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Introduction Long noncoding RNAs (lncRNAs) are profoundly dysregulated in multiple myeloma (MM), yet their tumor-promoting features remain to be defined. Here, we have coupled extensive transcriptomic profiling of patient samples (n=360) with a genome-wide CRISPR interference (CRISPRi) viability screen (4 cell lines) to generate a map of lncRNA "dependencies" in MM; moreover, we describe the molecular and functional role – as well as its therapeutic potential – of the screen top hit RNA Regulator of Lipogenesis (RROL), a nuclear-retained lncRNA generated by alternative splicing of MIR17HG. Methods Loss-of-function (LOF) studies using antisense oligonucleotides (ASO) confirmed the RROL dependency in a large panel of MM cell lines and CD138+ patient cells, in vitro and in vivo in animal models. This effect was not antagonized in DROSHA KO MM cells or by ectopic expression of MIR17HG-derived miR-17-92, indicating a microRNA-independent role of RROL. Results Transcriptomic analysis after RROL depletion indicated a significant impact on the de novo lipogenesis (DNL) gene networks. RROL occupancy at DNL gene loci was detected by chromatin isolation by RNA precipitation followed by qRT-PCR (ChIRP-qPCR) and confirmed by DUAL RNA FISH analysis. At functional level, we demonstrated that RROL depletion reduces DNL in MM cells using an unbiased lipidomic profiling as well as by measuring the incorporation of C14-radiolabeled glucose into lipids. Importantly, we also proved that exogenous palmitate, the downstream product of DNL pathway, can significantly rescue the growth inhibitory effect of RROL depletion in MM cells; thus implicating a role for DNL in the growth promoting effect of this lncRNA. RNA-Protein Pull Down (RPPD) and in vivo RNA yeast three-hybrid (Y3H) assays led to identify c-MYC (MYC) as a relevant protein interactor of RROL; a finding validated by RIP-qPCR. Coupling RNA FISH with immunofluorescence, we co-detected RROL and MYC at the ACC1 promoter. Importantly, neither RROL or MYC could occupy ACC1 promoter and exert regulatory control in the absence of the other factor. Moreover, using in vitro (Co-IP/MS) and in vivo (BioID) assays, we identified WDR82 as an RROL-dependent MYC partner implicated in the transcriptional control of ACC1 expression. Finally, to therapeutically antagonize RROL, we have screened >100 ASOs and provided the basis to develop a first-in-class RROL inhibitor. This inhibitor has shown a very strong anti-MM activity in vitro (IC50 Conclusion In conclusion, we here report a unique regulatory function of a novel lncRNA supporting MM cell growth via its control of the lipogenic metabolic axis. The ongoing development of RROL inhibitors may allow clinical application of this unique targeted therapy in MM.

Background Multiple Myeloma (MM) is a disease driven by numerous genetic and epigenetic alterations, however true drivers of the disease have yet to be identified. Methods To identify new dependencies and actionable therapeutic targets in MM we integrated gene expression and genetic dependency (CRISPR KO). Results We found that many of the specific and potent dependencies in MM are transcription factors, especially those establishing plasma cell identity. Among others, the POU2AF1 gene, which encodes the OCA-B/BOB-1, a B cell transcriptional coactivator protein, represented the most striking dependency in MM. Although BOB-1 is expressed throughout B-cell development, we found it to be highly expressed in CD138+ plasma cells from patients with precursor conditions (MGUS and SMM) as well and established MM compared to normal plasma cells (NPC). Loss-of-function studies using shRNA, siRNAs as well as antisense GapMers specific for BOB-1 confirmed significant impact on MM cell viability. Transcriptomic analysis by RNA-sequencing revealed a small set of genes commonly modulated in MM cell lines upon BOB-1 depletion, including the XBP1- BHLHA15 axis involved in lipid synthesis and unfolded protein response (UPR). Interestingly, among the genes most significantly upregulated by BOB-1 depletion was heme oxygenase 1 (HMOX1), that was affected via the NRF2/Keap1 pathway. We observed that HMOX1 expression is significantly lower in MM cells from patients compared to normal plasma cells and correlates with poor clinical outcome, suggesting important role in MM. Moreover, we found that siRNA depletion of HMOX1 reverted the inhibition of MM cell growth caused by BOB-1 KD, confirming significant role for HMOX1 in the BOB-1 addiction observed in MM cells. Next, we performed gene set enrichment analysis (GSEA) and observed ribosome biogenesis pathways and mRNA translation and elongation processes, along with WNT and senescence pathways, to be significantly enriched among genes modulated by BOB-1 depletion in MM cells. Since high protein load is a feature of MM, we evaluated the role of BOB-1 in the translational efficiency of MM cells. In MM cell lines, BOB-1 knockdown decreased de novo protein synthesis, while its overexpression significantly enhances protein synthesis compared to control cells. As MM is characterized by excess production of monoclonal immunoglobulins, we evaluated impact of BOB-1 perturbation on intracellular kappa and lambda light chains production. We observed changes in the intracellular abundance of the light chains with BOB-1 modulation in all MM cell lines tested. As a result, BOB-1 depletion was associated with induction of resistance to proteasome inhibition. Conclusion In conclusion, we report BOB1 as a specific dependency in MM cells with potential role on modulating the protein load/capacity balance in MM cells and therefore the sensitivity to proteasome inhibition.

Background On average 10% of SMM patients progress to symptomatic MM per year with in first 5 years of diagnosis. However, a subset of SMM patients re-classified as high risk patients on the basis of risk markers which identify risk of progression within 2 years. Although recent studies have evaluated the high-risk SMM, genomic background of SMM patients who do not progress to MM after long-term follow-up (>=5 years) has not been described. Methods Here, we evaluated transcriptomic and genomic changes enriched in non-progressor (NP) (no progression after 5 years of follow-up) precursor conditions (N=31) with those progressed within short period time (N=71) and compared them with changes observed in newly diagnosed MM (N=192). Additionally, using transcriptome, epigenome and whole genome profiling we also studied additional unique samples from 18 patients at their precursor stage as well as when progressed to MM. Results Overall, we have observed significantly lower mutational load for NP SMM (median SNV 5460 vs. 7018, p 90% accuracy and >0.80 area under the curve on ROC using ten-fold cross validation. This indicated that not only the load but also the patterns of mutations (type, location, frequency) are different between two conditions. We also found that NP samples have significantly lower heterogeneity (p 80%) with AUC score 0.80. Our transcriptomic analysis measured the distance between progressor and NP SMM as well as MM and found that NP SMM are less similar to MM which is closer to progressor SMM. Epigenomic analysis yield that 75 SEs regions were differentially utilized between precursor and symptomatic MM stage. The targeted genes included BMP6, PRDM1, STAT1 and RAB21 and possibly regulating genes related to oncogenic KRAS activities. Conclusions In conclusion, our results now provide the basis to develop genomic definition of SMM as well as risk driving features.

Background Immunotherapies in MM targeting BCMA, have shown remarkable clinical benefits. However, two recent reports highlighted that biallelic loss of BCMA cause resistance to anti-BCMA therapy. In both studies BCMA locus was deleted bringing in focus importance of del16p. We have evaluated 2883 MM patients at diagnosis and relapse to understand frequency of BCMA targeting events and characteristics of MM patients with BCMA deletion. Methods We observed del16p in 8.58% (7.6% to14.6% in individual studies) of newly-diagnosed patients (n=2458).Frequency of 16p loss in both HMM and NHMM were similar, suggesting its independence from MM subtypes. Overall CN loss was significantly higher in patients with BCMA loss compared to rest of the MM patients. High risk deletion events such as del1p and del17p were more likely to be observed in patients with loss of BCMA locus (OR [95% CI] 19.3(13.1-25.8), FDR = 1.5e-65; and 8.8(6.3-12.1), FDR = 5.5E-39, respectively)]. MM patients with loss of BCMA locus have increased mutational load (8202 with 95% HDI 6921 and 9535) compared to those without BCMA locus loss (6975 with 95% HDI 6626 – 7343); probability of difference greater than 0 was 96.8% and difference of the means were 1222 [95% CI 112-2589]. To understand the risk profile of patients with loss of BCMA locus, we next focused on the observation that BCMA loss frequently co-occurs with other deletions. Results We observed that when BCMA and TP53 or BCMA and del1p loss are present in the same patient, they are likely to have same clonality. These data suggested a possibility of co-occurrence of these events in same cell. To further investigate this observation, we used single cell DNA sequencing data from patients with sub clonal and clonal BCMA locus loss. Interestingly, almost all cells with BCMA loss also had p53 loss, while not all p53 loss cells had BCMA loss suggesting that the chronology of this copy number alternation may suggest first p53 loss followed by BCMA loss. Our data from a patient with BCMA targeting therapy also indicated that BCMA loss tend to co-occur with TP53 deletions (OR=5.67 [95% CI 4.12-7.84], p value Conclusions Our data from large scale copy number profiles showed that even without treatment pressure, monoallelic BCMA deletions are frequent events. Moreover, patients with these events show increased genomic loss. Such behavior potentially make these cells vulnerable for biallelic loss of other genes. Our results highlight that by looking at mRNA or protein expressions at bulk sample would not directly indicate the presence or absence of cells with target loss and therefore evaluating single cell level data are necessary. These results suggest the need to study del16p in patients being targeted for BCMA-directed therapy and its association with del17p raises question about the role of BCMA targeted therapy in high-risk myeloma.

Aims The hemoglobin HbA1C (HbA1C) value, translated into estimated average glucose concentration (eAG), is commonly used to assess glycaemic control and manage treatment regimens in people with diabetes. However, the relationships among HbA1C-derived eAG, and mean glucose concentration derived from continuous glucose monitoring (CGM) in different populations have not been well studied. We examined this relationship in older people with diabetes and compared the results to those currently used in clinical practice. Methods Data from three studies evaluating CGM in older adults (≥70 years of age), with stable glycaemic control were analyzed retrospectively. Mean glucose and mean amplitude of glucose excursion (MAGE) were calculated from CGM data and correlated with HbA1C and HbA1C-derived eAG using the ADAG study formula. Results HbA1C and CGM data were analyzed from 90 patients with mean age 76±5 years, HbA1C 7.9±1.2% (63±13mmol/mol) and 77% with Type 2 diabetes. The HbA1C and HbA1C-derived eAG correlated significantly with CGM-measured mean glucose ( r 2 =0.30, p r 2 =0.16, p =0.00013) in this population and all its subgroups, but the slopes of the relationship between HbA1C and eAG or CGM-measured mean glucose were significantly different. Conclusions HbA1C-derived eAG values may not accurately reflect CGM-measured mean glucose or MAGE in older adults with diabetes. Wide glucose excursions should be considered and HbA1C should be interpreted cautiously when making treatment changes based on HbA1C.

Background Nitrous oxide anesthesia increases postoperative homocysteine concentrations. Renal transplantation candidates present with higher homocysteine levels than patients with no renal disease. We designed this study to investigate if homocysteine levels are higher in subjects receiving nitrous oxide for renal transplantation compared with subjects undergoing nitrous oxide free anesthesia. Methods Data from 59 patients scheduled for living-related donor renal transplantation surgery were analyzed in this randomized, controlled, blinded, parallel-group, longitudinal trial. Patients were assigned to receive general anesthesia with (flowmeter was set at 2 L/min nitrous oxide and 1 L/min oxygen) or without nitrous oxide (2 L/min air and 1 L/min oxygen). We evaluated levels of total homocysteine and known determinants, including creatinine, folate, vitamin B12, albumin, and lipids. We evaluated factor V and von Willebrand factor (vWF) to determine endothelial dysfunction and creatinine kinase myocardial band (CKMB)-mass, troponin T to show myocardial ischemia preoperatively in the holding area (T1), after discontinuation of anesthetic gases (T2), and 24 hours after induction (T3). Results Compared with baseline, homocysteine concentrations significantly decreased both in the nitrous oxide (22.3 ± 16.3 vs 11.8 ± 9.9; P

Convulsions are one of the frequently seen problems for a neurologist in the daily routine. It is difficult to distinguish the seizure from pseudo-seizure because of lack of conclusive tests. The aim of this study is to investigate the relationship between seizure types and seizure periods by studying IMA serum levels in children having seizure. Two groups were included (patients and control) in our study. The patient group consisted of the children admitted to Pediatric Emergency Care during January 2008–January 2010 with seizure and the control group consisted of healthy children. Serum Ischemia modified albumin (IMA) level in the group having seizures was 99.7 and 83.2 U/ml in the control group. In the comparison of the patient and control groups, significant differences were found between their IMA values (p = 0.000). There was a significant difference between IMA values of the group having generalized tonic–clonic seizures and those of the control group (p = 0.001). In comparison of the IMA values of the group having febrile convulsions and those of the control group, a significant difference was determined (p = 0.011). It has been shown that if the seizure was prolonged over 5 min, IMA level increased, and there was a significant difference between the groups experiencing over 5 min of seizures and the groups experiencing less than 5 min of seizures (p = 0.001). An increase in IMA levels in febrile convulsion supports the hypoxia development in the brain during the seizure. Serum IMA levels increased with the elongation of the seizure period and may be an indicator for status epilepticus. 2012 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.