Pro-inflammatory cells sustain leukemic clonal expansion in T-cell large granular lymphocyte leukemia.

T-cell large granular lymphocyte leukemia (T-LGLL) is a chronic lymphoproliferative disorder characterized by the clonal expansion of T-cell large granular lymphocytes (T-LGL). Immunophenotypic and genotypic features contribute to discriminate symptomatic (CD8+ STAT3-mutated T-LGLL) from clinically indolent patients, this latter group including CD8+ wildtype (wt), CD4+ STAT5B-mutated and wt cases. T-LGL lymphoproliferation is sustained both by somatic gain-offunction mutations (i.e., STAT3 and STAT5B) and by pro-inflammatory cytokines, but little information is available on the activity of T-LGLL non-leukemic cells. In this study, we characterized pro-inflammatory cells in the peripheral blood of T-LGLL patients and analyzed their role in supporting the leukemic growth. In symptomatic patients we found that cell populations not belonging to the leukemic component showed a discrete pro-inflammatory pattern. In particular, CD8+ STAT3-mutated cases showed a skewed Th17/Treg ratio and an abnormal distribution of monocyte populations characterized by increased intermediate and non-classical monocytes. We also demonstrated that monocytes released high levels of interleukin-6 after CCL5 stimulation, a chemokine specifically expressed only by leukemic LGL. Conversely, in asymptomatic cases an altered distribution of monocyte populations was not detected. Moreover, T-LGLL patients' monocytes showed abnormal activation of signaling pathways, further supporting the different pathogenic role of monocytes in patients in discrete clinical settings. Altogether, our data contribute to deepening the knowledge on the different cell subtypes in T-LGLL, focusing particularly on non-leukemic cell populations and thus offering the rationale for new therapeutic strategies.

Focal adhesion kinase activation by calcium-dependent calpain is involved in chronic lymphocytic leukaemia cell aggressiveness.

Signalling events downstream the B-cell receptor (BCR) are central for the survival and progression of chronic lymphocytic leukaemia (CLL) cells. Focal adhesion kinase (FAK), regulated through calpain, interacts with molecules of BCR signalling, cytoskeletal modelling and disease progression, such as Src/Lyn, cortactin and HS1. Hypothesizing that FAK might play a key role in CLL pathogenesis, we observed a down-modulation of FAK whole form, associated with FAK cleavage due to calpain activity upon BCR stimulation. Patients, whose cells were able to release Ca++ after BCR stimulation, had less amount of full-length FAK, which translated into a higher presence of cleaved/activated form of the protein phosphorylated at Y397, these features being mostly shown by immunoglobulin heavy chain (IGHV)-unmutated poor-prognosis patients. Moreover, we found that cortactin and HS1 proteins were overexpressed in those cells, suggesting a possible interplay with FAK. Treatment with the FAK inhibitor Defactinib was able to induce apoptosis in CLL cells. In conclusion, the malignant phenotype in unfavourable-prognosis patients seems to be encouraged by the overexpression of cortactin and HS1, that, together with FAK, may be involved in a druggable pathogenetic pathway in CLL.

Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed "satellites," were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

The complex karyotype landscape in chronic lymphocytic leukemia allows the refinement of the risk of Richter syndrome transformation.

Complex karyotype (CK) at chronic lymphocytic leukemia (CLL) diagnosis is a negative biomarker of adverse outcome. Since the impact of CK and its subtypes, namely type-2 CK (CK with major structural abnormalities) or high-CK (CK with ≥5 chromosome abnormalities), on the risk of developing Richter syndrome (RS) is unknown, we carried out a multicenter real-life retrospective study to test its prognostic impact. Among 540 CLL patients, 107 harbored a CK at CLL diagnosis, 78 were classified as CK2 and 52 as high-CK. Twenty-eight patients developed RS during a median follow-up of 6.7 years. At the time of CLL diagnosis, CK2 and high-CK were more common and predicted the highest risk of RS transformation, together with advanced Binet stage, unmutated (U)-IGHV, 11q-, and TP53 abnormalities. We integrated these variables into a hierarchical model: high-CK and/or CK2 patients showed a 10-year time to RS (TTRS) of 31%; U-IGHV/11q- /TP53 abnormalities/Binet stage B-C patients had a 10-year TTRS of 12%; mutated (M)-IGHV without CK and TP53 disruption a 10-year TTRS of 3% (P<0.0001). We herein demonstrate that CK landscape at CLL diagnosis allows the risk of RS transformation to be refined and we recapitulated clinico-biological variables into a prognostic model.

Identification of a miR-146b-Fas ligand axis in the development of neutropenia in T large granular lymphocyte leukemia.

Tlarge granular lymphocyte leukemia (T-LGLL) is characterized by the expansion of several large granular lymphocyte clones, among which a subset of large granular lymphocytes showing constitutively activated STAT3, a specific CD8+/CD4- phenotype and the presence of neutropenia has been identified. Although STAT3 is an inducer of transcription of a large number of oncogenes, so far its relationship with miRNAs has not been evaluated in T-LGLL patients. Here, we investigated whether STAT3 could carry out its pathogenetic role in T-LGLL through an altered expression of miRNAs. The expression level of 756 mature miRNA was assessed on purified T large granular lymphocytes (T-LGLs) by using a TaqMan Human microRNA Array. Hierarchical Clustering Analysis of miRNA array data shows that the global miRNome clusters with CD8 T-LGLs. Remarkably, CD8 T-LGLs exhibit a selective and STAT3-dependent repression of miR-146b expression, that significantly correlated with the absolute neutrophil counts and inversely correlated with the expression of Fas ligand (FasL), that is regarded as the most relevant factor in the pathogenesis of neutropenia. Experimental evidence demonstrates that the STAT3-dependent reduction of miR-146b expression in CD8 T-LGLs occurs as a consequence of miR-146b promoter hypermethylation and results in the disruption of the HuR-mediated post-transcriptional machinery controlling FasL mRNA stabilization. Restoring miR-146b expression in CD8 T-LGLs lead to a reduction of HuR protein and, in turn, of FasL mRNA expression, thus providing mechanistic insights for the existence of a STAT3-miR146b-FasL axis and neutropenia in T-LGLL.

Ibrutinib in relapsed hairy cell leukemia variant: A case report and review of the literature.

Hairy cell leukemia variant (HCLv) is a provisional disease in the 2016 WHO classification of lymphomas, characterized by unfavorable prognosis and early relapse following conventional purine analog-based regimens. In this study, we report 2 patients with relapsed HCLv treated with ibrutinib. The first patient achieved a partial response following ibrutinib treatment and received the drug for 16 months, without severe adverse events. However, at disease progression venetoclax was not clinically active. The second patient discontinued the drug early due to intolerance. Ibrutinib was active in our patients with HCLv and deserve further investigations.

HSP70/HSF1 axis, regulated via a PI3K/AKT pathway, is a druggable target in chronic lymphocytic leukemia.

Considering the role played by the heat shock protein of 70 kDa (HSP70) in cancer, we characterized this protein and its major regulator, the heat shock factor 1 (HSF1), in chronic lymphocytic leukemia (CLL). We found both HSP70 and HSF1 overexpressed in CLL patients, correlated to poor prognosis and abnormally localized in the nucleus of leukemic B cells. The two proteins were strictly correlated each other and their levels decreased consensually in those patients responding to in vivo therapeutic regimens. HSP70 and HSF1 inhibition was proved to be effective in inducing a dose-dependent in vitro apoptosis of CLL B cells. Considering that HSF1 is finely regulated by kinases belonging to pathways triggered by rat sarcoma (RAS), we benefited from a previous proteomic study performed in CLL patients aiming to assess the activation/expression of key signaling proteins. We found that patients showing high levels of HSP70 also expressed high Akt-Ser473, thus activating HSF1. Inhibition of PI3K, which activates AKT, reduced the expression of HSF1 and HSP70. By contrast, HSP70-low patients displayed high activation of MEK1/2 and ERK1/2, known to negatively regulate HSF1. These data demonstrate that the HSP70 expression is regulated by the modulation of HSF1 activity through the activation of RAS-regulated pathways and suggest the HSP70/HSF1 interplay as an interesting target for antileukemic therapies. Finally, inhibition of PI3K, that activates AKT, reduced the expression of HSF1 and HSP70.

The combination of complex karyotype subtypes and IGHV mutational status identifies new prognostic and predictive groups in chronic lymphocytic leukaemia.

BACKGROUND: Complex karyotype (CK) is a heterogeneous category with a negative impact in chronic lymphocytic leukaemia (CLL). Our group has recently reported that CK patients with major structural abnormalities (i.e. CK2) are characterised by a worse prognosis, as compared to other lesions within CK(CK1). METHODS: We performed a multicentre retrospective study to test whether the combination of CK subtypes with IGHV status could be a relevant prognostic and predictive tool. RESULTS: Among 522 patients 13% harboured CK2, 41% CK1 and/or U-IGHV (U-CK1) and 46% M-IGHV without any CK subtypes (M-noCK). After a median follow-up of 5.8 years, CK2 patients had the shortest TTFT (5-year TTFT 31%, 39 and 81%, p < 0.0001) and OS (5-year OS 67%, 85 and 93%, p < 0.0001) as compared to U-CK1 or M-noCK cases, regardless of TP53 abnormalities. CK2 patients also had the worst outcome after chemoimmunotherapy. In fact, the median TTNT after FCR or BR was 1.86 and 4.79 years for CK2 and U-CK1, but not reached for M-noCK patients (p < 0.0005). CONCLUSIONS: We herein suggest that the combined assessment of the IGHV mutational status and CK subtypes refines the prognostication of CLL, allowing to identify M-IGHV patients without any CK subtypes who are characterised by an indolent disease and excellent outcome after chemoimmunotherapy.

Cortactin, a Lyn substrate, is a checkpoint molecule at the intersection of BCR and CXCR4 signalling pathway in chronic lymphocytic leukaemia cells.

Cortactin (CTTN) is a substrate of the Src kinase Lyn that is known to play an actin cytoskeletal regulatory role involved in cell migration and cancer progression following its phosphorylation at Y421. We recently demonstrated that Cortactin is overexpressed in patients with chronic lymphocytic leukaemia (CLL). This work was aimed at defining the functional role of Cortactin in these patients. We found that Cortactin is variably expressed in CLL patients both in the peripheral blood and lymph nodes and that its expression correlates with the release of matrix metalloproteinase 9 (MMP-9) and the motility of neoplastic cells. Cortactin knockdown, by siRNA, induced a reduction in MMP-9 release as well as a decrease of migration capability of leukaemic B cells in vitro, also after chemotactic stimulus. Furthermore, Cortactin phosphorylation was lowered by the Src kinase-inhibitor PP2 with a consequent decrease of MMP-9 release in culture medium. An impaired migration, as compared to control experiments without Cortactin knockdown, was observed following CXCL12 triggering. Reduced Cortactin expression and phosphorylation were also detected both in vivo and in vitro after treatment with Ibrutinib, a Btk inhibitor. Our results highlight the role of Cortactin in CLL as a check-point molecule between the BCR and CXCR4 signalling pathways.

Lyn sustains oncogenic signaling in chronic lymphocytic leukemia by strengthening SET-mediated inhibition of PP2A.

Aberrant protein kinase activities, and the consequent dramatic increase of Ser/Thr and -Tyr phosphorylation, promote the deregulation of the survival pathways in chronic lymphocytic leukemia (CLL), which is crucial to the pathogenesis and progression of the disease. In this study, we show that the tumor suppressor protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases, is in an inhibited form because of the synergistic contribution of 2 events, the interaction with its physiologic inhibitor SET and the phosphorylation of Y307 of the catalytic subunit of PP2A. The latter event is mediated by Lyn, a Src family kinase previously found to be overexpressed, delocalized, and constitutively active in CLL cells. This Lyn/PP2A axis accounts for the persistent high level of phosphorylation of the phosphatase's targets and represents a key connection linking phosphotyrosine- and phosphoserine/threonine-mediated oncogenic signals. The data herein presented show that the disruption of the SET/PP2A complex by a novel FTY720-analog (MP07-66) devoid of immunosuppressive effects leads to the reactivation of PP2A, which in turn triggers apoptosis of CLL cells. When used in combination with SFK inhibitors, the action of MP07-66 is synergistically amplified, providing a new option in the therapeutic strategy for CLL patients.

Not all IGHV3-21 chronic lymphocytic leukemias are equal: prognostic considerations.

An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset #2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset #2. Within subset #2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non-subset #2/IGHV3-21 was enriched for IGHV-unmutated cases (P = .002). Subset #2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non-subset #2/IGHV3-21 (22 vs 60 months, P = .001). No such difference was observed between non-subset #2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset #2 emerges as uniformly aggressive, contrasting non-subset #2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL.

Targeted activation of the SHP-1/PP2A signaling axis elicits apoptosis of chronic lymphocytic leukemia cells.

Lyn, a member of the Src family of kinases, is a key factor in the dysregulation of survival and apoptotic pathways of malignant B cells in chronic lymphocytic leukemia. One of the effects of Lyn's action is spatial and functional segregation of the tyrosine phosphatase SHP-1 into two pools, one beneath the plasma membrane in an active state promoting pro-survival signals, the other in the cytosol in an inhibited conformation and unable to counter the elevated level of cytosolic tyrosine phosphorylation. We herein show that SHP-1 activity can be elicited directly by nintedanib, an agent also known as a triple angiokinase inhibitor, circumventing the phospho-S591-dependent inhibition of the phosphatase, leading to the dephosphorylation of pro-apoptotic players such as procaspase-8 and serine/threonine phosphatase 2A, eventually triggering apoptosis. Furthermore, the activation of PP2A by using MP07-66, a novel FTY720 analog, stimulated SHP-1 activity via dephosphorylation of phospho-S591, which unveiled the existence of a positive feedback signaling loop involving the two phosphatases. In addition to providing further insights into the molecular basis of this disease, our findings indicate that the PP2A/SHP-1 axis may emerge as an attractive, novel target for the development of alternative strategies in the treatment of chronic lymphocytic leukemia.

Intrinsic and extrinsic mechanisms contribute to maintain the JAK/STAT pathway aberrantly activated in T-type large granular lymphocyte leukemia.

The JAK/STAT pathway is altered in T-cell large granular lymphocytic leukemia. In all patients, leukemic LGLs display upregulation of phosphorylated STAT3 (P-STAT3) that activates expression of many antiapoptotic genes. To investigate the mechanisms maintaining STAT3 aberrantly phosphorylated using transcriptional protein and functional assays, we analyzed interleukin (IL)-6 and suppressor of cytokine signaling-3 (SOCS3), 2 key factors of the JAK/STAT pathway that induce and inhibit STAT3 activation, respectively. We showed that IL-6 was highly expressed and released by the patients' peripheral blood LGL-depleted population, accounting for a trans-signaling process. By neutralizing IL-6 or its specific receptor with specific antibodies, a significant reduction of P-STAT3 levels and, consequently, LGL survival was demonstrated. In addition, we found that SOCS3 was down-modulated in LGL and unresponsive to IL-6 stimulation. By treating neoplastic LGLs with a demethylating agent, IL-6-mediated SOCS3 expression was restored with consequent P-STAT3 and myeloid cell leukemia-1 down-modulation. Methylation in the SOCS3 promoter was not detectable, suggesting that an epigenetic inhibition mechanism occurs at a different site. Our data indicate that loss of the inhibitor SOCS3 cooperates with IL-6 to maintain JAK/STAT pathway activation, thus contributing to leukemic LGL survival, and suggest a role of demethylating agents in the treatment of this disorder.

Reduced levels of circulating progenitor cells in juvenile idiopathic arthritis are counteracted by anti TNF-α therapy.

BACKGROUND: Endothelial progenitor cells (EPC) promote angiogenesis and vascular repair. Though reduced EPC levels have been shown in rheumatoid arthritis, no study has so far evaluated EPCs in children with juvenile idiopathic arthritis (JIA). We aimed to study circulating EPCs in children with JIA, their relation to disease activity, and effects of anti TNF-α treatment. METHODS: Circulating EPCs were quantified by flow cytometry based on CD34, CD133 and KDR expression in peripheral blood of 22 patients with oligoarticular JIA and 29 age-matched controls. EPCs were re-assessed in children with methotrexate-resistant oligo-extended JIA before and up to 12 month after initiation of anti-TNF-alpha therapy. Plasma concentrations of inflammatory and EPC-regulating factors were measured using a multiplex array. Confocal immunofluorescence was used to demonstrate EPCs in synovial tissues. RESULTS: Children with active JIA showed a significant reduction of relative and absolute counts of circulating progenitor cells and EPCs compared to age-matched healthy controls. CD34(+) cell levels were modestly and inversely correlated to disease activity. A strong inverse correlation was found between serum TNF-α and EPC levels. In 8 patients treated with anti TNF-α agents, the number of EPCs rose to values similar to healthy controls. CD34(+)KDR(+) EPCs were found in the synovial tissue of JIA children, but not in control. CONCLUSIONS: Children with JIA have reduced levels of the vasculoprotective and proangiogenic EPCs. While EPCs may contribute to synovial tissue remodelling, EPC pauperization may indicate an excess cardiovascular risk if projected later in life.

Ex Vivo Signaling Protein Mapping in T Lymphocytes in the Psoriatic Arthritis Joints.

We assessed signaling protein mapping in total T cells, to analyze the proportions of T regulatory (Treg) and TCD4+ effector (Teff) cell phenotypes, and the respective interleukin 6Rα (IL-6Rα) expression in the inflammatory microenvironment of synovial fluid (SF) of patients with sustained psoriatic arthritis (PsA). Our approach was to measure the IL-6 level in SF using a multiplex bead immunoassay. Reverse-phase protein array was used to assess Janus kinase (JAK) 1 and JAK2, extra-cellular regulated kinase (ERK) 1 and 2, protein kinase Cδ (PKCδ), signal transducer and activator and transcription (STAT) 1, STAT3, and STAT5 phosphoproteins in total T cell lysates from SF of patients with PsA. Frequencies of CD4+IL-17A-F+IL-23+ CD4+ Th cells producing IL-17A and IL-17F (Th17) and CD4+CD25high intracellular forkhead box transcription factor+ (FOXP3+) phenotypes, and the percentage of Treg- and Teff- cells were quantified in SF and matched peripheral blood (PB) of patients with PsA and PB of healthy controls (HC) by flow cytometry. Our results were the following: In PsA SF samples, a coordinate increase of JAK1, ERK1/2, STAT1, STAT3, and STAT5 phosphoproteins was found in total T cells in SF of PsA; where IL-6 levels were higher than in PB from HC. Expanded CD4+IL-17A-F+IL-23+ Th17, CD4+ CD25- Teff- and CD4+CD25(high) FoxP3+Treg subsets, showing similar levels of enhanced IL-6Rδ expression, were confined to PsA joints. In our studies, the transcriptional network profile identified by ex vivo signaling protein mapping in T lymphocytes in PsA joints revealed the complex interplay between IL-1, IL-6, and IL-23 signaling and differentiation of Th17 cells and CD4+Tregs in sustained joint inflammation in PsA.

Leukaemic cells from chronic lymphocytic leukaemia patients undergo apoptosis following microtubule depolymerization and Lyn inhibition by nocodazole.

Functional abnormalities of chronic lymphocytic leukaemia (CLL) cells may be related to the microtubular network of cell cytoskeleton; specifically tubulin involvement in cells after B-cell receptor engagement. As microtubule inhibitors could represent a therapeutic strategy for CLL, this study investigated the capability of nocodazole, a synthetic depolymerizing agent, to kill CLL leukaemic cells. We demonstrated that nocodazole was highly specific for the in vitro induction of apoptosis in leukaemic cells from 90 CLL patients, without affecting the viability of T-cells and/or mesenchymal stromal cells (MSCs) recovered from the same patients. Nocodazole was observed to overcome the pro-survival signals provided by MSCs. Competing with ATP for the nucleotide-binding site, nocodazole has been observed to turn off the high basal tyrosine phosphorylation of leukaemic cells mediated by the Src-kinase Lyn. Considering that most anti-microtubule drugs have limited clinical use because of their strong toxic effects, the high selectivity of nocodazole for leukaemic cells in CLL and its capability to bypass microenvironmental pro-survival stimuli, suggests the use of this inhibitor for designing new therapeutic strategies in CLL treatment.

Cortactin, another player in the Lyn signaling pathway, is over-expressed and alternatively spliced in leukemic cells from patients with B-cell chronic lymphocytic leukemia.

Cortactin, an actin binding protein and Lyn substrate, is up-regulated in several cancers and its level is associated with increased cell migration, metastasis and poor prognosis. The identification that the Src kinase Lyn and its substrate HS1 are over-expressed in B-cell chronic lymphocytic leukemia and involved in resistance to chemotherapy and poor prognosis, prompted us to investigate the role of cortactin, an HS1 homolog, in the pathogenesis and progression of this disorder. In this study, we observed that cortactin is over-expressed in leukemic cells of patients (1.10 ± 0.12) with respect to normal B lymphocytes (0.19 ± 0.06; P=0.0065). Fifty-three percent of our patients expressed the WT mRNA and p80/85 protein isoforms, usually lacking in normal B lymphocytes which express the SV1 variant and the p70/75 protein isoforms. Moreover, we found an association of the cortactin overexpression and negative prognostic factors, including ZAP-70 (P<0.01), CD38 (P<0.01) and somatic hypermutations in the immunoglobulin heavy-chain variable region (P<0.01). Our results show that patients with B-cell chronic lymphocytic leukemia express high levels of cortactin with a particular overexpression of the WT isoform that is lacking in normal B cells, and a correlation to poor prognosis, suggesting that this protein could be relevant in the pathogenesis and aggressiveness of the disease.