK Y223 and at PLCG2 Y753. As expected, the C481S variant was resistant to ibrutinib, but not to pirtobrutinib, while the T474I/L variants were sensitive to ibrutinib but resistant to pirtobrutinib. Interestingly, phosphorylation of AKT and ERK were retained downstream, even with mutations that failed to activate proximal BCR signaling. Furthermore, phosphorylation of AKT and ERK was also observed in the B7.10 cell line which lacks endogenous BTK, demonstrating significant activation of these pathways independent of BTK. In this study, we demonstrate that ex vivo efficacy of pirtobrutinib declines as patients’ CLL starts to progress, in concert with the development of gatekeeper and alternative site BTK mutations that lead to resistance to pirtobrutinib. Interestingly, many of the second-site BTK mutations fail to activate BTK phosphorylation but are still associated with downstream activation of phospho-AKT and phospho-ERK; the mechanism of this activation remains to be elucidated.

Irreversible inhibitors of Bruton tyrosine kinase (BTK), pioneered by ibrutinib, have become breakthrough drugs in the treatment of leukemias and lymphomas. Resistance variants (mutations) occur, but in contrast to those identified for many other tyrosine kinase inhibitors, they affect less frequently the "gatekeeper" residue in the catalytic domain. In this study we carried out variation scanning by creating 11 substitutions at the gatekeeper amino acid, threonine 474 (T474). These variants were subsequently combined with replacement of the cysteine 481 residue to which irreversible inhibitors, such as ibrutinib, acalabrutinib and zanubrutinib, bind. We found that certain double mutants, such as threonine 474 to isoleucine (T474I) or methionine (T474M) combined with catalytically active cysteine 481 to serine (C481S), are insensitive to ≥16-fold the pharmacological serum concentration, and therefore defined as super-resistant to irreversible inhibitors. Conversely, reversible inhibitors showed a variable pattern, from resistance to no resistance, collectively demonstrating the structural constraints for different classes of inhibitors, which may affect their clinical application.

Resistance to the irreversible Bruton´s tyrosine kinase (BTK) inhibitors is the main cause of disease progression in patients with Chronic Lymphocytic Leukemia (Quinquenel et. al. Blood 2019). Cysteine to serine substitution at the position 481 in BTK, is the most common resistance mutation. Other less frequent mutations like the constitutively active phospholipase C-gamma 2 (PLCG2) variant also occur (Woyach et. al. J. Clin. Oncology 2017). Different from many other tyrosine kinase inhibitors, BTK mutations less frequently affect the gatekeeper residue in the kinase domain (Maddocks et. al. JAMA Oncology 2015). In this study, we have performed mutation scanning with substitutions replacing the gatekeeper residue. We have generated all the possible amino acid substitutions requiring a single nucleotide change in the gatekeeper and several variants requiring 2 or 3 nucleotide substitutions. Selected variants were also combined with substitutions at the C481, which is the binding site of irreversible BTK inhibitors, such as ibrutinib, acalabrutinib and zanubrutinib. Our results show unexpected, super-resistant variants and demonstrate that concomitant mutations, such as cysteine 481 to serine combined with threonine 474 to isoleucine or methionine, enhanced the resistance to irreversible BTK inhibitors. On the other hand, reversible BTK inhibitors displayed different inhibitory responses against the super-resistant mutants. Binding of the BTK inhibitors was subjected to molecular dynamics predictions, which correlated with the experimental binding data. Based on the available clinical and experimental results, the mechanisms underlying the spectrum of resistance mutations in BTK are presented. Disclosures No relevant conflicts of interest to declare.

A novel immunodeficiency, frequently accompanied by high serum-IgE, and caused by mutations in the PGM3 gene was described in 2014. To date there are no unique phenotype characteristics for PGM3 deficiency. PGM3 encodes a carbohydrate-modifying enzyme, phosphoglucomutase 3. Null-mutations are quite likely lethal, and to date only missense mutations or small deletions have been reported. Such mutations frequently cause a combination of reduced enzyme activity and protein instability, complicating determination of the enzyme level needed for survival. Here we present the first patient with a homozygous splice-modifying mutation in the PGM3 gene. An A > G substitution at position c.871 + 3 (transcript NM_001199917) is causing a deletion of exon 7 in the majority of PGM3 transcripts. In addition, this case further increases the clinical phenotypes of immunodeficiency caused by PGM3 mutations.

In chronic lymphocytic leukaemia (CLL) patients, treatment with the Bruton tyrosine kinase inhibitor ibrutinib induces a rapid shift of tumour cells from lymph nodes (LN) to peripheral blood (PB). Here, we characterized in depth the dynamics of ibrutinib-induced inflammatory, transcriptional and cellular changes in different compartments immediately after treatment initiation in seven relapsed/refractory CLL patients. Serial PB and LN samples were taken before start and during the first 29 days of treatment. Changes in plasma inflammation-related biomarkers, CLL cell RNA expression, B-cell activation and migration markers expression, and PB mononuclear cell populations were assessed. A significant reduction of 10 plasma inflammation markers, the majority of which were chemokines and not CLL-derived, was observed within hours, and was paralleled by very early increase of CD19+ circulating cells. At the RNA level, significant and continuous changes in transcription factors and signalling molecules linked to B-cell receptor signalling and CLL biology was observed in both PB and LN CLL cells already after 2 days of treatment. In conclusion, ibrutinib seems to instantly shut off an ongoing inflammatory response and interfere with diverse sensitive pathways in the LN.

Bruton’s Tyrosine Kinase (BTK) is a cytoplasmic protein tyrosine kinase with a fundamental role in B-lymphocyte development and activation. The nucleocytoplasmic shuttling of BTK is specifically modulated by the Ankyrin Repeat Domain 54 (ANKRD54) protein and the interaction is known to be exclusively SH3-dependent. To identify the spectrum of the ANKRD54 SH3-interactome, we applied phage-display screening of a library containing all the 296 human SH3 domains. The BTK-SH3 domain was the prime interactor. Quantitative western blotting analysis demonstrated the accuracy of the screening procedure. Revealing the spectrum and specificity of ANKRD54-interactome is a critical step toward functional analysis in cells and tissues.

In this study we analyzed the very early effects of the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib on tumor and immune cells in 7 symptomatic, relapsed or refractory CLL patients during the first four weeks of treatment. Five of the patients had Rai stage IV at study entry and four patients had 17p deletion or TP53 mutation. Median number of previous treatment regimens was 2 (range 1-4). Peripheral blood (PB) samples were collected before (< 1 week) treatment start and at six different time points during the treatment (9 hours after treatment start on day 1; on day 2; day 4; day 8; day 15 and day 29). Fine needle aspiration of two pathological lymph nodes (LN) identified by ultrasound was performed before (< 1 week) treatment start and at day 2, day 8 and day 29. Flow-cytometry was performed to analyze the changes in the peripheral blood mononuclear cell populations in PB including CLL cells, Natural Killer (NK) cells, monocytes, dendritic cells (DC) and T cells memory subsets, helper subpopulations (Ths) and regulatory T cells (Tregs). Moreover, changes in expression of 18 B-cell activation and migration markers on CLL cells as well as T-cell activation and proliferation markers, were analyzed in paired LN and PB samples. Finally, plasma levels of 92 inflammation-related protein biomarkers were assessed by Multiplex Proximity Extension Assay (PEA).

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations in the gene coding for Bruton’s tyrosine kinase (BTK). Deficiency of BTK leads to a developmental block in B cell differentiation; hence, the patients essentially lack antibody-producing plasma cells and are susceptible to various infections. A substantial portion of the mutations in BTK results in splicing defects, consequently preventing the formation of protein-coding mRNA. Antisense oligonucleotides (ASOs) are therapeutic compounds that have the ability to modulate pre-mRNA splicing and alter gene expression. The potential of ASOs has been exploited for a few severe diseases, both in pre-clinical and clinical studies. Recently, advances have also been made in using ASOs as a personalized therapy for XLA. Splice-correction of BTK has been shown to be feasible for different mutations in vitro, and a recent proof-of-concept study demonstrated the feasibility of correcting splicing and restoring BTK both ex vivo and in vivo in a humanized bacterial artificial chromosome (BAC)-transgenic mouse model. This review summarizes the advances in splice correction, as a personalized medicine for XLA, and outlines the promises and challenges of using this technology as a curative long-term treatment option.