In BTK, phosphorylated Y223 in the SH3 domain mirrors catalytic activity, but does not influence biological function.

ABSTRACT: Bruton's tyrosine kinase (BTK) is an enzyme needed for B-cell survival, and its inhibitors have become potent targeted medicines for the treatment of B-cell malignancies. The initial activation event of cytoplasmic protein-tyrosine kinases is the phosphorylation of a conserved regulatory tyrosine in the catalytic domain, which in BTK is represented by tyrosine 551. In addition, the tyrosine 223 (Y223) residue in the SRC homology 3 (SH3) domain has, for more than 2 decades, generally been considered necessary for full enzymatic activity. The initial recognition of its potential importance stems from transformation assays using nonlymphoid cells. To determine the biological significance of this residue, we generated CRISPR-Cas-mediated knockin mice carrying a tyrosine to phenylalanine substitution (Y223F), maintaining aromaticity and bulkiness while prohibiting phosphorylation. Using a battery of assays to study leukocyte subsets and the morphology of lymphoid organs, as well as the humoral immune responses, we were unable to detect any difference between wild-type mice and the Y223F mutant. Mice resistant to irreversible BTK inhibitors, through a cysteine 481 to serine substitution (C481S), served as an additional immunization control and mounted similar humoral immune responses as Y223F and wild-type animals. Collectively, our findings suggest that phosphorylation of Y223 serves as a useful proxy for phosphorylation of phospholipase Cγ2 (PLCG2), the endogenous substrate of BTK. However, in contrast to a frequently held conception, this posttranslational modification is dispensable for the function of BTK.

T-RHEX-RNAseq - a tagmentation-based, rRNA blocked, random hexamer primed RNAseq method for generating stranded RNAseq libraries directly from very low numbers of lysed cells.

BACKGROUND: RNA sequencing has become the mainstay for studies of gene expression. Still, analysis of rare cells with random hexamer priming - to allow analysis of a broader range of transcripts - remains challenging. RESULTS: We here describe a tagmentation-based, rRNA blocked, random hexamer primed RNAseq approach (T-RHEX-RNAseq) for generating stranded RNAseq libraries from very low numbers of FACS sorted cells without RNA purification steps. CONCLUSION: T-RHEX-RNAseq provides an easy-to-use, time efficient and automation compatible method for generating stranded RNAseq libraries from rare cells.

Three Adult Cases of STAT1 Gain-of-Function with Chronic Mucocutaneous Candidiasis Treated with JAK Inhibitors.

PURPOSE: 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. METHODS: 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. RESULTS: 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-É£ and CXCL10 were downregulated. CONCLUSIONS: 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.

Immune Biomarkers in the Peripheral Blood and Tumor Microenvironment of Classical Hodgkin Lymphoma Patients in Relation to Tumor Burden and Response to Treatment.

In classical Hodgkin lymphoma (cHL), the malignant cells represent only a small fraction of the tumor. Yet, they orchestrate a lymphocyte-dominated tumor microenvironment (TME) that supports their survival and growth. The systemic effects of this local immunomodulation are not fully elucidated. Here, we aimed at characterizing circulating lymphocytes and plasma proteins in relation to clinical parameters and treatment effect. Peripheral blood (PB) samples were obtained from 48 consecutive patients at diagnosis and at 2 time points after successful primary treatment. Single-cell suspensions were prepared from lymph node (LN) biopsies obtained for routine diagnostic purposes. Twenty healthy individuals were included as controls. Cells from PB and LN were analyzed by flow cytometry, and plasma proteins by Proximity Extension Assay. We found that the frequencies of T and B cells positively correlated between the LN and the PB compartments. Compared to controls, cHL patients had higher frequencies of proliferating T cells as well as higher expression of programmed death (PD)-1 and cytotoxic T lymphocyte antigen (CTLA)-4 in circulating T cells, and lower naive T-cell frequencies. Advanced-stage patients had fewer NK cells with a functionally impaired phenotype. Differences in the immune profile were observed in patients with a high tumor burden and with high inflammation, respectively. Most of these deviations disappeared after standard first-line treatment. Patients who received radiotherapy involving the mediastinum had low T-cell counts for a prolonged period. Our findings suggest that the immunomodulation of lymphocytes in the TME of cHL might affect immune biomarkers in the PB.

Whole-genome sequencing of chronic lymphocytic leukemia identifies subgroups with distinct biological and clinical features.

The value of genome-wide over targeted driver analyses for predicting clinical outcomes of cancer patients is debated. Here, we report the whole-genome sequencing of 485 chronic lymphocytic leukemia patients enrolled in clinical trials as part of the United Kingdom's 100,000 Genomes Project. We identify an extended catalog of recurrent coding and noncoding genetic mutations that represents a source for future studies and provide the most complete high-resolution map of structural variants, copy number changes and global genome features including telomere length, mutational signatures and genomic complexity. We demonstrate the relationship of these features with clinical outcome and show that integration of 186 distinct recurrent genomic alterations defines five genomic subgroups that associate with response to therapy, refining conventional outcome prediction. While requiring independent validation, our findings highlight the potential of whole-genome sequencing to inform future risk stratification in chronic lymphocytic leukemia.

Postmitotic differentiation of human monocytes requires cohesin-structured chromatin.

Cohesin is a major structural component of mammalian genomes and is required to maintain loop structures. While acute depletion in short-term culture models suggests a limited importance of cohesin for steady-state transcriptional circuits, long-term studies are hampered by essential functions of cohesin during replication. Here, we study genome architecture in a postmitotic differentiation setting, the differentiation of human blood monocytes (MO). We profile and compare epigenetic, transcriptome and 3D conformation landscapes during MO differentiation (either into dendritic cells or macrophages) across the genome and detect numerous architectural changes, ranging from higher level compartments down to chromatin loops. Changes in loop structures correlate with cohesin-binding, as well as epigenetic and transcriptional changes during differentiation. Functional studies show that the siRNA-mediated depletion of cohesin (and to a lesser extent also CTCF) markedly disturbs loop structures and dysregulates genes and enhancers that are primarily regulated during normal MO differentiation. In addition, gene activation programs in cohesin-depleted MO-derived macrophages are disturbed. Our findings implicate an essential function of cohesin in controlling long-term, differentiation- and activation-associated gene expression programs.

FOXO1 and FOXO3 Cooperatively Regulate Innate Lymphoid Cell Development.

Natural killer (NK) cells play roles in viral clearance and early surveillance against malignant transformation, yet our knowledge of the underlying mechanisms controlling their development and functions remain incomplete. To reveal cell fate-determining pathways in NK cell progenitors (NKP), we utilized an unbiased approach and generated comprehensive gene expression profiles of NK cell progenitors. We found that the NK cell program was gradually established in the CLP to preNKP and preNKP to rNKP transitions. In line with FOXO1 and FOXO3 being co-expressed through the NK developmental trajectory, the loss of both perturbed the establishment of the NK cell program and caused stalling in both NK cell development and maturation. In addition, we found that the combined loss of FOXO1 and FOXO3 caused specific changes to the composition of the non-cytotoxic innate lymphoid cell (ILC) subsets in bone marrow, spleen, and thymus. By combining transcriptome and chromatin profiling, we revealed that FOXO TFs ensure proper NK cell development at various lineage-commitment stages through orchestrating distinct molecular mechanisms. Combined FOXO1 and FOXO3 deficiency in common and innate lymphoid cell progenitors resulted in reduced expression of genes associated with NK cell development including ETS-1 and their downstream target genes. Lastly, we found that FOXO1 and FOXO3 controlled the survival of committed NK cells via gene regulation of IL-15Rß (CD122) on rNKPs and bone marrow NK cells. Overall, we revealed that FOXO1 and FOXO3 function in a coordinated manner to regulate essential developmental genes at multiple stages during murine NK cell and ILC lineage commitment.

Linked-read whole-genome sequencing resolves common and private structural variants in multiple myeloma.

Multiple myeloma (MM) is an incurable and aggressive plasma cell malignancy characterized by a complex karyotype with multiple structural variants (SVs) and copy-number variations (CNVs). Linked-read whole-genome sequencing (lrWGS) allows for refined detection and reconstruction of SVs by providing long-range genetic information from standard short-read sequencing. This makes lrWGS an attractive solution for capturing the full genomic complexity of MM. Here we show that high-quality lrWGS data can be generated from low numbers of cells subjected to fluorescence-activated cell sorting (FACS) without DNA purification. Using this protocol, we analyzed MM cells after FACS from 37 patients with MM using lrWGS. We found high concordance between lrWGS and fluorescence in situ hybridization (FISH) for the detection of recurrent translocations and CNVs. Outside of the regions investigated by FISH, we identified >150 additional SVs and CNVs across the cohort. Analysis of the lrWGS data allowed for resolution of the structure of diverse SVs affecting the MYC and t(11;14) loci, causing the duplication of genes and gene regulatory elements. In addition, we identified private SVs causing the dysregulation of genes recurrently involved in translocations with the IGH locus and show that these can alter the molecular classification of MM. Overall, we conclude that lrWGS allows for the detection of aberrations critical for MM prognostics and provides a feasible route for providing comprehensive genetics. Implementing lrWGS could provide more accurate clinical prognostics, facilitate genomic medicine initiatives, and greatly improve the stratification of patients included in clinical trials.

CD49b identifies functionally and epigenetically distinct subsets of lineage-biased hematopoietic stem cells.

Hematopoiesis is maintained by functionally diverse lineage-biased hematopoietic stem cells (HSCs). The functional significance of HSC heterogeneity and the regulatory mechanisms underlying lineage bias are not well understood. However, absolute purification of HSC subtypes with a pre-determined behavior remains challenging, highlighting the importance of continued efforts toward prospective isolation of homogeneous HSC subsets. In this study, we demonstrate that CD49b subdivides the most primitive HSC compartment into functionally distinct subtypes: CD49b- HSCs are highly enriched for myeloid-biased and the most durable cells, while CD49b+ HSCs are enriched for multipotent cells with lymphoid bias and reduced self-renewal ability. We further demonstrate considerable transcriptional similarities between CD49b- and CD49b+ HSCs but distinct differences in chromatin accessibility. Our studies highlight the diversity of HSC functional behaviors and provide insights into the molecular regulation of HSC heterogeneity through transcriptional and epigenetic mechanisms.

FOXO Dictates Initiation of B Cell Development and Myeloid Restriction in Common Lymphoid Progenitors.

The development of B cells relies on an intricate network of transcription factors critical for developmental progression and lineage commitment. In the B cell developmental trajectory, a temporal switch from predominant Foxo3 to Foxo1 expression occurs at the CLP stage. Utilizing VAV-iCre mediated conditional deletion, we found that the loss of FOXO3 impaired B cell development from LMPP down to B cell precursors, while the loss of FOXO1 impaired B cell commitment and resulted in a complete developmental block at the CD25 negative proB cell stage. Strikingly, the combined loss of FOXO1 and FOXO3 resulted in the failure to restrict the myeloid potential of CLPs and the complete loss of the B cell lineage. This is underpinned by the failure to enforce the early B-lineage gene regulatory circuitry upon a predominantly pre-established open chromatin landscape. Altogether, this demonstrates that FOXO3 and FOXO1 cooperatively govern early lineage restriction and initiation of B-lineage commitment in CLPs.

Autologous NK cells as consolidation therapy following stem cell transplantation in multiple myeloma.

Few approaches have been made toward exploring autologous NK cells in settings of cancer immunotherapy. Here, we demonstrate the feasibility of infusing multiple doses of ex vivo activated and expanded autologous NK cells in patients with multiple myeloma (MM) post-autologous stem cell transplantation. Infused NK cells were detected in circulation up to 4 weeks after the last infusion. Elevations in plasma granzyme B levels were observed following each consecutive NK cell infusion. Moreover, increased granzyme B levels were detected in bone marrow 4 weeks after the last infusion. All measurable patients had objective, detectable responses after NK cell infusions in terms of reduction in M-component and/or minimal residual disease. The present study demonstrates that autologous NK cell-based immunotherapy is feasible in a setting of MM consolidation therapy. It opens up the possibility for usage of autologous NK cells in clinical settings where patients are not readily eligible for allogeneic NK cell-based immunotherapies.

Bhlhe40 function in activated B and TFH cells restrains the GC reaction and prevents lymphomagenesis.

The generation of high-affinity antibodies against pathogens and vaccines requires the germinal center (GC) reaction, which relies on a complex interplay between specialized effector B and CD4 T lymphocytes, the GC B cells and T follicular helper (TFH) cells. Intriguingly, several positive key regulators of the GC reaction are common for both cell types. Here, we report that the transcription factor Bhlhe40 is a crucial cell-intrinsic negative regulator affecting both the B and T cell sides of the GC reaction. In activated CD4 T cells, Bhlhe40 was required to restrain proliferation, thus limiting the number of TFH cells. In B cells, Bhlhe40 executed its function in the first days after immunization by selectively restricting the generation of the earliest GC B cells but not of early memory B cells or plasmablasts. Bhlhe40-deficient mice with progressing age succumbed to a B cell lymphoma characterized by the accumulation of monoclonal GC B-like cells and polyclonal TFH cells in various tissues.

Limited access to antigen drives generation of early B cell memory while restraining the plasmablast response.

Cell fate decisions during early B cell activation determine the outcome of responses to pathogens and vaccines. We examined the early B cell response to T-dependent antigen in mice by single-cell RNA sequencing. Early after immunization, a homogeneous population of activated precursors (APs) gave rise to a transient wave of plasmablasts (PBs), followed a day later by the emergence of germinal center B cells (GCBCs). Most APs rapidly exited the cell cycle, giving rise to non-GC-derived early memory B cells (eMBCs) that retained an AP-like transcriptional profile. Rapid decline of antigen availability controlled these events; provision of excess antigen precluded cell cycle exit and induced a new wave of PBs. Fate mapping revealed a prominent contribution of eMBCs to the MBC pool. Quiescent cells with an MBC phenotype dominated the early response to immunization in primates. A reservoir of APs/eMBCs may enable rapid readjustment of the immune response when failure to contain a threat is manifested by increased antigen availability.

Low dose venetoclax as a single agent treatment of plasma cell malignancies harboring t(11;14).

Approximately 20% of newly diagnosed multiple myeloma (NDMM) patients harbor t(11;14), a marker of inferior prognosis, resulting in up-regulation of CCND1. These patients respond to BCL2 inhibitor experimental drug venetoclax. Furthermore, t(11;14) is reported to be associated with increased BCL2/MCL1 ratio. We investigated the use of venetoclax (400 mg daily) in a cohort of 25 multiple myeloma (MM) and AL-amyloidosis patients harboring t(11;14) and assessed safety and efficacy. Efficacy was assessed by response rate (RR) and time on treatment. Furthermore, immunohistochemistry (IHC), for BCL2 family member expression was assessed at diagnosis and relapse in the venetoclax-treated group and analyzed for correlation with clinical RR. Additionally, patient material from venetoclax non-treated group including non-t(11;14) diagnosis (n = 27), t(11;14) diagnosis (n = 17), t(11;14) relapse (n = 7), hyperdiploidy (n = 6) and hyperdiploidy + t(11;14) (n = 6) was used for RNA sequencing (RNASeq) and validation by qPCR. Venetoclax treatment in t(11;14) patients demonstrated manageable safety and promising efficacy. Partial responses or better were observed in eleven patients (44%). Responding patients had significantly higher BCL2/MCL1 (p = 0.031) as well as BCL2/BCL-XL (p = 0.021) ratio, regardless of time of measurement before venetoclax treatment. Furthermore, an IRF5 motif was enriched (p < .001) in the downregulated genes in t(11;14) relapses vs diagnoses. The RR with single agent venetoclax was 71% in AL-amyloidosis and 33% in MM, and IHC proved useful in prediction of treatment outcome. We could also demonstrate possible resistance mechanisms of t(11;14), downregulation of IRF5 targeted genes, which can be exploited for therapeutic advantages.

Ibrutinib Has Time-dependent On- and Off-target Effects on Plasma Biomarkers and Immune Cells in Chronic Lymphocytic Leukemia.

Ibrutinib is a covalently binding inhibitor of the B-cell receptor signaling-mediator Bruton's tyrosine kinase (BTK) with great efficacy in chronic lymphocytic leukemia (CLL). Common side effects like atrial fibrillation (AF), bleeding and infections might be caused by ibrutinib's inhibition of other kinases in non-B cells. Five-year follow-up of plasma biomarkers by proximity extension assay and immune cell numbers by flow cytometry during ibrutinib treatment revealed that 86 of the 265 investigated plasma biomarkers significantly changed during treatment, 74 of which decreased. Among the 12 markers that increased, 6 are associated with cardiovascular diseases and therefore potentially involved in ibrutinib-induced AF. Comparison between healthy donors and X-linked agammaglobulinemia (XLA) patients, who have nonfunctional BTK and essentially lack B cells, showed indicative changes in 53 of the 265 biomarkers while none differed significantly. Hence, neither B cells nor BTK-dependent pathways in other cells seem to influence the levels of the studied plasma biomarkers in healthy donors. Regarding immune cells, the absolute number of T cells, including subsets, decreased, paralleling the decreasing tumor burden. T helper 1 (Th1) cell numbers dropped strongly, while Th2 cells remained relatively stable, causing Th2-skewing. Thus, long-term ibrutinib treatment has a profound impact on the plasma proteome and immune cells in patients with CLL.

The transcription factor Bcl11b promotes both canonical and adaptive NK cell differentiation.

Epigenetic landscapes can provide insight into regulation of gene expression and cellular diversity. Here, we examined the transcriptional and epigenetic profiles of seven human blood natural killer (NK) cell populations, including adaptive NK cells. The BCL11B gene, encoding a transcription factor (TF) essential for T cell development and function, was the most extensively regulated, with expression increasing throughout NK cell differentiation. Several Bcl11b-regulated genes associated with T cell signaling were specifically expressed in adaptive NK cell subsets. Regulatory networks revealed reciprocal regulation at distinct stages of NK cell differentiation, with Bcl11b repressing RUNX2 and ZBTB16 in canonical and adaptive NK cells, respectively. A critical role for Bcl11b in driving NK cell differentiation was corroborated in BCL11B-mutated patients and by ectopic Bcl11b expression. Moreover, Bcl11b was required for adaptive NK cell responses in a murine cytomegalovirus model, supporting expansion of these cells. Together, we define the TF regulatory circuitry of human NK cells and uncover a critical role for Bcl11b in promoting NK cell differentiation and function.

Genomic and transcriptomic correlates of Richter transformation in chronic lymphocytic leukemia.

The transformation of chronic lymphocytic leukemia (CLL) to high-grade B-cell lymphoma is known as Richter syndrome (RS), a rare event with dismal prognosis. In this study, we conducted whole-genome sequencing (WGS) of paired circulating CLL (PB-CLL) and RS biopsies (tissue-RS) from 17 patients recruited into a clinical trial (CHOP-O). We found that tissue-RS was enriched for mutations in poor-risk CLL drivers and genes in the DNA damage response (DDR) pathway. In addition, we identified genomic aberrations not previously implicated in RS, including the protein tyrosine phosphatase receptor (PTPRD) and tumor necrosis factor receptor-associated factor 3 (TRAF3). In the noncoding genome, we discovered activation-induced cytidine deaminase-related and unrelated kataegis in tissue-RS affecting regulatory regions of key immune-regulatory genes. These include BTG2, CXCR4, NFATC1, PAX5, NOTCH-1, SLC44A5, FCRL3, SELL, TNIP2, and TRIM13. Furthermore, differences between the global mutation signatures of pairs of PB-CLL and tissue-RS samples implicate DDR as the dominant mechanism driving transformation. Pathway-based clonal deconvolution analysis showed that genes in the MAPK and DDR pathways demonstrate high clonal-expansion probability. Direct comparison of nodal-CLL and tissue-RS pairs from an independent cohort confirmed differential expression of the same pathways by RNA expression profiling. Our integrated analysis of WGS and RNA expression data significantly extends previous targeted approaches, which were limited by the lack of germline samples, and it facilitates the identification of novel genomic correlates implicated in RS transformation, which could be targeted therapeutically. Our results inform the future selection of investigative agents for a UK clinical platform study. This trial was registered at www.clinicaltrials.gov as #NCT03899337.

Novel mouse model resistant to irreversible BTK inhibitors: a tool identifying new therapeutic targets and side effects.

Pharmacological inhibitors of Bruton tyrosine kinase (BTK) have revolutionized treatment of B-lymphocyte malignancies and show great promise for dampening autoimmunity. The predominant BTK inhibitors tether irreversibly by covalently binding to cysteine 481 in the BTK catalytic domain. Substitution of cysteine 481 for serine (C481S) is the most common mechanism for acquired drug resistance. We generated a novel C481S knock-in mouse model and, using a battery of tests, no overt B-lymphocyte phenotype was found. B lymphocytes from C481S animals were resistant to irreversible, but sensitive to reversible, BTK inhibitors. In contrast, irreversible inhibitors equally impaired T-lymphocyte activation in mice, mimicking the effect of treatment in patients. This demonstrates that T-lymphocyte blockage is independent of BTK. We suggest that the C481S knock-in mouse can serve as a useful tool for the study of BTK-independent effects of irreversible inhibitors, allowing for the identification of novel therapeutic targets and pinpointing potential side effects.

Bhlhe40 and Bhlhe41 transcription factors regulate alveolar macrophage self-renewal and identity.

Tissues in multicellular organisms are populated by resident macrophages, which perform both generic and tissue-specific functions. The latter are induced by signals from the microenvironment and rely on unique tissue-specific molecular programs requiring the combinatorial action of tissue-specific and broadly expressed transcriptional regulators. Here, we identify the transcription factors Bhlhe40 and Bhlhe41 as novel regulators of alveolar macrophages (AMs)-a population that provides the first line of immune defense and executes homeostatic functions in lung alveoli. In the absence of these factors, AMs exhibited decreased proliferation that resulted in a severe disadvantage of knockout AMs in a competitive setting. Gene expression analyses revealed a broad cell-intrinsic footprint of Bhlhe40/Bhlhe41 deficiency manifested by a downregulation of AM signature genes and induction of signature genes of other macrophage lineages. Genome-wide characterization of Bhlhe40 DNA binding suggested that these transcription factors directly repress the expression of lineage-inappropriate genes in AMs. Taken together, these results identify Bhlhe40 and Bhlhe41 as key regulators of AM self-renewal and guardians of their identity.