Potentiation of （-）-epigallocatechin-3-gaUate-induced apoptosis by bortezomib. in multiple myeloma cells

The green tea constituent, （-）-epigallocatechin-3- gallate （EGCG）, has chemopreventive and anticancer effects. This is partially because of the selective ability of EGCG to induce apoptosis and death in cancer cells without affecting normal cells. In the present study, the activity of EGCG against the myeloma cell line, KM3, was examined. Our results demonstrated, for the first time, that the treatment of the KM3 cell line with EGCG inhibits cell proliferation and induces apoptosis, and there is a synergistic effect when EGCG and borte- zomib are combined. Further experiments showed that this effect involves the NF-KB pathway. EGCG inhibits the expression of the P65 mRNA and P65/pP65 protein, meanwhile it downregulates pIKBα expression and upregulates IKBα expression. EGCG also activates caspase-3, -8, cleaved caspase-9, and poly-ADP-ribose polymerase （PARP） and subsequent apoptosis. These findings provided experimental evidence for efficacy of EGCG alone or in combination with bortezomib in multiple myeloma therapy.     

TrxR1 inhibition overcomes both hypoxia-induced and acquired bortezomib resistance in multiple myeloma through NF-кβ inhibition

Multiple myeloma (MM) is a B-cell malignancy characterized by an accumulation of abnormal clonal plasma cells in the bone marrow. Introduction of the proteasome-inhibitor bortezomib has improved MM prognosis and survival; however hypoxia-induced or acquired bortezomib resistance remains a clinical problem. This study highlighted the role of thioredoxin reductase 1 (TrxR1) in the hypoxia-induced and acquired bortezomib resistance in MM. Higher TrxR1 gene expression correlated with high-risk disease, adverse overall survival, and poor prognosis in myeloma patients. We demonstrated that hypoxia induced bortezomib resistance in myeloma cells and increased TrxR1 protein levels. Inhibition of TrxR1 using auranofin overcame hypoxia-induced bortezomib resistance and restored the sensitivity of hypoxic-myeloma cells to bortezomib. Hypoxia increased NF-кβ subunit p65 nuclear protein levels and TrxR1 inhibition decreased hypoxia-induced NF-кβ p65 protein levels in the nucleus and reduced the expression of NF-кβ-regulated genes. In addition, higher TrxR1 protein levels were observed in bortezomib-resistant myeloma cells compared to the naïve cells, and its inhibition using either auranofin or TrxR1-specific siRNAs reversed bortezomib resistance. TrxR1 inhibition reduced p65 mRNA and protein expression in bortezomib-resistant myeloma cells, and also decreased the expression of NF-кβ-regulated anti-apoptotic and proliferative genes. Thus, TrxR1 inhibition overcomes both hypoxia-induced and acquired bortezomib resistance by inhibiting the NF-кβ signaling pathway. Our findings demonstrate that elevated TrxR1 levels correlate with the acquisition of bortezomib resistance in MM. We propose considering TrxR1-inhibiting drugs, such as auranofin, either for single agent or combination therapy to circumvent bortezomib-resistance and improve survival outcomes of MM patients.     

Mechanism of immunomodulatory drugs＇ action in the treatment of multiple myeloma

Although immunomodulatory drugs （IMiDs）, such as thal- idomide, lenalidomide, and pomalidomide, are widely used in the treatment of multiple myeloma （MM）, the molecular mechanism of IMiDs＇ action is largely unknown. In this review, we will summarize recent advances in the application of IMiDs in MM cancer treatment as well as their effects on immunomodulatory activities, anti-angiogenic activities, intervention of cell surface adhesion molecules between myeloma cells and bone marrow stromal cells, anti-inflam- matory activities, anti-proliferation, pro-apoptotic effects, cell cycle arrest, and inhibition of cell migration and metastasis. In addition, the potential IMiDs＇ target protein, IMiDs＇ target protein＇s functional role, and the potential molecular mechanisms of IMiDs resistance will be discussed. We wish, by presentation of our naive discussion, that this review article will facilitate further investigation in these fields.     

Drug inhibition profile prediction for NFκB pathway in multiple myeloma

Nuclear factor κB (NFκB) activation plays a crucial role in anti-apoptotic responses in response to the apoptotic signaling during tumor necrosis factor (TNFα) stimulation in Multiple Myeloma (MM). Although several drugs have been found effective for the treatment of MM by mainly inhibiting NFκB pathway, there are not any quantitative or qualitative results of comparison assessment on inhibition effect between different drugs either used alone or in combinations. Computational modeling is becoming increasingly indispensable for applied biological research mainly because it can provide strong quantitative predicting power. In this study, a novel computational pathway modeling approach is employed to comparably assess the inhibition effects of specific drugs used alone or in combinations on the NFκB pathway in MM and to predict the potential synergistic drug combinations.     

Identify multiple myeloma stem cells: Utopia?

Multiple myeloma (MM) is a hematologic malignancy of monoclonal plasma cells which remains incurable despite recent advances in therapies. The presence of cancer stem cells (CSCs) has been demonstrated in many solid and hematologic tumors, so the idea of CSCs has been proposed for MM, even if MM CSCs have not been define yet. The existence of myeloma CSCs with clonotypic B and clonotypic non B cells was postulated by many groups. This review aims to focus on these distinct clonotypic subpopulations and on their ability to develop and sustain MM. The bone marrow microenvironment provides to MM CSCs self-renewal, survival and drug resistance thanks to the presence of normal and cancer stem cell niches. The niches and CSCs interact each other through adhesion molecules and the interplay between ligands and receptors activates stemness signaling (Hedgehog, Wnt and Notch pathways). MM CSCs are also supposed to be responsible for drug resistance that happens in three steps from the initial cancer cell homing microenvironment-mediated to development of microenvironment-independent drug resistance. In this review, we will underline all these aspects of MM CSCs.     

Curcusone D,a novel ubiquitin–proteasome pathway inhibitor via ROS-induced DUB inhibition,is synergistic with bortezomib against multiple myeloma cell growth

Background

Ubiquitin–proteasome pathway (UPP) plays a very important role in the degradation of proteins. Finding novel UPP inhibitors is a promising strategy for treating multiple myeloma (MM).

Methods

Ub-YFP reporter assays were used as cellular UPP models. MM cell growth, apoptosis and overall death were evaluated with the MTS assay, Annexin V/PI dual-staining flow cytometry, poly (ADP-ribose) polymerase (PARP) cleavage, and PI uptake, respectively. The mechanism of UPP inhibition was analyzed by western blotting for ubiquitin, in vitro and cellular proteasomal and deubiquitinases (DUBs) activity assays. Cellular reactive oxygen species (ROS) were measured with H₂DCFDA.

Results

Curcusone D, identified as a novel UPP inhibitor, causes cell growth inhibition and apoptosis in MM cells. Curcusone D induced the accumulation of poly-ubiquitin-conjugated proteins but could not inhibit proteasomal activity in vitro or in cells. Interestingly, the mono-ubiquitin level and the total cellular DUB activity were significantly downregulated following curcusone D treatment. Furthermore, curcusone D could induce ROS, which were closely correlated with DUB inhibition that could be nearly completely reversed by NAC. Finally, curcusone D and the proteasomal inhibitor bortezomib showed a strong synergistic effect against MM cells.

Conclusions

Curcusone D is novel UPP inhibitor that acts via the ROS-induced inhibition of DUBs to produce strong growth inhibition and apoptosis of MM cells and synergize with bortezomib.

General significance

The anti-MM molecular mechanism study of curcusone D will promote combination therapies with different UPP inhibitors against MM and further support the concept of oxidative stress regulating the activity of DUBs.     

The impact of new emerging drugs in the treatment of multiple myeloma: is there still a role for PBSC transplantation?

High-dose therapy with the rescue of autologous stem cells represents today the standard approach for multiple myeloma patients aged <65 years. Several studies, in fact, have demonstrated the superiority of high-dose therapy with respect to conventional chemotherapy in younger patients. Peripheral blood stem cells (PBSCs) provide a rapid and effective hematopoietic recovery after the administration of supra maximal chemotherapy and mainly for this reason have become the preferred source of stem cells for autologous transplantation. Recently, however, a number of new drugs have appeared in the armamentarium of the hematologist. Among these, thalidomide has been the first antiangiogenetic drug effectively adopted firstly in refractory-relapsed patients and now also as first line treatment with better results respect to VAD or VAD-like regimens. Inhibitors of proteasome, such as bortezomib, and other immunomodulatory agents, such as lenalidomide, have been also studied more recently in myeloma patients. In particular, bortezomib has shown to be very effective as single agent or in combination with high-dose dexamethasone. In this review, we try to define the potential role of these new drugs, how and when they can be included in the therapeutic program designed for younger and older patients, and mostly if and how these new agents could jeopardize the central role of autologous stem cell transplantation in the treatment of multiple myeloma.     

Fractal characteristics of May-Grünwald-Giemsa stained chromatin are independent prognostic factors for survival in multiple myeloma

Background

The use of computerized image analysis for the study of nuclear texture features has provided important prognostic information for several neoplasias. Recently fractal characteristics of the chromatin structure in routinely stained smears have shown to be independent prognostic factors in acute leukemia. In the present study we investigated the influence of the fractal dimension (FD) of chromatin on survival of patients with multiple myeloma.

Methodology

We analyzed 67 newly diagnosed patients from our Institution treated in the Brazilian Multiple Myeloma Study Group. Diagnostic work-up consisted of peripheral blood counts, bone marrow cytology, bone radiograms, serum biochemistry and cytogenetics. The International Staging System (ISS) was used. In every patient, at least 40 digital nuclear images from diagnostic May-Grünwald-Giemsa stained bone marrow smears were acquired and transformed into pseudo-3D images. FD was determined by the Minkowski-Bouligand method extended to three dimensions. Goodness-of-fit of FD was estimated by the R² values in the log-log plots. The influence of diagnostic features on overall survival was analyzed in Cox regressions. Patients that underwent autologous bone marrow transplantation were censored at the day of transplantation.

Principal Findings

Median age was 56 years. According to ISS, 14% of the patients were stage I, 39% were stage II and 47% were stage III. Additional features of a bad prognosis were observed in 46% of the cases. When stratifying for ISS, both FD and its goodness-of-fit were significant prognostic factors in univariate analyses. Patients with higher FD values or lower goodness-of-fit showed a worse outcome. In the multivariate Cox-regression, FD, R², and ISS stage entered the final model, which showed to be stable in a bootstrap resampling study.

Conclusions

Fractal characteristics of the chromatin texture in routine cytological preparations revealed relevant prognostic information in patients with multiple myeloma.     

KSP inhibitor SB743921 induces death of multiple myeloma cells via inhibition of the NF-κB signaling pathway

SB743921 is a potent inhibitor of the spindle protein kinesin and is being investigated in ongoing clinical trials for the treatment of myeloma. However, little is known about the molecular events underlying the induction of cell death in multiple myeloma (MM) by SB743921, alone or in combination treatment. Here, we report that SB743921 induces mitochondria-mediated cell death via inhibition of the NF-κB signaling pathway, but does not cause cell cycle arrest in KMS20 MM cells. SB743921-mediated inhibition of the NF-κB pathway results in reduced expression of SOD2 and Mcl-1, leading to mitochondrial dysfunction. We also found that combination treatment with SB743921 and bortezomib induces death in bortezomib-resistant KMS20 cells. Altogether, these data suggest that treatment with SB743921 alone or in combination with bortezomib offers excellent translational potential and promises to be a novel MM therapy. [BMB Reports 2015; 48(10): 571-576]     

Immunodominance in the immune response to “multiple” histocompatibility antigens

Cytotoxic effector T cells putatively specific for multiple non-H-2 histocompatibility (H) antigens were generated by immunizing and boosting C57BL/6 and B6.C-H-2 ^dmice with BALB.B and BALB/c stimulator cells, respectively. The generated effectors were tested for cell-mediated lympholysis on a panel of targets whose BALB/c-derived non-H-2 H antigens were donated by CXB recombinant inbred mice. The spectrum of reactivity of cytotoxic effector T cells with CXB targets demonstrated that the effectors did not recognize multiple H antigens but rather preferentially recognized a single immunodominant non-H-2 H antigen. The identity of the immunodominant H antigen was determined by the H-2 genotype of the stimulator cells when (B6 × B6.C-H-2 ^d)F ₁ cytotoxic effectors were tested. These observations indicate that despite the fact that responders were challenged with more than 40 individual non-H-2 H antigens, they preferentially responded to a single immunodominant antigen.     

Fbxw7α- and GSK3-mediated degradation of p100 is a pro-survival mechanism in multiple myeloma

Fbxw7α is a member of the F-box family of proteins, which function as the substrate-targeting subunits of SCF (Skp1/Cul1/F-box protein) ubiquitin ligase complexes. Using differential purifications and mass spectrometry, we identified p100, an inhibitor of NF-κB signalling, as an interactor of Fbxw7α. p100 is constitutively targeted in the nucleus for proteasomal degradation by Fbxw7α, which recognizes a conserved motif phosphorylated by GSK3. Efficient activation of non-canonical NF-κB signalling is dependent on the elimination of nuclear p100 through either degradation by Fbxw7α or exclusion by a newly identified nuclear export signal in the carboxy terminus of p100. Expression of a stable p100 mutant, expression of a constitutively nuclear p100 mutant, Fbxw7α silencing or inhibition of GSK3 in multiple myeloma cells with constitutive non-canonical NF-κB activity results in apoptosis both in cell systems and xenotransplant models. Thus, in multiple myeloma, Fbxw7α and GSK3 function as pro-survival factors through the control of p100 degradation.     

Do treatment protocols improve end results? A study of survival of patients with multiple myeloma in Finland.

OBJECTIVE--To determine whether patients with multiple myeloma treated in three consecutive clinical trials of chemotherapy of the Finnish Leukaemia Group during 1979-85 had a more favourable prognosis than both patients treated in the trial area before the trials and those treated in the rest of Finland. DESIGN--Comparison of the time trend in survival of patients living in the trial area with that of patients living in the rest of Finland. SETTING--Trial area covered 17 of the 21 main hospital districts in Finland (serving more than two thirds of patients with multiple myeloma). PATIENTS--663 Men and 690 women in the trial area and 318 men and 307 women in the reference area aged under 71 in whom multiple myeloma was diagnosed during 1959-85. In the trial area the disease was diagnosed in 455 men and 493 women in 1959-78 and in 208 men and 197 women in 1979-85; in the reference area it was diagnosed in 234 men and 227 women in 1959-78 and in 84 men and 80 women in 1979-85. MAIN OUTCOME MEASURES--Five year cumulative relative survival rates during 1959-85, annual relative survival rates in the first seven years of follow up during 1979-85, and fitted annual relative survival rates in the first five years of follow up during 1959-85. RESULTS--During the first two years of follow up the annual relative survival rates did not differ between the two areas, but in the next five years of follow up patients in the trial area did better than those in the reference area. For cases diagnosed in 1979-85 the difference in the five year cumulative relative survival rates between patients in the trial area and those in the reference area was 10%, those in the trial area doing better. Generalised proportional hazards regression analysis of the first five years of follow up showed that the patients in the trial area had a survival advantage over those in the reference area. The model with the best fit included year of follow up, time of diagnosis, the joint effect of year of follow up and time of diagnosis, and the joint effect of area and time of diagnosis. CONCLUSION--The patients in the trial area benefited from the clinical trials, which suggests that the use of a treatment protocol improves the end results of treatment. In other words, the results favour a systematic treatment schedule in preference to a schedule determined by the free choice of a clinician.     

BTK inhibitor ibrutinib is cytotoxic to myeloma and potently enhances bortezomib and lenalidomide activities through NF-κB

Ibrutinib (previously known as PCI-32765) has recently shown encouraging clinical activity in chronic lymphocytic leukaemia (CLL) effecting cell death through inhibition of Bruton's tyrosine kinase (BTK). In this study we report for the first time that ibrutinib is cytotoxic to malignant plasma cells from patients with multiple myeloma (MM) and furthermore that treatment with ibrutinib significantly augments the cytotoxic activity of bortezomib and lenalidomide chemotherapies. We describe that the cytotoxicity of ibrutinib in MM is mediated via an inhibitory effect on the nuclear factor-κB (NF-κB) pathway. Specifically, ibrutinib blocks the phosphorylation of serine-536 of the p65 subunit of NF-κB, preventing its nuclear translocation, resulting in down-regulation of anti-apoptotic proteins Bcl-xL, FLIP_L and survivin and culminating in caspase-mediated apoptosis within the malignant plasma cells. Taken together these data provide a platform for clinical trials of ibrutinib in myeloma and a rationale for its use in combination therapy, particularly with bortezomib.