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.
Chronic lymphocytic leukemia (CLL) is an incurable B-cell neoplasm characterized by highly variable clinical outcomes. In recent years, genomic and molecular studies revealed a remarkable ...heterogeneity in CLL, which mirrored the clinical diversity of this disease. These studies profoundly enhanced our understanding of leukemia cell biology and led to the identification of new biomarkers with potential prognostic and therapeutic significance. Accumulating evidence indicates a key role of deregulated NOTCH1 signaling and
mutations in CLL. This review highlights recent discoveries that improve our understanding of the pathophysiological NOTCH1 signaling in CLL and the clinical impact of
mutations in retrospective and prospective trials. In addition, we discuss the rationale for a therapeutic strategy aiming at inhibiting NOTCH1 signaling in CLL, along with an overview on the currently available NOTCH1-directed approaches.
CAR-T therapy has revolutionized the treatment of relapsed/refractory B-cell malignancies. Patients who are receiving such therapy are susceptible to an increased incidence of infections due to ...post-treatment immunosuppression. The need for antifungal prophylaxis during the period of neutropenia remains to be determined. The clinical outcome of a 55-year-old patient with relapsed/refractory DLBCL who received axicabtagene ciloleucel is described here. The patient developed CRS grade II and ICANS grade IV requiring tocilizumab, prolonged use of steroids and anakinra. An invasive pulmonary aspergillosis arose after 1 month from CAR-T reinfusion, resolved with tracheal sleeve pneumonectomy. The patient is now in Complete Remission. This case suggests that antifungal prophylaxis should be considered. We have now included micafungin as a standard prophylaxis in our institution.
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.
Introduction
Constitutive activation of
NOTCH1
-wild-type (NT1-WT) signaling is associated with poor outcomes in chronic lymphocytic leukemia (CLL), and
NOTCH1
mutation (c.7541_7542delCT), which ...potentiates NOTCH1 signaling, worsens the prognosis. However, the specific mechanisms of NOTCH1 deregulation are still poorly understood. Accumulative evidence mentioned endoplasmic reticulum (ER) stress/unfolded protein response (UPR) as a key targetable pathway in CLL. In this study, we investigated the impact of NOTCH1 deregulation on CLL cell response to ER stress induction, with the aim of identifying new therapeutic opportunities for CLL.
Methods
We performed a bioinformatics analysis of
NOTCH1
-mutated (NT1-M) and NT1-WT CLL to identify differentially expressed genes (DEGs) using the rank product test. Quantitative real-time polymerase chain reaction (qPCR), Western blotting, cytosolic Ca
2+
, and annexin V/propidium iodide (PI) assay were used to detect curcumin ER stress induction effects. A median-effect equation was used for drug combination tests. The experimental mouse model Eμ-TCL1 was used to evaluate the impact of ER stress exacerbation by curcumin treatment on the progression of leukemic cells and NOTCH1 signaling.
Results and discussion
Bioinformatics analysis revealed gene enrichment of the components of the ER stress/UPR pathway in NT1-M compared to those in NT1-WT CLL. Ectopic expression of
NOTCH1
mutation upregulated the levels of ER stress response markers in the PGA1 CLL cell line. Primary NT1-M CLL was more sensitive to curcumin as documented by a significant perturbation in Ca
2+
homeostasis and higher expression of ER stress/UPR markers compared to NT1-WT cells. It was also accompanied by a significantly higher apoptotic response mediated by C/EBP homologous protein (CHOP) expression, caspase 4 cleavage, and downregulation of NOTCH1 signaling in NT1-M CLL cells. Curcumin potentiated the apoptotic effect of venetoclax in NT1-M CLL cells. In Eμ-TCL1 leukemic mice, the administration of curcumin activated ER stress in splenic B cells
ex vivo
and significantly reduced the percentage of CD19
+
/CD5
+
cells infiltrating the spleen, liver, and bone marrow (BM). These cellular effects were associated with reduced NOTCH1 activity in leukemic cells and resulted in prolonged survival of curcumin-treated mice. Overall, our results indicate that ER stress induction in NT1-M CLL might represent a new therapeutic opportunity for these high-risk CLL patients and improve the therapeutic effect of drugs currently used in CLL.
Abstract
The coexistence of chronic myeloid leukemia (CML) and multiple myeloma (MM) is a rare clinical condition. By means of FISH and molecular analysis on both sorted CD138 plasma cells and ...cryopreserved CD34 stem cells, a distinct clonal origin of the hematological malignancies was demonstrated in our case. We report on the first patient diagnosed with CML and MM treated with daratumumab, bortezomib, thalidomide, and dexamethasone (Dara‐VTd) induction, stem‐cell collection, and autologous stem cell transplantation (ASCT). The co‐administration of Dara‐VTd and imatinib proved feasible and highly effective in the management of both CML and MM. Despite concerns with stem cell mobilization and collection in patients exposed to daratumumab, in our experience the use of higher cyclophosphamide dose 4 g/m
2
together with plerixafor granted optimal stem cell mobilization and collection, irrespective of daratumumab, concomitant myeloid neoplasm, and imatinib. Moreover, ASCT was easily performed with a rapid hematological reconstitution.
Pelvic Melanoma relapse occurs in 15% of patients with loco regional metastases, and 25% of cases do not respond to new target-therapy and/or immunotherapy. Melphalan hypoxic pelvic perfusion may, ...therefore, be an option for these non-responsive patients. Overall median survival time (MST), stratified for variables, including BRAF V600E mutation and eligibility for treatments with new immunotherapy drugs, was retrospectively assessed in 41 patients with pelvic melanoma loco regional metastases. They had received a total of 175 treatments with Melphalan hypoxic perfusion and cytoreductive excision. Among the 41 patients, 22 (53.7%) patients exhibited a wild-type
genotype, 11 of which were not eligible for immunotherapy. The first treatment resulted in a 97.5% response-rate in the full cohort and a 100% response-rate in the 22 wild-type
patients. MST was 18 months in the full sample, 20 months for the 22 wild-type
patients and 21 months for the 11 wild-type
patients not eligible for immunotherapy. Melphalan hypoxic perfusion is a potentially effective treatment for patients with pelvic melanoma loco regional metastases that requires confirmation in a larger multicenter study.
Chronic lymphocytic leukemia (CLL) is an incurable disorder associated with alterations in several pathways essential for survival and proliferation. Despite the advances made in CLL therapy with the ...new target agents, in some cases, relapses and resistance could occur, making the discovery of new alternatives to manage CLL refractoriness necessary. To provide new therapeutic strategies for CLL, we investigated the anti-leukemic activity of silver nanoparticles (AgNPs), whose impact on CLL cells has been poorly explored.
We studied the action mechanisms of AgNPs in vitro through flow cytometry and molecular analyses. To improve the bioavailability of AgNPs, we generated AgNPs coated with the anti-CD20 antibody Rituximab (AgNPs@Rituximab) and carried out imaging-based approaches and in vivo experiments to evaluate specificity, drug uptake, and efficacy.
AgNPs reduced the viability of primary CLL cells and the HG-3 cell line by inducing an intrinsic apoptotic pathway characterized by Bax/Bcl-2 imbalance, caspase activation, and PARP degradation. Early apoptotic events triggered by AgNPs included enhanced Ca
influx and ROS overproduction. AgNPs synergistically potentiated the cytotoxicity of Venetoclax, Ibrutinib, and Bepridil. In vitro, the AgNPs@Rituximab conjugates were rapidly internalized within CLL cells and strongly prolonged the survival of CLL xenograft models compared to each unconjugated single agent.
AgNPs showed strong anti-leukemic activity in CLL, with the potential for clinical translation in combination with agents used in CLL. The increased specificity of AgNPs@Rituximab toward CLL cells could be relevant for overcoming in vivo AgNPs' non-specific distribution and increasing their efficacy.
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