CD20 expression regulates CD37 levels in B-cell lymphoma - implications for immunotherapies.

Rituximab (RTX) plus chemotherapy (R-CHOP) applied as a first-line therapy for lymphoma leads to a relapse in approximately 40% of the patients. Therefore, novel approaches to treat aggressive lymphomas are being intensively investigated. Several RTX-resistant (RR) cell lines have been established as surrogate models to study resistance to R-CHOP. Our study reveals that RR cells are characterized by a major downregulation of CD37, a molecule currently explored as a target for immunotherapy. Using CD20 knockout (KO) cell lines, we demonstrate that CD20 and CD37 form a complex, and hypothesize that the presence of CD20 stabilizes CD37 in the cell membrane. Consequently, we observe a diminished cytotoxicity of anti-CD37 monoclonal antibody (mAb) in complement-dependent cytotoxicity in both RR and CD20 KO cells that can be partially restored upon lysosome inhibition. On the other hand, the internalization rate of anti-CD37 mAb in CD20 KO cells is increased when compared to controls, suggesting unhampered efficacy of antibody drug conjugates (ADCs). Importantly, even a major downregulation in CD37 levels does not hamper the efficacy of CD37-directed chimeric antigen receptor (CAR) T cells. In summary, we present here a novel mechanism of CD37 regulation with further implications for the use of anti-CD37 immunotherapies.

PRDX-1 Supports the Survival and Antitumor Activity of Primary and CAR-Modified NK Cells under Oxidative Stress.

Oxidative stress, caused by the imbalance between reactive species generation and the dysfunctional capacity of antioxidant defenses, is one of the characteristic features of cancer. Here, we quantified hydrogen peroxide in the tumor microenvironment (TME) and demonstrated that hydrogen peroxide concentrations are elevated in tumor interstitial fluid isolated from murine breast cancers in vivo, when compared with blood or normal subcutaneous fluid. Therefore, we investigated the effects of increased hydrogen peroxide concentration on immune cell functions. NK cells were more susceptible to hydrogen peroxide than T cells or B cells, and by comparing T, B, and NK cells' sensitivities to redox stress and their antioxidant capacities, we identified peroxiredoxin-1 (PRDX1) as a lacking element of NK cells' antioxidative defense. We observed that priming with IL15 protected NK cells' functions in the presence of high hydrogen peroxide and simultaneously upregulated PRDX1 expression. However, the effect of IL15 on PRDX1 expression was transient and strictly dependent on the presence of the cytokine. Therefore, we genetically modified NK cells to stably overexpress PRDX1, which led to increased survival and NK cell activity in redox stress conditions. Finally, we generated PD-L1-CAR NK cells overexpressing PRDX1 that displayed potent antitumor activity against breast cancer cells under oxidative stress. These results demonstrate that hydrogen peroxide, at concentrations detected in the TME, suppresses NK cell function and that genetic modification strategies can improve CAR NK cells' resistance and potency against solid tumors.

Effects of Low-Dose Atorvastatin on the Peripheral Blood Mononuclear Cell Secretion of Angiogenic Factors in Type 2 Diabetes.

The aim of this study was to investigate the influence of statins on the secretion of angiogenesis mediators by the peripheral blood mononuclear cells (PBMCs) derived from patients suffering from type 2 diabetes. The study group comprised 30 participants and included: 10 statin-treated patients with diabetes, 10 statin-free diabetic subjects, and 10 statin-free non-diabetic individuals. PBMCs isolated from the blood were cultured in vitro in standard conditions and in an environment mimicking hyperglycemia. Culture supernatants were evaluated for VEGF, MCP-1, Il-10, and Il-12 by flow cytometry using commercial BDTM. Cytometric Bead Array tests. The secretion of VEGF, MCP-1 and Il-12 by PBMCs, cultured both in standard and hyperglycemic conditions, was significantly lower in the statin-treated patients with type 2 diabetes in comparison with the statin-free diabetic patients. Conversely, the secretion of Il-10 was higher in the statin-treated than in the statin-free diabetic patients. VEGF, MCP-1 and Il-12 levels in PBMCs supernatants from the glucose-containing medium were higher than those from the standard medium in each of the diabetic groups. The results of the study suggest that statins in low doses exhibit an antiangiogenic activity, reducing the secretion of potent proangiogenic factors, such as VEGF and MCP-1, and increasing the secretion of antiangiogenic Il-10 by PBMCs, also under hyperglycemic conditions characteristic for type 2 diabetes.

Inhibition of PIM Kinases in DLBCL Targets MYC Transcriptional Program and Augments the Efficacy of Anti-CD20 Antibodies.

The family of PIM serine/threonine kinases includes three highly conserved oncogenes, PIM1, PIM2, and PIM3, which regulate multiple prosurvival pathways and cooperate with other oncogenes such as MYC. Recent genomic CRISPR-Cas9 screens further highlighted oncogenic functions of PIMs in diffuse large B-cell lymphoma (DLBCL) cells, justifying the development of small-molecule PIM inhibitors and therapeutic targeting of PIM kinases in lymphomas. However, detailed consequences of PIM inhibition in DLBCL remain undefined. Using chemical and genetic PIM blockade, we comprehensively characterized PIM kinase-associated prosurvival functions in DLBCL and the mechanisms of PIM inhibition-induced toxicity. Treatment of DLBCL cells with SEL24/MEN1703, a pan-PIM inhibitor in clinical development, decreased BAD phosphorylation and cap-dependent protein translation, reduced MCL1 expression, and induced apoptosis. PIM kinases were tightly coexpressed with MYC in diagnostic DLBCL biopsies, and PIM inhibition in cell lines and patient-derived primary lymphoma cells decreased MYC levels as well as expression of multiple MYC-dependent genes, including PLK1. Chemical and genetic PIM inhibition upregulated surface CD20 levels in an MYC-dependent fashion. Consistently, MEN1703 and other clinically available pan-PIM inhibitors synergized with the anti-CD20 monoclonal antibody rituximab in vitro, increasing complement-dependent cytotoxicity and antibody-mediated phagocytosis. Combined treatment with PIM inhibitor and rituximab suppressed tumor growth in lymphoma xenografts more efficiently than either drug alone. Taken together, these results show that targeting PIM in DLBCL exhibits pleiotropic effects that combine direct cytotoxicity with potentiated susceptibility to anti-CD20 antibodies, justifying further clinical development of such combinatorial strategies. SIGNIFICANCE: These findings demonstrate that inhibition of PIM induces DLBCL cell death via MYC-dependent and -independent mechanisms and enhances the therapeutic response to anti-CD20 antibodies by increasing CD20 expression.

The Tumor Microenvironment-A Metabolic Obstacle to NK Cells' Activity.

NK cells have unique capabilities of recognition and destruction of tumor cells, without the requirement for prior immunization of the host. Maintaining tolerance to healthy cells makes them an attractive therapeutic tool for almost all types of cancer. Unfortunately, metabolic changes associated with malignant transformation and tumor progression lead to immunosuppression within the tumor microenvironment, which in turn limits the efficacy of various immunotherapies. In this review, we provide a brief description of the metabolic changes characteristic for the tumor microenvironment. Both tumor and tumor-associated cells produce and secrete factors that directly or indirectly prevent NK cell cytotoxicity. Here, we depict the molecular mechanisms responsible for the inhibition of immune effector cells by metabolic factors. Finally, we summarize the strategies to enhance NK cell function for the treatment of tumors.

CD20 is dispensable for B-cell receptor signaling but is required for proper actin polymerization, adhesion and migration of malignant B cells.

Surface protein CD20 serves as the critical target of immunotherapy in various B-cell malignancies for decades, however its biological function and regulation remain largely elusive. Better understanding of CD20 function may help to design improved rational therapies to prevent development of resistance. Using CRISPR/Cas9 technique, we have abrogated CD20 expression in five different malignant B-cell lines. We show that CD20 deletion has no effect upon B-cell receptor signaling or calcium flux. Also B-cell survival and proliferation is unaffected in the absence of CD20. On the contrary, we found a strong defect in actin cytoskeleton polymerization and, consequently, defective cell adhesion and migration in response to homeostatic chemokines SDF1α, CCL19 and CCL21. Mechanistically, we could identify a reduction in chemokine-triggered PYK2 activation, a calcium-activated signaling protein involved in activation of MAP kinases and cytoskeleton regulation. These cellular defects in consequence result in a severely disturbed homing of B cells in vivo.

Selective inhibition of HDAC6 sensitizes cutaneous T-cell lymphoma to PI3K inhibitors.

Histone deacetylase (HDAC) inhibitors, approved for the treatment of cutaneous T-cell lymphoma (CTCL), are non-selective agents associated with an unsatisfactory response and considerable side-effects. Targeting single HDAC isoforms is considered to provide novel therapeutic options. HDAC6 is overexpressed in primary samples from patients with CTCL and preclinical studies using transgenic mice that spontaneously develop a CTCL-like disease, have suggested that combinations including HDAC6 inhibitors may be successful in the treatment of CTCL. PI3K inhibition is currently being tested in clinical trials for CTCL with promising results. Since HDAC6 is known to diminish the activity of Akt via its deacetylation, the aim of the present study was to evaluate the therapeutic potential of selective HDAC6 inhibitors in combination with PI3K inhibitors in CTCL. Through the genetic and pharmacological inhibition of HDAC6, it was demonstrated that combining HDAC6 with PI3K inhibition may be an attractive therapeutic option for patients with CTCL.

Monoclonal Antibodies in Dermatooncology-State of the Art and Future Perspectives.

Monoclonal antibodies (mAbs) targeting specific proteins are currently the most popular form of immunotherapy used in the treatment of cancer and other non-malignant diseases. Since the first approval of anti-CD20 mAb rituximab in 1997 for the treatment of B-cell malignancies, the market is continuously booming and the clinically used mAbs have undergone a remarkable evolution. Novel molecular targets are constantly emerging and the development of genetic engineering have facilitated the introduction of modified mAbs with improved safety and increased capabilities to activate the effector mechanisms of the immune system. Next to their remarkable success in hematooncology, mAbs have also an already established role in the treatment of solid malignancies. The recent development of mAbs targeting the immune checkpoints has opened new avenues for the use of this form of immunotherapy, also in the immune-rich milieu of the skin. In this review we aim at presenting a comprehensive view of mAbs' application in the modern treatment of skin cancer. We present the characteristics and efficacy of mAbs currently used in dermatooncology and summarize the recent clinical trials in the field. We discuss the side effects and strategies for their managing.

Continuous femoral nerve block is more effective than continuous adductor canal block for treating pain after total knee arthroplasty: A randomized, double-blind, controlled trial.

OBJECTIVES: Previous studies comparing adductor canal block (ACB) with femoral nerve block (FNB) are inconclusive with regard to patient-controlled analgesia (PCA) induced by opioids. Moreover, some postoperative pain severity results differ between previous randomized controlled trials (RCTs). The primary aim of the current study was to compare total intravenous morphine consumption administered via PCA during the first postoperative day in continuous FNB and ACB groups after total knee arthroplasty (TKA). Secondary aims included evaluation of postoperative pain via a visual analog scale, degree of knee extension, quadriceps muscle strength, and ability to sit, stand upright, and walk. METHODS: The study was a RCT. Inclusion criteria were presence of gonarthrosis, age >18 and <75 years, and scheduled for TKA under single-shot spinal anesthesia. RESULTS: A number of morphine uses was lower in the FNB group than in the ACB group (14, range 12-15 vs 20, range 18-22; P = .0001), and they perceived less severe pain at the 8th (P = .00003) and 24th hours. However, ACB was significantly superior with regard to most of the other parameters pertaining to mobility, including muscle strength at the 8th and 24th hours, degree of knee extension at the 8th hour, sitting at the 8th hour, standing upright at the 24th hour, and walking at the 24th and 48th hours. DISCUSSION: FNB was associated with the perception of less severe pain after TKAs. However, ACB was associated with earlier mobility rehabilitation.

Mechanistic Information on the Redox Cycling of Nickel(II/III) Complexes in the Presence of Sulfur Oxides and Oxygen. Correlation with DNA Damage Experiments.

The reactions of the water-soluble complexes [NiCR](2+) (where CR = 2,12-dimethyl-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),2,11,13,15-pentaene) and [NiKGH-CONH(2)](+) (where KGH-CONH(2) = lysylglycylhistidinecarboxamide) with sulfite/O(2) and peroxymonosulfate have been investigated using spectrophotometric and rapid-scan techniques. In most cases, the spectral changes suggest the formation of an intermediate Ni(III) species, followed by decomposition reactions which involve a back-reaction to Ni(II). Only in the case of the [NiCR](2+)-S(IV)-O(2) system is the formed Ni(III) species stable in solution. When sulfite and oxygen are used to oxidize Ni(II) to Ni(III), the reaction is oxygen dependent and an induction period could be observed, whereas the use of the strong oxidizing agent peroxymonosulfate resulted in no induction period and no oxygen dependence. In addition, the oxidation of Ni(II) to Ni(III) was faster if peroxymonosulfate was used instead of sulfite/O(2). The [NiKGH-CONH(2)](+) complex reacts much faster with sulfite/O(2) and peroxymonosulfate than the [NiCR](2+) does. Rate constants for the oxidation process and possible reaction mechanisms, based on available literature data, that can account for the observed kinetic observations in a qualitative way are presented, and the results are correlated with previously obtained data on DNA modification using these systems.