Deregulated microRNA (miR)/transcription factor (TF)-based networks represent a hallmark of cancer. We report here a novel c-Myc/miR-23b/Sp1 feed-forward loop with a critical role in multiple myeloma (MM) and Waldenstrom's macroglobulinemia (WM) cell growth and survival. We have found miR-23b to be downregulated in MM and WM cells especially in the presence of components of the tumor bone marrow milieu. Promoter methylation is one mechanism of miR-23b suppression in myeloma. In gain-of-function studies using miR-23b mimics-transfected or in miR-23b-stably expressing MM and WM cell lines, we observed a significant decrease in cell proliferation and survival, along with induction of caspase-3/7 activity over time, thus supporting a tumor suppressor role for miR-23b. At the molecular level, miR-23b targeted Sp1 3'UTR and significantly reduced Sp1-driven nuclear factor-κB activity. Finally, c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, transcriptionally repressed miR-23b. Thus MYC-dependent miR-23b repression in myeloma cells may promote activation of oncogenic Sp1-mediated signaling, representing the first feed-forward loop with critical growth and survival role in myeloma.

Nucleotide excision repair (NER) is involved in the removal of bulky adducts and DNA crosslinks induced by various genotoxins including alkylating agents. In cancer, somatic mutations, genes' up-regulation, and epigenetic silencing of NER may lead to abnormal DNA damage responses. Little is known about the role of NER in biology of multiple myeloma (MM), a heterogeneous disease characterized by genomic instability which is often treated by alkylating agents.In our genome sequencing data we did not observe recurrent mutation in NER although non silent mutations affected 13 genes involved in NER in 20out of 272 distinct patients. Using a functional assay based on the purified DNA-Damage Binding protein 2 (DDB2) proteo-probe complex that allows monitoring of photoproducts removal after UV irradiation, we observed NER variability in MM cell lines (MMCL) allowing to separate the cell lines in 2 groups according to their ability to repair: namely rapid (repair>90% after 2 hours) versus slow (repair < 85% after 2 hours).The separation in two NER phenotypes did not correlate with P53 loss of function or any cytogenetic event with the exception of t (4;14) translocation which was associated with the group showing a repair rate > 90%. Interestingly, the LR5 melphalan resistant cell line harbored a rapid repair phenotype as compared to the parental RPMI-8226 cell line which exhibited a slow repair.We evaluated melphalan sensitivity in relationship to NER activity in rapid and slow MMCL and observed that MMCL with slow NER were more sensitive to melphalan. We next evaluated whether inhibiting NER modified drug sensitivity. We utilized spironolactone which is known to inhibit NER. With the DDB2 proteo-probe assay, we confirmed the ability of spironolactone to inhibit NER in MMCL by degrading the Xeroderma Pigmentosum group B (XPB) protein. Combination of melphalan with NER inhibition in the 20 MMCL induced a significant increase in melphalan sensitivity in MMCL (increase from 15 to 79%).We have also confirmed similar NER activity in CD138 positive patient plasma cells. We then developed a gene expression signature using rapid and slow NER cell lines. In a multivariate analysis, the rapid NER signature was significantly associated with low survival in the IFM 2005-01 trial dataset that included 170 patients treated with high-dose melphalan.In conclusion, we have observed that NER affects MM cells and plays a significant role in drug sensitivity; we have also shown that NER can be targeted to increase melphalan sensitivity and potentially improve patient outcome.Disclosures Avet-Loiseau: onyx: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Anderson: Celgene: Consultancy; Millennium: Consultancy; BMS: Consultancy; Gilead: Consultancy; Oncopep: Equity Ownership; Acetylon: Equity Ownership.↵* Asterisk with author names denotes non-ASH members.

The 3' untranslated region (3'UTR) of mRNA contains elements that play regulatory roles in polyadenylation, localization, translation efficiency, and mRNA stability. Although relative contributions of different regulatory mechanisms remain poorly understood, several recent studies showed that alterations in 3'UTRs may affect protein localization as well as their interactions with other proteins.Here, we obtained global measurements of 3'UTR sequencing and RNA-seq in MM and normal plasma cells to study the effects of 3'UTR alterations in MM. The transcriptomes of 15 MM samples and two samples from normal plasma cells were assessed by RNA-seq and alternative 3'UTR isoforms were measured using 3'-seq, an established quantitative 3' end sequencing method.We first studied gene expression results from two different platforms to estimate the overlap of mRNA abundance. For each individual sample there was a high level of correlation between 3'-seq and RNA-seq in terms of identifying genes that were up or down regulated. We identified a common set of 195 genes that were significantly different between normal plasma cells and the 15 MM samples as measured with two independent platforms and this list was enriched in genes regulated by NF-kB in response to TNF and genes up-regulated by KRAS activation pathways.In addition to finding differences in mRNA abundance levels, with 3'-seq, we found 187 genes that had shorter 3'UTRs and 170 genes that had longer 3'UTRs in MM compared to normal plasma cells. Genes that had shorter 3'UTR sequences were enriched for those localized to nucleus and endoplasmic reticulum. They were also enriched in RNA metabolic processes, RNA splicing as well as DNA repair. Genes that had longer 3'UTRs were enriched for genes involved in signal transduction affecting a change in the level or activity of a second messenger or other downstream targets, and ultimately affecting a change in cellular functions. We observed enrichment for G2/M checkpoint, as in progression through the cell division cycle and activation of mTORC1 complex and MYC targets for both shorter and longer 3'UTR genes, which suggests we should investigate these genes deeply to understand their individual regulatory roles in MM development. We found five oncogenes that had shorter 3'UTRs, whereas genes with longer 3'UTRs contained two oncogenes and two tumor suppressor genes.Interestingly amongst 15 patients, five patients had UTR pattern similar to normal plasma cells, 6 had higher frequency of longer UTRs and 4 patients had relatively shorter UTRs compared to normal plasma cells. This pattern did not correlate with common cytogenetic abnormalities. Further investigation is now required to understand the functional consequences of UTR changes and to correlate clinical and prognostic impact of such UTR differences. In conclusion, we report that significant 3'UTR alterations are observed in MM with impact on MM cell pathobiology and suggesting need for further investigation to understand their precise role in regulation and translation of proteins in MM.Disclosures Munshi: millenium: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees.↵* Asterisk with author names denotes non-ASH members.

Recently we reported the concomitant inhibition of MYC and E2F activity in multiple myeloma (MM) upon treatment with the BET bromodomain inhibitor JQ1. BET bromodomains (BETs) are transcriptional co-activators that occupy active promoters and enhancers, but are asymmetrically localized to a small number of "super-enhancer" domains. JQ1 treatment results in disproportionate displacement of BETs from super-enhancers leading to potent and selective downregulation of super-enhancer target genes, with MYC levels > 90% depleted after 6 hours. In contrast, E2F protein levels are relatively unperturbed by JQ1 treatment. Prompted by these observations, we here explored the global genomic occupancy and interaction of E2F and BET bromodomains in regulating MM cell state.We have previously reported that E2F1 and its heterodimerization partner DP1 promote MM tumor proliferation both in vitro and in vivo with a statistically significant inverse correlation between DP1 expression and patient outcome suggesting a role of E2F1/DP1 in the pathogenesis of MM. To better understand how E2F1 and DP1 drive proliferation, we mapped the global occupancy of E2F1/DP1 in MM. Integration of E2F1 and DP1 genomic localization in MM reference epigenome revealed specific co-occupancy of the factors at promoters of active genes marked by H3K4me3 promoters, with a strong positive correlation between E2F and RNA Polymerase II (RNA Pol II) levels at transcription start sites. In contrast, active enhancers, as defined by promoter distal Mediator (MED1) peaks and marked by H3K27ac, showed virtually no E2F binding.Using unbiased hierarchical clustering, we organized different factors/epigenetic modifications in MM1.S by spatial similarity of binding patterns. From this analysis, we identify two distinct active regulatory axes in MM1.S. The first comprised Mediator, P-TEFb (CDK9), RNA Pol II, BRD4, IRF4, and H3K27ac - factors/epigenetic modifications that are found at both promoters and enhancers. The second comprised MYC/MAX, E2F1/DP1, and the transcription start site specific histone modification H3K4me3. Although MYC is also found at enhancers in MM1.S, these data are consistent with MYC and E2F collaborative regulation of E2F target genes at promoters and suggest a role for E2F in regulation of MM transcription separate from BETs. Functional analysis of genes governed by superenhancers (SE) or E2F revealed divergent functionality, with SE-associated genes involved in cell signaling, apoptosis, and hypoxia, and E2F associated genes involved in cell cycle regulation and canonical E2F/MYC regulation. Therefore, our global chromatin analysis reveals two distinct regulatory axes for E2F and BETs in the MM epigenome, with E2F predominantly localized to active gene promoters and BETs disproportionately found at super-enhancers.We hypothesized that the presence of BETs and E2F in distinct regulatory axes divides active genes in MM into those that can be selectively influenced by BET inhibition or E2F perturbation, but not both. In line with this we have observed that dual E2F and BET inhibition is synergistic for MM cell growth. Moreover, we observed that MM cell lines absent of IgH/MYC translocations fail to place MYC directly under SE control, and consequently BET inhibition fails to rapidly downregulate MYC or E2F transcriptional activity. In these non MYC translocated lines, direct E2F inhibition either by genetic approach or by a dimerization inhibiting stapled peptide is synergistic with BET inhibition.In conclusions, our results implicate E2F and superenhancer-driven transcriptional programs as dependencies in MM and suggest an unexplored collaboration between Myc, E2F and BET in maintenance of MM that can be synergistically targeted.Disclosures Anderson: Gilead: Consultancy; Millennium: Consultancy; BMS: Consultancy; acetylon pharmaceuticals: Equity Ownership; Oncocorp: Equity Ownership; Celgene Corporation: Consultancy. Munshi: millenium: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees.↵* Asterisk with author names denotes non-ASH members.

Introduction: Immunoglobulin (Ig) gene rearrangement is a hallmark of early B-cell development. Multiple myeloma (MM), a malignancy of plasma cells, typically have mutated Ig sequences but are thought to be stable throughout the course of the disease. We previously observed that multiple Ig sequences related by somatic hypermutation (SHM) may be present in some MM patients at diagnosis. Here we provide an expanded observation and investigate whether there is ongoing evolution in Ig sequences over the course of the disease.Methods: 550 MM patientsenrolled in IFM/DFCI study were included in this analysis. The next-generation sequencing (NGS)-based immunosequencing platform was used to detect evidence of oligoclonality at the Ig heavy chain loci. Using universal primer sets, we amplified IGH variable, diversity, and joining gene segments from DNA and/or RNA isolated from purified CD138+ MM cells collected at the time of diagnosis. Amplified products were sequenced and analyzed (Faham et al., Blood 2012). MM-specific clonotypes were identified for each patient based on their high frequency (5%) within the B-cell repertoire in the diagnostic (dx) sample. The highest frequency MM clonotype in a dx sample is termed the Òindex clonotype.Ó DNA and/or RNA isolated from dx AND post-treatment bone marrow samples were assessed for evidence of evolved MM clonotypes. A clonotype was considered ÒevolvedÓ based on CDR3 sequence homology to the dx Òindex clonotype.ÓResults: We identified Ig clones in 340 RNA samples and 311 DNA samples from the IFM/DFCI cohort.We first looked at V segment usage in these MM clones comparedto a database of ~30 million Ig VDJ sequences derived from normal B cells. The frequencies for 6 V segments were found to be significantly different from this dataset compared to the database.We then looked for cases with evidence that Myeloma cells have two unrelated origins. We found 9/550 (1.6%) cases which had evidence of unrelated clones as evident by having three IgH or two functional sequences. We then considered cases where we find two IgH sequences that are related to each other by SHM. 128/340 (37.6%) of RNA dx samples showed evidence of evolved clones via SHM, with 69/128 (53.9%) having 3 or more related clones, while 15/311 patients (4.8%) showed evidence of evolved clones related to the index clone via SHM in DNA samples from diagnosis. Of note, the majority of RNA evolved clones were found at low frequency (<10-3) which would have been impossible to observe in the limited cell input DNA samples available for testing. Out of the 15 patients with evidence of evolved clones related to the index clone, we tested RNA from 8 of them. In 4/8 cases, the index and the related clones were present at a similar ratio in the DNA and RNA, while in 3/8 cases the index clone was found in the RNA but not the related clone. 249 post-treatment samples from 164 patients were MRD positive and were assessed for the presence of clonal evolution. In 19/249 follow-up samples (7.6%), an evolved clone related to the index clone was observed. In 6/19 patients, a substantial change in the relative index and evolved clone frequencies was observed from the dx to post-treatment time points suggesting a differential sensitivity to treatment. For example, in one case, the evolved ÒnewÓ clonotype was not observed at diagnosis but appears in the post-maintenance sample only. In another case, the evolved clonotype either increased or decreased in the post-maintenance sample relative to the index clonotype (Fig 1).Conclusions: We observed multiple evolved clonotypes in a substantial percentage of dx MM samples (37.6%). The presence of multiple clonotypes related by SHM indicates that this mechanism remains active after myeloma development in at least a portion of the cells. We also found marked changes in the relative frequency of the MM clonotypes in post-treatment samples and emergence of new Ig clones which may not be due to selective advantage of the newly acquired mutations in the Ig gene, but rather some other ongoing genomic mutation process. Thus, these evolved myeloma clonotypes may be useful as surrogate markers for other oncogenic mutations providing resistance to therapy.Figure 1. Evolution of related clones in the post-maintenance time point. Below are the sequences of two related clones, with one base difference bolded and underlined.Disclosures Richardson: Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees. Attal: celgene: Membership on an entity's Board of Directors or advisory committees; jansen: Honoraria. Anderson: Celgene Corporation: Consultancy; Millennium: Consultancy; BMS: Consultancy; Gilead: Consultancy; acetylon pharmaceuticals: Equity Ownership; Oncocorp: Equity Ownership. Faham: Adaptive Biotechnologies Corp.: Employment, Other: Stockholder. Avet-Loiseau: BMS: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.↵* Asterisk with author names denotes non-ASH members.

Recent advances in high-throughput whole transcriptome analysis of human genome has allowed for the identifications of more than 13,000 long intergenic non-coding RNA transcripts (lincRNAs), with potential central roles in gene regulation and tumorigenesis. Using RNA-seq data from newly-diagnosed multiple myeloma (MM) patients, we identified linc00936 (chr 12) to be highly downregulated in CD138+ MM cells from 320 MM patients compared to 16 normal bone marrow plasma cells. Decreased expression of lincRNA was also observed in a panel of MM cells lines when compared with normal PBMC by both quantitative RT-PCR (qRT-PCR) and RNA-seq (N=66).Interestingly, linc00936 overexpression significantly inhibited MM cell line growth (N=7), associated with G1 cell cycle arrest, decreased S phase, and induction of apoptotis via caspase 3 activation. Enforced expression of linc00936 was associated with induction of various gene sets, as measured by gene expression profiling. Importantly, linc00936 overexpression had a negative impact on genes involved in the mTOR pathway. WB analysis confirmed reduction in p-mTOR, along with a decrease in p-p70 S6 kinase and p-4E BP1, following linc00936 overexpression. Due to the role of mTOR pathway regulating autophagy, we next analyzed the effect of linc00936 on the autophagic response: autophagy was induced, evidenced by autophagic vacuoles and associated with decreased p62 and increased LC3A/B protein levels. Chloroquine, an autophagy inhibitor, partially abrogated the anti-MM activity of linc00936. RNA pull-down assay identified spleen tyrosine kinase (Syk), an E3 ubiquitin ligase (Rnf2), and serine/threonine-protein kinase (Taok3) as partner proteins. These interactions were validated by WB analysis of the lincRNA-pull-down proteins. Of note, the strongest interaction was with Syk, a well known modulator of mTOR activity. RNA-immunoprecipitation assay (RIP) followed by RT-PCR confirmed an increase in linc00936 levels in RNA-Syk complex compared to immunoglobulin G (IgG)-bound sample. Finally, treatment with demethylating agent 5-aza-deoxycitidine significantly increases the expression of linc00936 in RPMI-8226, MM1S and H929 MM cells, implicating promoter methylation as a mechanism of lincRNA suppression in MM. Based on these data, we analyzed the prognostic significance of linc00936 expression in MM patients enrolled on IFM/DFCI 2009 clinical study (N=296): low linc00936 expression was associated with significantly shorter event free survival compared to higher expression (p= 0.018). Collectively, our data provide the first evidence of a differentially expressed lincRNA in MM with a significant impact on tumor cell growth and survival via inhibition of mTOR pathway, with potential biological, prognostic and therapeutic implications.Disclosures No relevant conflicts of interest to declare.↵* Asterisk with author names denotes non-ASH members.

Genomic instability leads to acquisition of mutational changes which underlie development and progression of cancer, including development of drug resistance and poor clinical outcome. Understanding mechanisms of genomic instability is therefore necessary to develop promising strategies for prevention and treatment of disease. Homologous recombination (HR), the most precise DNA repair mechanism, has been previously described to be dysregulated in multiple myeloma (MM) mediating genomic instability. Since nuclease activity, by producing free ends of DNA, can induce DNA recombination leading to genomic rearrangements, we investigated prognostic significance of nuclease activity and nuclease gene expression in MM. We first developed a nuclease gene signature correlating with both the genomic instability and survival in myeloma patients. We used two different myeloma patient datasets (gse26863, n=246 and IFM 170 patient dataset) which had both the gene expression and SNP/CGH array-based copy number information for each patient. Genomic instability in each patient was determined by counting the total number of amplification and/or deletion events; an event was defined as a change in ≥3 and/or 5 consecutive SNPs/probes. We identified 34 nucleases whose elevated expression correlated with increased genomic instability in gse26863 dataset. Of these, the elevated expression of 21 nucleases also correlated with increased genomic instability in 170 dataset. Elevated expression of seven of these genes also correlated with poor overall survival (p=0.00005) as well as event free survival (P=0.0003) in myeloma patients (n=170). We further tested one of these nucleases (APEX2) in both the loss and gain of function studies and found that its suppression significantly reduces DNA breaks and dysregulated HR, an important activity underlying ongoing genomic rearrangements and instability in myeloma. Upregulation of APEX2 was associated with excessive DNA breaks, dysregulation of HR, acquisition of new genomic changes over time in myeloma cells.We also investigated the prognostic significance of nucleolytic activity in cell lines and patient samples using a plasmid degradation assay in which supercoiled DNA is converted to open circular and linear forms by the MM cell lystae prepared from purified CD138+ patient MM cells or MM cell lines. The ratio of supercoiled to total DNA per lane was graphed across successive time points (0, 3, 6, 12, 24 minutes) and analyzed via nonlinear regression using PRISM (statistical software) to calculate a half-life and k-constant. The longer half life suggests lower nuclease activity in MM cells. This assay was able to differentiate MGUS and smoldering myeloma (SMM) patients with long half-life of plasmid (9499 minutes) versus newly diagnosed MM (9 minutes) in which there was variability with some patients with plasmid degradation pattern closer to SMM versus some with significantly higher activity. A large number of (N = 410) clinically annotated sample patients are currently being evaluted for both functional and clinical correlation of nuclease activity. In summary, we show correlation between nucleases activity and genomic instability with impact on survival in MM. The genes in this signature not only provide novel markers to predict clinical outcome but also potential targets for prevention/reduction of genomic evolution. Investigation of the role of each of the seven genes, separately and in combination, in the overall nucleolytic activity, genomic instability, and pathways involved in the regulation of cell cycle, DNA repair/maintenance checkpoints, apoptosis and survival is currently ongoing.Disclosures Avet-Loiseau: jansen: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.↵* Asterisk with author names denotes non-ASH members. This icon denotes a clinically relevant abstract

Whole genome and exome sequencing (WGS, WES) have enabled the identification of mutational signatures in Multiple Myeloma (MM) and other cancer types. In studies that assess the impact of coding mutations on protein structure and function, only reads mapping to the exome are pertinent. Thus, WES is typically preferred over WGS, as it provides deeper coverage given the same amount of total reads. However, exome enrichment - a necessary step in WES, limits the ability to call mutations, as coverage is restricted to the capture regions and affected by their GC content. Furthermore, without transcriptional information, it is not possible to determine which coding mutations found by WGS or WES are expressed and, therefore, more likely to be relevant. As an alternative, RNA-seq data directly targets the transcriptome, providing deep coverage, not requiring an enrichment step and intrinsically omitting non-expressed mutations. Moreover, when RNA-seq data is already available for evaluation of gene expression profiles, one can further leverage the data to explore expressed mutational profiles. However, limitations in pipelines to analyze RNA-seq data have restricted their applicability so far.Using paired WES and RNA-seq data from MM patient samples, we have observed that the majority of recurrent mutations in MM occur within genes with very low or no detectable expression (only 27% of mutated genes express). Here, we have further analyzed a large RNA-seq sample set to describe a comprehensive transcriptional mutational landscape in MM and identify potential mutational driver genes. Specifically, we performed RNA-seq on CD138+ MM cells from 292 newly-diagnosed patients and 16 normal bone marrow plasma cell (NBM) samples. The unstranded 50bp paired-end reads were mapped to the human genome using MapSplice followed by a workflow for variant analysis based on GATK. Output was filtered for germline variants and technical artifacts, then evaluated computationally for functional impact, and finally further filtered at the gene level. Using this workflow we were able to identify most reported recurrently mutated genes in MM, including but not limited to TP53 (14%), NRAS (14%), KRAS (11%), ACTG1 (4%), CCND1 (4%), TRAF3 (3%), FAM46C (3%), CYLD (3%) and DIS3 (2%). Importantly, we were also able to identify novel putative mutational driver genes of lower frequency, including several genes involved in the NF-κB pathway (BCR, TAOK2, NFKBIA, PIM1) and genes coding for proteins forming the mTORC2 complex (SIN1, RICTOR, MTOR). We observe that the average mutational frequency, which is a convolution of clonality and relative allelic expression, is slightly below 0.5. Yet, we find diverse mutational frequencies across samples for each given gene. For instance, FAM46C shows a pattern representative of highly subclonal mutations, whereas CCND1 presents mostly bi-allelic and clonal mutations, and others such as TRAF3 show a wide spectrum of mutational frequencies. Further developments will be needed to deconvolve these frequencies.We also applied the workflow to 10 of the samples for which we reported mutations at the DNA level, and observe CCND1, TP53 and KRAS to be recurrently mutated using either WES or RNA-seq. Nevertheless, some mutations are not shared, including 3 WES-exclusive BRAF mutations and one seen in CCND1 through RNA-seq only.In conclusion, we report the first computational analysis to identify mutational driver genes using RNA-seq data, providing additional insight into the mutational landscape of MM. Our findings demonstrate that RNA-seq of unpaired tumor samples can suffice to characterize the most salient characteristics of cancer mutational landscapes.Disclosures Campbell: 14M genomics: Other: Co-founder and consultant. Munshi: celgene: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees.↵* Asterisk with author names denotes non-ASH members.

Transcriptome modifiers such as alternative pre-mRNA splicing (AS), long non-coding RNA and microRNA (miRNA) need to be considered in order to provide a more accurate genomic framework for clinical correlation, as well as for high value therapeutic target discovery. Aberrant splicing of numerous genes has been reported in other malignancies, including a small number of genes reported in MM. We have evaluated AS in MM by analyzing clinically annotated high throughput RNA-seq data from 410 newly-diagnosed patients and 18 normal donor plasma cells. We observed a profound and significant AS in MM with over 600 genes showing significant changes in relative isoform abundances (isoform switching) between MM and normal samples. Importantly, unsupervised analysis identified clinically relevant MM subgroups with high and low splicing index respectively and showed significant impact of alternate splicing on overall clinical outcome. Based on these data, we next focused on understanding the molecular mechanisms driving aberrant alternate splicing in myeloma. Several studies provide evidence that an abnormally expressed splicing factor (SF) can have oncogenic properties by impacting alternative splicing of cancer-associated genes.We detected dysregulated expression of several SFs, including SF3B1, Fox2, SRSF1, NONO, in patients with MM compared to normal plasma cells with impact on outcome, highlighting for the first time the prognostic significance of splicing related factors in myeloma. We further observed that overexpression of some of these SFs increased cell proliferation, enhanced anchorage independent growth in semi-solid medium, and affected tumorigenic potential. We have further investigated role of Serine/Arginine Splicing Factor 1 (SRSF1) in MM by gain of- and loss of- function studies. Enforced expression of SRSF1 in MM cells significantly increased proliferation, especially in the presence of bone marrow stromal cells. Conversely, transient or stable downregulation of SRSF1 with specific siRNA and shRNAs respectively, significantly inhibited MM cell proliferation and cell survival. We have also investigated a small molecular inhibitor of SRSF1 (TG003) and observed inhibition of MM cell growth and survival. The impact of this inhibitor on allelic isoforms of specific gene targets is undergoing.To dissect the mechanisms involved in the SRSF1-mediated MM growth induction, we used SRSF1 mutants lacking either of the two RNA-recognition motifs (ΔRRM1 or ΔRRM2 mutants) or the serine/argine-rich C-terminal domain (ΔRS mutant) involved in protein-protein interactions, subcellular localization, and recruitment of spliceosome components. We also used a C-terminal fusion of SRSF1 with the nuclear-retention signal of SRSF2 (NRS1 mutant), to force SRSF1 retention in the nucleus and assess the role of its nuclear versus cytoplasmic functions. We surprisingly found that only NRS1 mutant failed to promote MM growth, suggesting an important role of cytoplasmic SRSF1 in promoting MM cells proliferation.Finally, using genome wide chromatin and transcription landscape mapping techniques, we have found SRSF1 to be under the transcriptional control of E2F1, a transcription factor with significant impact on MM cell growth and survival. A significant reduction in SRSF1 at mRNA and protein levels was observed after E2F1 and/or E2F1 heterodimerization partner Dp1 gene silencing. Moreover, peptide-based strategy to abrogate interaction between Dp1-E2F1 led to decreased SRSF1 expression levels.These results indicate a functional role and clinical significance of a gene involved in regulation of alternate splicing in MM. The study highlights the need to further understand the splicing pattern in myeloma and also supports the emerging concept that splicing programs, together with transcriptional programs participate in the altered cellular function during tumor initiation and progression.Disclosures Munshi: onyx: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees.↵* Asterisk with author names denotes non-ASH members.

Homologous recombination (HR) is a DNA repair mechanism based on extensive sequence homology betyween DNA molecules. In a normal cellular environment, the branch of HR which is utilized to copy missing or altered information from a sister chromatid in the G2 phase of the cell cycle, is one of the most tightly regulated and error-free DNA repair mechanism. Thus HR, especially the one associated with G2 phase of cell cycle, has a unique role in the maintenance of genomic integrity and stability. However, we have previously observed that, in vitro, elevated or dysregulated HR activity mediates genomic instability and development of drug resistance in multiple myeloma (MM). In this study we have now evaluated clinical significance of elevated HR activity in MM. We optimized an in vitro HR assay using cell lysates and demonstrated that evaluation of HR with this assay is consistent with assay conducted with intact cells (R=0.97; P=0.0005). Using this assay, we evaluated HR activity in 100 patient specimens. We found that HR activity was elevated ³2-fold relative to normal PBMC in 69% and ³4-fold in 20% of MM samples. At 47 months 74% of patients with very high HR (³4-fold) had event where as 45% of patients with lower HR had event (P=0.049) To further define genomic signature of elevated HR activity, we performed RNASeq on these patient samples (N=65) and identified 345 genes whose expression correlated with HR activity in MM. Expression of 147 genes correlated positively with HR activity (R, ≥ 0.3; P ≤ 0.01). Higher expression of 65 of these genes significantly associated with poor event free survival (EFS). Expression of 198 genes correlated negatively with HR activity (R, ≥ -0.3; P ≤ 0.01). TP53, a known negative regulator of HR, was among top five in this list. Lower expression of 26 of these potential negative regulators of HR associated with poor EFS. The genes correlating with HR activity in myeloma include novel genes (previously not shown to have association with HR), genes previously known to regulate HR, as well as the genes recently identified as HR regulators in other cancers. Gene network analyses showed that the novel HR genes identified in our signature belonged to a variety of functional groups including those involved in signal transduction by phosphorylation, chromatin organization, chromosome function, cytoskeleton function, cellular response to stimulus, response to stress, DNA/nucleic acid binding, DNA/nucleic acid metabolic process, nuclear metabolic process, nucleolus function, cell cycle, and proliferation. The network analysis is consistent with the view that the novel genes identified in this signature may have roles in DNA repair and genome maintenance. We also tested HR correlating genes for correlation with genomic instability (by investigating copy number changes) in a unique MM dataset (gse26863) and found that 50% of the genes significantly correlated with genomic instability. Elevated expression of MCM5, one of the genes in myeloma HR signature, significantly correlated with hyperdiploidy in MM (P≤0.004). Some of the novel genes, including negative regulators of HR, are currently being confirmed for their impact on HR and genome stability in loss and gain of function studies. In summary, we have developed a novel clinically applicable assay for HR activity and present evidence of prognostic significance of high HR activity in myeloma and have identified novel targets with potential to overcome/reduce dyregulated HR and genomic instability.Disclosures No relevant conflicts of interest to declare.↵* Asterisk with author names denotes non-ASH members.

Background: Gene fusions play an important role in aberrant cellular biology as well as development and progression of cancer. Expression of fusion genes such as PML-RAR drives the transformation in APL and provides important targets for therapy. However, in multiple Myeloma (MM), a heterogeneous disease characterized by genomic instability, frequent gains and losses of DNA, and a diverse mutational landscape, only the well characterized MMSET-IGH fusion product has been reported. Here we investigate the fusion gene landscape in multiple myeloma, and its possible impact on survival.Method: Deep RNA-Seq was performed on purified MM cells from 430 newly-diagnosed MM patients, 20 normal individuals and 71 cell-lines; data were analyzed for gene expression profiles, long-non coding RNA signatures, and both novel and known fusion genes using two common algorithms: TopHat and MapSplice. MM characteristics, cytogenetic and FISH as well as clinical survival outcomes were also analyzed and correlated with genomic data.Results: After filtering candidate fusions linking genes belonging to the same family, we identified 416 different candidates in myeloma patients, 40 % of which identified either IGH or Kappa as a partner. IGH fusion partners included the previously described and validated WHSC1 and B2M genes, as well as over 50 new candidates, while more than 70 different partners were found to be fused with Kappa. These genes exhibit functional enrichment of positive regulators of the cytokine-mediated signaling pathway, negative regulation of myeloid cell differentiation, negative regulation of interleukin-6 production, as well as others. 31% of patients presented no fusions, and another 32% presented a single fusion event. The other 37% presented at least 2 fusion candidates, with up to 27 different candidates. Similar patterns were observed in cell-lines, with 196 unique candidates identified, only 16% of which involving IGH or kappa. However, all partners were found in at least one patient as well. Only 12% of cell-lines exhibited no fusion, and another 14% presented only one fusion. On average, 3 fusions were identified per cell-line, with a maximum of 10. Validation of some these fusion genes is required to understand their functional role. Importantly, although having IgH-or kappa-related fusions did not affect patient outcome by themselves, patients with high numbers of fusion candidates had worse event-free survival.Conclusion: Our data describes a diverse and rich fusion gene landscape in Multiple Myeloma. Similar to mutational profiles, there is no predominant fusion gene driving the disease process. Association of poor prognosis with a higher number of fusions may indicate that genomic instability plays an important role in the biology of Multiple Myeloma.Disclosures No relevant conflicts of interest to declare.↵* Asterisk with author names denotes non-ASH members.

Long intergenic non-coding RNA (lincRNA) are transcripts longer than 200 nucleotides which have a diverse sets of regulatory functions but do not get translated into protein. lincRNAs are located between the protein coding genes and do not overlap exons of either protein-coding or other non-lincRNA. However precise role of individual lincRNA in disease biology remains unclear. Here, we have evaluated the lincRNA expression and their potential biological functions in MM.We performed RNA-seq on CD138+ MM cells from 296 newly diagnosed patients and 16 normal bone marrow plasma cells (NBM) and analyzed for lincRNA expression. Data from paired-end RNAseq reads were mapped to the latest human genome, differentially expressed lincRNAs were identified and for each expressed lincRNA event free survival was examined with univariate cox regression model and support vector machine. Finally, we identified protein coding genes that are strongly correlated (cor > 0.5) with lincRNAs with significant altered expression in MM and impact on EFS to identify their biological role.lincRNA and protein coding genes that have more than 10 reads/million reads for at least 15 normal samples or 62 MM samples (20% all MM samples) were included in the analysis. We identified 60 differentially expressed lincRNA (adj p value <0.05), 51 of those had at least 1.5 fold change difference. The differentially expressed lncRNAs were in close proximity of Ig-related genes, genome stability related genes, hosting miRNAs such as mir222 and mir22 and previously reported for other cancers (PVT and TTY15). We evaluated relation of these lincRNAs with event free survival (EFS) and observed 6 lincRNAs associated with shorter EFS. We have developed multivariate signature model to predict EFS by using these 6 lincRNAs. We divided our dataset into training (n=99) and test (n=156) dataset and we utilized support vector machine classification to divide samples into 2 groups using six lincRNAs. This model was able to predict good and poor survival groups in training dataset (p val < 0.001) as well as test dataset (p val = 0.002) (Figure).We examined genome wide correlation between these six differentially expressed and prognostically significant lincRNAs to expressed protein coding genes to identify their biological functions in MM. Four of these lincRNAs strongly correlated with 47 to 504 genes (abs(cor) > 0.5), affecting immune system pathways and pathways in cancer including Jak-STAT signaling pathway. We also found that these lincRNAs are also highly correlated with tumor development genes such as TNFRSF1B,FGR,TP53BP2,TNF and T or B cells related genes PIK3CD, BCL6. In addition, two of these lincRNAs (LINC00936 and CTB-61M7.2) were found highly correlated with their protein coding neighbor genes ATP2B1(cor = 0.45) and FCAR (cor = 0.95) respectively and MIR22HG was host gene for mir22 which may indicate lincRNAs are using different machinery in MM to regulate protein coding genes.In summary, we report that lincRNA is differentially expressed and prognostically significant in myeloma and may function through their impact on immune system and tumor progression. Our ongoing integrative approach will provide further evidence of their regulatory role in MM with potential therapeutic application.Figure 1. Disclosures Anderson: acetylon pharmaceuticals: Equity Ownership; Celgene Corporation: Consultancy; Gilead: Consultancy; Oncocorp: Equity Ownership; Millennium: Consultancy; BMS: Consultancy. Munshi: onyx: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees.↵* Asterisk with author names denotes non-ASH members.

Progress in the treatment of multiple myeloma (MM) has increased extent and frequency of response, as well as prolonged progression-free (PFS) and overall survival (OS). Today complete remission (CR) rates up to 70% are achieved with new drug combinations. This has lead to development of sensitive next generation sequencing (NGS) -based methods to predict deeper responses that may more accurately predict survival outcomes in MM. Our large recent study has confirmed the clinical impact of achieving MRD- status in MM. Here we are evaluating the genomic alterations that may predict attainment of MRD negative status in MM.MRD status was evaluted in 279 patients from IFM/DFCI 2009 trial. We obtained gene expression by RNA-seq, and copy number profile by cytoScan HD array to evaluate genomic differences between MRD negative and MRD positive groups. We generated copy number data for 175 / 279 patients (72 MRD- and 103 MRD+) with Affymetrix Cytoscan HD array and compared genome wide copy number alterations. We observed statistically significant copy number alterations in chromosome 1p, 2, 4q, 11q, 13, 14 and 20 between MRD- and MRD+ patients. However, the extent of alterations in these regions is limited. The largest difference was on chromosome 11q arm where MRD- patients had 2.2 copies on average and MRD+ had 2.4 (p value < 0.001).Similarly, we generated gene expression profiles with RNAseq for 69 MRD- patients and 92 MRD+ patients to study gene expression alterations that may predict attainment of MRD negative status and to examine possible biological pathways. Although first two component of principle component analysis (PCA) showed that two groups have similar expression profile, we were able to identify 586 differentially expressed genes; 333 of those were up and 253 were down regulated in MRD+ compared to MRD- groups. We found that seven oncogenes (CCND1, CD79B, IDH1, PATZ1, PAX5, POU2AF1, RUNX1) were significantly high in MRD+ and two (CCND2 and MYCN) were high in MRD-. Additional genes that were high in MRD+ samples were enriched in genes regulated by NF-kB in response to TNF, P53 pathway, KRAS signaling and genes down-regulated in response to ultraviolet (UV) radiation. Genes that were high in MRD- compared to MRD+ were also enriched in genes up-regulated by STAT5 in response to IL2 stimulation, p53 pathways and networks, and genes up-regulated in response to ultraviolet (UV) radiation pathways.Finally, we have created a signature to predict MRD+ and MRD- in MM samples from differentially expressed genes. We used 40 genes that has at least 2 fold change difference between MRD+ and MRD- groups as a predictor and we randomly separated 161 RNAseq samples into train (n=99) and test group (n=62). We developed our classifiers with diagonal discriminant analysis and we achieved 0.79 classifier performance on test dataset. Then we tested our signature against 1000 random signature and it was significantly different than random signatures (Figure).In conclusion, we here report a first genomic landscape predictive of minimal residual disease (MRD) in Multiple Myeloma (MM). This analysis will help understand genomic and molecular correlates of achieving minimal residula disease and confirms feasibility of using RNAseq data from diagnosis sample to predict MRD status. The ongoing integration of other genomic correlates such as copy number status as well as alternate splicing may allow further improvement in the performance of prediction.Figure 1. Disclosures Anderson: Gilead: Consultancy; acetylon pharmaceuticals: Equity Ownership; Oncocorp: Equity Ownership; Celgene Corporation: Consultancy; BMS: Consultancy; Millennium: Consultancy. Attal: jansen: Honoraria; celgene: Membership on an entity's Board of Directors or advisory committees. Munshi: onyx: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees.↵* Asterisk with author names denotes non-ASH members.

While inflammation has been associated with the development and progression of colorectal cancer, the exact role of the inflammatory Th17 pathway remains unclear. In this study, we aimed to determine the relative importance of IL-17A and the master regulator of the Th17 pathway, the transcription factor RORγt, in the sporadic intestinal neoplasia of APC(MIN/+) mice and in human colorectal cancer. We show that levels of IL-17A are increased in human colon cancer as compared to adjacent uninvolved colon. Similarly, naïve helper T cells from colorectal cancer patients are more inducible into the Th17 pathway. Furthermore, IL-17A, IL-21, IL-22, and IL-23 are all demonstrated to be directly mitogenic to human colorectal cancer cell lines. Nevertheless, deficiency of IL-17A but not RORγt is associated with decreased spontaneous intestinal tumorigenesis in the APC(MIN/+) mouse model, despite the fact that helper T cells from RORγt-deficient APC(MIN/+) mice do not secrete IL-17A when subjected to Th17-polarizing conditions and that Il17a expression is decreased in the intestine of RORγt-deficient APC(MIN/+) mice. Differential expression of Th17-associated cytokines between IL-17A-deficient and RORγt-deficient APC(MIN/+) mice may explain the difference in adenoma development.

PURPOSE: PD-1/PD-L1 signaling promotes tumor growth while inhibiting effector cell-mediated antitumor immune responses. Here, we assessed the impact of single and dual blockade of PD-1/PD-L1, alone or in combination with lenalidomide, on accessory and immune cell function as well as multiple myeloma cell growth in the bone marrow (BM) milieu. EXPERIMENTAL DESIGN: Surface expression of PD-1 on immune effector cells, and PD-L1 expression on CD138(+) multiple myeloma cells and myeloid-derived suppressor cells (MDSC) were determined in BM from newly diagnosed (ND) multiple myeloma and relapsed/refractory (RR) multiple myeloma versus healthy donor (HD). We defined the impact of single and dual blockade of PD-1/PD-L1, alone and with lenalidomide, on autologous anti-multiple myeloma immune response and tumor cell growth. RESULTS: Both ND and RR patient multiple myeloma cells have increased PD-L1 mRNA and surface expression compared with HD. There is also a significant increase in PD-1 expression on effector cells in multiple myeloma. Importantly, PD-1/PD-L1 blockade abrogates BM stromal cell (BMSC)-induced multiple myeloma growth, and combined blockade of PD-1/PD-L1 with lenalidomide further inhibits BMSC-induced tumor growth. These effects are associated with induction of intracellular expression of IFNγ and granzyme B in effector cells. Importantly, PD-L1 expression in multiple myeloma is higher on MDSC than on antigen-presenting cells, and PD-1/PD-L1 blockade inhibits MDSC-mediated multiple myeloma growth. Finally, lenalidomide with PD-1/PD-L1 blockade inhibits MDSC-mediated immune suppression. CONCLUSION: Our data therefore demonstrate that checkpoint signaling plays an important role in providing the tumor-promoting, immune-suppressive microenvironment in multiple myeloma, and that PD-1/PD-L1 blockade induces anti-multiple myeloma immune response that can be enhanced by lenalidomide, providing the framework for clinical evaluation of combination therapy. Clin Cancer Res; 1-12. ©2015 AACR.