Beata Pyrzynska

TRAIL Triggers Apoptosis in Human Malignant Glioma Cells Through Extrinsic and Intrinsic Pathways

Many malignant glioma cells express death receptors for tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL), yet some of these cells are resistant to TRAIL. Here, we examined signaling events in TRAIL‐induced apoptosis and searched for therapeutic agents that could overcome TRAIL resistance in glioma cells. TRAIL induced apoptosis through death receptor 5 (DR5) and was mediated by caspase‐8‐initiated extrinsic and intrinsic mitochondrial pathways in sensitive glioma cell lines. TRAIL also triggered apoptosis in resistant glioma cell lines through the same pathways, but only if the cells were pretreated with chemotherapeutic agents, cisplatin, camptothecin and etoposide. Previous studies suggested that this was due to an increase in DR5 expression in wild‐type TP53 cells, but this mechanism did not account for cells with mutant TP53. Here, we show that a more general effect of these agents is to down regulate caspase‐8 inhibitor c‐FLIPS (the short form of cellular Fasassociated death domain‐like interleukin‐1‐converting enzyme‐inhibitory protein) and up‐regulate Bak, a pro‐apoptotic Bcl‐2 family member, independently of cells TP53 status. Furthermore, we showed that TRAIL alone or in combination with chemotherapeutic agents, induced apoptosis in primary tumor cultures from patients with malignant gliomas, reinforcing the potential of TRAIL as an effective therapeutic agent for malignant gliomas.

Proteomic identification of the wt-p53-regulated tumor cell secretome.

Tumor–stroma interactions play a major role in tumor development, maintenance and progression. Yet little is known on how the genetic alterations that underlie cell transformation elicit cell extrinsic changes modulating heterotypic cell interactions. We hypothesized that these events involve a modification in the complement of secreted proteins by the cell, acting as mediators of intercellular communication. To test this hypothesis, we examined the role of wt-p53, a major tumor suppressor, on the tumor microenvironment through its regulation of secreted factors. Using a combination of 2-DE and cICAT proteomic techniques, we found a total of 111 secreted proteins, 39 of which showed enhanced and 21 inhibited secretion in response to wt-p53 expression. The majority of these were not direct targets of p53 transcription factor activity, suggesting a novel role for wt-p53 in the control of intracellular protein trafficking and/or secreted protein stability. Evidence for p53-controlled post-translational modifications on nine secreted proteins was also found. These findings will enhance our understanding of wt-p53 modulated interactions of the tumor with its environment.

B-cell receptor pathway inhibitors affect CD20 levels and impair antitumor activity of anti-CD20 monoclonal antibodies

B-cell receptor pathway inhibitors affect CD20 levels and impair antitumor activity of anti-CD20 monoclonal antibodies

P14ARF inhibits human glioblastoma–induced angiogenesis by upregulating the expression of TIMP3

Malignant gliomas are the most common and the most lethal primary brain tumors in adults. Among malignant gliomas, 60%-80% show loss of P14ARF tumor suppressor activity due to somatic alterations of the INK4A/ARF genetic locus. The tumor suppressor activity of P14ARF is in part a result of its ability to prevent the degradation of P53 by binding to and sequestering HDM2. However, the subsequent finding of P14ARF loss in conjunction with TP53 gene loss in some tumors suggests the protein may have other P53-independent tumor suppressor functions. Here, we report what we believe to be a novel tumor suppressor function for P14ARF as an inhibitor of tumor-induced angiogenesis. We found that P14ARF mediates antiangiogenic effects by upregulating expression of tissue inhibitor of metalloproteinase-3 (TIMP3) in a P53-independent fashion. Mechanistically, this regulation occurred at the gene transcription level and was controlled by HDM2-SP1 interplay, where P14ARF relieved a dominant negative interaction of HDM2 with SP1. P14ARF-induced expression of TIMP3 inhibited endothelial cell migration and vessel formation in response to angiogenic stimuli produced by cancer cells. The discovery of this angiogenesis regulatory pathway may provide new insights into P53-independent P14ARF tumor-suppressive mechanisms that have implications for the development of novel therapies directed at tumors and other diseases characterized by vascular pathology.

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.

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.

Dissection of CD20 regulation in lymphoma using RNAi.

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Sorafenib improves rituximab and ofatumumab efficacy by decreasing the expression of complement regulatory proteins

Sorafenib improves rituximab and ofatumumab efficacy by decreasing the expression of complement regulatory proteins

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

ABSTRACT 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.

Abstract LB-242: PTEN regulates the CD20 antigen expression and affects rituximab-based therapy of lymphoma malignancies

The therapeutic strategies currently used in B cell malignancies include the treatment with monoclonal antibodies (rituximab and ofatumumab) directed against CD20 antigen. These antibodies specifically eliminate B cells by triggering indirect effector mechanisms of the immune system, like complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. In many patients, the reduced level of CD20 antigen on the surface of tumor B cells leads to the resistance to anti-CD20 therapy. The aim of this study was to explore the molecular mechanisms governing the regulation of CD20 expression in lymphoma cells as a potential explanation of the resistance to rituximab/ofatumumab therapy. We previously observed that CD20 mRNA expression is significantly affected by the SRC family inhibitors. Here, we report that also PTEN tumor suppressor is a negative regulator of CD20 expression. To uncover the transcriptional mechanisms governing the CD20 expression we employed the construct encoding the promoter region of CD20 cloned upstream of the firefly luciferase gene. Overexpression of wild-type PTEN (but not the phosphatase-deficient mutant) strongly affected the promoter activity and the expression of CD20, leading to decreased binding of rituximab and ofatumumab and increased resistance of tumor cells to complement-dependent cytotoxicity. Using the truncated versions of the CD20 promoter we identified a particular region (-313/-198) as the major region sensitive to PTEN overexpression. We found that the negative regulation of CD20 promoter activity by PTEN was mediated by inhibition of AKT signaling. We observed that the overexpression of constitutively active AKT1 (CA-AKT1) overcame the negative effect of PTEN and sensitized cells to rituximab/ofatumumab treatment. The results of our studies indicate that PTEN status in tumor cells should therefore be considered when analyzing the mechanisms of resistance of B cell malignancies to anti-CD20 therapies. Citation Format: Beata Pyrzynska, Kamil Bojarczuk, Marta Siernicka, Michal Dwojak, Malgorzata Bobrowicz, Nina Miazek, Piotr Zapala, Agnieszka Zagozdzon, Jakub Golab, Magdalena Winiarska. PTEN regulates the CD20 antigen expression and affects rituximab-based therapy of lymphoma malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-242. doi:10.1158/1538-7445.AM2015-LB-242