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PDF file - 2.9MB, In-vitro kinase assay, Histology, Cell proliferation, MANOVA analysis for genomic correlates of drug sensitivity

Dysregulation of the PI3K/mTOR pathway, either through amplifications, deletions, or as a direct result of mutations, has been closely linked to the development and progression of a wide range of cancers. Moreover, this pathway activation is a poor prognostic marker for many tumor types and confers resistance to various cancer therapies. Here, we describe VS-5584, a novel, low-molecular weight compound with equivalent potent activity against mTOR (IC50 = 37 nmol/L) and all class I phosphoinositide 3-kinase (PI3K) isoforms IC50: PI3Kα = 16 nmol/L; PI3Kβ = 68 nmol/L; PI3Kγ = 25 nmol/L; PI3Kδ = 42 nmol/L, without relevant activity on 400 lipid and protein kinases. VS-5584 shows robust modulation of cellular PI3K/mTOR pathways, inhibiting phosphorylation of substrates downstream of PI3K and mTORC1/2. A large human cancer cell line panel screen (436 lines) revealed broad antiproliferative sensitivity and that cells harboring mutations in PI3KCA are generally more sensitive toward VS-5584 treatment. VS-5584 exhibits favorable pharmacokinetic properties after oral dosing in mice and is well tolerated. VS-5584 induces long-lasting and dose-dependent inhibition of PI3K/mTOR signaling in tumor tissue, leading to tumor growth inhibition in various rapalog-sensitive and -resistant human xenograft models. Furthermore, VS-5584 is synergistic with an EGF receptor inhibitor in a gastric tumor model. The unique selectivity profile and favorable pharmacologic and pharmaceutical properties of VS-5584 and its efficacy in a wide range of human tumor models supports further investigations of VS-5584 in clinical trials. Mol Cancer Ther; 12(2); 151–61. ©2012 AACR.

PDF file - 663K, Ambit kinome tree, Cell proliferation data, Pharmacokinetics, Body weight changes in PC3 efficacy study, Active vessel stain in COLO-205 efficacy study, MV4-11 efficacy model, Drug sensitivity profiling and correlation to mutation status

XLS file - 167K, Table 1: Cell line drug sensitivity data for SB2343 and mutations in cancer genes. Table 2: Results from MANOVA analysis of cancer genes as modifiers of drug sensitivity

Specifically blocking more than one oncogenic pathway simultaneously in a cancer cell with a combination of different drugs is the mainstay of the majority of cancer treatments. Being able to do this via two targeted pathways without inducing side effects through a general mechanism, such as chemotherapy, could bring benefit to patients. In this work we describe a new dual inhibitor of the JAK-STAT and HDAC pathways through designing and developing two types of molecule based on the JAK2 selective inhibitor XL019 and the pan-HDAC inhibitor, vorinostat. Both series of compounds had examples with low nanomolar JAK2 and HDAC1/6 inhibition. In some cases good HDAC1 selectivity was achieved while retaining HDAC6 activity. The observed potency is explained through molecular docking studies of all three enzymes. One example, 69c had 16–25 fold selectivity against the three other JAK-family proteins JAK1, JAK3 and TYK2. A number of compounds had sub-micromolar potencies against a panel of 4 solid tumor cell lines and 4 hematological cell lines with the most potent compound, 45h, having a cellular IC50 of 70 nM against the multiple myeloma cell line KMS-12-BM. Evidence of both JAK and HDAC pathway inhibition is presented in Hela cells showing that both pathways are modulated. Evidence of apoptosis with two compounds in 4 sold tumor cell lines is also presented.

Research demonstrates that the success of a service provider does not rely solely on sales volume but also in retaining existing customers. This research aims to propose a conceptual framework for improving customer satisfaction and thus facilitate service providers in retaining existing customers, specifically in the telecommunication industry in Malaysia. The Malaysian telecommunication and broadband industry is continuing growing each year and the demand for a better service and customer experience is increasing. All providers fight against each other to provide competitive packages with a good pricing plan. There are about 87% Internet users and 118% broadband penetration rate in Malaysia. It is not only good pricing plan that should be offered, it is crucial to know how to maintain the existing subscriber and to gain more trust among customers for increasing new subscribers. The main goal is to create better understanding of customer buying habits, good interaction (reactions or responses to something/helpful returned information) given during customer service and good experience during subscribing the product or services. A cloud-based customer support solution will be designed based on the proposed framework in order to offer a high quality service which satisfies customers in the said industry. Hence, this research focuses customer loyalty and retention marketing. Focus group study will be conducted to identify key determinants which influence customer satisfaction, followed by a survey and statistical analysis in order to determine the relationship between customer satisfaction (effect), existing services and customer experience (cause). The participants for this research will be obtained from one of the leading and largest broadband service provider in Malaysia – Telekom Malaysia (TM (M)) Berhad. As an empirical study, data of customer feedback about faulty restoration will be obtained from Voice of Customer (VOC) using questionnaire from a conducted survey. By providing a cloud-based customer support solution, it is believed that the customer satisfaction level will be lifted and negative feedback will be reduced.

A phase 2 clinical trial has demonstrated that daratumumab monotherapy was safe and well tolerated in relapsed/refractory Natural Killer/T-Cell Lymphoma (NKTL). However, no patients achieved complete response, and duration of response was short. Similarly, in Multiple Myeloma (MM), while daratumumab based combinations are approved for front line treatment, responses are heterogeneous and development of treatment resistance inevitable. Therefore, elucidation of mechanisms which can overcome daratumumab resistance is essential for the optimization of therapeutic response in patients. To this end, 2 pairs of isogenic daratumumab resistant and sensitive models of NKTL were developed in vitro via sequential exposure of cell lines to increasing concentrations of daratumumab in the presence of complement serum. A 3rd model was also studied in vivo whereby long-term administration of daratumumab in mice identified a sub-group of tumors which stopped responding to treatment and began to rapidly enlarge ('Resistant') as compared to others which remained similar to or smaller ('Sensitive') than the tumour volume at the initiation of treatment. RNA sequencing was performed on these models and genes commonly upregulated or downregulated analyzed. Differential gene expression analysis highlighted an enrichment for the upregulation of genes involved in exosome biogenesis and secretion in both cell line and mouse-derived daratumumab resistant NKTL models. An additional daratumumab resistant model was developed in an MM cell line to further validate these findings and extend the study in an MM model. Immunoblotting of the 3 pairs of isogenic sensitive and resistant cell line models demonstrate that there is indeed an upregulation of proteins regulating exosomal biogenesis and secretion; Alix, TSG101 and Rab27b in the daratumumab resistant phenotype. This is associated with a concomitant increase in secreted exosomes levels in the tumour microenvironment. The size and quantification of extracellular vesicles (EV) secreted in the media were studied by nanoparticle tracking analysis. Extracellular vesicles ranged in the size of 70-150nM corroborating with the size of exosomes and nanoparticle quantification revealed a higher concentration of exosomes present in the tumour microenvironment of resistant cells as compared to sensitive cells. Subsequently, exosomes were purified via ultracentrifugation and protein expression analysis confirmed elevation of exosome markers CD63 and CD81. To study the role of exosomal-mediated mechanisms in the survival of daratumumab resistant cells, we treated isogenic models with neticonazole and ketoconazole (azoles) which have been identified as selective inhibitors of exosomal biogenesis in a drug repurposing study for advanced cancers. Interestingly, azole treatment demonstrated a selective and more effective suppression of tumour cell viability in daratumumab resistant than sensitive cell lines. Immunoblot analysis showed that azole treatment at identical concentrations resulted in a more extensive downregulation of Alix, Rab27b and CD81 protein expression in resistant than sensitive cells. Additionally, depletion of Alix and Rab27b protein expression via siRNA knockdown induced cell death confirming that daratumumab-resistant cell lines are dependent on exosomal-mediated pathways for survival. Current research is focused mainly on intrinsic or immune cell-mediated mechanisms of daratumumab resistance, but little is known regarding the effect of extrinsic components in the tumour microenvironment. We demonstrate that daratumumab resistant models exhibit a distinct upregulation of proteins mediating exosome biogenesis resulting in enhanced exosome secretion. Daratumumab resistant cells are targeted more efficaciously with exosome biogenesis inhibitors than sensitive counterparts thereby suggesting an addiction to exosome-mediated mechanisms of survival. This is further supported by gene silencing studies. In future, we aim to perform miRNA profiling of exosomes purified from the tumour microenvironment of isogenic daratumumab-resistant and sensitive cell line models as well as from bone marrow plasma of daratumumab treated patients. miRNA which are enriched in the exosomes of resistant phenotypes will be characterized, unique biomarkers of response identified and in depth mechanisms of resistance studied. Disclosures No relevant conflicts of interest to declare.

BackgroundThe potential therapeutic efficacy of daratumumab in natural killer T-cell lymphoma (NKTL) was highlighted when its off-label usage produced sustained remission in a patient with highly refractory disease. This is corroborated recently by a phase II clinical trial which established that daratumumab monotherapy is well tolerated and displayed encouraging response in relapsed/refractory NKTL patients. However, little is known regarding the molecular factors central to the induction and regulation of the daratumumab-mediated antitumor response in NKTL.MethodsCD38 expression was studied via immunohistochemistry, multiplex immunofluorescence and correlated with clinical characteristics of the patient. The therapeutic efficacy of daratumumab was studied in vitro via CellTiter-Glo (CTG) assay, complement-dependent cytotoxicity (CDC), antibody-dependent cell cytotoxicity (ADCC), and in vivo, via a patient-derived xenograft mouse model of NKTL, both as a single agent and in combination with L-asparaginase. Signaling mechanisms were characterized via pharmacologic treatment, RNA silencing, flow cytometry and corroborated with public transcriptomic data of NKTL.ResultsEpstein-Barr virus-positive NKTL patients significantly express CD38 with half exhibiting high expression. Daratumumab effectively triggers Fc-mediated ADCC and CDC in a CD38-dependent manner. Importantly, daratumumab monotherapy and combination therapy with L-asparaginase significantly suppresses tumor progression in vivo. Ablation of complement inhibitory proteins (CIP) demonstrate that CD55 and CD59, not CD46, are critical for the induction of CDC. Notably, CD55 and CD59 expression were significantly elevated in the late stages of NKTL. Increasing the CD38:CIP ratio through sequential CIP knockdown, followed by CD38 upregulation via All-Trans Retinoic Acid treatment, potently augments complement-mediated lysis in cells previously resistant to daratumumab. The CD38:CIP ratio consistently demonstrates a statistically superior correlation to antitumor efficacy of daratumumab than CD38 or CIP expression alone.ConclusionThis study characterizes CD38 as an effective target for a subset of NKTL patients and the utilization of the CD38:CIP ratio as a more robust identifier for patient stratification and personalisation of treatment. Furthermore, elucidation of factors which sensitize the complement-mediated response provides an alternative approach toward optimizing therapeutic efficacy of daratumumab where CDC remains a known limiting factor. Altogether, these results propose a strategic rationale for further evaluation of single or combined daratumumab treatment in the clinic for NKTL.

// Nurulhuda Mustafa 1 , Jeannie Xue Ting Lee 2 , Huey Fang Adina Nee 2 , Chonglei Bi 1 , Tae-Hoon Chung 2 , Stefan Hart 4 and Wee Joo Chng 1, 2, 3 1 Yong Loo Lin School of Medicine, National University of Singapore, Singapore 2 Cancer Science Institute of Singapore, National University of Singapore, Singapore 3 Department of Haematology-Oncology, National University Cancer Institute of Singapore, National University Health System, Singapore 4 S*BIO Pte Ltd., Singapore Correspondence to: Wee Joo Chng, email: mdccwj@nus.edu.sg Keywords: myeloma, dual PI3K/mTOR inhibitor, RARRES3, panobinostat, therapeutics Received: March 28, 2017 Accepted: September 22, 2017 Published: October 20, 2017 ABSTRACT The PI3K/mTOR/AKT pathway is an integral regulator of survival and drug resistance in multiple myeloma (MM). VS-5584 was synthesized with dual-specific and equipotent activity against mTORC1/2 and all four Class I PI3K isoforms so as to durably inhibit this pathway. We show that VS-5584 is highly efficacious against MM cell lines even in the presence of IL-6 and IGF-1 and that this growth inhibition is partially dependent on Bim. Importantly, VS-5584 triggers apoptosis in patient cells with a favorable therapeutic index. Gene expression profiling revealed a VS-5584-induced upregulation of RARRES3, a class II tumor suppressor gene. MM patient databases, UAMS and APEX, show that RARRES3 is under-expressed in 11q13 subsets which correlates with the reduced effectiveness of VS-5584 in 11q13 cell lines. Silencing RARRES3 expression significantly rescues VS-5584-induced cell death and increases cyclin D2 expression but not cyclin D1 or other cyclins implying a role for RARRES3 in cell cycle arrest. In vivo , VS-5584 significantly reduces the tumor burden of MM mouse xenografts. We further identified that VS-5584 synergised with Dexamethasone, Velcade, and exceptionally so with HDAC inhibitor, Panobinostat. Interestingly, this was consistently observed in several patient samples, proposing a promising novel clinical strategy for combination treatment especially in relapsed/refractory patients.

Concomitant inhibition of multiple oncogenic pathways is a desirable goal in cancer therapy. To achieve such an outcome with a single molecule would simplify treatment regimes. Herein the core features of ruxolitinib (1), a marketed JAK1/2 inhibitor, have been merged with the HDAC inhibitor vorinostat (2), leading to new molecules that are bispecific targeted JAK/HDAC inhibitors. A preferred pyrazole substituted pyrrolopyrimidine, 24, inhibits JAK1 and HDACs 1, 2, 3, 6, and 10 with IC50 values of less than 20 nM, is

Herein, we report the discovery of a dual histone deacetylase inhibitor displaying a unique HDAC3/6 selectivity profile. An initial strategy to merge two epigenetic pharmacophores resulted in the discovery of potent HDAC6 inhibitors with selectivity over HDAC1. Screening in an HDAC panel revealed additional low nanomolar inhibition only against HDAC3. Low micromolar antiproliferative activities against two breast cancer and four hematological cancer cell lines was supported by pharmacodynamic studies on a preferred molecule, 24c, substantiating the HDAC inhibitory profile in cells. Apoptosis was identified as one of the main cell death pathways. Modelling studies of 24c against HDAC1,2,3 and 6 further provided insights on the orientation of specific residues relevant to compound potency, explaining the observed HDAC3/6 selectivity. A subset of the compounds also exhibited good antimalarial activities, particularly against the chloroquine-resistant strain K1 of P.falciparum. In vitro studies revealed a favourable DMPK profile warranting further investigation of the therapeutic potential of these compounds.

Oncogenic EZH2 is overexpressed and extensively involved in the pathophysiology of different cancers including extranodal natural killer/T-cell lymphoma (NKTL). However, the mechanisms regarding EZH2 upregulation is poorly understood, and it still remains untargetable in NKTL. In this study, we examine EZH2 protein turnover in NKTL and identify MELK kinase as a regulator of EZH2 ubiquitination and turnover. Using quantitative mass spectrometry analysis, we observed a MELK-mediated increase of EZH2 S220 phosphorylation along with a concomitant loss of EZH2 K222 ubiquitination, suggesting a phosphorylation-dependent regulation of EZH2 ubiquitination. MELK inhibition through both chemical and genetic means led to ubiquitination and destabilization of EZH2 protein. Importantly, we determine that MELK is upregulated in NKTL, and its expression correlates with EZH2 protein expression as determined by tissue microarray derived from NKTL patients. FOXM1, which connected MELK to EZH2 signaling in glioma, was not involved in mediating EZH2 ubiquitination. Furthermore, we identify USP36 as the deubiquitinating enzyme that deubiquitinates EZH2 at K222. These findings uncover an important role of MELK and USP36 in mediating EZH2 stability in NKTL. Moreover, MELK overexpression led to decreased sensitivity to bortezomib treatment in NKTL based on deprivation of EZH2 ubiquitination. Therefore, modulation of EZH2 ubiquitination status by targeting MELK may be a new therapeutic strategy for NKTL patients with poor bortezomib response.

Multiple myeloma (MM) is a B cell malignancy characterized by clonal proliferation of plasma cells in the bone marrow. With the advent of novel targeted agents, the median survival rate has increased to 5 -7 years. However, majority of patients with myeloma suffer relapse or develop chemoresistance to existing therapeutic agents. Thus, there is a need to develop novel alternative therapies for the treatment of MM. Thus in the present study, we investigated whether thymoquinone (TQ), a bioactive constituent of black seed oil, could suppress the proliferation and induce chemosensitization in human myeloma cells and xenograft mouse model. Our results show that TQ inhibited the proliferation of MM cells irrespective of their sensitivity to doxorubicin, melphalan or bortezomib. Interestingly, TQ treatment also resulted in a significant inhibition in the proliferation of CD138+ cells isolated from MM patient samples in a concentration dependent manner. TQ also potentiated the apoptotic effects of bortezomib in various MM cell lines through the activation of caspase-3, resulting in the cleavage of PARP. TQ treatment also inhibited chemotaxis and invasion induced by CXCL12 in MM cells. Furthermore, in a xenograft mouse model, TQ potentiated the antitumor effects of bortezomib (p < 0.05, vehicle versus bortezomib + TQ; p < 0.05, bortezomib versus bortezomib + TQ), and this correlated with modulation of various markers for survival and angiogenesis, such as Ki-67, vascular endothelial growth factor (VEGF), Bcl-2 and p65 expression. Overall, our results demonstrate that TQ can enhance the anticancer activity of bortezomib in vitro and in vivo and may have a substantial potential in the treatment of MM.

Complement-dependent cytotoxicity (CDC) is one of the major mechanisms mediating the anti-tumor efficacy of Daratumumab. We have previously demonstrated that a majority of Natural Killer/T- Cell Lymphoma (NKTL) patient samples express CD38 and Daratumumab is highly effective against NKTL cell lines expressing mid-high levels of CD38. In this report we show that subsequent testing in an NKTL mouse xenograft model confirms the potency of Daratumumab in vivo as evidenced by the inhibition in tumour progression and prolongation of mouse survival. When treatment was continued over a month, some tumors began to rapidly enlarge ('Resistant') while the rest remained similar or smaller ('Sensitive') than the tumour volume at the initiation of Daratumumab treatment. An mRNA analysis comparing 'Resistant' and 'Sensitive' tumors showed that while both tumours bore similar levels of CD38 expression, resistant tumours displayed an upregulation of complement inhibitory proteins (CIP), CD55 and CD59 but not CD46. This led us to hypothesize that CD59 and CD55 may play a critical role in Daratumumab-mediated CDC in NKTL. FACS analyses demonstrated that the number of membrane molecules of CD55 and CD59 appeared inversely correlated to Daratumumab-mediated CDC. A single CIP knockdown was first performed to delineate the role of each CIP. Silencing CD46 confirmed that it does not have any effect on CDC in NKTL. However, single knockdown of CD55 or CD59 was able to induce cytotoxicity in CDC-resistant cell line CD38midCD55hiCD59lo NKYS, and promote NKS1 CD38hiCD55hiCD59mid to further lysis. Both single and double knockdown of CD55 and CD59 could not enhance Daratumumab-induced CDC in CD38loCD55hiCD59hi HuT78 which recapitulates the importance of CD38 levels. Unexpectedly, the double knockdown did not sensitize CD38hiCD55hiCD59hi KMS12BM either. This led us to conjecture that it may be the ratio of CD38:CIPs which is predictive of response to Daratumumab than CD38 or CIPs alone. All-Trans Retinoic Acid (ATRA) binds the RARE element in CD38 gene leading to upregulation of mRNA and protein expression of CD38. We thus downregulated the expression of CIPs with siRNA followed by amplification of CD38 expression with ATRA in NKTL. This strategy resulted in a significant increase in the CD38:CIP ratio and induced almost a total lysis of NKS1 cells, as well as sensitised HuT78 to a massive amount of Daratumumab-mediated CDC. These experiments suggest that by increasing the CD38: CIP, ratio we can overcome resistance to Daratumumab-mediated CDC. To further statistically study this, a Spearman's rank correlation analyses was performed. The Spearman correlation coefficient shows that the number of surface molecules of CD38 positively correlates to CDC while that of CD55 displays an inverse correlation. CD46 and CD59 do not show any significant correlation. Notably, when correlating the CD38:CIP ratio instead to CDC, the CD38:CD46, CD38:CD55 and CD38:CD59 ratios always show a significant positive correlation coefficient. This suggests that the potential efficacy of Daratumumab can be predicted more accurately based on the ratio of CD38:CIP than any of the molecules alone. Detection of a low CD38:CIP ratio in patient samples could be a biomarker for potentially poorer response to Daratumumab treatment. Daratumumab-resistant NKTL cell lines are being developed in our lab and RNA sequencing comparing sensitive and resistance cells will be subsequently performed in order to gain further insights to mechanisms that may lead to resistance. Preliminary analyses on CD38 and CIP expression so far has shown that CD38 protein and mRNA expression are prominently downregulated in resistant cell lines while the level of CIPs remain similar or increased. The total outcomes of these studies will contribute valuable insights to clinical trials that currently involve Daratumumab treatment. Disclosures Zhou: Janssen R&D: Employment. Yang:Janssen R&D: Employment. Chng:Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Aslan: Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Merck: Research Funding.

Blockage of more than one oncoprotein or pathway is now a standard approach in modern cancer therapy. Multiple inhibition is typically achieved with two or more drugs. Herein, we describe a pharmacophore merging strategy combining the JAK2/FLT3 inhibitor pacritnib with the pan-HDAC inhibitor, vorinostat, to create bispecific single molecules with both JAK and HDAC targeted inhibition. A preferred ether hydroxamate, 51, inhibits JAK2 and HDAC6 with low nanomolar potency, is100 nM potent against HDACs 2 and 10, submicromolar potent against HDACs 1, 8, and 11, and50-fold selective for JAK2 in a panel of 97 kinases. Broad cellular antiproliferative potency is supported by demonstration of JAK-STAT and HDAC pathway blockade in several hematological cell lines, inhibition of colony formation in HEL cells, and analysis of apoptosis. This study provides new tool compounds for further exploration of dual JAK-HDAC pathway inhibiton achieved with a single molecule.

Dysregulation of the PI3K/mTOR pathway, either through amplifications, deletions, or as a direct result of mutations, has been closely linked to the development and progression of a wide range of cancers. Moreover, this pathway activation is a poor prognostic marker for many tumor types and confers resistance to various cancer therapies. Here, we describe VS-5584, a novel, low-molecular weight compound with equivalent potent activity against mTOR (IC50 = 37 nmol/L) and all class I phosphoinositide 3-kinase (PI3K) isoforms IC50: PI3Kα = 16 nmol/L; PI3Kβ = 68 nmol/L; PI3Kγ = 25 nmol/L; PI3Kδ = 42 nmol/L, without relevant activity on 400 lipid and protein kinases. VS-5584 shows robust modulation of cellular PI3K/mTOR pathways, inhibiting phosphorylation of substrates downstream of PI3K and mTORC1/2. A large human cancer cell line panel screen (436 lines) revealed broad antiproliferative sensitivity and that cells harboring mutations in PI3KCA are generally more sensitive toward VS-5584 treatment. VS-5584 exhibits favorable pharmacokinetic properties after oral dosing in mice and is well tolerated. VS-5584 induces long-lasting and dose-dependent inhibition of PI3K/mTOR signaling in tumor tissue, leading to tumor growth inhibition in various rapalog-sensitive and -resistant human xenograft models. Furthermore, VS-5584 is synergistic with an EGF receptor inhibitor in a gastric tumor model. The unique selectivity profile and favorable pharmacologic and pharmaceutical properties of VS-5584 and its efficacy in a wide range of human tumor models supports further investigations of VS-5584 in clinical trials. Mol Cancer Ther; 12(2); 151–61. ©2012 AACR.

CD137L (4-1BBL) is a member of the TNFSF and is expressed on APCs as a transmembrane protein. Reverse signaling by CD137L in monocytes causes cell activation and differentiation to mature inflammatory DCs that can stimulate T cell proliferation. However, CD137L agonists have also been reported to induce apoptosis in PBMCs. This study aimed at clarifying these seemingly opposing activities. We find that the dying cells within PBMCs are T cells and that this T cell death is dependent on monocytes and correlates with the monocyte:T cell ratio. This CD137L-induced, monocyte-mediated T cell apoptosis is reminiscent of MDCD, and both are cell contact-dependent. T cell death is not mediated by CD95 or DR4 or -5 but by ROS produced by the T cells. T cell apoptosis is restricted to the first 24 h of stimulation, and at later time-points, the monocytes differentiate to inflammatory DCs under the influence of CD137L signaling and acquire the capacity to stimulate T cell proliferation from Day 4 onward. This biphasic activity may contribute to infection-induced T cell attrition, where in the early phase (

Multiple myeloma (MM) is a genetically complex disease that is becoming more common in today’s ageing population. Approximately 50% of MM harbour recurrent translocations involving the immunoglobulin heavy chain (IgH) locus on chromosome 14q32. The translocation t(4;14)(p16;q32) is one of the most common translocation in MM and is associated with very poor prognosis. The multiple myeloma SET domain (MMSET), involved in the fusion to the IgH locus in t(4;14) MM, is universally overexpressed and has been suggested to play an important role in tumorigenicity in t(4;14) MM. In order to identify downstream functional targets of MMSET, we knocked down MMSET expression with shRNAs in KMS11, a t(4;14) MM cell line, and identified differentially expressed genes by gene expression microarray analysis. The results identified 321 down-regulated and 375 up-regulated genes upon MMSET knockdown. Of interest, the knockdown reduced the transcription levels of the transcription factor IRF4 (interferon regulatory factor 4), which has been shown recently to be required for myeloma cell survival. Quantitative PCR (QPCR) analysis confirmed the transcription levels of IRF4 were reduced upon MMSET knockdown in t(4;14) MM cells (KMS11, KMS18, KMS28BM). Western-blots analysis indicated MMSET knockdown reduced protein levels of IRF4, c-Myc and PARP, and IRF4 knockdown could also reduce protein levels of c-Myc and PARP. Flow cytometric analysis indicated silencing of MMSET or IRF4 could induce apoptosis in t(4;14) MM cells. QPCR and Western-blot analysis revealed that ectopic expression of MMSET could increase IRF4 expression in KMS11 cells. Using chromatin immunoprecipitation (ChIP), both MMSET and NFκB can bind to the promoter region of IRF4, and the binding region of MMSET is located 1 kb upstream of that of NFκB. Furthermore, endogenous NFκB p65 could be coprecipitated by anti-MMSET antibody, indicating the association of MMSET and NFκB. These data suggest that MMSET may be a coactivator for NFkB in the regulation of IRF4 expression. Luciferase reporter assays in KMS11 cells showed the activity of IRF4 promoters were decreased significantly upon MMSET knockdown, suggesting MMSET is an important functional element for IRF4 promoter. Cell proliferation assays and apoptosis analysis showed that silencing of MMSET could sensitize t(4;14) MM cells to Bortezomib and Melphalan killing. In myeloma xenograft, combined MMSET silencing with Melphalan or Bortezomib resulted in significantly greater inhibition of t(4;14) MM tumorigeness than treatments with either drugs alone. Importantly, we found both of Melphalan and Bortezomib could also reduce protein levels of MMSET and IRF4. QPCR analysis indicated that they inhibited MMSET and IRF4 expression through transcription control. These results might partly explain the additive mechanism of combination of MMSET knockdown and Melphalan or Bortezomib. Overall, our data indicated MMSET is involved in the regulatory network of IRF4 that is critical for t(4:14) MM cell survival, and its silence potentiates the effect of antimyeloma agents in vitro and in vivo. Disclosures No relevant conflicts of interest to declare.

Currently, most multiple myeloma (MM) patients experience relapse and develop resistance to standard treatments. A recent study showed that patients who relapsed had poor outcomes, with an overall survival of only 6 months and an event-free survival of 1 month. The PI3K/mTOR/AKT pathway represents a critical target in MM because it stimulates proliferation, survival, and drug resistance of MM cells. VS-5584 is a novel agent, with specific and equipotent activity against mTOR and all 4 Class I PI3K isoforms, without relevant activity on 400 other lipid and protein kinases. Here we report that VS-5584 is highly efficacious against a wide panel of MM cells including Velcade- and Doxorubucin- resistant cell lines. This efficacy is maintained even in the presence of additional MM growth factors, IL-6 and IGF-1, and seems independent of PTEN status in the cell lines. Importantly, VS-5584 shows similar efficacy in patient myeloma cells and preferential tumor cell targeting compared to healthy peripheral blood mononuclear cells. Further testing in a myeloma xenograft mouse model further confirmed the potency of this compound in vivo. We have also observed synergistic activity in combination with both MM clinical therapeutic Dexamethasone, and novel anti-MM candidate Panabinostat. Comparing the basal expression profile of hypersensitive (H929) vs less sensitive (OPM2, U266) cell lines have identified the interferon alpha/beta pathway as a marker for association with sensitivity. Just recently, VS-5584 has been reported to evidence very favourable pharmaceutical and pharmacological properties in a wide range of solid tumors resistant to standard care therapies. Taken together with our data, this offers a compelling rationale for its clinical development as a single or combination therapy in multiple myeloma. Disclosures: No relevant conflicts of interest to declare.