Anna Colpo

Protein Kinase CK2 Protects Multiple Myeloma Cells from ER Stress–Induced Apoptosis and from the Cytotoxic Effect of HSP90 Inhibition through Regulation of the Unfolded Protein Response

Purpose: Protein kinase CK2 promotes multiple myeloma cell growth by regulating critical signaling pathways. CK2 also modulates proper HSP90-dependent client protein folding and maturation by phosphorylating its co-chaperone CDC37. Because the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) is central in myeloma pathogenesis, we tested the hypothesis that the CK2/CDC37/HSP90 axis could be involved in UPR in myeloma cells. Experimental Design: We analyzed CK2 activity upon ER stress, the effects of its inactivation on the UPR pathways and on ER stress–induced apoptosis. The consequences of CK2 plus HSP90 inhibition on myeloma cell growth in vitro and in vivo and CK2 regulation of HSP90-triggered UPR were determined. Results: CK2 partly localized to the ER and ER stress triggered its kinase activity. CK2 inhibition reduced the levels of the ER stress sensors IRE1α and BIP/GRP78, increased phosphorylation of PERK and EIF2α, and enhanced ER stress–induced apoptosis. Simultaneous inactivation of CK2 and HSP90 resulted in a synergic anti-myeloma effect (combination index = 0.291) and in much stronger alterations of the UPR pathways as compared with the single inhibition of the two molecules. Cytotoxicity from HSP90 and CK2 targeting was present in a myeloma microenvironment model, on plasma cells from patients with myeloma and in an in vivo mouse xenograft model. Mechanistically, CK2 inhibition led to a reduction of IRE1α/HSP90/CDC37 complexes in multiple myeloma cells. Conclusions: Our results place CK2 as a novel regulator of the ER stress/UPR cascades and HSP90 function in myeloma cells and offer the groundwork to design novel combination treatments for this disease. Clin Cancer Res; 18(7); 1888–900. ©2012 AACR.

Protein Kinase CK2 Inhibition Down Modulates the NF-κB and STAT3 Survival Pathways, Enhances the Cellular Proteotoxic Stress and Synergistically Boosts the Cytotoxic Effect of Bortezomib on Multiple Myeloma and Mantle Cell Lymphoma Cells

CK2 is a pivotal pro-survival protein kinase in multiple myeloma that may likely impinge on bortezomib-regulated cellular pathways. In the present study, we investigated CK2 expression in multiple myeloma and mantle cell lymphoma, two bortezomib-responsive B cell tumors, as well as its involvement in bortezomib-induced cytotoxicity and signaling cascades potentially mediating bortezomib resistance. In both tumors, CK2 expression correlated with that of its activated targets NF-κB and STAT3 transcription factors. Bortezomib-induced proliferation arrest and apoptosis were significantly amplified by the simultaneous inhibition of CK2 with two inhibitors (CX-4945 and K27) in multiple myeloma and mantle cell lymphoma cell lines, in a model of multiple myeloma bone marrow microenvironment and in cells isolated from patients. CK2 inhibition empowered bortezomib-triggered mitochondrial-dependent cell death. Phosphorylation of NF-κB p65 on Ser529 (a CK2 target site) and rise of the levels of the endoplasmic reticulum stress kinase/endoribonuclease Ire1α were markedly reduced upon CK2 inhibition, as were STAT3 phospho Ser727 levels. On the contrary, CK2 inhibition increased phospho Ser51 eIF2α levels and enhanced the bortezomib-dependent accumulation of poly-ubiquitylated proteins and of the proteotoxic stress-associated chaperone Hsp70. Our data suggest that CK2 over expression in multiple myeloma and mantle cell lymphoma cells might sustain survival signaling cascades and can antagonize bortezomib-induced apoptosis at different levels. CK2 inhibitors could be useful in bortezomib-based combination therapies.

Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

BackgroundGlycogen Synthase Kinase-3 (GSK-3) α and β are two serine-threonine kinases controlling insulin, Wnt/β-catenin, NF-κB signaling and other cancer-associated transduction pathways. Recent evidence suggests that GSK-3 could function as growth-promoting kinases, especially in malignant cells. In this study, we have investigated GSK-3α and GSK-3β function in multiple myeloma (MM).MethodsGSK-3 α and β expression and cellular localization were investigated by Western blot (WB) and immunofluorescence analysis in a panel of MM cell lines and in freshly isolated plasma cells from patients. MM cell growth, viability and sensitivity to bortezomib was assessed upon treatment with GSK-3 specific inhibitors or transfection with siRNAs against GSK-3 α and β isoforms. Survival signaling pathways were studied with WB analysis.ResultsGSK-3α and GSK-3β were differently expressed and phosphorylated in MM cells. Inhibition of GSK-3 with the ATP-competitive, small chemical compounds SB216763 and SB415286 caused MM cell growth arrest and apoptosis through the activation of the intrinsic pathway. Importantly, the two inhibitors augmented the bortezomib-induced MM cell cytotoxicity. RNA interference experiments showed that the two GSK-3 isoforms have distinct roles: GSK-3β knock down decreased MM cell viability, while GSK-3α knock down was associated with a higher rate of bortezomib-induced cytotoxicity. GSK-3 inhibition caused accumulation of β-catenin and nuclear phospho-ERK1, 2. Moreover, GSK-3 inhibition and GSK-3α knockdown enhanced bortezomib-induced AKT and MCL-1 protein degradation. Interestingly, bortezomib caused a reduction of GSK-3 serine phosphorylation and its nuclear accumulation with a mechanism that resulted partly dependent on GSK-3 itself.ConclusionsThese data suggest that in MM cells GSK-3α and β i) play distinct roles in cell survival and ii) modulate the sensitivity to proteasome inhibitors.

Current Status of Autologous Stem Cell Transplantation in Relapsed and Refractory Hodgkin's Lymphoma

Despite the relatively high long-term disease-free survival (DFS) rate for patients with Hodgkin lymphoma (HL) with modern combination chemotherapy or combined modality regimens, ∼20% of patients die from progressive or relapsed disease. The standard treatment for relapsed and primary refractory HL is salvage chemotherapy followed by high-dose chemotherapy and autologous stem cell transplantation (ASCT), which has shown a 5-year progression-free survival rate of ∼50%-60%. Recent developments in a number of diagnostic and therapeutic modalities have begun to improve these results. Functional imaging, refinement of clinical prognostic factors, and development of novel biomarkers have improved the predictive algorithms, allowing better patient selection and timing for ASCT. In addition, these algorithms have begun to identify a group of patients who are candidates for more aggressive treatment beyond standard ASCT. Novel salvage regimens may potentially improve the rate of complete remission prior to ASCT, and the use of maintenance therapy after ASCT has become a subject of current investigation. We present a summary of developments in each of these areas.

Therapeutic apheresis in pregnancy: General considerations and current practice

It is widely known that pregnancy does not represent a contraindication to therapeutic apheresis (TA) techniques. In fact, since the first experiences of TA in pregnancy for the prevention of hemolytic disease of the newborn, several diseases are at present treated with TA, mainly within 6 clinical categories: (a) TA is a priority and has no alternative equally effective treatment (e.g., thrombotic thrombocytopenic purpura); (b) TA is a priority but there are alternative therapies not contraindicated in pregnancy (e.g., myasthenia gravis); (c) TA is an effective tool of saving/avoiding drugs contraindicated in pregnancy (e.g., systemic lupus erythematosus); (d) TA is a treatment of specific conditions/complications of pregnancy with maternal and/or fetal risk (e.g., antiphospholipid syndrome); (e) TA is a treatment of specific conditions of pregnancy with exclusive fetal risk (e.g., hemolytic disease of the newborn); (f) TA is a treatment of disease which is strongly indicated and can exceptionally occur during pregnancy (e.g., Goodpastures syndrome). When dealing with TA pregnant patients, some technical aspects due to the physiological changes of gestation have to be carefully considered, in particular the increase of the circulating blood volume. Moreover a multidisciplinary medical team, including an obstetrician, a clinical consultant, specialist in TA and in transfusion medicine, and a neonatologist stand as a basic requirement for the proper management of some clinical conditions that may be characterized by high maternal and fetal risk.

The mechanisms of rejection in solid organ transplantation

Organ transplantation represents the preferred treatment option for many patients in terminal organ failure. The half-life of transplanted organs, however, is still far from being satisfactory with the vast majority of the organs failing within the first two decades following transplantation. At this stage, it has become apparent that rejection (prevalently mediated by humoral events) remains the primary cause of graft loss after the first year. In this light, studies are underway to better comprehend the immune events underlying graft rejection and novel immunosuppressive strategies are being explored. In this context, therapeutic apheresis techniques, that include therapeutic plasma exchange (TPE), immunoadsorption (IA) and extracorporeal photochemotherapy (ECP), represent an important adjunct in the current immunosuppressive armamentarium. This article briefly reviews our current understanding of the immune process underlying rejection of a solid organ transplant and describes the principal areas of application of therapeutic apheresis techniques in transplantation.

Successful treatment of pyoderma gangrenosum with granulocyte and monocyte adsorption apheresis

Pyoderma gangrenosum is a neutrophilic dermatosis clinically characterised by the presence of painful skin ulcerations with erythematous and undetermined borders and histologically by the presence of neutrophilic infiltrates in the dermis. Granulocyte and monocyte adsorption apheresis, also called granulocytapheresis, is a therapeutic strategy for extracorporeal immunomodulation that selectively removes activated granulocytes and monocytes/macrophages from the peripheral blood. Here, we report a case of a 73‐year‐old patient affected by a severe form of pyoderma gangrenosum presenting with multiple painful ulcers and pustules on his trunk and extremities. The disease was resistant to high doses of methylprednisolone and methotrexate and successfully treated by granulocyte and monocyte adsorption apheresis. To the best of our knowledge, this is the first report on the efficacy of granulocyte and monocyte adsorption apheresis in pyoderma gangrenosum in Europe.

Administration of vincristine in a patient with Machado-Joseph disease.

Chemotherapy-induced peripheral neurotoxicity is a major problem because it represents the dose-limiting side effect of a significant number of antineoplastic drugs, such as vinca alkaloids. Hereditary neuropathies usually predispose to severe vincristine neurotoxicity. Here, we report the case of a 56-year-old man with Machado-Joseph disease, also known as spinocerebellar ataxia type 3, treated with a vinca alkaloid without exacerbation of neurological symptoms.

Personalized treatment with immunoadsorption and intravenous immunoglobulin in a case of severe Rh alloimmunization during pregnancy unresponsive to plasma – exchange

INTRODUCTION Despite prophylaxis, a small proportion of RhD-negative women may develop anti-D antibodies after a sensitizing event occurring during pregnancy or delivery of a D-positive baby. Intrauterine transfusion (IUT) is the treatment of choice in case of fetal anemia, but it cannot be performed early during pregnancy. Combined treatment with therapeutic plasma-exchange (TPE) and intravenous immunoglobulin (IVIG) can avoid or delay IUT. Immunoadsorption (IA) could represent a more effective treatment in selected cases. CASE REPORT We report a D-negative female with a history of induced abortion and hydrops fetalis, referred at 8 weeks of gestation with a high anti-D titer. Despite implementing a TPE-IVIG protocol, the patient experienced a spontaneous abortion. At the beginning of her fourth pregnancy, only after a partially effective intensive TPE course, cycles of IA-IVIG were performed. Despite a suboptimal response on the anti-D titer, Doppler ultrasonographic measurements of the fetal middle cerebral artery peak systolic velocity first showed evidence of anemia at 30 weeks of gestation and a IUT was required. After the IUT, anemia persisted with a subsequent dramatic rise in titer, requiring an emergent cesarean section. The infant subsequently underwent successful treatment with IVIG, phototherapy and exchange transfusion and was discharged 7 weeks later without neurological deficits. DISCUSSION The treatment of high titer anti-D antibodies during pregnancy may require a multidisciplinary approach with utilization of different apheresis strategies in order to have a successful pregnancy outcome.

My pregnancy in the apheresis unit

The Apheresis Unit is a multidisciplinary service, and the application of therapeutic apheresis during pregnancy may treat several high-risk conditions, as reviewed by Marson et al. [1] in this issue of the journal. Pregnancy per se is a crucial life transitional event with physical, psychological, relational and emotional changes, with an impact on all aspects of life. Anxiety and concerns about one’s self and the baby are common during pregnancy. However, in highrisk conditions, a pre-existing medical illness or the new onset of a disease in the expectant mother may increase fears and stress. Apart from physiological and pathological knowledge and technical expertise, apheresis unit personnel should deal with the psychological aspect of a high-risk pregnancy. Moreover, in cases of pre-existing conditions in which apheresis treatment might be indicated to mitigate maternal and/or fetal risks, the therapeutic apheresis specialists are involved in pre-conception counseling. For the above mentioned reasons, in this particular patient population, it is very important to establish a positive caring relationship at the beginning of the therapeutic process. My experience as an apheresis physician working during my own pregnancy (Fig. 1) put me in a position to discover an emotional intimacy and empathy with other expectant mothers less fortunate than me. I could thoroughly understand their fears and their uncertainties, in addition to their need to identify credible and similar representative with whom they could establish a positive relationship, based on trust and respect. Pregnancy thereby became a common denominator between the doctor and the patient. Ordinary roles were altered and the physician got closer to the patient through a natural instinct composed of compassion, solidarity and emotional sharing. The experience of pregnancy and subsequent motherhood is a revolution that removes the distance between mothers and emphasizes social and professional instinct of solidarity and collegiality. During that period, I experienced “new eyes” to look at the other mothers-in therapy, and that is the reason why I tried to invest in my institutional role, offering competence, understanding, willingness and support to expectant mothers. At that moment, even more than in the past, competence and compassion were intertwined, I was emptied of my “positions of strength”, acquiring greater affinity with patients. Because at a certain time, when fears increase or when certainties waver, one can feel a kick coming from the womb. And in those moments, moms are moms, whether they are physicians or patients.