Targeting peroxiredoxin 1 impairs growth of breast cancer cells and potently sensitises these cells to prooxidant agents.

BACKGROUND: Our previous work has shown peroxiredoxin-1 (PRDX1), one of major antioxidant enzymes, to be a biomarker in human breast cancer. Hereby, we further investigate the role of PRDX1, compared to its close homolog PRDX2, in mammary malignant cells. METHODS: CRISPR/Cas9- or RNAi-based methods were used for genetic targeting PRDX1/2. Cell growth was assessed by crystal violet, EdU incorporation or colony formation assays. In vivo growth was assessed by a xenotransplantation model. Adenanthin was used to inhibit the thioredoxin-dependent antioxidant defense system. The prooxidant agents used were hydrogen peroxide, glucose oxidase and sodium L-ascorbate. A PY1 probe or HyPer-3 biosensor were used to detect hydrogen peroxide content in samples. RESULTS: PRDX1 downregulation significantly impaired the growth rate of MCF-7 and ZR-75-1 breast cancer cells. Likewise, xenotransplanted PRDX1-deficient MCF-7 cells presented a retarded tumour growth. Furthermore, genetic targeting of PRDX1 or adenanthin, but not PRDX2, potently sensitised all six cancer cell lines studied, but not the non-cancerous cells, to glucose oxidase and ascorbate. CONCLUSIONS: Our study pinpoints the dominant role for PRDX1 in management of exogeneous oxidative stress by breast cancer cells and substantiates further exploration of PRDX1 as a target in this disease, especially when combined with prooxidant agents.

FOXO1 promotes resistance of non-Hodgkin lymphomas to anti-CD20-based therapy.

Diminished overall survival rate of non-Hodgkin lymphoma (NHL) patients treated with a combination regimen of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) has been recently linked to recurrent somatic mutations activating FOXO1. Despite of the clinical relevance of this finding, the molecular mechanism driving resistance to R-CHOP therapy remains largely unknown. Herein, we investigated the potential role of FOXO1 in the therapeutic efficacy of rituximab, the only targeted therapy included in the R-CHOP regimen. We found CD20 transcription is negatively regulated by FOXO1 in NHL cell lines and in human lymphoma specimens carrying activating mutations of FOXO1. Furthermore, both the expression of exogenous mutants of FOXO1 and the inhibition of AKT led to FOXO1 activation in lymphoma cells, increased binding to MS4A1 promoter and diminished CD20 expression levels. In contrast, a disruption of FOXO1 with CRISPR/Cas9 genome-editing (sgFOXO1) resulted in CD20 upregulation, improved the cytotoxicity induced by rituximab and the survival of mice with sgFOXO1 tumors. Accordingly, pharmacological inhibition of FOXO1 activity in primary samples upregulated surface CD20 levels. Importantly, FOXO1 was required for the downregulation of CD20 levels by the clinically tested inhibitors of BTK, SYK, PI3K and AKT. Taken together, these results indicate for the first time that the AKT-unresponsive mutants of FOXO1 are important determinant of cell response to rituximab-induced cytotoxicity, and suggest that the genetic status of FOXO1 together with its transcriptional activity need further attention while designing anti-CD20 antibodies based regimens for the therapy of pre-selected lymphomas.

Intrinsic Functional Potential of NK-Cell Subsets Constrains Retargeting Driven by Chimeric Antigen Receptors.

Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19+ targets, and maintained their intrinsic degranulation response toward CD19- K562 cells. The response of redirected NK-cell subsets against CD19+ targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA-specific KIR(s), displayed superior ability to kill CD19+, HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells. Cancer Immunol Res; 6(4); 467-80. ©2018 AACR.

Synergistic antitumor effects of histone deacetylase inhibitor scriptaid and bortezomib against ovarian cancer cells.

Despite debulking surgery and good initial response to chemotherapy, the majority of patients with advanced ovarian cancer relapse and succumb to their disease. Thus, there is a pressing need to improve treatment outcome. In the present study, the antitumor activity of histone deacetylase (HDAC) inhibitor scriptaid in combination with bortezomib or conventional chemotherapeutics was tested in vitro against representative ovarian cancer cell lines: SKOV­3, MDAH 2774, and OVP­10. Incubation of ovarian cancer cells with scriptaid and bortezomib (or doxorubicin) led to synergistic antitumor effects. As shown in the Annexin V-FITC/PI assay and western blot analysis of caspase­3/-9 and p21 protein expression, these synergistic antitumor effects were due to both induction of apoptosis and inhibition of proliferation. Since synergistic antitumor activity of scriptaid and bortezomib appeared in suboptimal concentrations, one can assume that the administration of the combination of these agents to ovarian cancer patients can exert the therapeutic effect in parallel with limited general toxicity of the treatment.

HDAC6 inhibition upregulates CD20 levels and increases the efficacy of anti-CD20 monoclonal antibodies.

Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene (MS4A1) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors.

New insights into redox homeostasis as a therapeutic target in B-cell malignancies.

PURPOSE OF REVIEW: The goal of this review is to summarize recent advances in our understanding of the regulation of redox homeostasis and the subtype-specific role of antioxidant enzymes in B-cell-derived malignancies. Furthermore, it presents selected prooxidative therapeutic strategies against B-cell neoplasms. RECENT FINDINGS: Recent reports have shown that the disturbed redox homeostasis in B-cell malignancies is regulated by cancer-specific signaling pathways and therefore varies between the individual subtypes. For instance, in a subtype of diffuse large B-cell lymphoma with increased oxidative phosphorylation, elevated reactive oxygen species are accompanied by higher levels of thioredoxin and glutathione and inhibition of either of these systems is selectively toxic to this subtype. In addition, growing number of small molecule inhibitors targeting antioxidant enzymes, such as auranofin, SK053, adenanthin, or decreasing glutathione level, such as imexon, buthionine sulfoximine, and L-cysteinase, trigger specific cytotoxic effects against B-cell malignancies. Lastly, attention is drawn to recent reports of effective treatment modalities involving prooxidative agents and interfering with redox homeostasis provided by stromal cells. SUMMARY: Recent findings reveal important differences in redox homeostasis within the distinct subsets of B-cell-derived malignancies that can be therapeutically exploited to improve existing treatment and to overcome drug resistance.

B-cell receptor signaling in the pathogenesis of lymphoid malignancies.

B-cell receptor (BCR) signaling pathway plays a central role in B-lymphocyte development and initiation of humoral immunity. Recently, BCR signaling pathway has been shown as a major driver in the pathogenesis of B-cell malignancies. As a result, a vast array of BCR-associated kinases has emerged as rational therapeutic targets changing treatment paradigms in B cell malignancies. Based on high efficacy in early-stage clinical trials, there is rapid clinical development of inhibitors targeting BCR signaling pathway. Here, we describe the essential components of BCR signaling, their function in normal and pathogenic signaling and molecular effects of their inhibition in vitro and in vivo.

Adenanthin, a new inhibitor of thiol-dependent antioxidant enzymes, impairs the effector functions of human natural killer cells.

Natural killer (NK) cells are considered critical components of the innate and adaptive immune responses. Deficiencies in NK cell activity are common, such as those that occur in cancer patients, and they can be responsible for dysfunctional immune surveillance. Persistent oxidative stress is intrinsic to many malignant tumours, and numerous studies have focused on the effects of reactive oxygen species on the anti-tumour activity of NK cells. Indeed, investigations in animal models have suggested that one of the most important thiol-dependent antioxidant enzymes, peroxiredoxin 1 (PRDX1), is essential for NK cell function. In this work, our analysis of the transcriptomic expression pattern of antioxidant enzymes in human NK cells has identified PRDX1 as the most prominently induced transcript out of the 18 transcripts evaluated in activated NK cells. The change in PRDX1 expression was followed by increased expression of two other enzymes from the PRDX-related antioxidant chain: thioredoxin and thioredoxin reductase. To study the role of thiol-dependent antioxidants in more detail, we applied a novel compound, adenanthin, to induce an abrupt dysfunction of the PRDX-related antioxidant chain in NK cells. In human primary NK cells, we observed profound alterations in spontaneous and antibody-dependent NK cell cytotoxicity against cancer cells, impaired degranulation, and a decreased expression of activation markers under these conditions. Collectively, our study pinpoints the unique role for the antioxidant activity of the PRDX-related enzymatic chain in human NK cell functions. Further understanding this phenomenon will prospectively lead to fine-tuning of the novel NK-targeted therapeutic approaches to human disease.

Inhibitors of SRC kinases impair antitumor activity of anti-CD20 monoclonal antibodies.

Clinical trials with SRC family kinases (SFKs) inhibitors used alone or in a combination with anti-CD20 monoclonal antibodies (mAbs) are currently underway in the treatment of B-cell tumors. However, molecular interactions between these therapeutics have not been studied so far. A transcriptional profiling of tumor cells incubated with SFKs inhibitors revealed strong downregulation of MS4A1 gene encoding CD20 antigen. In a panel of primary and established B-cell tumors we observed that SFKs inhibitors strongly affect CD20 expression at the transcriptional level, leading to inhibition of anti-CD20 mAbs binding and increased resistance of tumor cells to complement-dependent cytotoxicity. Activation of the AKT signaling pathway significantly protected cells from dasatinib-triggered CD20 downregulation. Additionally, SFKs inhibitors suppressed antibody-dependent cell-mediated cytotoxicity by direct inhibition of natural killer cells. Abrogation of antitumor activity of rituximab was also observed in vivo in a mouse model. Noteworthy, the effects of SFKs inhibitors on NK cell function are largely reversible. The results of our studies indicate that development of optimal combinations of novel treatment modalities with anti-CD20 mAbs should be preceded by detailed preclinical evaluation of their effects on target cells.