ALBERT

Description: © The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 9 (2018): 189, doi:10.3389/fmicb.2018.00189.

Description: Only select prokaryotes can biosynthesize vitamin B12 (i.e., cobalamins), but these organic co-enzymes are required by all microbial life and can be vanishingly scarce across extensive ocean biomes. Although global ocean genome data suggest cyanobacteria to be a major euphotic source of cobalamins, recent studies have highlighted that 〉95% of cyanobacteria can only produce a cobalamin analog, pseudo-B12, due to the absence of the BluB protein that synthesizes the α ligand 5,6-dimethylbenzimidizole (DMB) required to biosynthesize cobalamins. Pseudo-B12 is substantially less bioavailable to eukaryotic algae, as only certain taxa can intracellularly remodel it to one of the cobalamins. Here we present phylogenetic, metagenomic, transcriptomic, proteomic, and chemical analyses providing multiple lines of evidence that the nitrogen-fixing cyanobacterium Trichodesmium transcribes and translates the biosynthetic, cobalamin-requiring BluB enzyme. Phylogenetic evidence suggests that the Trichodesmium DMB biosynthesis gene, bluB, is of ancient origin, which could have aided in its ecological differentiation from other nitrogen-fixing cyanobacteria. Additionally, orthologue analyses reveal two genes encoding iron-dependent B12 biosynthetic enzymes (cbiX and isiB), suggesting that iron availability may be linked not only to new nitrogen supplies from nitrogen fixation, but also to B12 inputs by Trichodesmium. These analyses suggest that Trichodesmium contains the genus-wide genomic potential for a previously unrecognized role as a source of cobalamins, which may prove to considerably impact marine biogeochemical cycles.

Description: This work was funded by NSF research grants OCE-1260233, OCE-1260490, OCE-1657757, and OCE-143566.

Description: Gene expression profiling (GEP) of purified plasma cells 48 hours after thalidomide and dexamethasone test doses showed these agents' mechanisms of action and provided prognostic information for untreated myeloma patients on Total Therapy 2 (TT2). Bortezomib was added in Total Therapy 3 (TT3), and 48 hours after bortezomib GEP analysis identified 80 highly survival-discriminatory genes in a training set of 142 TT3A patients that were validated in 128 patients receiving TT3B. The 80-gene GEP model (GEP80) also distinguished outcomes when applied at baseline in both TT3 and TT2 protocols. In context of our validated 70-gene model (GEP70), the GEP80 model identified 9% of patients with a grave prognosis among those with GEP70-defined low-risk disease and 41% of patients with favorable prognosis among those with GEP70-defined high-risk disease. PMSD4 was 1 of 3 genes common to both models. Residing on chromosome 1q21, PSMD4 expression is highly sensitive to copy number. Both higher PSMD4 expression levels and higher 1q21 copy numbers affected clinical outcome adversely. GEP80 baseline-defined high risk, high lactate dehydrogenase, and low albumin were the only independent adverse variables surviving multivariate survival model. We are investigating whether second-generation proteasome inhibitors (eg, carfilzomib) can overcome resistance associated with high PSMD4 levels.

Description: Introduction - Multiple Myeloma (MM) is a hematologic malignancy characterized by clonal growth of differentiated plasma cells (PCs). Despite improvement in MM therapy, the disease remains mostly incurable and is characterized by recurrent relapses with development of resistant clones that eventually lead to patient death. The pathways that lead to resistant and aggressive MM are not fully understood highlighting the need to improve our understanding of MM biology to identify potential new pathways and therapeutical targets. PHD Finger Protein 19 (PHF19) is a regulator of Polycomb Repressive Complex 2 (PRC2), the sole methyltransferase complex capable of catalyzing H3K27me3 to induce and enforce gene repression. PRC2 employs enhancer of zeste homolog 1 and 2 (EZH1/EZH2) as enzymatic subunits to hypermethylate H3K27. While overexpression and gain of function mutations of EZH1/2 have been observed in many cancers the role of this particular pathway in MM remains poorly understood. In the present study, we report on PHF19 as a new candidate gene to play a potential crucial role in MM oncogenesis. Methods- Gene expression profiling (GEP; Affymetrix U133 Plus 2.0) was performed on 739 MM patients (from total therapy trials [TT] 3-5; low risk MM n=636, high risk MM n=103), 42 patients with monoclonal gammopathy of undetermined significance (MGUS), 73 smoldering MM patients, 42 patients with primary plasma cell leukemia and 34 healthy donors. Myeloma risk was determined by the GEP 70 signature as previously defined. To test the implications of functional PHF19 knock down (KD) we used TRIPZ inducible PHF19 shRNA vs. scrambled control (Dharmacon) in two MM cell lines (JJN3 and ARP1). Real time PCR as well as western blotting was used to confirm PHF19 KD as well as to elucidate the effect on H3K27me3 (Cell Signaling). Functional in vitro studies included proliferation (Promega), clonogenic assays (StemCell), cell cycle and apoptosis assays (both Invitrogen). In vivo studies were performed using SCID mice that were subjected to tail vain injection with PHF19 KD JJN3 cells (n=10) or scrambled shRNA control (n=10). Weekly ELISA (Bethyl) and in vivo imaging (Xenogen) were performed and survival was recorded. Results- GEP of the previously mentioned patient populations and healthy controls identified PHF19 (chr9q33.2) as a candidate gene that was consistently dysregulated in MM patients. Mean expression levels at different MM stages correlated with disease aggressiveness (ANOVA, p10.46) at diagnosis correlated significantly with adverse clinical parameters, including ISS III, anemia and elevated LDH, as well as worse overall survival (5 yr OS = 29% for patients with high PHF19 expression vs 77% for patients with low PHF19 expression [log275% reduction in both cell lines, p

Description: Key Points CYR61/CCN1 is a bone marrow microenvironmental biomarker for myeloma progression and for transformation of MGUS and asymptomatic disease to overt myeloma. CCN1 reduces myeloma bone disease and tumor growth and is a potential therapeutic target for myeloma.

Description: Abstract 2896 Background: Focal lesions (FL) are a well-recognized consequence of active multiple myeloma (MM), and distinguish it from its antecedent monoclonal gammopathy of undetermined significance (MGUS). We have recently reported on the superior performance of gene expression profiling (GEP) based on random trephine bone marrow aspirate sampling (RS) in distinguishing between 85% of patients with low-risk (LO) MM and 15% with high-risk (HI) MM compared to conventional prognostic variables. MM occasionally presents as macro-focal disease, in which cases RS may be inconclusive because of paucity of malignant cells in the sample. Here we report the comparison of GEP data from paired FL and RS samples from 106 untreated patients. Methods: We identified 106 newly diagnosed patients with paired samples, who were treated on Total Therapy (TT) protocols (3 in TT2, 19 in TT3, 75 in TT4, and 9 in TT5) in our multiple myeloma database. GEP risk scores, molecular subgroup classifications, overall survival (OS), and event-free survival (EFS) were compared and tested with the RS-derived 70-gene risk prediction and molecular subgroup classification models. Results: GEP defined molecular subgroups were correlated in 90 of 106 patients (85%). Looking at GEP-defined risk designation, we found a high degree of correlation between RS and FL samples with 95 of 106 samples showing the same designation (90%). For the 11 patients with divergent GEP designations, 8 (73%) were located at the boundary of the RS GEP risk score cutoff of +0.66 (range +0.43 to +0.84). For these risk designation-divergent patients, FL- but not RS-defined risk determined clinical outcome. Conclusion: Both the 70-gene risk prediction and molecular subgroup classification models can be used in FL-GEP samples. But, more importantly, for patients with RS-GEP risk score close to cutoff boundary (about 14% in our current data sets), FL-GEP provides better risk stratification, suggesting that the FL signal more adequately reflects to disease biology, progression and treatment response in MM. We therefore recommend that, for patients with borderline RS-based GEP risk scores, FL-GEP be used for staging and prognosis assessment in myeloma. Studies are in progress to determine, among multiple FL samples from the same patient, the variability in risk score compared to multiple RS samples. Disclosures: Shaughnessy: Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties. Barlogie:Celgene: Consultancy, Honoraria, Research Funding; IMF: Consultancy, Honoraria; MMRF: Consultancy; Millennium: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy; Novartis: Research Funding; NCI: Research Funding; Johnson & Johnson: Research Funding; Centocor: Research Funding; Onyx: Research Funding; Icon: Research Funding.

Description: Abstract 303 Background: Gene expression profiling (GEP) of purified plasma cells identifies 15% of newly diagnosed MM as high-risk with a median survival of 2yr compared to 10+yr for the remainder. A validated 70-gene GEP risk model (GEP70) making such determinations is related to copy number increases in chromosome 1q21. Moreover, FISH-defined gains of 1q21 at diagnosis are associated with poor outcome and serial studies have shown that both the percentage of cells with 1q21 gains and 1q21 copies in these cells invariably increase at relapse. Combined with the fact that 1q21 is the only recurrent high-level amplicon in MM, these data suggests that 1q21 harbors a copy number sensitive gene or genes that confer resistance to apoptosis. PSMD4 and CKS1B are the only genes in the GEP70 model that map to the 1q21 amplicon. PSMD4 is the polyubiquitin receptor for the proteasome and the only component of the proteasome that exists free of the proteasome complex. High levels of free cytoplasmic PSMD4 and a small proteolytic fragment of PSMD4, known as anti-anti-secretoy factor, may be able to reduce proteasome load thereby reducing sensitivity of MM cells to proteasome inhibition-induced apoptosis. Patients and Method: In TT3, we added BOR to TT2 and performed GEP at baseline and 48hr after BOR test-dosing (1.0mg/m2). We correlated post BOR GEP (TT3), baseline GEP (TT2 and TT3), and baseline 1q21 FISH (TT2 and TT3) with outcomes in over 600 cases. Result: PSMD4 and 14 other proteasome genes were among 80 genes in a post-BOR GEP model (GEP80) created in TT3 and validated in TT3B, whose post-BOR elevated expression was related to poor outcome. The absence of hyper-activation of PSMD4 and proteasome genes after in-vivo thalidomide, dexamethasone or lenalidomide test dosing suggested that this effect was BOR-specific. There was strong but not complete overlap between risk designations by the GEP70 and GEP80 models in TT2 and TT3. We combined the risk predictions of the two models in baseline samples creating four risk combinations. Kaplan Meier analysis revealed a dramatic improvement in outcomes of GEP70 high-risk/GEP80 low-risk cases in TT3 relative to TT2. Similarly, while a significant improvement in outcomes were observed in cases with 3 copies of 1q21, there was no difference for cases with 4+ copies of 1q21. To determine if 1q21 copy number-driven expression changes could account for these differences, we correlated GEP of candidate genes with the presence of 2, 3 or 4+ copies of 1q21. Using FISH-defined tertiles we discovered that intermediate levels of PSMD4, corresponding to 3 copies of 1q21, was associated with significant improvement in outcome in TT3. Conclusion: BOR incorporated into TT3 overcomes GEP70 high-risk disease with 3, but not 4+ copies of 1q21. PSMD4, is a copy number dependent gene at 1q21 and appears to be a strong prognostic biomarker for BOR-containing therapies. We propose that TT3-like therapies can overcome the anti-apoptotic effects of modest increases in PSMD4 levels in MM, but that novel therapeutic strategies specifically targeting PSMD4 function might be needed to improve the currently dismal outcomes associated with high-level expression of PSMD4. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction In multiple myeloma (MM), deletion of chromosome 17 p13 (del17p) is a poor prognostic feature. The percentage of cells carrying an abnormality has been reported to be important with thresholds of 20% being taken generally but thresholds as high as 60% being suggested more recently. We have reported previously in the Total Therapy (TT)-2 trial (NCT00083551) for newly diagnosed (ND) MM that del17p is an adverse prognostic factor (Blood 112: 4235). The TT3 trial (NCT00081939) incorporated Brtezomib into tandem Melphalan-based autotransplants with DT-PACE for induction/consolidation and Thalidomide and Dexamethasone for maintenance to treat patients with newly diagnosed MM. In more recent iterations of these trials following the introduction of novel agents in induction and during maintenance the impact of carrying del17p has not been studied. In particular we have stratified patients into low- or high-risk molecular subgroups based on the GEP-70 (TT4 protocol [NCT00734877] or TT5 protocol [NCT00869232], respectively). We have used interphase FISH (iFISH) to detect the presence of del17p in baseline bone marrow samples. Method The iFISH slides were prepared with bone marrow aspirates after removing erythrocytes. A specific TP53 probe at chromosome 17 arm p13 combined with a control probe for the ERBB3 locus (HER2, 17q12), in different colors, were hybridized to bone marrow cells. Myeloma PCs were identified by restricted Kappa or Lambda immunoglobulin light-chain staining. We investigated role of 20% cutoffs per ≥100 tumor cells for significant deletion of the TP53 probe. Kaplan-Meier analysis was used to estimate the distributions of overall survival (OS) and progression-free survival (PFS) during the follow-ups. OS was calculated from registration until the date of decease. PFS was similarly calculated, but also incorporated progressive disease as an event. Results We examined 709 baseline samples from TT3, 4, and 5 trials with the two probes at chromosome 17. Overall, 66 of 709 patients (9.3%) had deletion of TP53 locus, including 44 of the 591 (7.5%) of low-risk patients and 20 of the 118 (17.0%) high-risk patients (Table). The range of TP53-deleted cells among newly diagnosed patients is 20-99% (median=75%) overall; 35-100% (median=62%) in TT3-low-risk; 30-97% (median=80%) in TT3-high-risk; 21-99% (median=76%) in TT4; and 20-97% (median=81%) in TT5. Deletion of TP53 was associated with significant shorter OS and PFS in HR patients treated on TT3. The 3 year estimated OS of patients for TT3-HR with del17p was 33% compared with 56% for TT3-LR with del17p, and PFS of patients for TT3-HR with del17p was 25% compared with 51% for TT3-LR with del17p (Figure). The comparison of TT4 to TT5 continued showing short OS in HR patients treated on TT5. The 3 year estimated OS of patients for HRMM with del17p was 17% compared with 75% for TT5 patients without deletion (p=0.0008). But, del17p was neutral in LR patients treated on TT4 (Figure). Conclusion Since the introduction of novel agents during various stages of the disease and a focus on HRMM and LRMM defined by GEP70 we show that while TP53 deletion is an adverse prognostic factor for patients with HRMM it is no longer prognostically relevant in LRMM. Table 1. Patients with iFISH results GEP-70 riskLow ≤0.66 High 〉0.66 Deletion TP53 in 20-59% PCs (n/N [%]) Deletion TP53 in ≥60% PCs (n/N, [%]) Total TT3 (N=329) Low=256 9/329, [2.7%] 9/329, [2.7%] 18/329, [5.5%] High=73 3/329, [0.9%] 9/329, [2.7%] 12/329, [3.7%] TT4 (N=313) Low=313 5/313, [1.6%] 21/313, [6.7%] 26/313, [8.3%] High=0 0 0 0 TT5 (N=67) Low=22 2/67, [3.0%] 0 2/67, [3.0%] High=45 0 8/67, [11.9%] 8/67, [11.9%] Sum (N=709) Low=591 (83.4%) 14/709, [2.0%] 30/709, [4.2%] High=118 (16.6%) 3/709, [0.4%] 17/709, [2.4%] 66/709 (9.3%) Figure 1. Figure 1. Disclosures Tian: University of Arkansas for Medical Sciecnes: Employment. Epstein:University of Arkansas for Medical Sciences: Employment. Qu:Cancer Research and Biostatistics: Employment. Heuck:Millenium: Other: Advisory Board; Janssen: Other: Advisory Board; Celgene: Consultancy; Foundation Medicine: Honoraria; University of Arkansas for Medical Sciences: Employment. van Rhee:University of Arkansa for Medical Sciences: Employment. Zangari:University of Arkansas for Medical Sciences: Employment; Millennium: Research Funding; Onyx: Research Funding; Novartis: Research Funding. Hoering:Cancer Research and Biostatistics: Employment. Sawyer:University of Arkansas for Medical Sciences: Employment. Barlogie:University of Arkansas for Medical Sciences: Employment. Morgan:Weismann Institute: Honoraria; CancerNet: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; MMRF: Honoraria; University of Arkansas for Medical Sciences: Employment; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Introduction: We have previously used global gene expression (GEP)-based clinical pharmacogenomics of dexamethasone, thalidomide, pomalidomide and bortezomib (Bor) to understand their mechanism of actions and how this impacts their clinical efficacy in multiple myeloma (MM) patients. High dose therapy with melphalan (Mel) followed by autologous stem cell transplantation is a major treatment regimen for MM and consequently assessing the pharmacogenomics of Mel is clinically relevant. Importantly not all patients benefit from Mel exposure and, therefore, it remains important to understand the molecular mechanism of its actions and how they underlie treatment resistance. Materials and methods: 252 newly diagnosed MM patients randomized to Total Therapy (TT)4 (GEP70 low-risk, N=210) and TT5 (GEP70 high-risk, n=42) clinical trials received single administration of Bor (1.0mg/m2) followed after 48 hrs latter by a single administration of Mel (10mg/m2). BM aspirates were obtained at baseline, 48 hrs post-Bor and 48 hrs Post-Mel. Purified CD138-selected MM cells underwent GEP analysis using the Affymetrix U133plus2 microarrays and the results generated were analyzed using Gene Set Enrichment Analysis (GSEA) and Ingenuity. The effect of Mel on expression of cell surface receptors in MM cell lines was validated by flow cytometry. Results: Expression of 176 probe sets was changed 48 hours after a single Bor administration at FDR 〈 0.01, and expression of 5117 additional probe sets was further changed after Mel. Expression of 6309 probe sets was changed when comparing post-Mel to baseline, of these 108 were also changed post-Bor and 4043 overlapped with changes observed between post-Mel and post-Bor. By utilizing GSEA and Ingenuity we identified the top pathways associated with Mel activity including activation of p53, nitrogen metabolism and metabolism of xenobiotics by p450, whereas Bor was associated with proteasome activation. Bor and Mel both downregulated pathways related to cell cycle and DNA replication and damage response. Top listed genes differentially expressed between baseline and post-Mel and/or post-Bor and post-Mel and reportedly linked to MM pathogenesis include underexpression of IRF4, ASPM, MYC and NEK2, and upregulation of TNFSF10 (TRAIL), MDM2, BAX and KLF9. Among the top upregulated genes by Mel were also set of 7 cell surface receptors (MERTK, CXCR4, OGFRL1, INSR, TGFBR2, S1PR1, IL1R2) and 5 cytokines (AREG, TNFSF8, BDNF, IGF1, TNFSF15). We initially focused our analysis on MERTK and CXCR4 which have been previously implicated in MM and whose expression was upregulated by 2.5 (FDR 〈 4.6x10-39) and 1.8 (FDR 〈 3.9x10-36) folds, respectively. Increased expression of MERTK and CXCR4 was associated with shorter progression-free survival (PFS) and over survival (OS) in our Total Therapy trials, including analysis restricted to GEP70 high-risk patients in TT3 and TT5. Moreover, GEP of paired MM cell samples obtained from focal lesion and random BM sites of the same patient (n=170) showed reduced CXCR4 expression in MM cells residing within focal lesions (FDR 〈 6.1x10-5), further implicating intra-patient heterogeneity of CXCR4 expression in distinct BM niches with response to Mel. To validate direct effect of Mel on these factors at the protein level, flow cytometry analysis for MERTK and CXCR4 was performed on MM cell lines (n=3) following treatment with Mel (5-10µM) or Bor (2.5-5nM) for 72 hrs. Mel but not Bor increased mean fluorescent intensity of MERTK and CXCR4 by 3.1±0.5 (p 〈 0.05) and 5.1±0.5 (p

Description: Introduction Extramedullary disease (EMD) is a primary disease manifestation of MM, which while not seen frequently at presentation increases in incidence at relapse where its incidence seems to be increasing following the introduction of novel agents. Patients with EMD have a shorter overall survival as well as an increased incidence of anemia, thrombocytopenia, elevated serum lactate dehydrogenase, cytogenetic abnormalities, and high-risk features as determined by gene expression profiling. There is also an increased incidence of the high risk MAF subtypes t (14:16 or 14; 20). Understanding the biology of EMD and identifying its present could give important information about how to improve the outcome of this group. In this work we have used GEP analysis of bone marrow derived plasma cells to predict the presence of EMD so that we can identify the genomic risk factors that define the features of a plasma cell clone, which can develop the capacity to metastasize outside the BM. Materials and Methods We focused on patients treated on TT protocols, at the UAMS, Myeloma Institute between 1989 - 2010, a total of 1154 patients, of which 46 developed EMD before the start of therapy (EMD-1), and 91 developed EMD after registration to UAMS for MM treatment EMD-2. Results We show that most EMD2 cases (57.14%) develop within 3 years after initiation of therapy at the UAMS with few cases developing after this time. Predicting the risk of EMD Combining patients with EMD1 and EMD2 diagnosis within 3 years gave a total of 98 EMD cases. We used 824 samples from 1017 myeloma patients who never developed EMD and had follow up at least 3 years as a comparator group. The data were divided into training (n=619 with 66 EMD cases and 553 controls) and test sets (n=303 with 32 EMD cases and 271 controls). Using the training set, we identified 5 significant gene probes (with a q value 〈 0.001) and made a score to predict cases and controls. The sensitivity and specificity turned out to be 74.24% and 77.40% in the training set, and 56.25% and 76.75% in the test set, respectively. Predicting the time to EMD2 We tested whether we could predict time to EMD2 based on using baseline GEP samples. In this analysis, all EMD2 cases and controls were included. We divided the data into training (n=743 with 61 EMD2 and 682 controls) and test sets (n=365 with 30 EMD2 and 335 controls). By fitting a uniform Cox regression model to each gene in the training set, we identified 68 gene probes that are associated with time to EMD2 (with a q-value

Description: Abstract 2985 Background: The prognosis of patients with MM is best captured by GEP-defined risk, distinguishing ~15% of patients with a median overall survival (OS) in TT2 of ~2yr as opposed to 〉10yr in low-risk disease. Serum LDH elevation has remained an independent adverse feature, along with the presence of metaphase cytogenetic abnormalities (CA). Here we examined whether GEP features within CD138-selected plasma cells can identify MM-associated serum LDH elevation and CA. Patients and Methods: The training set consisted of 621 cases and the test set consisted of 325 TT patients who had both baseline clinical and GEP data from whole genome Affymetrix U133Plus2.0 microarrays on CD138-enriched plasma cells. Using the training data and the scoring approach described in Shaughnessy et al (2007), we defined a GEP score based on 50 genes to predict serum LDH 〉190U/L and a GEP score with 15 genes to predict the presence of CA. The gene scores were then tested in univariate and multivariate Cox regression models to assess their clinical utilities in both training and test sets. Results: In the training set, sensitivity and specificity of the GEP 50-gene score for serum LDH 〉190 were 65% and 76%, respectively; those of the GEP 15-gene score predicting CA were 75% and 78%, respectively. While both GEP scores were significantly associated with overall and event-free survival (p 〈 .001), they were not selected in multivariate stepwise Cox regression analysis. However, the gene scores seem comparable to the clinical variables they try to predict. When serum LDH 〉190 and CA were replaced by the GEP scores in multivariate models, both gene scores were significant at the .1 level, and the R-squared (R2) statistic decreased by only 2.4% (Table 1). In the test set, sensitivity and specificity of the GEP 50-gene score for serum LDH 〉190 were 59% and 71%, respectively. Those of the GEP 15-gene score for CA were 58% and 70%, respectively. The GEP scores behaved similarly overall as in the training set with the LDH gene score showing a little higher association than the CA gene score (p