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In chronic lymphocytic leukaemia (CLL) the efficacy of SARS-CoV-2 vaccination remains unclear as most studies have focused on humoral responses. Here we comprehensively examined humoral and cellular responses to vaccine in CLL patients. Seroconversion was observed in 55.2% of CLL with lower rate and antibody titres in treated patients. T-cell responses were detected in a significant fraction of patients. CD4 + and CD8 + frequencies were significantly increased independent of serology with higher levels of CD4 + cells in patients under a Bruton tyrosine kinase (BTK) or a B-cell lymphoma 2 (BCL-2) inhibitor. Vaccination skewed CD8 + cells towards a highly cytotoxic phenotype, more pronounced in seroconverted patients. A high proportion of patients showed spike-specific CD4 + and CD8 + cells producing interferon gamma (IFNγ) and tumour necrosis factor alpha (TNFα). Patients under a BTK inhibitor showed increased production of IFNγ and TNFα by CD4 + cells. Vaccination induced a Th1 polarization reverting the Th2 CLL T-cell profile in the majority of patients with lower IL-4 production in untreated and BTK-inhibitor-treated patients. Such robust T-cell responses may have contributed to remarkable protection against hospitalization and death in a cohort of 540 patients. Combining T-cell metrics with seroprevalence may yield a more accurate measure of population immunity in CLL, providing consequential insights for public health., (© 2022 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

NOTCH1 alterations have been associated with chronic lymphocytic leukemia (CLL), but the molecular mechanisms underlying NOTCH1 activation in CLL cells are not completely understood. Here, we show that GSK3β downregulates the constitutive levels of the active NOTCH1 intracellular domain (N1-ICD) in CLL cells. Indeed, GSK3β silencing by small interfering RNA increases N1-ICD levels, whereas expression of an active GSK3β mutant reduces them. Additionally, the GSK3β inhibitor SB216763 enhances N1-ICD stability at a concentration at which it also increases CLL cell viability. We also show that N1-ICD is physically associated with GSK3β in CLL cells. SB216763 reduces GSK3β/N1-ICD interactions and the levels of ubiquitinated N1-ICD, indicating a reduction in N1-ICD proteasomal degradation when GSK3β is less active. We then modulated the activity of two upstream regulators of GSK3β and examined the impact on N1-ICD levels and CLL cell viability. Specifically, we inhibited AKT that is a negative regulator of GSK3β and is constitutively active in CLL cells. Furthermore, we activated the protein phosphatase 2 A (PP2A) that is a positive regulator of GSK3β, and has an impaired activity in CLL. Results show that either AKT inhibition or PP2A activation reduce N1-ICD expression and CLL cell viability in vitro, through mechanisms mediated by GSK3β activity. Notably, for PP2A activation, we used the highly specific activator DT-061, that also reduces leukemic burden in peripheral blood, spleen and bone marrow in the Eµ-TCL1 adoptive transfer model of CLL, with a concomitant decrease in N1-ICD expression. Overall, we identify in GSK3β a key component of the network regulating N1-ICD stability in CLL, and in AKT and PP2A new druggable targets for disrupting NOTCH1 signaling with therapeutic potential., (© 2022. The Author(s).)

Key features of chronic lymphocytic leukemia (CLL) are defects in the immune system and the ability of leukemic cells to evade immune defenses and induce immunosuppression, resulting in increased susceptibility to infections and disease progression. Several immune effectors are impaired in CLL, including T and natural killer (NK) cells. The role of T cells in defense against CLL and in CLL progression and immunotherapy has been extensively studied. Less is known about the role of NK cells in this leukemia, and data on NK cell alterations in CLL are contrasting. Besides studies showing that NK cells have intrinsic defects in CLL, there is a large body of evidence indicating that NK cell dysfunctions in CLL mainly depend on the escape mechanisms employed by leukemic cells. In keeping, it has been shown that NK cell functions, including antibody-dependent cellular cytotoxicity (ADCC), can be retained and/or restored after adequate stimulation. Therefore, due to their preserved ADCC function and the reversibility of CLL-related dysfunctions, NK cells are an attractive source for novel immunotherapeutic strategies in this disease, including chimeric antigen receptor (CAR) therapy. Recently, satisfying clinical responses have been obtained in CLL patients using cord blood-derived CAR-NK cells, opening new possibilities for further exploring NK cells in the immunotherapy of CLL. However, notwithstanding the promising results of this clinical trial, more evidence is needed to fully understand whether and in which CLL cases NK cell-based immunotherapy may represent a valid, alternative/additional therapeutic option for this leukemia. In this review, we provide an overview of the current knowledge about phenotypic and functional alterations of NK cells in CLL and the mechanisms by which CLL cells circumvent NK cell-mediated immunosurveillance. Additionally, we discuss the potential relevance of using NK cells in CLL immunotherapy.

Purpose: Ibrutinib, a Bruton tyrosine kinase inhibitor (BTKi), has improved the outcomes of chronic lymphocytic leukemia (CLL), but primary resistance or relapse are issues of increasing significance. While the predominant mechanism of action of BTKi is the B-cell receptor (BCR) blockade, many off-target effects are unknown. We investigated potential interactions between BCR pathway and NOTCH1 activity in ibrutinib-treated CLL to identify new mechanisms of therapy resistance and markers to monitor disease response., Experimental Design: NOTCH activations was evaluated either in vitro and ex vivo in CLL samples after ibrutinib treatment by Western blotting. Confocal proximity ligation assay (PLA) experiments and analyses of down-targets of NOTCH1 by qRT-PCR were used to investigate the cross-talk between BTK and NOTCH1., Results: In vitro ibrutinib treatment of CLL significantly reduced activated NOTCH1/2 and induced dephosphorylation of eIF4E, a NOTCH target in CLL. BCR stimulation increased the expression of activated NOTCH1 that accumulated in the nucleus leading to HES1, DTX1, and c-MYC transcription. Results of in situ PLA experiments revealed the presence of NOTCH1-ICD/BTK complexes, whose number was reduced after ibrutinib treatment. In ibrutinib-treated CLL patients, leukemic cells showed NOTCH1 activity downregulation that deepened over time. The NOTCH1 signaling was restored at relapse and remained activated in ibrutinib-resistant CLL cells., Conclusions: We demonstrated a strong clinical activity of ibrutinib in a real-life context. The ibrutinib clinical efficacy was associated with NOTCH1 activity downregulation that deepened over time. Our data point to NOTCH1 as a new molecular partner in BCR signaling with potential to further improve CLL-targeted treatments., (©2019 American Association for Cancer Research.)

As previously reported, chronic lymphocytic leukemia (CLL) cells show constitutive Notch1/2 activation and express the Notchligand Jagged1. Despite increasing knowledge of the impact of Notch alterations on CLL biology and pathogenesis, the role of Jagged1 expressed in CLL cells remains undefined. In other cell types, it has been shown that after Notch engagement, Jagged1 not only activates Notch in signal-receiving cell, but also undergoes proteolytic activation in signal-sending cell, triggering a signaling with biological effects. We investigated whether Jagged1 expressed in CLL cells undergoes proteolytic processing and/or is able to induce Notch activation through autocrine/paracrine loops, focusing on the effect that CLL prosurvival factor IL-4 could exert on the Notch-Jagged1 system in these cells. We found that Jagged1 was constitutively processed in CLL cells and generated an intracellular fragment that translocated into the nucleus, and an extracellular fragment released into the culture supernatant. IL-4 enhanced expression of Jagged1 and its intracellular fragments, as well as Notch1/2 activation. The IL-4-induced increase in Notch1/2 activation was independent of the concomitant upregulated Jagged1 levels. Indeed, blocking Notch-Jagged1 interactions among CLL cells with Jagged1 neutralizing antibodies did not affect the expression of the Notch target Hes1. Notably, anti-Jagged1 antibodies partially prevented the IL-4-induced increase in Jagged1 processing and cell viability, suggesting that Jagged1 processing is one of the events contributing to IL-4-induced CLL cell survival. Consistent with this, Jagged1 silencing by small interfering RNA partially counteracted the capacity of IL-4 to promote CLL cell survival. Investigating the pathways whereby IL-4 promoted Notch1/2 activation in CLL cells independent of Jagged1, we found that PI3Kδ/AKT and PKCδ were involved in upregulating Notch1 and Notch2 proteins, respectively. Overall, this study provides new insights into the Notch-ligand system in CLL cells and suggests that targeting this system may be exploited as a novel/additional therapy approach for CLL.

Dysregulated NOTCH1 signaling, by either gene mutations or microenvironment interactions, has been increasingly linked to chronic lymphocytic leukemia (CLL). Thus, inhibiting NOTCH1 activity represents a potential therapeutic opportunity for this disease. Using gene expression-based screening, we identified the calcium channel modulator bepridil as a new NOTCH1 pathway inhibitor. In primary CLL cells, bepridil induced selective apoptosis even in the presence of the protective stroma. Cytotoxic effects of bepridil were independent of NOTCH1 mutation and other prognostic markers. The antitumor efficacy of bepridil was associated with inhibition of NOTCH1 activity through a decrement in trans-membrane and activated NOTCH1 protein levels with unchanged NOTCH2 protein levels. In a CLL xenotransplant model, bepridil significantly reduced the percentage of leukemic cells infiltrating the spleen via enhanced apoptosis and decreased NOTCH1 activation. In conclusion, we report in vitro and in vivo anti-leukemic activity of bepridil associated with inhibition of the NOTCH1 pathway in CLL. These data provide a rationale for the clinical development of bepridil as anti-NOTCH1 targeted therapy for CLL patients., (© 2018 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

In chronic lymphocytic leukemia (CLL), Notch1 and Notch2 signaling is constitutively activated and contributes to apoptosis resistance. We show that genetic inhibition of either Notch1 or Notch2, through small-interfering RNA, increases apoptosis of CLL cells and is associated with decreased levels of the anti-apoptotic protein Mcl-1. Thus, Notch signaling promotes CLL cell survival at least in part by sustaining Mcl-1 expression. In CLL cells, an enhanced Notch activation also contributes to the increase in Mcl-1 expression and cell survival induced by IL-4.Mcl-1 downregulation by Notch targeting is not due to reduced transcription or degradation by caspases, but in part, to increased degradation by the proteasome. Mcl-1 downregulation by Notch targeting is also accompanied by reduced phosphorylation of eukaryotic translation initiation factor 4E (eIF4E), suggesting that this protein is another target of Notch signaling in CLL cells.Overall, we show that Notch signaling sustains CLL cell survival by promoting Mcl-1 expression and eIF4E activity, and given the oncogenic role of these factors, we underscore the therapeutic potential of Notch inhibition in CLL.

The NOTCH and nuclear factor kappa B (NF-κB) pathways are both constitutively activated in Chronic Lymphocytic Leukemia (CLL). We first described the NOTCH1 PEST domain mutation in a CLL subgroup, but the activation of the NOTCH pathway in NOTCH1-unmutated cases remains unexplained. Here, we investigated whether genetic lesions in the NF-κB/NOTCH loop might support the NOTCH activation status by sequencing negative (TNFAIP3/A20) and positive (TRAF2, TRAF5, TNFRSF11A/RANK, MAP3K7/TAK1, and CARD11) regulators of NF-κB together with NF-κB targets on the NOTCH pathway, the NOTCH ligands Jagged1 and Jagged2, in CLL patients. The sequence analysis revealed four missense mutations for A20, TRAF2, TRAF5 and RANK1 genes, all causing a change in amino acid group from polar to non-polar, but functional domains were not involved. Specific predictive software analyses confirmed that the amino acid changes have a low-functional impact on the protein. Our results show that in CLL, NF-κB regulators and Jagged are both unmutated, suggesting that the Jagged-mediated interplay between NF-κB and NOTCH is independent of genetic lesions.

γ-Secretase inhibitors (GSIs) have been proposed for combined therapies of malignancies with a dysregulated Notch signaling. GSI I (Z-Leu-Leu-Nle-CHO) induces apoptosis of some tumor cells by inhibiting proteasome and Notch activity. Alterations in these two cell survival regulators contribute to apoptosis resistance of chronic lymphocytic leukemia (CLL) cells. Here, we investigated the mechanisms whereby GSI I increases apoptosis of primary CLL cells. Time-course studies indicate that initial apoptotic events are inhibition of proteasome activity, concomitant with an increased endoplasmic reticulum (ER) stress apoptotic signaling, and a consistent Noxa protein up-regulation. These events precede, and some of them contribute to, mitochondrial alterations, which occur notwithstanding Mcl-1 accumulation induced by GSI I. In CLL cells, GSI I inhibits Notch1 and Notch2 activation only in the late apoptotic phases, suggesting that this event does not initiate CLL cell apoptosis. However, Notch inhibition may contribute to amplify GSI I-induced CLL cell apoptosis, given that Notch activation sustains the survival of these cells, as demonstrated by the evidence that both Notch1 and Notch2 down-regulation by small-interfering RNA accelerates spontaneous CLL cell apoptosis. Overall, our results show that GSI I triggers CLL cell apoptosis by inhibiting proteasome activity and enhancing ER stress, and amplifies it by blocking Notch activation. These findings suggest the potential relevance of simultaneously targeting these three important apoptosis regulators as a novel therapeutic strategy for CLL., (Copyright © 2012 UICC.)

A better understanding of apoptotic signaling in B-chronic lymphocytic leukemia (B-CLL) cells may help to define new therapeutic strategies. This study investigated endoplasmic reticulum (ER) stress signaling in spontaneous apoptosis of B-CLL cells and whether manipulating ER stress increases their apoptosis. Results show that a novel ER stress-triggered caspase cascade, initiated by caspase-4 and involving caspase-8 and -3, plays an important role in spontaneous B-CLL cell apoptosis. ER stress-induced apoptosis in B-CLL cells also involves CHOP/GADD153 up-regulation, increased JNK1/2 phosphorylation, and caspase-8-mediated cleavage of Bap31 to Bap20, known to propagate apoptotic signals from ER to mitochondria. In ex vivo B-CLL cells, some apoptotic events associated with mitochondrial pathway also occur, including mitochondrial cytochrome c release and caspase-9 processing. However, pharmacologic inhibition studies show that caspase-9 plays a minor role in B-CLL cell apoptosis. ER stress also triggers survival signals in B-CLL cells by increasing BiP/GRP78 expression. Manipulating ER signaling by siRNA down-regulation of BiP/GRP78 or treating B-CLL cells with 2 well-known ER stress-inducers, tunicamycin and thapsigargin, increases their apoptosis. Overall, our findings show that ER triggers an essential pathway for B-CLL cell apoptosis and suggest that genetic and pharmacologic manipulation of ER signaling could represent an important therapeutic strategy.

Notch signaling is involved in tumorigenesis, but its role in B-chronic lymphocytic leukemia (B-CLL) pathogenesis is not completely defined. This study examined the expression and activation of Notch receptors in B-CLL cells and the role of Notch signaling in sustaining the survival of these cells. Our results show that B-CLL cells but not normal B cells constitutively express Notch1 and Notch2 proteins as well as their ligands Jagged1 and Jagged2. Notch signaling is constitutively activated in B-CLL cells, and its activation is further increased in B-CLL cells, which resist spontaneous apoptosis after 24-hour ex vivo culture. Notch stimulation by a soluble Jagged1 ligand increases B-CLL cell survival and is accompanied by increased nuclear factor-kappa B (NF-kappaB) activity and cellular inhibitor of apoptosis protein 2 (c-IAP2) and X-linked inhibitor of apoptosis protein (XIAP) expression. In contrast, Notch-signaling inhibition by the gamma-secretase inhibitor I (GSI; z-Leu-Leu-Nle-CHO) and the specific Notch2 down-regulation by small-interfering RNA accelerate spontaneous B-CLL cell apoptosis. Apoptotic activity of GSI is accompanied by reduction of NF-kappaB activity and c-IAP2 and XIAP expression. Overall, our findings show that Notch signaling plays a critical role in B-CLL cell survival and apoptosis resistance and suggest that it could be a novel potential therapeutic target.

Background Aims: The impact of chronic lymphatic leukemia (CLL) tumor burden on the autologous immune system has already been demonstrated. This study attempted to elucidate the molecular mechanisms underlying T-cell immunologic deficiencies in CLL., Methods: Freshly isolated CD3(+) T cells from patients with a diagnosis of CLL and healthy donors were analyzed by gene expression profiling. Activated T cells from 20 patients with CLL were tested in vitro for cytotoxicity against mutated and unmutated autologous B cells and DAUDI, K562 and P815 cell lines. To investigate T-cell mediated cytotoxicity in vivo, we co-transplanted OKT3-activated T lymphocytes and autologous B-cell CLL (B-CLL) cells into NOD/SCID mice., Results: Gene expression profiles of peripheral blood T cells from B-CLL patients showed 25 down-regulated, and 31 up-regulated, genes that were mainly involved in cell differentiation, proliferation, survival, apoptosis, cytoskeleton formation, vesicle trafficking and T-cell activation. After culture, the T-cell count remained unchanged, CD8 cells expanded more than CD4 and a cytotoxicity index >30% was present in 5/20 patients. Cytotoxicity against B autologous leukemic cells did not correlate with B-cell mutational status. Only activated T cells exerting cytotoxicity against autologous leukemic B cells prevented CLL in a human-mouse chimera., Conclusions: This study indicates that patients with CLL are affected by a partial immunologic defect that might be somewhat susceptible to repair. This study identifies the molecular pathways underlying T-cell deficiencies in CLL and shows that cytotoxic T-cell functions against autologous B-CLL can be rebuilt at least in part in vitro and in vivo.

Tumor necrosis factor alpha (TNFalpha) is constitutively produced by B-chronic lymphocytic leukemia (B-CLL) cells and may act as an autocrine factor for their growth and survival. However, very few data are available on the possible cytotoxic effect of TNFalpha produced by B-CLL cells. This study investigated whether B-CLL cells exert in vitro cytotoxicity by TNFalpha and if so, whether this cytotoxicity can be modulated by cytokines. In 8 of 12 patients (66.6%), B-CLL cells in vitro constitutively produced TNFalpha and exerted a TNFalpha-mediated cytotoxicity, evaluated in an 18-h 51Cr release assay, against the TNFalpha-sensitive Jurkat, U937 and K562 cell lines but not against the TNFalpha-resistant Raji cell line. Involvement of TNFalpha in B-CLL cell cytotoxicity is demonstrated by the fact that anti-TNFalpha antibodies strongly inhibited it and supernatants of cytotoxic cultures contained TNFalpha and mediated a completely TNFalpha-dependent cytotoxicity. When the cytotoxic B-CLL cells were stimulated with interleukin (IL)-2 plus IL-12, there was increased TNFalpha mRNA expression, TNFalpha production and TNFalpha-mediated cytotoxicity. All eight patients with cytotoxic leukemic cells had progressive disease and six of these also expressed high levels of ZAP-70 protein. In the other four patients (33.3%), B-CLL cells did not produce TNFalpha in vitro and were not cytotoxic, either spontaneously or after IL-2 plus IL-12 stimulation. Of these four patients, three had stable disease and one had progressive disease. The patient with progressive disease and one of the three with stable disease expressed low levels of ZAP-70 protein. We conclude that a group of B-CLL patients with progressive disease have leukemic B cells able to exert in vitro a TNFalpha-mediated cytotoxicity, which is modulated by cytokines.