Dysfunction of Gpl1-Gih35-Wdr83 Complex in S. pombe Affects the Splicing of DNA Damage Repair Factors Resulting in Increased Sensitivity to DNA Damage.

Pre-mRNA splicing plays a key role in the regulation of gene expression. Recent discoveries suggest that defects in pre-mRNA splicing, resulting from the dysfunction of certain splicing factors, can impact the expression of genes crucial for genome surveillance mechanisms, including those involved in cellular response to DNA damage. In this study, we analyzed how cells with a non-functional spliceosome-associated Gpl1-Gih35-Wdr83 complex respond to DNA damage. Additionally, we investigated the role of this complex in regulating the splicing of factors involved in DNA damage repair. Our findings reveal that the deletion of any component within the Gpl1-Gih35-Wdr83 complex leads to a significant accumulation of unspliced pre-mRNAs of DNA repair factors. Consequently, mutant cells lacking this complex exhibit increased sensitivity to DNA-damaging agents. These results highlight the importance of the Gpl1-Gih35-Wdr83 complex in regulating the expression of DNA repair factors, thereby protecting the stability of the genome following DNA damage.

Investigation of the Interaction between Mechanosynthesized ZnS Nanoparticles and Albumin Using Fluorescence Spectroscopy.

In this paper, ZnS nanoparticles were bioconjugated with bovine serum albumin and prepared in a form of nanosuspension using a wet circulation grinding. The stable nanosuspension with monomodal particle size distribution (d50 = 137 nm) and negative zeta potential (-18.3 mV) was obtained. The sorption kinetics and isotherm were determined. Interactions between ZnS and albumin were studied using the fluorescence techniques. The quenching mechanism, describing both static and dynamic interactions, was investigated. Various parameters were calculated, including the quenching rate constant, binding constant, stoichiometry of the binding process, and accessibility of fluorophore to the quencher. It has been found that tryptophan, in comparison to tyrosine, can be closer to the binding site established by analyzing the synchronous fluorescence spectra. The cellular mechanism in multiple myeloma cells treated with nanosuspension was evaluated by fluorescence assays for quantification of apoptosis, assessment of mitochondrial membrane potential and evaluation of cell cycle changes. The preliminary results confirm that the nontoxic nature of ZnS nanoparticles is potentially applicable in drug delivery systems. Additionally, slight changes in the secondary structure of albumin, accompanied by a decrease in α-helix content, were investigated using the FTIR method after analyzing the deconvoluted Amide I band spectra of ZnS nanoparticles conjugated with albumin. Thermogravimetric analysis and long-term stability studies were also performed to obtain a complete picture about the studied system.

Topical application of local anesthetics to melanoma increases the efficacy of anti-PD-1 therapy.

Experimental and clinical data have shown that the nervous system can significantly stimulate the initiation and progression of melanoma. In support of this, approaches that reduce the transmission of signals from peripheral nerves to effector tissues reduce the recurrence of melanoma. Therefore, we investigated the effect of topical application of the local anesthetic Pliaglis (7% lidocaine and 7% tetracaine) on the growth of melanoma induced by intradermal application of B16F0 cells in mice without treatment and in mice treated with the anti-PD-1 antibody. We found that application of Pliaglis to melanoma significantly reduced its growth and this effect was even pronounced in mice treated with the anti-PD-1 antibody. To determine the mechanisms and pathways responsible for the observed effect, the in vitro effect of incubating melanoma cells with lidocaine and/or tetracaine and the in vivo gene expression of cancer and immune-related factors, percentage of immune cells, gene expression of selected neurotransmitter receptors and nerve growth factors in melanoma tissue were studied. We found that lidocaine and tetracaine significantly reduced the viability of B16F0 cells in vitro. In mice with melanoma, Pliaglis potentiated the effect of anti-PD-1 antibody on gene expression of COX-2, IL-1ß, IL-6, CCL11, F4/80, CD206, and NCR1. In addition, Pliaglis increased the gene expression of α9nACHR and 5-HT2a receptors and decreased the gene expression of nerve growth factor receptor (p75NTR) and p53. We also observed Pliaglis-mediated changes in myeloid populations. Topical application of this local anesthetic cream decreased the CD11b+Gr1- population and increased the CD11b+Gr1high population. Our data suggest that Pliaglis reduces melanoma growth through a direct effect on melanoma cells as well as through modulation of the immune response. The involvement of nervous system-related signaling in the inhibitory effect of Pliaglis on melanoma is inconclusive from our data.

Nano-bio interface between As4S4 nanoparticles and albumin influenced by wet stirred media milling.

Arsenic sulfide (As4S4) nanoparticles have been intensively researched as a promising drug in a cancer treatment. For the first time, the interaction between As4S4 and bovine serum albumin has been studied in this paper. Initially, the sorption kinetics of albumin on the surface of nanoparticles was investigated. Subsequently, its structural changes influenced by interaction with the As4S4 nanoparticles during wet stirred media milling were studied in deep. Both the dynamic and static quenching were detected after analyzing the fluorescence quenching spectra. From the synchronous fluorescence spectra it was investigated, that the fluorescence intensity for tyrosine residues decreased by about 55%, and for tryptophan it was about 80%. It indicates the fluorescence from tryptophan is more intense and gets more efficiently quenched than those from tyrosine residues in presence of As4S4, implying that the tryptophan can be closer to the binding site. From the circular dichroisms and FTIR spectra it was observed that conformation of the protein remains almost unchanged. The content of appropriate secondary structures was determined by deconvolution of the absorption peak attributed to the amide I band in FTIR spectra. The preliminary anti-tumor cytotoxic effect of prepared albumin-As4S4 system was also tested on multiple myeloma cell lines.

Phytochemical-based nanodrugs going beyond the state-of-the-art in cancer management-Targeting cancer stem cells in the framework of predictive, preventive, personalized medicine.

Cancer causes many deaths worldwide each year, especially due to tumor heterogeneity leading to disease progression and treatment failure. Targeted treatment of heterogeneous population of cells - cancer stem cells is still an issue in protecting affected individuals against associated multidrug resistance and disease progression. Nanotherapeutic agents have the potential to go beyond state-of-the-art approaches in overall cancer management. Specially assembled nanoparticles act as carriers for targeted drug delivery. Several nanodrugs have already been approved by the US Food and Drug Administration (FDA) for treating different cancer types. Phytochemicals isolated from plants demonstrate considerable potential for nanomedical applications in oncology thanks to their antioxidant, anti-inflammatory, anti-proliferative, and other health benefits. Phytochemical-based NPs can enhance anticancer therapeutic effects, improve cellular uptake of therapeutic agents, and mitigate the side effects of toxic anticancer treatments. Per evidence, phytochemical-based NPs can specifically target CSCs decreasing risks of tumor relapse and metastatic disease manifestation. Therefore, this review focuses on current outlook of phytochemical-based NPs and their potential targeting CSCs in cancer research studies and their consideration in the framework of predictive, preventive, and personalized medicine (3PM).

Heterogeneity of B cell lymphopoiesis in patients with premalignant and active myeloma.

To better characterize the heterogeneity of multiple myeloma (MM), we profiled plasma cells (PCs) and their B cell lymphopoiesis in the BM samples from patients with monoclonal gammopathy of undetermined significance, smoldering MM, and active MM by mass cytometry (CyTOF) analysis. Characterization of intra- and interneoplastic heterogeneity of malignant plasmablasts and PCs revealed overexpression of the MM SET domain (MMSET), Notch-1, and CD47. Variations in upregulation of B cell signaling regulators (IFN regulatory factor 4 [IRF-4], CXCR4, B cell lymphoma 6 [Bcl-6], c-Myc, myeloid differentiation primary response protein 88 [MYD88], and spliced X box-binding protein 1 [sXBP-1]) and aberrant markers (CD319, CD269, CD200, CD117, CD56, and CD28) were associated with different clinical outcomes in clonal PC subsets. In addition, prognosis was related to heterogeneity in subclonal expression of stemness markers, including neuroepithelial stem cell protein (Nestin), SRY-box transcription factor 2 (Sox2), Krüppel-like factor 4 (KLF-4), and Nanog. Furthermore, we have defined significantly elevated levels of MMSET, MYD88, c-Myc, CD243, Notch-1, and CD47 from hematopoietic stem cells to PCs in myeloma B cell lymphopoiesis, noted even in premalignant conditions, with variably modulated expression of B cell development regulators, including IRF-4, Bcl-2, Bcl-6, and sXBP-1; aberrant PC markers (such as CD52, CD44, CD200, CD81, CD269, CD117, and CXCR4); and stemness-controlling regulators, including Nanog, KLF-4, octamer-binding transcription factor 3/4 (Oct3/4), Sox2, and retinoic acid receptor α2 (RARα2). This study provides the rationale for precise molecular profiling of patients with MM by CyTOF technology to define disease heterogeneity and prognosis.

Dysfunctions of innate and adaptive immune tumor microenvironment in Waldenström macroglobulinemia.

Waldenström macroglobulinemia (WM) is a rare subtype of non-Hodgkin lymphoma characterized by malignant lymphoplasmacytic cells in the bone marrow (BM). To dissect the pathophysiology of WM, we evaluated clonal cells by mapping of B cell lymphomagenesis with adaptive and innate immune tumor microenvironment (TME) in the BM of WM patients using mass cytometry (CyTOF). In-depth immunophenotypic profiling of WM cells exhibited profound expansion of clonal cells in both unswitched and switched memory B cells and also plasma cells with aberrant expression variations. WM B lymphomagenesis was associated with reduction of most B cell precursors assessed with the same clonally restricted light chain and phenotypic changes. The immune TME was infiltrated by mature monocytes, neutrophils and adaptive T cells, preferentially subsets of effector T helper, effector CTL and effector memory CTL cells that were associated with superior overall survival (OS), in contrast to progenitors of T cells and myeloid/monocytic lineage subsets that were suppressed in WM cohort. Moreover, decrease in immature B and NKT cells was related to worse OS in WM patients. Innate and adaptive immune subsets of WM TME were modulated by immune checkpoints, including PD-1/PD-L1&PD-L2, TIGIT/PVR, CD137/CD137-L, CTLA-4, BTLA and KIR expression. The response of ibrutinib treatment to the reduction of clonal memory B cell was associated with high levels of immature B cells and effector memory CTL cells. Our study demonstrates that CyTOF technology is a powerful approach for characterizing the pathophysiology of WM at various stages, predicting patient risk and monitoring the effectiveness of treatment strategies.

In vitro and ex vivo anti-myeloma effects of nanocomposite As4S4/ZnS/Fe3O4.

Nanoparticles in medicine can integrate actively targeted imaging agents and drug delivery vehicles, and combining multiple types of therapeutics in a single particle has numerous advantages, especially in multiple myeloma. MM is an incurable hematological disorder characterized by clonal proliferation of plasma cells in the bone marrow. In this study, we evaluated the anti-myeloma activity of 3 nanocomposites (3NPs): As4S4/ZnS/Fe3O4 (1:4:1), As4S4/ZnS/Fe3O4 with folic acid (FA), and As4S4/ZnS/Fe3O4 with FA and albumin with reduced survival MM cell lines and primary MM samples by each of 3NP. Cytotoxic effects of 3NPs were associated with caspase- and mitochondria-dependent apoptosis induction and reduced c-Myc expression. Modulation of cell cycle regulators, such as p-ATM/ATM and p-ATR/ATR, and increases in p-Chk2, cyclin B1, and histones were accompanied by G2/M arrest triggered by 3NPs. In addition, 3NPs activated several myeloma-related signaling, including JNK1/2/3, ERK1/2 and mTOR. To overcome BM microenvironment-mediated drug resistance, nanocomposites retained its anti-MM activity in the presence of stroma. 3NPs significantly decreased the stem cell-like side population in MM cells, even in the context of stroma. We observed strong synergistic effects of 3NPs combined with lenalidomide, pomalidomide, or melphalan, suggesting the potential of these combinations for future clinical studies.

Tracking Clonal Evolution of Multiple Myeloma Using Targeted Next-Generation DNA Sequencing.

Clonal evolution drives treatment failure in multiple myeloma (MM). Here, we used a custom 372-gene panel to track genetic changes occurring during MM progression at different stages of the disease. A tumor-only targeted next-generation DNA sequencing was performed on 69 samples sequentially collected from 30 MM patients. The MAPK/ERK pathway was mostly affected with KRAS mutated in 47% of patients. Acquisition and loss of mutations were observed in 63% and 37% of patients, respectively. Four different patterns of mutation evolution were found: branching-, mutation acquisition-, mutation loss- and a stable mutational pathway. Better response to anti-myeloma therapy was more frequently observed in patients who followed the mutation loss-compared to the mutation acquisition pathway. More than two-thirds of patients had druggable genes mutated (including cases of heavily pre-treated disease). Only 7% of patients had a stable copy number variants profile. Consequently, a redistribution in stages according to R-ISS between the first and paired samples (R-ISS″) was seen. The higher the R-ISS″, the higher the risk of MM progression and death. We provided new insights into the genetics of MM evolution, especially in heavily pre-treated patients. Additionally, we confirmed that redefining R-ISS at MM relapse is of high clinical value.

Imaging flow cytometry and fluorescence microscopy in assessing radiation response in lymphocytes from umbilical cord blood and cancer patients.

DNA double strand breaks (DSB) induced by ionizing radiation (IR) are usually measured using γH2AX/53BP1 DNA repair foci, that is considered to be the most sensitive assay for DSB analysis. While fluorescence microscopy (FM) is the gold standard for this analysis, imaging flow cytometry (IFC) may offer number of advantages such as lack of the fluorescence background, higher number of cells analyzed, and higher sensitivity in detection of DNA damage induced by IR at low doses. Along with appearance of γH2AX foci, the variable fraction of the cells exhibits homogeneously stained γH2AX signal resulting in so-called γH2AX pan-staining, which is believed to appear at early stages of apoptosis. Here, we investigated incidence of γH2AX pan-staining at different time points after irradiation with γ-rays using IFC and compared the obtained data with the data from FM. Appearance of γH2AX pan-staining during the apoptotic process was further analyzed by fluorescence-activated cell sorting (FACS) of cells at different stages of apoptosis and subsequent immunofluorescence analysis. Our results show that IFC was able to reveal dose dependence of pan-staining, while FM failed to detect all pan-staining cells. Moreover, we found that γH2AX pan-staining could be induced by therapeutic, but not low doses of γ-rays and correlate well with percentage of apoptotic cells was analyzed using flow cytometric Annexin-V/7-AAD assay. Further investigations showed that γH2AX pan-staining is formed in the early phases of apoptosis and remains until later stages of apoptotic process. Apoptotic DNA fragmentation as detected with comet assay using FM correlated with the percentage of live and late apoptotic/necrotic cells as analyzed by flow cytometry. Lastly, we successfully tested IFC for detection of γH2AX pan-staining and γH2AX/53BP1 DNA repair foci in lymphocyte of breast cancer patients after radiotherapy, which may be useful for assessing individual radiosensitivity in a clinically relevant cohort of patients.

Genoprotective activities of plant natural substances in cancer and chemopreventive strategies in the context of 3P medicine.

Severe durable changes may occur to the DNA structure caused by exogenous and endogenous risk factors initiating the process of carcinogenesis. By evidence, a large portion of malignancies have been demonstrated as being preventable. Moreover, the targeted prevention of cancer onset is possible, due to unique properties of plant bioactive compounds. Although genoprotective effects of phytochemicals have been well documented, there is an evident lack of articles which would systematically present the spectrum of anticancer effects by phytochemicals, plant extracts, and plant-derived diet applicable to stratified patient groups at the level of targeted primary (cancer development) and secondary (cancer progression and metastatic disease) prevention. Consequently, clinical implementation of knowledge accumulated in the area is still highly restricted. To stimulate coherent co-development of the dedicated plant bioactive compound investigation on one hand and comprehensive cancer preventive strategies on the other hand, the current paper highlights and deeply analyses relevant evidence available in the area. Key molecular mechanisms are presented to detail genoprotective and anticancer activities of plants and phytochemicals. Clinical implementation is discussed. Based on the presented evidence, advanced chemopreventive strategies in the context of 3P medicine are considered.

SDS-Stabilized CuInSe2/ZnS Multinanocomposites Prepared by Mechanochemical Synthesis for Advanced Biomedical Application.

The CuInSe2/ZnS multiparticulate nanocomposites were first synthesized employing two-step mechanochemical synthesis. In the first step, tetragonal CuInSe2 crystals prepared from copper, indium and selenium precursors were co-milled with zinc acetate dihydrate and sodium sulfide nonahydrate as precursors for ZnS in different molar ratios by mechanochemical route in a planetary mill. In the second step, the prepared CuInSe2/ZnS nanocrystals were further milled in a circulation mill in sodium dodecyl sulphate (SDS) solution (0.5 wt.%) to stabilize the synthesized nanoparticles. The sodium dodecyl sulphate capped CuInSe2/ZnS 5:0-SDS nanosuspension was shown to be stable for 20 weeks, whereas the CuInSe2/ZnS 4:1-SDS one was stable for about 11 weeks. After sodium dodecyl sulphate capping, unimodal particle size distribution was obtained with particle size medians approaching, respectively, 123 nm and 188 nm for CuInSe2/ZnS 5:0-SDS and CuInSe2/ZnS 4:1-SDS nanocomposites. Successful stabilization of the prepared nanosuspensions due to sodium dodecyl sulphate covering the surface of the nanocomposite particles was confirmed by zeta potential measurements. The prepared CuInSe2/ZnS 5:0-SDS and CuInSe2/ZnS 4:1-SDS nanosuspensions possessed anti-myeloma sensitizing potential assessed by significantly reduced viability of multiple myeloma cell lines, with efficient fluorescence inside viable cells and higher cytotoxic efficacy in CuInSe2/ZnS 4:1-SDS nanosuspension.

Synthesis and Anticancer Activity of Novel 9-O-Substituted Berberine Derivatives.

Berberine is a bioactive isoquinoline alkaloid derived from many plants. Although berberine has been shown to inhibit growth and induce apoptosis of several tumor cell lines, its poor absorption and moderate activity hamper its full therapeutic potential. Here, we describe the synthesis of a series of 9-O-substituted berberine derivatives with improved antiproliferative and apoptosis-inducing activities. An analysis of novel berberine derivatives by EPR spectroscopy confirmed their similar photosensitivity and analogous behavior upon UVA irradiation as berberine, supporting their potential to generate ROS. Improved antitumor activity of novel berberine derivatives was revealed by MTT assay, by flow cytometry and by detection of apoptotic DNA fragmentation and caspase-3 activation, respectively. We showed that novel berberine derivatives are potent inhibitors of growth of HeLa and HL-60 tumor cell lines with IC50 values ranging from 0.7 to 16.7 µM for HL-60 cells and 36 to >200 µM for HeLa cells after 48 h treatment. Further cell cycle analysis showed that the observed inhibition of growth of HL-60 cells treated with berberine derivatives was due to arresting these cells in the G2/M and S phases. Most strikingly, we found that berberine derivative 3 (9-(3-bromopropoxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquino[3,2-a] isoquinolin-7-ylium bromide) possesses 30-fold superior antiproliferative activity with an IC50 value of 0.7 µM and 6-fold higher apoptosis-inducing activity in HL-60 leukemia cells compared to berberine. Therefore, further studies are merited of the antitumor activity in leukemia cells of this berberine derivative.

Realgar nanoparticles versus ATO arsenic compounds induce in vitro and in vivo activity against multiple myeloma.

Multiple myeloma (MM), a B cell malignancy characterized by clonal proliferation of plasma cells in the bone marrow, remains incurable despite the use of novel and conventional therapies. In this study, we demonstrated MM cell cytotoxicity triggered by realgar (REA; As4 S4 ) nanoparticles (NREA) versus Arsenic trioxide (ATO) against MM cell lines and patient cells. Both NREA and ATO showed in vivo anti-MM activity, resulting in significantly decreased tumour burden. The anti-MM activity of NREA and ATO is associated with apoptosis, evidenced by DNA fragmentation, depletion of mitochondrial membrane potential, cleavage of caspases and anti-apoptotic proteins. NREA induced G2 /M cell cycle arrest and modulation of cyclin B1, p53 (TP53), p21 (CDKN1A), Puma (BBC3) and Wee-1 (WEE1). Moreover, NREA induced modulation of key regulatory molecules in MM pathogenesis including JNK activation, c-Myc (MYC), BRD4, and histones. Importantly, NREA, but not ATO, significantly depleted the proportion and clonogenicity of the MM stem-like side population, even in the context of the bone marrow stromal cells. Finally, our study showed that both NREA and ATO triggered synergistic anti-MM activity when combined with lenalidomide or melphalan. Taken together, the anti-MM activity of NREA was more potent compared to ATO, providing the preclinical framework for clinical trials to improve patient outcome in MM.

A novel 3D mesenchymal stem cell model of the multiple myeloma bone marrow niche: biologic and clinical applications.

Specific niches within the tumor bone marrow (BM) microenvironment afford a sanctuary for multiple myeloma (MM) clones due to stromal cell-tumor cell interactions, which confer survival advantage and drug resistance. Defining the sequelae of tumor cell interactions within the MM niches on an individualized basis may provide the rationale for personalized therapies. To mimic the MM niche, we here describe a new 3D co-culture ex-vivo model in which primary MM patient BM cells are co-cultured with mesenchymal stem cells (MSC) in a hydrogel 3D system. In the 3D model, MSC with conserved phenotype (CD73+CD90+CD105+) formed compact clusters with active fibrous connections, and retained lineage differentiation capacity. Extracellular matrix molecules, integrins, and niche related molecules including N-cadherin and CXCL12 are expressed in 3D MSC model. Furthermore, activation of osteogenesis (MMP13, SPP1, ADAMTS4, and MGP genes) and osteoblastogenic differentiation was confirmed in 3D MSC model. Co-culture of patient-derived BM mononuclear cells with either autologous or allogeneic MSC in 3D model increased proliferation of MM cells, CXCR4 expression, and SP cells. We carried out immune profiling to show that distribution of immune cell subsets was similar in 3D and 2D MSC model systems. Importantly, resistance to novel agents (IMiDs, bortezomib, carfilzomib) and conventional agents (doxorubicin, dexamethasone, melphalan) was observed in 3D MSC system, reflective of clinical resistance. This 3D MSC model may therefore allow for studies of MM pathogenesis and drug resistance within the BM niche. Importantly, ongoing prospective trials are evaluating its utility to inform personalized targeted and immune therapy in MM.

The novel compound STK405759 is a microtubule-targeting agent with potent and selective cytotoxicity against multiple myeloma in vitro and in vivo.

Despite advances in treatment, multiple myeloma (MM) remains incurable. Here we propose the use of STK405759, a novel microtubule targeting agent (MTA) and member of the furan metotica family for MM therapy.STK405759 inhibited tubulin polymerization in a cell-free system and in myeloma cells. This molecule had potent cytotoxic activity against several MM cell lines and patient-derived MM cells. Moreover, STK405759 demonstrated cytotoxicity against drug-resistant myeloma cells that overexpressed the P-glycoprotein drug-efflux pump. STK405759 was not cytotoxic to peripheral blood mononuclear cells, including activated B and T lymphocytes. This compound caused mitotic arrest and apoptosis of myeloma cells characterized by cleavage of poly (ADP-ribose) polymerase-1 and caspase-8, as well as decreased protein expression of mcl-1. The combination of STK405759 with bortezomib, lenalidomide or dexamethasone had synergistic cytotoxic activity. In in vivo studies, STK405759-treated mice had significantly decreased MM tumor burden and prolonged survival compared to vehicle treated- mice.These results provide a rationale for further evaluation of STK405759 as monotherapy or part of combination therapy for treating patients with MM.

Combination of a Selective HSP90α/ß Inhibitor and a RAS-RAF-MEK-ERK Signaling Pathway Inhibitor Triggers Synergistic Cytotoxicity in Multiple Myeloma Cells.

Heat shock protein (HSP)90 inhibitors have shown significant anti-tumor activities in preclinical settings in both solid and hematological tumors. We previously reported that the novel, orally available HSP90α/ß inhibitor TAS-116 shows significant anti-MM activities. In this study, we further examined the combination effect of TAS-116 with a RAS-RAF-MEK-ERK signaling pathway inhibitor in RAS- or BRAF-mutated MM cell lines. TAS-116 monotherapy significantly inhibited growth of RAS-mutated MM cell lines and was associated with decreased expression of downstream target proteins of the RAS-RAF-MEK-ERK signaling pathway. Moreover, TAS-116 showed synergistic growth inhibitory effects with the farnesyltransferase inhibitor tipifarnib, the BRAF inhibitor dabrafenib, and the MEK inhibitor selumetinib. Importantly, treatment with these inhibitors paradoxically enhanced p-C-Raf, p-MEK, and p-ERK activity, which was abrogated by TAS-116. TAS-116 also enhanced dabrafenib-induced MM cytotoxicity associated with mitochondrial damage-induced apoptosis, even in the BRAF-mutated U266 MM cell line. This enhanced apoptosis in RAS-mutated MM triggered by combination treatment was observed even in the presence of bone marrow stromal cells. Taken together, our results provide the rationale for novel combination treatment with HSP90α/ß inhibitor and RAS-RAF-MEK-ERK signaling pathway inhibitors to improve outcomes in patients with in RAS- or BRAF-mutated MM.

Selective and potent Akt inhibition triggers anti-myeloma activities and enhances fatal endoplasmic reticulum stress induced by proteasome inhibition.

The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we, therefore, examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patient MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of C/EBP homologous protein (CHOP), a fatal ER stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM.

Accessory cells of the microenvironment protect multiple myeloma from T-cell cytotoxicity through cell adhesion-mediated immune resistance.

PURPOSE: Cellular immunotherapy frequently fails to induce sustained remissions in patients with multiple myeloma, indicating the ability of multiple myeloma cells to evade cellular immunity. Toward a better understanding and effective therapeutic modulation of multiple myeloma immune evasion mechanisms, we here investigated the role of the tumor microenvironment in rendering multiple myeloma cells resistant to the cytotoxic machinery of T cells. EXPERIMENTAL DESIGN: Using a compartment-specific, bioluminescence imaging-based assay system, we measured the lysis of luciferase-transduced multiple myeloma cells by CD4(+) or CD8(+) CTLs in the presence versus absence of adherent accessory cells of the bone marrow microenvironment. We simultaneously determined the level of CTL activation by measuring the granzyme B release in culture supernatants. RESULTS: Bone marrow stromal cells from patients with multiple myeloma and healthy individuals, as well as vascular endothelial cells, significantly inhibited the lysis of multiple myeloma cells in a cell-cell contact-dependent manner and without substantial T-cell suppression, thus showing the induction of a cell adhesion-mediated immune resistance (CAM-IR) against CTL lysis. Further analyses revealed that adhesion to accessory cells downregulated Fas and upregulated the caspase-3 inhibitor survivin in multiple myeloma cells. Reconstitution of Fas expression with bortezomib enhanced the CTL-mediated lysis of multiple myeloma cells. Repressing survivin with the small-molecule YM155 synergized with CTLs and abrogated CAM-IR in vitro and in vivo. CONCLUSION: These results reveal the cell adhesion-mediated induction of apoptosis resistance as a novel immune escape mechanism and provide a rationale to improve the efficacy of cellular therapies by pharmacologic modulation of CAM-IR.

The interaction of bortezomib with multidrug transporters: implications for therapeutic applications in advanced multiple myeloma and other neoplasias.

PURPOSE: Bortezomib is an important agent in multiple myeloma treatment, but resistance in cell lines and patients has been described. The main mechanisms of resistance described in cancer fall into one of two categories, pharmacokinetic resistance (PK), e.g. over expression of drug efflux pumps and pharmacodynamic resistance, e.g. apoptosis resistance or altered survival pathways, where the agent reaches an appropriate concentration, but this fails to propagate an appropriate cell death response. Of the known pump mechanisms, P-glycoprotein (P-gp) is the best studied and considered to be the most important in contributing to general PK drug resistance. Resistance to bortezomib is multifactorial and there are conflicting indications that cellular overexpression of P-gp may contribute to resistance agent. Hence, better characterization of the interactions of this drug with classical resistance mechanisms should identify improved treatment applications. METHODS: Cell lines with different P-gp expression levels were used to determine the relationship between bortezomib and P-gp. Coculture system with stromal cells was used to determine the effect of the local microenvironment on the bortezomib-elacridar combination. To further assess P-gp function, intracellular accumulation of P-gp probe rhodamine-123 was utilised. RESULTS: In the present study, we show that bortezomib is a substrate for P-gp, but not for the other drug efflux transporters. Bortezomib activity is affected by P-gp expression and conversely, the expression of P-gp affect bortezomib's ability to act as a P-gp substrate. The local microenvironment did not alter the cellular response to bortezomib. We also demonstrate that bortezomib directly affects the expression and function of P-gp. CONCLUSIONS: Our findings strongly support a role for P-gp in bortezomib resistance and, therefore, suggest that combination of a P-gp inhibitor and bortezomib in P-gp positive myeloma would be a reasonable treatment combination to extend efficacy of this important drug.