Piotr Mrowka

Statins impair antitumor effects of rituximab by inducing conformational changes of CD20.

Background Rituximab is used in the treatment of CD20+ B cell lymphomas and other B cell lymphoproliferative disorders. Its clinical efficacy might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising antilymphoma effects. The objective of this study was to evaluate the influence of statins on rituximab-induced killing of B cell lymphomas. Methods and Findings Complement-dependent cytotoxicity (CDC) was assessed by MTT and Alamar blue assays as well as trypan blue staining, and antibody-dependent cellular cytotoxicity (ADCC) was assessed by a 51Cr release assay. Statins were found to significantly decrease rituximab-mediated CDC and ADCC of B cell lymphoma cells. Incubation of B cell lymphoma cells with statins decreased CD20 immunostaining in flow cytometry studies but did not affect total cellular levels of CD20 as measured with RT-PCR and Western blotting. Similar effects are exerted by other cholesterol-depleting agents (methyl-β-cyclodextrin and berberine), but not filipin III, indicating that the presence of plasma membrane cholesterol and not lipid rafts is required for rituximab-mediated CDC. Immunofluorescence microscopy using double staining with monoclonal antibodies (mAbs) directed against a conformational epitope and a linear cytoplasmic epitope revealed that CD20 is present in the plasma membrane in comparable amounts in control and statin-treated cells. Atomic force microscopy and limited proteolysis indicated that statins, through cholesterol depletion, induce conformational changes in CD20 that result in impaired binding of anti-CD20 mAb. An in vivo reduction of cholesterol induced by short-term treatment of five patients with hypercholesterolemia with atorvastatin resulted in reduced anti-CD20 binding to freshly isolated B cells. Conclusions Statins were shown to interfere with both detection of CD20 and antilymphoma activity of rituximab. These studies have significant clinical implications, as impaired binding of mAbs to conformational epitopes of CD20 elicited by statins could delay diagnosis, postpone effective treatment, or impair anti-lymphoma activity of rituximab.

Proteasome Inhibition Potentiates Antitumor Effects of Photodynamic Therapy in Mice through Induction of Endoplasmic Reticulum Stress and Unfolded Protein Response

Photodynamic therapy (PDT) is an approved therapeutic procedure that exerts cytotoxic activity toward tumor cells by inducing production of reactive oxygen species such as singlet oxygen. PDT leads to oxidative damage of cellular macromolecules, including proteins that undergo multiple modifications such as fragmentation, cross-linking, and carbonylation that result in protein unfolding and aggregation. Because the major mechanism for elimination of carbonylated proteins is their degradation by proteasomes, we hypothesized that a combination of PDT with proteasome inhibitors might lead to accumulation of carbonylated proteins in endoplasmic reticulum (ER), aggravated ER stress, and potentiated cytotoxicity toward tumor cells. We observed that Photofrin-mediated PDT leads to robust carbonylation of cellular proteins and induction of unfolded protein response. Pretreatment of tumor cells with three different proteasome inhibitors, including bortezomib, MG132, and PSI, gave increased accumulation of carbonylated and ubiquitinated proteins in PDT-treated cells. Proteasome inhibitors effectively sensitized tumor cells of murine (EMT6 and C-26) as well as human (HeLa) origin to PDT-mediated cytotoxicity. Significant retardation of tumor growth with 60% to 100% complete responses was observed in vivo in two different murine tumor models (EMT6 and C-26) when PDT was combined with either bortezomib or PSI. Altogether, these observations indicate that combination of PDT with proteasome inhibitors leads to potentiated antitumor effects. The results of these studies are of immediate clinical application because bortezomib is a clinically approved drug that undergoes extensive clinical evaluations for the treatment of solid tumors.

Effect of substrate stiffness on the osteogenic differentiation of bone marrow stem cells and bone-derived cells.

There is a profound dependence of cell behaviour on the stiffness of its microenvironment. To gain a better understanding of the regulation of cellular differentiation by mechanical cues, we investigated the influence of matrix stiffness (E = 1.46 kPa and E = 26.12 kPa) on differentiated osteogenic cell lineage of bone marrow stem cells (BM‐MSCs) and bone‐derived cells (BDCs) using flexible collagen‐coated polyacrylamide substrates. Differentiation potential was determined by measuring alkaline phosphatase activity, expression of osteoblast‐specific markers including alkaline phosphatase, osteocalcin, Runx2 and collagen type I, as well as assessment of mineralisation (Alizarin Red S staining). We found that osteogenic differentiation can be regulated by the rigidity of the substrate, which may depend on the commitment in multi‐ or uni‐potent targeting cells. Osteogenic differentiation of BM‐MSCs was enhanced on a stiff substrate compared to a soft one, whereas BDCs osteogenic differentiation did not vary depending on the substrate stiffness. The data help in understanding the role of the external mechanical determinants in stem cell differentiation, and can also be useful in translational approach in functional tissue engineering.

PPARγ ligands increase antileukemic activity of second- and third-generation tyrosine kinase inhibitors in chronic myeloid leukemia cells

BCR-ABL1 tyrosine kinase inhibitors (TKIs) have revolutionized the therapy of chronic myeloid leukemia (CML) and converted it into a truly chronic disease. However, there is still a significant group of patients who do not fully benefit from this success, as they fail to achieve remission, suffer from serious adverse effects of the therapy or undergo relapse or progression. Failure to complete eradication of CML cells with the current state-of-the-art treatment results from insensitivity of leukemia stem cells (LSCs) to TKIs.1 Knowing that more efficient inhibition of BCR-ABL1 with newer generations of TKIs is not able to cure the disease, a significant part of research effort has been redirected to find a way to effectively target LSCs. Therefore, many research groups have turned their interest into combination therapies, thereby allowing for interference with various signaling pathways.2, 3

α2β1 integrin-mediated mechanical signals during osteodifferentiation of stem cells from the Wharton’s jelly of the umbilical cord

INTRODUCTION The formation and maintenance of tissues is regulated by various signals triggered by biological, chemical, and physical factors. Data increasingly confirm that matrix or tissue elasticity plays an influential role in regulating numerous cell functions. The aim of the present study was to better understand the regulation of cellular differentiation by mechanical cues. We studied the influence of matrix stiffness on the osteodifferentiation of two cell lineages characterized by different responses: mesenchymal stromal/stem cells isolated from the Whartons jelly of the umbilical cord (UC-MSCs) with strong stiffness-dependent responses; and bone-derived cells (BDCs), which are insensitive to changes in matrix rigidity. The study also aimed to delineate how matrix stiffness affects intracellular signaling through focal adhesion kinase (FAK) activity—one of the key components in integrin-mediated signaling pathways. MATERIAL AND METHODS The effect of substrate stiffness on the expression of α2, α5, and β1 integrin was studied using real time PCR and Western blot using cells cultured in an osteogenic medium on tunable polyacrylamide gels coated with type I collagen, with elasticities corresponding to Youngs moduli of 1.46 kPa and 26.12 kPa. FAK activity was monitored using ELISA assays. RESULTS We demonstrate for the first time the changes in the expression of α2, α5, and β1 integrin subunits in perinatal stem cells and in adult osteoblast precursor cells during in vitro osteogenic differentiation on surfaces characterized by different stiffness. We found that matrix rigidity significantly affects the osteogenic differentiation of UC-MSCs through α2 integrin-mediated mechanotransduction events, though not through the α5 integrin subunit. In BDCs, there were no significant changes in the expression levels of the tested protein associated with varying stiffness. CONCLUSIONS Our results provide evidence that matrix rigidity affects the osteogenic differentiation of UC-MSCs via mechanotransduction events mediated by α2 integrin subunits.

Differential expression of BIRC family genes in chronic myeloid leukaemia – BIRC3 and BIRC8 as potential new candidates to identify disease progression

Although the therapy of chronic myeloid leukaemia (CML) with tyrosine kinase inhibitors (TKI) is considered a major advance in oncology, a significant group of patients still develops drug resistance and does not benefit from targeted therapy (Marin et al, 2013). These patients are at high risk of progression from chronic phase (CML-CP) to almost inevitably fatal blastic phase (CML-BP) (or blast crisis). Therefore, there is a need to identify suboptimal responders to imatinib who should be switched to secondor third-generation TKI as early as possible (Marin et al, 2013). Although the Sokal and Hasford risk scores remain the most used prognostic indicators at diagnosis, reliable markers of suboptimal response and/or progression of the disease are needed. BCR-ABL1 transcript level monitoring is used to evaluate response to the therapy, and its predictive value at 3 and/or 6 months after starting TKI therapy has recently been shown (Neelakantan et al, 2013). Among other possible candidates, high levels of cancerous inhibitor of PP2A (CIP2A) may be an independent determinant of progression to blast crisis (Lucas et al, 2011). Searching for potential candidates of disease progression we have focused on the BIRC (baculoviral IAP repeat-containing; BIRC) gene family expression in various stages of CML. To date, there is no comprehensive analysis of the whole family of BIRC genes in CML. The BIRC family comprises eight functionallyand structurally-related members. Their common structural feature, a motif termed the baculovirus IAP repeat, is required for their cytoprotective function, hence the majority of BIRC serve as endogenous inhibitors of apoptosis (Deveraux & Reed, 1999). While the first human BIRC gene (originally termed BIRC1, now NAIP, encoding the NLR family, apoptosis inhibitory protein) was described in the neurodegenerative disorder, spinal muscular atrophy (Roy et al, 1995), most recent studies focus on the role of BIRC genes in various types of neoplasms. Overexpression of BIRC genes has been associated with cancer progression, multidrug resistance, poor prognosis and short survival in several cancers including haematological malignancies. One of the most extensively studied members of BIRC family – BIRC5 (BIRC5, also known as survivin) is upregulated in haematological malignancies and solid tumours, and was also shown to be overexpressed in CML-BP in comparison to CML-CP (Hernandez-Boluda et al, 2005). Higher expression of BIRC5 was also linked to higher Sokal score and was positively correlated with P-glycoprotein expression in late CML-CP (Reis et al, 2011). Recently, the disruption of BIRC3, leading to truncated protein expression has been implicated in resistance to fludarabine in chronic lymphocytic leukaemia (CLL) (Rossi et al, 2012). To elucidate the potential role of the BIRC family of genes in CML we investigated the relative expression of all eight known BIRC family members. We analysed sequential samples of cDNA from peripheral blood obtained from CML patients at various stages of the disease. Blood samples were taken after informed consent was obtained and reverse transcription (RT)-quantitative real time polymerase chain reaction (qPCR) experiments were performed according to MIQE (Minimum Information for Publication of qPCR Experiments) guidelines (Bustin et al, 2009). Initially we looked at samples from CML-CP patients at diagnosis and after development of TKI resistance (confirmed as a loss of cytogenetic response, n = 5). Four patients developed resistance to imatinib (one had V299L mutation); the fifth was resistant to imatinib, dasatinib and nilotinib (no mutation detected) (Fig. 1A). We then analysed sequential samples from patients with CML who progressed to either accelerated phase (CMLAccPh) (n = 2) or to CML-BP (n = 4). Contrary to the general view of the role of BIRC genes in tumour progression, we found a significant decrease in BIRC3 and BIRC8 expression in CML-CP after development of TKI resistance (Fig. 1A) and also in progression to CML-AccPh/BP (Fig. 1B). We also observed a marked increase in BIRC5 expression after progression to CML-AccPh/BP (as previously shown by Hernandez-Boluda et al, 2005) (Fig. 1B) but not in CML-CP samples obtained after development of TKI resistance (Fig. 1A). Expression of other BIRC genes, namely: NAIP (BIRC1), BIRC2, XIAP (BIRC4), BIRC6 and BIRC7 was comparable in all the studied stages of the disease (data not shown). To confirm these results analysis of a larger group of patients was performed. Samples were obtained from patients in either CML-CP at diagnosis prior to any treatment (n = 15) or in CML-BP (n = 11) (Fig. 2). To compare BIRC family genes expression in CML with that of normal haematopoietic cells, cDNA from healthy blood donors was also used (n = 10). In accordance with paired samples analysis, we observed downregulation of BIRC3 and BIRC8 expression in CML-BP, while BIRC5 was upregulated in CML-BP patients (as compared to CML-CP and healthy blood donors) (Fig. 2). There was no significant difference in the relative expression of other BIRC family members (data not shown). correspondence