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Recurrent cytogenetic abnormalities are the main hallmark of multiple myeloma (MM) and patients having 2 or more high-risk prognostic events are associated with extremely poor outcome. 17p13(del) and 1q21(gain) are critical and independent high-risk cytogenetic markers, however, the biological significance underlying the poor outcome in MM patients having co-occurrence of both these chromosomal aberrations has never been interrogated. Herein, we identified that patients harbouring concomitant 17p13(del) with 1q21(gain) demonstrated the worst prognosis as compared to patients with single- (either 17p13(del) or 1q21(gain)) and with no chromosomal events (WT for both chromosomal loci); and they are highly enriched for genomic instability (GI) signature. We discovered that the GI feature in the patients with concomitant 17p13(del)-1q21(gain) was recapitulating the biological properties of myeloma cells with co-existing p53-deficiency and NEIL1 mRNA-hyper-editing (associated with chromosome 17p and 1q, respectively) that have inherent DNA damage response (DDR) and persistent activation of Chk1 pathway. Importantly, this became a vulnerable point for therapeutic targeting whereby the cells with this co-abnormalities demonstrated hyper-sensitivity to siRNA- and pharmacological-mediated-Chk1 inhibition, as observed at both the in vitro and in vivo levels. Mechanistically, this was attributable to the synthetic lethal relationship between p53-NEIL1-Chk1 abnormalities. The Chk1 inhibitor (AZD7762) tested showed good synergism with standard-of-care myeloma drugs, velcade and melphalan, thus further reinforcing the translational potential of this therapeutic approach. In summary, combination of NEIL1-p53 abnormalities with an ensuing Chk1 activation could serve as an Achilles heel and predispose MM cells with co-existing 1q21(gain) and 17p13(del) to therapeutic vulnerability for Chk1 inhibition.

Supplementary Table 1 shows the list of 4C primers applied in the Circular Chromosome Conformation Capture assay.

Supplementary Materials and Methods shows detailed descriptions of methodology or materials and methods used in this manuscript.

Supplementary Figures, including Figures S1-6, and each figure legends.

Supplementary Table 4 shows the sequences of SE fragments inserted into pGL3 vector

Supplementary Table 5 shows the results of NSD2-MASPEC in KMS11 cells.

Supplementary Table 2 shows 26 SE-associated genes commonly acquired in both t(4:14)-positive HMCLs and patient-derived MM sample.

Multiple myeloma is an incurable malignancy with marked clinical and genetic heterogeneity. The cytogenetic abnormality t(4;14) (p16.3;q32.3) confers aggressive behavior in multiple myeloma. Recently, essential oncogenic drivers in a wide range of cancers have been shown to be controlled by super-enhancers (SE). We used chromatin immunoprecipitation sequencing of the active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) to profile unique SEs in t(4;14)-translocated multiple myeloma. The histone chaperone HJURP was aberrantly overexpressed in t(4;14)-positive multiple myeloma due to transcriptional activation by a distal SE induced by the histone lysine methyltransferase NSD2. Silencing of HJURP with short hairpin RNA or CRISPR interference of SE function impaired cell viability and led to apoptosis. Conversely, HJURP overexpression promoted cell proliferation and abrogated apoptosis. Mechanistically, the NSD2/BRD4 complex positively coregulated HJURP transcription by binding the promoter and active elements of its SE. In summary, this study introduces SE profiling as an efficient approach to identify new targets and understand molecular pathogenesis in specific subtypes of cancer. Moreover, HJURP could be a valuable therapeutic target in patients with t(4;14)-positive myeloma.Significance:A super-enhancer screen in t(4;14) multiple myeloma serves to identify genes that promote growth and survival of myeloma cells, which may be evaluated in future studies as therapeutic targets.

Supplementary Table 3 Uni- and multivariate Cox survival analysis using HJURP and NSD2 expression in myeloma dataset

Table S1: RT-PCR primers Figure S1: mRNA and protein expression of PRL-3 in HMCLs. Figure S2. IL-6 treatment of MM cell lines. Figure S3. Cellular effects of PRL-3 knockdown in HMCLs. Figure S4. Treatment of BTZ-resistant cells with PRL-3 inhibitor I. Figure S5. CoMMpass Dataset analysis

Overexpression of PRL-3, an oncogenic phosphatase, was identified as a novel cluster in patients with newly diagnosed multiple myeloma. However, the regulation and oncogenic activities of PRL-3 in multiple myeloma warrant further investigation. Here, we report that IL6 activates STAT3, which acts as a direct transcriptional regulator of PRL-3. Upregulation of PRL-3 increased myeloma cell viability and rephosphorylated STAT3 in a biphasic manner through direct interaction and deactivation of SHP2, thus blocking the gp130 (Y759)-mediated repression of STAT3 activity. Abrogation of PRL-3 reduced myeloma cell survival, clonogenicity, and tumorigenesis, and detailed mechanistic studies revealed “deactivation” of effector proteins such as Akt, Erk1/2, Src, STAT1, and STAT3. Furthermore, loss of PRL-3 efficiently abolished nuclear localization of STAT3 and reduced its occupancy on the promoter of target genes c-Myc and Mcl-1, and antiapoptotic genes Bcl2 and Bcl-xL. PRL-3 also played a role in the acquired resistance of myeloma cells to bortezomib, which could be overcome by PRL-3 silencing. Of clinical relevance, STAT3 and PRL-3 expression was positively correlated in five independent cohorts, and the STAT3 activation signature was significantly enriched in patients with high PRL-3 expression. Furthermore, PRL-3 could be used as a biomarker to identify high-risk patients with multiple myeloma that exhibited poor prognosis and inferior outcome even when treated with novel combinational therapeutics (proteasome inhibitors and immunomodulatory imide drugs). Conclusively, our results support a feedforward mechanism between STAT3 and PRL-3 that prolongs prosurvival signaling in multiple myeloma, and suggest targeting PRL-3 as a valid therapeutic opportunity in multiple myeloma.Significance:IL6 promotes STAT3-dependent transcriptional upregulation of PRL-3, which in turn re-phosphorylates STAT3 and aberrantly activates STAT3 target genes, leading to bortezomib resistance in multiple myeloma.

Supplementary Figure 2 from Molecular Dissection of Hyperdiploid Multiple Myeloma by Gene Expression Profiling

Supplementary Methods from Molecular Dissection of Hyperdiploid Multiple Myeloma by Gene Expression Profiling

Supplementary Figure 3 from Molecular Dissection of Hyperdiploid Multiple Myeloma by Gene Expression Profiling

Supplementary Tables 1-8 from Molecular Dissection of Hyperdiploid Multiple Myeloma by Gene Expression Profiling

Supplementary Figure 1 from Molecular Dissection of Hyperdiploid Multiple Myeloma by Gene Expression Profiling

Multiple myeloma is an incurable malignancy with marked clinical and genetic heterogeneity. The cytogenetic abnormality t(4;14) (p16.3;q32.3) confers aggressive behavior in multiple myeloma. Recently, essential oncogenic drivers in a wide range of cancers have been shown to be controlled by super-enhancers (SE). We used chromatin immunoprecipitation sequencing of the active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) to profile unique SEs in t(4;14)-translocated multiple myeloma. The histone chaperone HJURP was aberrantly overexpressed in t(4;14)-positive multiple myeloma due to transcriptional activation by a distal SE induced by the histone lysine methyltransferase NSD2. Silencing of HJURP with short hairpin RNA or CRISPR interference of SE function impaired cell viability and led to apoptosis. Conversely, HJURP overexpression promoted cell proliferation and abrogated apoptosis. Mechanistically, the NSD2/BRD4 complex positively coregulated HJURP transcription by binding the promoter and active elements of its SE. In summary, this study introduces SE profiling as an efficient approach to identify new targets and understand molecular pathogenesis in specific subtypes of cancer. Moreover, HJURP could be a valuable therapeutic target in patients with t(4;14)-positive myeloma. SIGNIFICANCE: A super-enhancer screen in t(4;14) multiple myeloma serves to identify genes that promote growth and survival of myeloma cells, which may be evaluated in future studies as therapeutic targets. Published version The work was supported by the National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centres of Excellence initiative (to W.J. Chng), the NMRC Clinician Scientist Investigator award (to W.J. Chng), the RNA Biology Center at CSI Singapore, NUS, from funding by the Singapore Ministry of Education’s Tier 3 grants, grant number MOE2014-T3–1-006 (to W.J. Chng), the National Natural Science Foundation of China (grant no. 82000212 to Y. Jia), Natural Science Foundation of Zhejiang Province (grant no. LQ21H160022 to Y. Jia), Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (grant no. 2021RC003 to Y. Jia). Y. Jia also thanks the China Scholarship Council (grant no. 201706320167) for financial support to visit National University of Singapore.

Photo(electro)catalysis methods have drawn significant attention for efficient, energy-saving, and environmental-friendly organic contaminant degradation in wastewater. However, conventional oxide-based powder photocatalysts are limited to UV-light absorption and are unfavorable in the subsequent postseparation process. In this paper, a large-area crystalline-semiconductor nitride membrane with a distinct nanoporous surface is fabricated, which can be scaled up to a full wafer and easily retrieved after photodegradation. The unique nanoporous surface enhances broadband light absorption, provides abundant reactive sites, and promotes the dye-molecule reaction with adsorbed hydroxyl radicals on the surface. The superior electric contact between the nickel bottom layer and nitride membrane facilitates swift charge carrier transportation. In laboratory tests, the nanostructure membrane can degrade 93% of the dye in 6 h under illumination with a small applied bias (0.5 V vs Ag/AgCl). Furthermore, a 2 inch diameter wafer-scale membrane is deployed in a rooftop test under natural sunlight. The membrane operates stably for seven cycles (over 50 h) with an outstanding dye degradation efficiency (92%) and satisfied average total organic carbon removal rate (≈50%) in each cycle. This demonstration thus opens the pathway toward the production of nanostructured semiconductor layers for large-scale and practical wastewater treatment using natural sunlight.

Introducing suitable electron/hole transport layers and transparent conductive layers (TCLs) into perovskite solar cells (PSCs) is key to enhancing the selective extraction of charge carriers and r...

Chromosomal rearrangement involving 14q32 region that results in TNF receptor associated factor 3 (TRAF3) dysfunctional mutation is the most frequent NF-κB pathway mutation in multiple myeloma (MM). Subsequent NF-κB inducing Kinase (NIK) stabilization plays a critical role in alternative NF-κB activation. However, disease progression resulting from TRAF3 dysregulation has not been well understood. In this study, we identified lymphocyte cellular protein 1 (LCP1) as a novel NIK-driven alternative NF-κB target in TRAF3 dysfunctional mutation using RNA-seq, ChIP-seq (RelA/p65 and p52 NF-κB) and other validation methods. LCP1 is exclusively activated in MM cells with TRAF3 loss-of-function mutation. In MM patients, higher LCP1 expression was significantly pronounced in poor prognosis groups such as 4p16 and MAF. CD138 negative MM patient cells showed elevated LCP1 expression and inhibition of LCP1 can sensitize proteasome inhibitor bortezomib in TRAF3 mutant MM cells in vitro. We report that LCP1 is a NIK-driven biomarker in TRAF3 dysfunctional MM and targeting LCP1 can provide a valuable therapeutic intervention in TRAF3 mutated MM.

Background Recurrent chromosomal translocations are central to the pathogenesis of Multiple Myeloma (MM), and the t(4;14) translocation is an adverse prognostic factor found in approximately 15% of MM patients. REIIBP is a t(4;14)-deregulated histone methyltransferase arising from alternative promoter usage within the MMSET locus. The role of this short isoform in t(4;14) myelomagenesis is poorly understood. Methods RPMI8266 was transfected with REIIBP vector to create stable isogenic cells using puromycin selection, and the oncogenic and histone methyltransferase activities were evaluated by RT-qPCR, immunoblotting, viability and colony-forming assays. Microarray and ChIP-sequencing were used to identify REIIBP targets, followed by genetic and biochemical inhibition to determine the underlying mechanisms in vitro and in vivo. Clinical relevance was validated with CoMMpass dataset. Results Despite sharing identical sequence with the C-terminus of MMSET, we found that REIIBP displayed distinct expression pattern with other MMSET breakpoint variants and gene products of the t(4;14) locus. Additionally, the expression of REIIBP can be regulated through microRNAs by another histone methyltransferase, EZH2 in a Dicer-dependent manner. Stable overexpression of REIIBP in the t(4;14)-negative RPMI8226 promoted oncogenic phenotypes and predominantly catalyzes the repressive H3K27me3 and active H3K4me3 histone modifications. Identification of differentially-regulated genes through microarray and correlation with H3K4me3/H3K27me3 ChIP-sequencing revealed that Toll-like receptor 7 (TLR7) and Bruton’s tyrosine kinase (BTK) are targets of REIIBP. Importantly, this led to a B-cell receptor (BCR)-independent activation of BTK and NF-ĸB effector signaling in t(4;14) myeloma cells. As myeloma cells do not express BCR, the re-activation of BTK signaling by TLR7 was functionally validated, conferring bortezomib resistance through the dysregulation of pro-inflammatory cytokine expression such as IL-6 and IL-27. Furthermore, comparison of BTK expression and dependency scores across lineages revealed that BTK is an important target in multiple myeloma, and correlated with REIIBP expression in the CoMMpass dataset. t(4;14)-translocated cells were amenable to BTK targeting using Ibrutinib, improving bortezomib response in myeloma cells and mice engrafted with RPMI8226-REIIBP tumors. Conclusion Altogether, our results provided a rationale for the blockade of REIIBP in t(4;14) cells through combining proteasome and BTK inhibitors as a therapeutic strategy for further clinical evaluation.

1q21 amplification is an important prognostic marker in multiple myeloma. In this study we identified that IL6R (the interleukin-6 membrane receptor) and ADAR1 (an RNA editing enzyme) are critical genes located within the minimally amplified 1q21 region. Loss of individual genes caused suppression to the oncogenic phenotypes, the magnitude of which was enhanced when both genes were concomitantly lost. Mechanistically, IL6R and ADAR1 collaborated to induce a hyper-activation of the oncogenic STAT3 pathway. High IL6R confers hypersensitivity to interleukin-6 binding, whereas, ADAR1 forms a constitutive feed-forward loop with STAT3 in a P150-isoform-predominant manner. In this respect, ADAR1-P150 acts as a direct transcriptional target for STAT3 and this STAT3-induced-P150 in turn directly interacts with and stabilizes the former protein, leading to a larger pool of proteins acting as oncogenic transcription factors for pro-survival genes. The importance of both IL6R and ADAR1-P150 in STAT3 signaling was further validated when concomitant knockdown of both genes impeded IL6-induced-STAT3 pathway activation. Clinical evaluation of various datasets of myeloma patients showed that low expression of either one or both genes was closely associated with a compromised STAT3 signature, confirming the involvement of IL6R and ADAR1 in the STAT3 pathway and underscoring their essential role in disease pathogenesis. In summary, our findings highlight the complexity of the STAT3 pathway in myeloma, in association with 1q21 amplification. This study therefore reveals a novel perspective on 1q21 abnormalities in myeloma and a potential therapeutic target for this cohort of high-risk patients.

Multiple myeloma is an incurable malignancy with marked clinical and genetic heterogeneity. The cytogenetic abnormality t(4;14) (p16.3;q32.3) confers aggressive behavior in multiple myeloma. Recently, essential oncogenic drivers in a wide range of cancers have been shown to be controlled by super-enhancers (SE). We used chromatin immunoprecipitation sequencing of the active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) to profile unique SEs in t(4;14)-translocated multiple myeloma. The histone chaperone

Background Multiple myeloma (MM) is an incurable malignancy characterized by complex heterogeneous cytogenetic abnormalities. Defined as large clusters of cis-acting enhancers, super-enhancers (SEs) could promote oncogenic transcription to which cancer cells highly addicted. Structural maintenance of chromosome 4 (SMC4) is a core subunit of condensin complexes, which contributes to chromosome condensation and segregation. Here, we identified SMC4 as a novel SE-associated oncogene of myeloma cells, whose functional roles in MM remain largely elusive. Methods We performed H3K27ac ChIP-seq and RNA-seq on primary MM patient samples, MM cell lines, normal CD138+ plasma cell and lymphoma cell lines as the controls, then the ROSE analysis was used to annotate SEs and their associated genes. THZ1 as a transcriptional CDK7 inhibitor was used to further filter THZ1-responsive genes. Combined analysis of THZ1-sensitive and SE-associated gene uncovered a number of promising MM oncogenes as SMC4. The public data mining, RNA interference, CRISPR/Cas9 gene editing system, overexpression and a variety of cellular functional assays were performed to determine the oncogenic effects of SMC4 on MM malignant. Transcriptome analysis with SMC4 silencing and overexpression, and the underlying molecular mechanism of SMC4 oncogenic role in MM are ongoing. Results We have demonstrated SMC4 as one of the SE-associated genes specific to MM patient samples and cell lines. SMC4 was related to myelomagenesis with increased expression from MGUS, SMM to MM cases, and overexpression of SMC4 predicted poor overall survival of MM patients, suggesting its clinical relevance. SMC4 silencing in MM cells suppressed cell proliferation and lead to cell apoptosis. Repression of the SMC4-SE region also led to impairment of cell viability and cell growth, consistent with an oncogenic function. Conclusion In summary, our integrative approached by combing H3H27ac ChIP-seq, RNA-seq and THZ1-sensitive transcripts identified the landscape of SEs and novel oncogenic transcripts as SMC4 in MM. Mapping these acquired SEs and their associated genes may provide novel insight into understanding MM biology, and SMC4 may serve as novel therapeutic targets in MM. Citation Format: Yunlu Jia, Jianbiao Zhou, Tze-King Tan, Tae-Hoon Chung, Yongxia Chen, Enfan Zhang, Zhen Cai, Takaomi Sanda, Wee-Joo Chng. Superenhancer-drvien SMC4 promote cell growth and proliferation in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2956.

Background Growing evidence suggests that alterations in epigenetic landscape contribute to pathogenesis of multiple myeloma (MM). MM cells are highly dependent on unfolded protein response signaling pathways due to high level of endoplasmic reticulum stress. Phosphorylation of eIF2a can attenuate protein translation. The PPP1R15B(denoted as R15B hereafter) gene encodes a regulatory subunit of eIF2a-specific phosphatase complex. In this study,we identified super enhancer (SE)-driven oncogenes specific in MM with a particular focus on a candidate gene R15B, whose functional roles in MM remain largely elusive. Methods We performed H3K27Ac ChIP-seq on MM cell lines, primary MM patient samples, normal CD138+ plasma cells and memory B cells. ROSE analysis was used to annotate SEs and their associated genes. A combination of public data mining, RNAi, overexpression and CRISPR/Cas9 technologies were conducted to determine the oncogenic effects of R15B in MM. Transcriptome analysis of MM cell line H929 with R15B KD and scrambled control was performed. To further study the interactions between SE and its promoters, we are currently working on HiChIP. Results We have identified R15B as one of the SE-associated genes specific to MM patient samples and cell lines. SE activity was correlated with the expression level of R15B. Higher expression of R15B predicted poor overall survival of MM patients, suggesting its clinical relevance in MM pathogenesis. R15B KD or KO significantly reduced cell viability, clonogenicity and induced G2/M arrest. ChIP-qPCR assays showed that C/EBP-β is strongly enriched at R15B SE region. We also found that salubrinal, a selective inhibitor of eIF2a phosphorylation, inhibited MM cell proliferation in a dose-dependent manner. Conclusions Our integrative approaches identified R15B as a novel SE-driven oncogene. We propose that targeting R15B may serve as a new approach for effective anti-myeloma therapy, which warrants further clinical investigation.

Background Multiple myeloma is an incurable hematological malignancy characterized by a heterogeneous genetic and epigenetic landscape. Although a number of genetic aberrations associated with myeloma pathogenesis, progression and prognosis have been well characterized, the role of many epigenetic aberrations in multiple myeloma remain elusive. G9a, a histone methyltransferase, has been found to promote disease progression, proliferation and metastasis via diverse mechanisms in several cancers. A role for G9a in multiple myeloma, however, has not been previously explored. Methods Expression levels of G9a/EHMT2 of multiple myeloma cell lines and control cells Peripheral Blood Mononuclear Cells (PBMCs) were analyzed. Correlation of G9a expression and overall survival of multiple myeloma patients were analyzed using patient sample database. To further study the function of G9a in multiple myeloma, G9a depleted multiple myeloma cells were built by lentiviral transduction, of which proliferation, colony formation assays as well as tumorigenesis were measured. RNA-seq of G9a depleted multiple myeloma with controls were performed to explore the downstream mechanism of G9a regulation in multiple myeloma. Results G9a is upregulated in a range of multiple myeloma cell lines. G9a expression portends poorer survival outcomes in a cohort of multiple myeloma patients. Depletion of G9a inhibited proliferation and tumorigenesis in multiple myeloma. RelB was significantly downregulated by G9a depletion or small molecule inhibition of G9a/GLP inhibitor UNC0642, inducing transcription of proapoptotic genes Bim and BMF. Rescuing RelB eliminated the inhibition in proliferation and tumorigenesis by G9a depletion. Conclusions In this study, we demonstrated that G9a is upregulated in most multiple myeloma cell lines. Furthermore, G9a loss-of-function analysis provided evidence that G9a contributes to multiple myeloma cell survival and proliferation. This study found that G9a interacts with NF-κB pathway as a key regulator of RelB in multiple myeloma and regulates RelB-dependent multiple myeloma survival. G9a therefore is a promising therapeutic target for multiple myeloma.

A discrete core-shell-like micro-light-emitting diode (micro-LED) array was grown on a 100 nm-thick sapphire nano-membrane array without harmful plasma etching for chip singulation. Due to proper design for the sapphire nano-membrane array, an array of multi-faceted micro-LEDs with size of 4 μm × 16 μm was grown. Threading dislocation density in the micro-LED formed on sapphire nano-membrane was reduced by 59.6% due to the sapphire nano-membranes, which serve as compliant substrates, compared to GaN formed on a planar substrate. Enhancements in internal quantum efficiency by 44% and 3.3 times higher photoluminescence intensity were also observed from it. Cathodoluminescence emission at 435 nm was measured from c-plane multiple quantum wells (MQWs), whereas negligible emissions were detected from semi-polar sidewall facets. A core-shell-like MQWs were formed on all facets, hopefully lowering concentration of non-radiative surface recombination centers and reducing leakage current paths. This study provides an attractive platform for micro-LEDs by using sapphire nano-membrane.

Primary mediastinal large B-cell lymphoma (PMBL), a distinct mature B-cell lymphoma, expresses CD20 and has recently been successfully treated with the combination of a type I anti-CD20 monoclonal antibody, rituximab, with multiple combination chemotherapy regimens. Obinutuzumab is a glycoengineered type II anti-CD20 monoclonal antibody (mAb), recognizing a unique CD20 extracellular membrane epitope with enhanced antibody dependent cellular cytotoxicity (ADCC) vs rituximab. We hypothesize that obinutuzumab vs rituximab will significantly enhance in-vitro and in-vivo cytotoxicity against PMBL. PMBL cells were treated with equal dose of obinutuzumab and rituximab for 24 hours (1-100 μg/ml). ADCC were performed with ex-vivo expanded natural killer cells at 10:1 E: T ratio. Mice were xenografted with intravenous injections of luciferase expressing Karpas1106P cells and treated every 7 days for 8 weeks. Tumor burden was monitored by IVIS spectrum system. Compared with rituximab, obinutuzumab significantly inhibited PMBL cell proliferation (p = 0.01), promoted apoptosis (p = 0.05) and enhanced ADCC (p = 0.0002) against PMBL. Similarly, in PMBL xenografted NOD scid gamma mice, obinutuzumab significantly enhanced survival than rituximab when treated with equal doses (p = 0.05). Taken together our results suggest that obinutuzumab significantly enhanced natural killer cytotoxicity, reduced PMBL proliferation and prolonged the overall survival in humanized PMBL xenografted NOD scid gamma mice.

Background: Multiple myeloma (MM) is a hematological malignancy arising from immunoglobulin-secreting plasma cells. Despite recent progress in stem-cell transplantation, high-dose chemotherapy and novel therapies, MM remains incurable and most patients experience relapse. In addition of genetic alterations, growing evidence has suggested that alterations in epigenetic landscape contribute to pathogenesis of MM. Super enhancers (SE) are large clusters of putative enhancers with aberrantly strong binding of mediators and transcription-regulating proteins. MM cells are highly dependent on unfolded protein response (UPR) signaling pathways due to high level of endoplasmic reticulum (ER) stress. Phosphorylation of eIF2α can attenuate protein translation. The PPP1R15B (denoted as R15B hereafter) gene encodes a regulatory subunit of an eIF2α-specific phosphatase complex. In this study, we identified SE-driven oncogenes specific in MM with a particular focus on a candidate SE-associated gene R15B, whose functional roles in MM remain largely elusive. Methods: We performed H3K27Ac ChIP-seq on MM cell lines, primary MM patient samples and normal CD138+ plasma cells, memory B cells (controls). ROSE analysis was used to systematically annotate SEs and their associated genes between tumors and controls. A combination of public data mining, RNA interference (RNAi), overexpression and CRISPR/Cas9 technologies followed by functional assays were conducted to determine the oncogenic effects of R15B in MM. Transcriptome analysis of MM cell line NCI-H929 with R15B knockdown and scrambled control was performed. To further study the interactions between SE and its promoters, we are currently working on highly integrative chromatin immunoprecipitation (HiChIP) followed by sequencing. Results: We have identified R15B as one of the SE-associated genes specific to MM patient samples and MM cell lines. The expression of R15B was 6- to 50-fold higher in a panel of 8 MM cell lines than normal controls. SE activity was correlated with the expression level of R15B. Higher expression of R15B predicted poor overall survival of MM patients, suggesting its clinical relevance in MM pathogenesis. Knockdown and knockout of R15B significantly reduced cell viability, clonogenicity and induced G2/M phase cell cycle arrest. Gene Ontology (GO) enrichment and pathway analysis of downregulated genes in response to R15B knockdown indicates that these genes are significantly associated with G2/M checkpoint hallmark, oxidative phosphorylation, unfolded protein response and E2F1-related pathways. Loss of R15B may induce apoptosis via activation of UPR pro-apoptotic executors PUMA and NOXA. ChIP-qPCR assays indicates that transcription factor C/EBP-β is strongly enriched at R15B SE region. Furthermore, we examined the therapeutic effects of Salubrinal, a selective inhibitor of eIF2α phosphorylation, on MM cells. Salubrinal inhibited MM cell proliferation in a dose-dependent manner, and MM cell lines with higher R15B expression were more sensitive to Salubrinal than those with lower R15B level. Conclusions: Our integrative approaches identified R15B as a novel SE-driven oncogene, and may form a positive feedback loop with C/EBP-β. Salubrinal selectively inhibits proliferation of MM cells with high R15B expression. Hence, we propose that targeting R15B may serve as a new approach for effective anti-myeloma therapy, which warrants further clinical investigation. Disclosures Chng: Novartis: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Abbvie: Honoraria; Amgen: Honoraria, Research Funding.

// 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.

We report the influences of a Si-doped graded-superlattice (SiGSL) on the structural, optical, and electrical properties of InGaN/GaN light-emitting diodes (LEDs). For comparison, LEDs fabricated on an undoped graded superlattice (unGSL-LED) and conventional LEDs without the superlattice (C-LED) were prepared. The dependences of the carrier behaviors on the surface inhomogeneity and the defect/dislocation distribution were investigated by using fluorescence microscopy images, wherein the dark features corresponding to the defects/threading dislocations for the unGSL-LED and the SiGSL-LED were significantly reduced compared to that for the C-LED. The photoluminescence intensities of the unGSL-LED and the SiGSL-LED were 1.15 and 1.24 times stronger than that of the C-LED, respectively. The output powers of the unGSL-LED and the SiGSL-LED at an injection current of 20 mA were measured to be 1.32 and 1.41 times higher than that of the C-LED, respectively. Moreover, the electrostatic discharge characteristics of the LED sample using the SiGSL structure were drastically improved.

Background The WHO defines three categories of NK cell malignancies; extra nodal NK/T cell lymphoma (NKTCL), aggressive NK cell leukemia, and the provisional entity chronic lymphoproliferative disorder of NK cells (CLPD-NK). Although the flow cytometric (FC) phenotype of CLPD-NK has been described, studies on FC phenotype of NKTCL are limited. To the best of our knowledge ours is the first study to compare the phenotype of NKTCL, CLPD-NK, reactive NK lymphocytosis (RNKL), and normal NK cells using eight color (8C) FC. Methods Specimens analyzed using the Euroflow8C NK Lymphoproliferative Disorder (NKLPD) panel between 2011 and 2014 were identified from our database. All samples were analyzed on the FACSCantoII cytometer. NK cells were identified as CD45+, smCD3−, CD19−, CD56+ and normal T-cells served as internal controls. Results The majority of NKTCL were CD56 bright, CD16 dim, CD57−, and CD94+. CLPD-NK and RNKL were predominantly CD56+ or dim with positive expression of CD16 and CD57 and weak CD94 expression. Antigen based statistical analyses showed robust division of samples along the NKTCL/normal CD56 bright NK cell and CLPD-NK/RNKL/normal CD56 positive NK cell groups. Conclusions It was concluded that FC can reliably distinguish NKTCL from CLPD-NK, normal NK cells of CD56+ phenotype, and RNKL. It was proposed that the typical phenotype for NKTCL is: CD56 bright, CD16 dim with positive CD2, CD7, CD94, HLADR, CD25, CD26, and absent CD57. This resembles the phenotype of the CD56 bright immunoregulatory subset of NK cells which we therefore hypothesize is the cell of origin of NKTCL. © 2017 International Clinical Cytometry Society

In this paper, the influence of a Si-doped graded superlattice (SiGSL) on the electrostatic discharge (ESD) characteristics of green InGaN/GaN light-emitting diodes (LEDs) is discussed. To investigate the effects of Si doping to the superlattice, green LEDs were also fabricated on undoped graded superlattice (unGSL). Furthermore, a conventional green InGaN/GaN LED without the superlattice (C-LED), emitting at a wavelength of 534 nm, was also prepared as a reference. The current-voltage (I-V) curves for the C-LED and the unGSL-LED subjected to the ESD treatments at surge voltages from 2000 to 8000 V showed a drastic increase in the leakage current. However, there was relatively small change in the I-V curves for the SiGSL-LED after the ESD treatments, even at the surge voltages of 6000 and 8000 V. After the ESD treatment at a surge voltage of 8000 V, the EL intensities for the C-LED, unGSL, and SiGSL, measured at a driving current of 120 mA, were reduced by 55, 53, and 30%, respectively, compared to those of the as-fabricated LEDs.

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.

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.

The photoelectrochemical devices with morphologically tuned photoelectrodes assist in addressing the existing sustainable energy challenges by generating hydrogen energy from sunlight and water. In the present work, morphologically tuned nanoporous GaN photoelectrodes are successfully fabricated via a low-damage electrochemical wet etching. Also, a facile halogen treatment is used to replace Ga-O bonds with Ga-halogen bonds at the nanopore region. The influence of halogen treatment on the nanoporous GaN photoelectrodes is investigated in comparison with planar and nanoporous GaN photoelectrode. From the conventional analysis, a maximum 4-fold improvement in the applied bias photon-to-current efficiency is obtained for the Br treated nanoporous GaN photoelectrode (0.43%) compared to planar GaN (0.11%). Based on the result of X-ray and ultraviolet photoemission spectroscopy along with density functional theory calculations, the superior performance owing to the modification in the space charge region and reconstruction of the Ga-halogen bonds enables more efficient extraction of photogenerated charge carriers. In addition, it is confirmed that the halogen treatment assists in suitably tuning the band bending and electronic characteristics for enhanced photoelectrochemical water splitting performance. Altogether, we concluded that the halogen treatment has great potential for the development of photoelectrochemical water splitting devices.

An array of InGaN-based flexible light-emitting diodes (FLEDs) was fabricated on a Ni-embedded electrical conducting flexible fabric with a full-scale 2-in. size. The FLED chip operation under current injection was realized using a single current probe as the negative electrode on the n-GaN surface; the conducting substrate was used as the positive electrode. The stability of the output power in the FLEDs was improved dramatically on the Ni-embedded conducting flexible fabric compared to that on the conventional polyimide flexible substrate. The former showed linear operation up to an input current 950 mA with no wavelength shift, whereas the latter exhibited rolling-over behavior after an input current of 200 mA.

Simple Summary T-cells defend the human body from pathogenic invasion via specific recognition by T-cell receptors (TCRs). The TCR genes undergo recombination (rearrangement) in a myriad of possible ways to generate different TCRs that can recognize a wide diversity of foreign antigens. However, in patients with T-cell lymphoma (TCL), a particular T-cell becomes malignant and proliferates, resulting in a population of genetically identical cells with same TCR rearrangement pattern. To help diagnose patients with TCL, a polymerase chain reaction (PCR)-based assay is currently used to determine if neoplastic cells in patient samples are of T-cell origin and bear identical (monoclonal) TCR rearrangement pattern. Herein, we report the application of a novel segmentation and copy number computation algorithm to accurately identify different TCR rearrangement patterns using data from the whole genome sequencing of patient materials. Our approach may improve the diagnostic accuracy of TCLs and can be similarly applied to the diagnosis of B-cell lymphomas. Abstract T-cell lymphomas arise from a single neoplastic clone and exhibit identical patterns of deletions in T-cell receptor (TCR) genes. Whole genome sequencing (WGS) data represent a treasure trove of information for the development of novel clinical applications. However, the use of WGS to identify clonal T-cell proliferations has not been systematically studied. In this study, based on WGS data, we identified monoclonal rearrangements (MRs) of T-cell receptors (TCR) genes using a novel segmentation algorithm and copy number computation. We evaluated the feasibility of this technique as a marker of T-cell clonality using T-cell lymphomas (TCL, n = 44) and extranodal NK/T-cell lymphomas (ENKTLs, n = 20), and identified 98% of TCLs with one or more TCR gene MRs, against 91% detected using PCR. TCR MRs were absent in all ENKTLs and NK cell lines. Sensitivity-wise, this platform is sufficiently competent, with MRs detected in the majority of samples with tumor content under 25% and it can also distinguish monoallelic from biallelic MRs. Understanding the copy number landscape of TCR using WGS data may engender new diagnostic applications in hematolymphoid pathology, which can be readily adapted to the analysis of B-cell receptor loci for B-cell clonality determination.

Metal-nanowire such as silver nanowire (AgNW) is widely studied as a promising candidate for replacement of indium-tin-oxide (ITO)-based transparent conducting electrodes (TCEs) in LEDs. However, the efficiency of LEDs with metal nanowire is inferior to that of LEDs with ITO due to high contact resistance, low current spreading and high absorption in visible range. To solve these problems, we demonstrate localized surface plasmon Ag NW + nanoparticle (NP)-based TCEs where internal quantum efficiency (IQE) is improved by quantum well (QW)-LSP coupling and light extraction efficiency (LEE) is enhanced by NPs working as leak mode and scattering center.

Overexpression of PRL-3, an oncogenic phosphatase, was identified as a novel cluster in patients with newly diagnosed multiple myeloma. However, the regulation and oncogenic activities of PRL-3 in multiple myeloma warrant further investigation. Here, we report that IL6 activates STAT3, which acts as a direct transcriptional regulator of PRL-3. Upregulation of PRL-3 increased myeloma cell viability and rephosphorylated STAT3 in a biphasic manner through direct interaction and deactivation of SHP2, thus blocking the gp130 (Y759)-mediated repression of STAT3 activity. Abrogation of PRL-3 reduced myeloma cell survival, clonogenicity, and tumorigenesis, and detailed mechanistic studies revealed “deactivation” of effector proteins such as Akt, Erk1/2, Src, STAT1, and STAT3. Furthermore, loss of PRL-3 efficiently abolished nuclear localization of STAT3 and reduced its occupancy on the promoter of target genes c-Myc and Mcl-1, and antiapoptotic genes Bcl2 and Bcl-xL. PRL-3 also played a role in the acquired resistance of myeloma cells to bortezomib, which could be overcome by PRL-3 silencing. Of clinical relevance, STAT3 and PRL-3 expression was positively correlated in five independent cohorts, and the STAT3 activation signature was significantly enriched in patients with high PRL-3 expression. Furthermore, PRL-3 could be used as a biomarker to identify high-risk patients with multiple myeloma that exhibited poor prognosis and inferior outcome even when treated with novel combinational therapeutics (proteasome inhibitors and immunomodulatory imide drugs). Conclusively, our results support a feedforward mechanism between STAT3 and PRL-3 that prolongs prosurvival signaling in multiple myeloma, and suggest targeting PRL-3 as a valid therapeutic opportunity in multiple myeloma. Significance: IL6 promotes STAT3-dependent transcriptional upregulation of PRL-3, which in turn re-phosphorylates STAT3 and aberrantly activates STAT3 target genes, leading to bortezomib resistance in multiple myeloma.

Microarrays allow for the analysis of transcription in a large number of genes simultaneously. They have played an important role in the identification of cancer cell signaling pathways. The ease of use and reliability of microarrays has been a result of standardization of assay protocols and consolidation of data analysis techniques. Online public repositories of gene expression profile data have been created, allowing for researchers to test their hypothesis regarding the link between gene signaling and disease. Microarrays have many different applications in the study of cancer. It allows for disease stratification of tumors into different molecular subgroups. They can also provide additional information on disease mechanism, cancer prognosis, and cancer metastasis. Multiple studies have used microarrays on cell lines to perform preclinical screening to predict anticancer drug responses. In addition, they can be used in real-life patient’s tissues to provide personalized prediction of drug response.

DNA alterations have been extensively reported in multiple myeloma (MM); however, they cannot yet fully explain all the biological and molecular abnormalities in MM, which remains to this day an incurable disease with eventual emergence of refractory disease. Recent years have seen abnormalities at the RNA levels being reported to possess potential biological relevance in cancers. ADAR1-mediated A-to-I editing is an important posttranscriptional mechanism in human physiology, and the biological implication of its abnormality, especially at the global level, is underexplored in MM. In this study, we define the biological implications of A-to-I editing and how it contributes to MM pathogenesis. Here, we identified that the MM transcriptome is aberrantly hyperedited because of the overexpression of ADAR1. These events were associated with patients' survival independent of 1q21 amplifications and could affect patients' responsiveness to different treatment regimes. Our functional assays established ADAR1 to be oncogenic, driving cellular growth and proliferation in an editing-dependent manner. In addition, we identified NEIL1 (base-excision repair gene) as an essential and a ubiquitously edited ADAR1 target in MM. The recoded NEIL1 protein showed defective oxidative damage repair capacity and loss-of-function properties. Collectively, our data demonstrated that ADAR1-mediated A-to-I editing is both clinically and biologically relevant in MM. These data unraveled novel insights into MM molecular pathogenesis at the global RNA level.