Flavopiridol is a small molecule inhibitor of cyclin-dependent kinases (CDK) known to impair global transcription via inactivation of positive transcription elongation factor b. It has been demonstrated to have significant activity predominantly in chronic lymphocytic leukemia and acute myeloid leukemia in phase I/II clinical trials while other similar CDK inhibitors are vigorously being pursued in pre-clinical and clinical studies. Although flavopiridol is a potent therapeutic agent against blood diseases, some patients still have primary or acquired resistance throughout their clinical course. Considering the limited knowledge of resistance mechanisms of flavopiridol, we investigated the potential mechanisms of resistance to flavopiridol in a cell line system, which gradually acquired resistance to flavopiridol in vitro, and then confirmed the mechanism in patient samples. Herein, we present that this resistant cell line developed resistance through up-regulation of phosphorylation of RNA polymerase II C-terminal domain, activation of CDK9 kinase activity, and prolonged Mcl-1 stability to counter flavopiridol's drug actions. Further analyses suggest MAPK/ERK activation-mediated Mcl-1 stabilization contributes to the resistance and knockdown of Mcl-1 in part restores sensitivity to flavopiridol-induced cytotoxicity. Altogether, these findings demonstrate that CDK9 is the most relevant target of flavopiridol and provide avenues to improve the therapeutic strategies in blood malignancies.

A growing body of evidence suggests that the resistance of CLL cells to apoptosis is partly mediated through the interactions between leukemia cells and adjacent stromal cells residing in the lymphatic tissue or bone marrow microenvironment. Mcl-1, an anti-apoptotic protein that is associated with failure to treatment is up-regulated in CLL lymphocytes after interaction with microenvironment. However, the regulation of its expression in context to microenvironment is unclear. We evaluated and compared changes in Mcl-1 in CLL B-cells in suspension culture and when co-cultured on stromal cells. The blockade of apoptosis in co-cultured CLL cells is associated with diminution in caspase-3 and PARP cleavage and is not dependent on cytogenetic profile or prognostic factors of the disease. Stroma-derived resistance to apoptosis is associated with a cascade of transcriptional events such as increase in levels of total RNA Pol II and its phosphorylation at Ser2 and Ser5, increase in the rate of global RNA synthesis, and amplification of Mcl-1 transcript levels. The latter is associated with increase in Mcl-1 protein level without an impact on the levels of Bcl-2 and Bcl-xL. Post-translational modifications of protein kinases show increased phosphorylation of Akt at Ser473, Erk at Thr202/Tyr204 and Gsk-3β at Ser9 and augmentation of total Mcl-1 accumulation along with phosphorylation at Ser159/Thr163 sites. Collectively, stroma-induced apoptosis resistance is mediated through signaling proteins that regulate transcriptional and translational expression and post-translational modification of Mcl-1 in CLL cells in context to bone marrow stromal microenvironment.

Results of RESONATE-2 (PCYC-1115/1116) supported approval of ibrutinib for first-line treatment of chronic lymphocytic leukemia. Extended analysis of RESONATE-2 was conducted to determine long-term efficacy and safety of ibrutinib in older patients with chronic lymphocytic leukemia. A total of 269 patients aged ≥65 years with previously untreated chronic lymphocytic leukemia without del(17p) were randomized 1:1 to ibrutinib (n=136) or chlorambucil (n=133) on days 1 and 15 of a 28-day cycle for 12 cycles. Median ibrutinib treatment duration was 28.5 months. Ibrutinib significantly prolonged progression-free survival versus chlorambucil (median, not reached vs 15 months; hazard ratio, 0.12; 95% confidence interval, 0.07-0.20; P<0.0001). The 24-month progression-free survival was 89% with ibrutinib (97% and 89% in patients with del[11q] and unmutated immunoglobulin heavy chain variable region gene, respectively). Progression-free survival rates at 24 months were also similar regardless of age (<75 years [88%], ≥75 years [89%]). Overall response rate was 92% (125/136). Rate of complete response increased substantially from 7% at 12 months to 18% with extended follow up. Greater quality of life improvements occurred with ibrutinib versus chlorambucil in Functional Assessment of Chronic Illness Therapy-Fatigue (P=0.0013). The most frequent grade ≥3 adverse events were neutropenia (12%), anemia (7%), and hypertension (5%). Rate of discontinuations due to adverse events was 12%. Results demonstrated that first-line ibrutinib for elderly patients with chronic lymphocytic leukemia provides sustained response and progression-free survival benefits over chemotherapy, with depth of response improving over time without new toxicity concerns. This trial was registered at clinicaltrials.gov identifier 01722487 and 01724346.

We performed an observational study on the efficacy of ben-damustine and rituximab (BR) as first salvage regimen in chronic lymphocytic leukemia (CLL). In an intention-to-treat analysis including 237 patients, the median progression-free survival (PFS) was 25 months. The presence of del(17p), unmutated IGHV and advanced stage were associated with a shorter PFS at multivariate analysis. The median time-to-next treatment was 31.3 months. Front-line treatment with a chemoimmunotherapy regimen was the only predictive factor for a shorter time to next treatment at multivariate analysis. The median overall survival (OS) was 74.5 months. Advanced disease stage (i.e. Rai stage III-IV or Binet stage C) and resistant disease were the only parameters significantly associated with a shorter OS. Grade 3-5 infections were recorded in 6.3% of patients. A matched-adjusted indirect comparison with ibrutinib given second-line within Named Patient Programs in the United Kingdom and in Italy was carried out with OS as objective end point. When restricting the analysis to patients with intact 17p who had received chemoimmunotherapy in first line, there was no difference in OS between patients treated with ibrutinib (63% alive at 36 months) and patients treated with BR (74.4% alive at 36 months). BR is an efficacious first salvage regimen in CLL in a real-life population, including the elderly and unfit patients. BR and ibrutinib may be equally effective in terms of OS when used as first salvage treatment in patients without 17p deletion.

Treatment of chronic lymphocytic leukemia (CLL) has shifted from chemo-immunotherapy to targeted agents. To define the evolutionary dynamics induced by targeted therapy in CLL, we perform serial exome and transcriptome sequencing for 61 ibrutinib-treated CLLs. Here, we report clonal shifts (change >0.1 in clonal cancer cell fraction, Q < 0.1) in 31% of patients during the first year of therapy, associated with adverse outcome. We also observe transcriptional downregulation of pathways mediating energy metabolism, cell cycle, and B cell receptor signaling. Known and previously undescribed mutations in BTK and PLCG2, or uncommonly, other candidate alterations are present in seventeen subjects at the time of progression. Thus, the frequently observed clonal shifts during the early treatment period and its potential association with adverse outcome may reflect greater evolutionary capacity, heralding the emergence of drug-resistant clones.

Human T cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a subset of infected subjects. Two viral proteins, Tax-1 and HTLV-1 basic leucine zipper factor (HBZ), play important roles in the pathogenesis of both diseases. We recently demonstrated that HBZ, previously considered a nuclear protein, is exclusively localized in the cytoplasm of peripheral blood mononuclear cells (PBMCs) of HAM/TSP patients. Here, the analysis of a larger panel of HAM/TSP cases confirmed that HBZ is a cytoplasmic protein, while Tax-1 preferentially localized in the cytoplasm with fewer speckle-like dots in the nucleus. More importantly, here we report for the first time that HBZ, when expressed in asymptomatic carriers (AC), is also confined in the cytoplasm. Similarly, Tax-1 was preferentially expressed in the cytoplasm in a significant proportion of AC. Interestingly, in both HAM/TSP and AC patients, the expression of HBZ and Tax-1 was rarely found in the same cell. We observed only few cases coexpressing the two oncoprotein in a very limited number of cells. In representative AC and HAM/TSP patients, cells expressing cytoplasmic HBZ were almost exclusively found in the CD4+ T cell compartment and very rarely in CD8+ T cells. Interestingly, at least in the cases analyzed, the expression of thymocite-expressed molecule involved in selection (THEMIS) is dispensable for the cytoplasmic localization of HBZ in both AC and HAM/TSP. The study of an HTLV-1-immortalized cell line established from an HAM/TSP patient confirmed HBZ as a resident cytoplasmic protein not shuttling between the cytoplasm and nucleus. These results extend our previous observation on the dichotomy of HBZ localization between HAM/TSP and ATL, pointing to the exclusive either cytoplasmic or nuclear localization in the two diseased states, respectively. Moreover, they show a rather selective expression in distinct cells of either HBZ or Tax-1. The unprecedented observation that HBZ is expressed only in the cytoplasm in AC strongly suggests a progressive modification of HBZ localization during the disease states associated to HTLV-1 infection. Future studies will clarify whether the distinct HBZ intracellular localization is a marker or a causative event of disease evolution.

The constitutive activation of B-cell receptor (BCR) signaling, together with the overexpression of the Bcl-2 family anti-apoptotic proteins, represents two hallmarks of chronic lymphocytic leukemia (CLL) that drive leukemia cell proliferation and sustain their survival. TG02 is a small molecule multi-kinase inhibitor that simultaneously targets both of these facets of CLL pathogenesis. First, its inhibition of cyclin-dependent kinase 9 blocked the activation of RNA polymerase II and transcription. This led to the depletion of Mcl-1 and rapid induction of apoptosis in the primary CLL cells. This mechanism of apoptosis was independent of CLL prognostic factors or prior treatment history, but dependent on the expression of BAX and BAK. Second, TG02, which inhibits the members of the BCR signaling pathway such as Lck and Fyn, blocked BCR-crosslinking-induced activation of NF-κB and Akt, indicating abrogation of BCR signaling. Finally, the combination of TG02 and ibrutinib demonstrated moderate synergy, suggesting a future combination of TG02 with ibrutinib, or use in patients that are refractory to the BCR antagonists. Thus, the dual inhibitory activity on both the CLL survival pathway and BCR signaling identifies TG02 as a unique compound for clinical development in CLL and possibly other B cell malignancies.

Ibrutinib, a once-daily oral inhibitor of Bruton's tyrosine kinase, is approved in the United States and Europe for treatment of patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). The phase 3 RESONATE study showed improved efficacy of single-agent ibrutinib over ofatumumab in patients with relapsed/refractory CLL/SLL, including those with high-risk features. Here we report the final analysis from RESONATE with median follow-up on study of 65.3 months (range, 0.3-71.6) in the ibrutinib arm. Median progression-free survival (PFS) remained significantly longer for patients randomized to ibrutinib vs ofatumumab (44.1 vs 8.1 months; hazard ratio [HR]: 0.148; 95% confidence interval [CI]: 0.113-0.196; P˂.001). The PFS benefit with ibrutinib vs ofatumumab was preserved in the genomic high-risk population with del(17p), TP53 mutation, del(11q), and/or unmutated IGHV status (median PFS 44.1 vs 8.0 months; HR: 0.110; 95% CI: 0.080-0.152), which represented 82% of patients. Overall response rate with ibrutinib was 91% (complete response/complete response with incomplete bone marrow recovery, 11%). Overall survival, censored for crossover, was better with ibrutinib than ofatumumab (HR: 0.639; 95% CI: 0.418-0.975). With up to 71 months (median 41 months) of ibrutinib therapy, the safety profile remained consistent with prior reports; cumulatively, all-grade (grade ≥3) hypertension and atrial fibrillation occurred in 21% (9%) and 12% (6%) of patients, respectively. Only 16% discontinued ibrutinib because of adverse events (AEs). These long-term results confirm the robust efficacy of ibrutinib in relapsed/refractory CLL/SLL irrespective of high-risk clinical or genomic features, with no unexpected AEs. This trial is registered at www.clinicaltrials.gov (NCT01578707).

Mutations in BTK, PLCG2, and BCL2 have been reported in patients with progressive disease (PD) on continuous single-agent BTK or BCL2 inhibitor treatment. We tested for these mutations in samples from patients with PD after completion of first-line treatment with fixed-duration ibrutinib plus venetoclax for chronic lymphocytic leukemia (CLL) in the phase II CAPTIVATE study.

We developed a simple, rapid and quantitative assay using the fluorescent probe PicoGreen to measure the concentration of ionizing radiation-induced double-stranded DNA (dsDNA) in mouse plasma, and we correlated this concentration with the radiation dose. With 70 μl of blood obtained by fingerstick, this 30 min assay reduces protein interference without extending sample processing time. Plasma from nonirradiated mice (BALB/c and NIH Swiss) was pooled, diluted and spiked with dsDNA to establish sensitivity and reproducibility of the assay to quantify plasma dsDNA. The assay was then used to directly quantify dsDNA in plasma at 0-48 h after mice received 0-10 Gy total-body irradiation (TBI). There are three optimal conditions for this assay: 1:10 dilution of plasma in water; 1:200 dilution of PicoGreen reagent in water; and calibration of radiation-induced dsDNA concentration through a standard addition method using serial spiking of samples with genomic dsDNA. Using the internal standard calibration curve of the spiked samples method, the signal developed within 5 min, exhibiting a linear signal (r(2) = 0.997). The radiation-induced elevation of plasma DNA in mice started at 1-3 h, peaked at 9 h and gradually returned to baseline at 24 h after TBI (6 Gy). DNA levels in plasma collected from mice 9 h after 0-10 Gy TBI correlated strongly with dose (r(2) = 0.991 and 0.947 for BALB/c and NIH Swiss, respectively). Using the PicoGreen assay, we observed a radiation dose-dependent response in extracellular plasma DNA 9 h after irradiation with an assay time ≤ 30 min.

Resistance to the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been attributed solely to mutations in BTK and related pathway molecules. Using whole-exome and deep-targeted sequencing, we dissect evolution of ibrutinib resistance in serial samples from five chronic lymphocytic leukaemia patients. In two patients, we detect BTK-C481S mutation or multiple PLCG2 mutations. The other three patients exhibit an expansion of clones harbouring del(8p) with additional driver mutations (EP300, MLL2 and EIF2A), with one patient developing trans-differentiation into CD19-negative histiocytic sarcoma. Using droplet-microfluidic technology and growth kinetic analyses, we demonstrate the presence of ibrutinib-resistant subclones and estimate subclone size before treatment initiation. Haploinsufficiency of TRAIL-R, a consequence of del(8p), results in TRAIL insensitivity, which may contribute to ibrutinib resistance. These findings demonstrate that the ibrutinib therapy favours selection and expansion of rare subclones already present before ibrutinib treatment, and provide insight into the heterogeneity of genetic changes associated with ibrutinib resistance.

HTLV-1 is the causative agent of a severe form of adult T cell leukemia/Lymphoma (ATL), and of a chronic progressive neuromyelopathy designated HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Two important HTLV-1-encoded proteins, Tax-1 and HBZ, play crucial roles in the generation and maintenance of the oncogenic process. Less information is instead available on the molecular and cellular mechanisms leading to HAM/TSP. More importantly, no single specific biomarker has been described that unambiguously define the status of HAM/TSP. Here we report for the first time the finding that HBZ, described until now as an exclusive nuclear protein both in chronically infected and in ATL cells, is instead exclusively localized in the cytoplasm of peripheral blood mononuclear cells (PBMC) from patients suffering of HAM/TSP. Interestingly, at the single cell level, HBZ and Tax-1 proteins are never found co-expressed in the same cell, suggesting the existence of mechanisms of expression uncoupling of these two important HTLV-1 viral products in HAM/TSP patients. Cells expressing cytoplasmic HBZ were almost exclusively found in the CD4+ T cell compartment that was not, at least in a representative HAM/TSP patient, expressing the CD25 marker. Less than 1 percent CD8+ T cells were fond positive for HBZ, while B cells and NK cells were found negative for HBZ in HAM/TSP patients. Our results identify the cytoplasmic localization of HBZ in HAM/TSP patient as a possible biomarker of this rather neglected tropical disease, and raise important hypotheses on the role of HBZ in the pathogenesis of the neuromyelopathy associated to HTLV-1 infection.

Anti-CD20 antibody treatments, such as obinutuzumab, have been associated with infusion-related reactions (IRRs). In the phase 3 iLLUMINATE study of ibrutinib-obinutuzumab versus chlorambucil-obinutuzumab in first-line chronic lymphocytic leukemia/small lymphocytic lymphoma, IRRs were substantially reduced with ibrutinib-obinutuzumab versus chlorambucil-obinutuzumab. We prospectively analyzed inflammatory cytokines to evaluate the impact of ibrutinib on circulating cytokine levels following obinutuzumab infusion. In iLLUMINATE, ibrutinib or chlorambucil was given approximately 30-120 min before the first obinutuzumab infusion. Cytokines evaluated were IFNγ, IL-6, IL-8, IL-10, IL-18, MCP-1, MIP-1α, MIP-1β, and TNFα. Changes in peak cytokine levels from baseline (immediately before obinutuzumab) to post-obinutuzumab infusion were compared between arms and between patients with versus without IRRs using Wilcoxon rank sum test. Of 228 treated patients, 95 on ibrutinib-obinutuzumab (15 with IRRs, 80 without) and 88 on chlorambucil-obinutuzumab (45 with IRRs, 43 without) with cytokine data were included. Irrespective of IRR occurrence, median increase in cytokines was lower with ibrutinib-obinutuzumab versus chlorambucil-obinutuzumab for all cytokines (P < 0.01) except MIP-1β. Across treatment arms, post-obinutuzumab median increase in all cytokines except MIP-1β was greater in patients with versus without IRRs (P < 0.001). IL-6 and IL-8 elevations were associated with IRRs in both treatment arms. Among patients with IRRs, those receiving ibrutinib-obinutuzumab had lower post-obinutuzumab increases in IL-6, IL-8, IL-10, and MCP-1 (P < 0.04) than patients receiving chlorambucil-obinutuzumab. For patients in the ibrutinib-treatment arm, we observed a reduction in both the rate of clinically apparent IRRs and the levels of IRR-related cytokines and chemokines. This observation supports an immunomodulatory mechanism of action for ibrutinib. Clinical Trial Registration: NCT02264574.

We report long-term follow-up from the RESONATE-2 phase 3 study of the once-daily Bruton's tyrosine kinase inhibitor ibrutinib, which is the only targeted therapy with significant progression-free survival (PFS) and overall survival (OS) benefit in multiple randomized chronic lymphocytic leukemia (CLL) studies. Patients (≥65 years) with previously untreated CLL, without del(17p), were randomly assigned 1:1 to once-daily ibrutinib 420 mg until disease progression/unacceptable toxicity (n = 136) or chlorambucil 0.5-0.8 mg/kg ≤12 cycles (n = 133). With up to 8 years of follow-up (range, 0.1-96.6 months; median, 82.7 months), significant PFS benefit was sustained for ibrutinib vs chlorambucil (hazard ratio [HR], 0.154; 95% confidence interval [CI], 0.108-0.220). At 7 years, PFS was 59% for ibrutinib vs 9% for chlorambucil. PFS benefit was also observed for ibrutinib- vs chlorambucil-randomized patients with high-risk genomic features: del(11q) (HR, 0.033; 95% CI, 0.010-0.107) or unmutated immunoglobulin heavy chain variable region (HR, 0.112; 95% CI, 0.065-0.192). OS at 7 years was 78% with ibrutinib. Prevalence of adverse events (AEs) was consistent with previous 5-year follow-up. Ibrutinib dosing was held (≥7 days) for 79 patients and reduced for 31 patients because of AEs; these AEs resolved or improved in 85% (67 of 79) and 90% (28 of 31) of patients, respectively. With up to 8 years of follow-up, 42% of patients remain on ibrutinib. Long-term RESONATE-2 data demonstrate sustained benefit with first-line ibrutinib treatment for CLL, including for patients with high-risk genomic features. These trials were registered at www.clinicaltrials.gov as #NCT01722487 and #NCT01724346.

CLL cell trafficking between blood and tissue compartments is an integral part of the disease process. Idelalisib, a phosphoinositide 3-kinase delta (PI3Kδ) inhibitor causes rapid lymph node shrinkage, along with an increase in lymphocytosis, prior to inducing objective responses in CLL patients. This characteristic activity presumably is due to CLL cell redistribution from tissues into the blood, but the underlying mechanisms are not fully understood. We therefore analyzed idelalisib effects on CLL cell adhesion to endothelial and bone marrow stromal cells (EC, BMSC). We found that idelalisib inhibited CLL cell adhesion to EC and BMSC under static and shear flow conditions. TNFα-induced VCAM-1 (CD106) expression in supporting layers increased CLL cell adhesion and accentuated the inhibitory effect of idelalisib. Co-culture with EC and BMSC also protected CLL from undergoing apoptosis, and this EC- and BMSC-mediated protection was antagonized by idelalisib. Furthermore, we demonstrate that CLL cell adhesion to EC and VLA-4 (CD49d) resulted in the phosphorylation of Akt, which was sensitive to inhibition by idelalisib. These findings demonstrate that idelalisib interferes with integrin-mediated CLL cell adhesion to EC and BMSC, providing a novel mechanism to explain idelalisib-induced redistribution of CLL cells from tissues into the blood.

We previously demonstrated that the HLA class II transactivator CIITA inhibits HIV-1 replication in T cells by competing with the viral transactivator Tat for the binding to Cyclin T1 subunit of the P-TEFb complex. Here, we analyzed the anti-viral function of CIITA in myeloid cells, another relevant HIV-1 target cell type. We sinvestigated clones of the U937 promonocytic cell line, either permissive (Plus) or non-permissive (Minus) to HIV-1 replication. This different phenotype has been associated with the expression of TRIM22 in U937 Minus but not in Plus cells.

After in vivo implantation of cell-loaded devices, only the cells close to the capillaries can obtain nutrients to maintain their functions. It is known that factors secreted by stem cells, rather than stem cells themselves, are fundamental to guarantee new vascularization in the area of implant.

Recognition of modified histone species by distinct structural domains within 'reader' proteins plays a critical role in the regulation of gene expression. Readers that simultaneously recognize histones with multiple marks allow transduction of complex chromatin modification patterns into specific biological outcomes. Here we report that chromatin regulator tripartite motif-containing 24 (TRIM24) functions in humans as a reader of dual histone marks by means of tandem plant homeodomain (PHD) and bromodomain (Bromo) regions. The three-dimensional structure of the PHD-Bromo region of TRIM24 revealed a single functional unit for combinatorial recognition of unmodified H3K4 (that is, histone H3 unmodified at lysine 4, H3K4me0) and acetylated H3K23 (histone H3 acetylated at lysine 23, H3K23ac) within the same histone tail. TRIM24 binds chromatin and oestrogen receptor to activate oestrogen-dependent genes associated with cellular proliferation and tumour development. Aberrant expression of TRIM24 negatively correlates with survival of breast cancer patients. The PHD-Bromo of TRIM24 provides a structural rationale for chromatin activation through a non-canonical histone signature, establishing a new route by which chromatin readers may influence cancer pathogenesis.

Waldenström Macroglobulinemia (WM) is a B-cell lymphoma characterized by the precursor condition IgM monoclonal gammopathies of undetermined significance (IgM MGUS). We performed a gene expression profiling study to compare the transcriptome signatures of bone marrow (BM) B-cells and plasma cells of 36 WM patients, 13 IgM MGUS cases, and 7 healthy subjects used as controls (CTRLs) by Affymetrix microarray. We determined 2038 differentially expressed genes (DEGs) in CD19+ cells and 29 DEGs genes in CD138+ cells, respectively. The DEGs identified in B-cells were associated with KEGG pathways, mainly involved in hematopoietic cell lineage antigens, cell adhesion/focal adhesion/transmembrane proteins, adherens junctions, Wnt-signaling pathway, BCR-signaling pathway, calcium signaling pathway, complement/coagulation cascade, platelet activation, cytokine-cytokine receptor interactions, and signaling pathways responsible for cell cycle, apoptosis, proliferation and survival. In conclusion, we showed the deregulation of groups of genes belonging to KEGG pathways in the comparison among WM vs. IgM MGUS vs. CTRLs in B-cells. Interestingly, a small set of genes in B-cells displayed a common transcriptome expression profile between WM and IgM MGUS compared to CTRLs, suggesting its possible role in the risk of transformation of IgM MGUS to WM.

In lymphoid organs, nurse-like cells (NLCs) show properties of tumor-associated macrophages, playing a crucial role in chronic lymphocytic leukemia (CLL) cell survival. Ibrutinib, a potent inhibitor of Bruton's tyrosine kinase (BTK), is able to counteract pro-survival signals in CLL cells. Since the effects on CLL cells have been studied in the last years, less is known about the influence of ibrutinib on NLCs properties. We sought to determine how ibrutinib modifies NLCs functions focusing on the balance between immunosuppressive and inflammatory features. Our data show that ibrutinib targets BTK expressed by NLCs modifying their phenotype and function. Treatment with ibrutinib reduces the phagocytic ability and increases the immunosuppressive profile of NLCs exacerbating the expression of M2 markers. Accordingly, ibrutinib hampers LPS-mediated signaling, decreasing STAT1 phosphorylation, while allows IL-4-mediated STAT6 phosphorylation. In addition, NLCs treated with ibrutinib are able to protect CLL cells from drug-induced apoptosis partially through the secretion of IL-10. Results from patient samples obtained prior and after 1 month of treatment with ibrutinib show an accentuation of CD206, CD11b and Tie2 in the monocytic population in the peripheral blood. Our study provides new insights into the immunomodulatory action of ibrutinib on monocyte/macrophage population in CLL.