Critical Role of Split Hand/Foot Malformation Type 1 (SHFM1) in Homologous Recombination and Cell Survival in Multiple Myeloma (MM)

Abstract Genomic instability is associated with development and progression of cancer. Our previous studies have demonstrated that myeloma cells display a marked genomic instability and the number of mutations correlates with clinical outcome. Based on published observation that dysregulated homologous recombination (HR) contributes to genomic instability in multiple myeloma (MM) we have investigated mechanisms underlying dysregulated HR. We conducted a functional high-throughput shRNA screen using HR assay and identified Split hand/foot malformation type 1 (SHFM1) as a novel regulator of HR in MM. SHFM1 has been shown to directly interact with BRCA2 and function in DNA binding and recombination. Based on these we have here investigated the role/s of SHFM1 in genomic evolution and impact on other oncogenic pathways/activities in MM. Using 2 different datasets (gse26863, n=246 and IFM n=170) with both expression data and copy number/SNP information, we analyzed the association of SHFM1 with genomic instability in vivo, Genomic instability in each patient was determined by counting the total number of copy number change events (deletions and amplifications) defined as changes in ≥ 5 consecutive SNPs. We observed that the higher SHFM1 expression correlated with increase in both the amplifications (p=0.0015) and deletions (p=0.0188) in gse26863 dataset, and correlated with increase in amplifications in 170 dataset (p=0.0006). When events were defined as changes in ≥ 3 consecutive SNPs, the increased SHFM1 correlated with increase in amplifications (p ≤ 0.0004) in both datasets. The increased SHFM1 expression also correlated with hyperdiploidy (P=0.0008) in MM patients. These observations suggest that elevated SHFM1 is associated with evolution, including hyperdiploidy, in myeloma. In a loss of function study, we suppressed SHFM1 in three different myeloma cell lines (ARP, RPMI, U266) using shRNA. In all three cell lines the SHFM1-KD was associated with reduction in live cell number (~50% of control non-targeting shRNA) at day 1 after selection, to complete cell death over a period of 1 - 3 weeks, in 3 different experiments. Both cell cycle and annexin V-labeling showed that mechanism of cell death was apoptotic. These data suggest that elevated SHFM1 has a critical role in survival and prevention of apoptosis in myeloma cells. To further understand the roles of SHFM1 in myeloma, we identified the genes whose expression correlated with SHFM1. In two different datasets, the expression of SHFM1 positively correlated with genes involved in DNA synthesis, homologous recombination (ATR, TOPBP1, RAD1, RAD50, POLD2, NBN, SSBP1, MRE11A), cell cycle progression and telomere maintenance, whereas negatively correlated with several apoptosis related genes (including TNFRSF12A, TNFRSF9 and DAPK2) and SUV420H2 (involved in epigenetic transcriptional repression). Investigation by real time PCR confirmed that SHFM1-KD was associated with ~2.0-fold increase in the expression of TNFRSF9; these and other associations are being further investigated by mass spectrometry. In summary, our data show that SHFM1 has critical roles in genomic evolution as well as prevention of apoptosis in myeloma cells, and can be targeted to make myeloma cells static. Disclosures Anderson: acetylon pharmaceuticals: Equity Ownership; BMS: Consultancy; Oncocorp: Equity Ownership; Celgene Corporation: Consultancy; Gilead: Consultancy; Millennium: Consultancy. 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; BMS: 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; onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.

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XRCC5 Plays an Important Role in Homologous Recombination, Genome Stability and Survival of Myeloma Cells

Blood ◽

10.1182/blood.v126.23.1218.1218 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1218-1218 ◽

Cited By ~ 1

Author(s):

Edward Laane ◽

Purushothama Nanjappa ◽

Subodh Kumar ◽

Florence Magrangeas ◽

Stephane Minvielle ◽

...

Keyword(s):

Cell Cycle ◽

Board Of Directors ◽

Genomic Instability ◽

Cell Lines ◽

Copy Number ◽

Genome Stability ◽

Myeloma Cell ◽

Advisory Committees ◽

Myeloma Cells ◽

Elevated Expression

Abstract Understanding mechanisms underlying genomic instability is critical in delineating pathogenesis and development of new treatments for prevention and treatment of cancer. We have previously shown that dysregulated homologous recombination (HR) significantly contributes to genomic instability and progression in multiple myeloma (MM). To identify the regulators of HR and genome stability in MM, we conducted a functional shRNA screen and identified XRCC5 (Ku80) as a novel regulator of HR in MM cells. XRCC5 has been known to work as part of DNA ligase IV-XRCC4 complex in the repair of DNA breaks by non-homologous end joining (NHEJ) and the completion of V(D)J recombination events. Evaluation by Western blotting showed that all myeloma cell lines tested (RPMI, MM1S, OPM2, MM1R, U266, ARP, H929) had elevated expression of XRCC5, ranging from 3- to 10-fold elevation relative to average expression in two normal PBMC samples. Expression profiling showed a wide range of XRCC5 expression in myeloma patients, with a subset of patients with very high expression. To investigate the role of XRCC5 in ongoing acquisition of genomic changes, we investigated the association of XRCC5 with genomic instability using two different patient datasets (gse26863, n=246 and IFM 170 pt dataset) in which both the gene expression and genomic copy number information for each patient was available. Copy events were defined as changes observed in ≥ 3 and/or 5 consecutive SNPs. Higher XRCC5 expression significantly correlated with increase in the number of copy number change events in both the 170 dataset (p ≤ 0.005 for amplifications and p = 0.0001 for deletions) as well as in gse26863 dataset (p ≤ 0.004 for amplifications and p ≤ 0.00003 for deletions). To understand mechanisms by which XRCC5 regulates HR in myeloma cells, we investigatedprotein-protein interactions using a custom protein array coated with antibodies against major DNA repair and cell cycle proteins. Array was sequentially incubated with MM cell lysate and HRP-conjugated anti-XRCC5 antibody, and interacting partners were then identified by their address on the array. Investigation in two different cell lines (RPMI and U266) showed that XRCC5 in myeloma interacts with XRCC4 (an NHEJ protein), a panel of major HR regulators (RAD51, RAD52, BRCA2, BRCA1, BARD1, P73, P53, C-ABL) and with components of cell cycle including CDC42, CDK1 (which controls entry from G2 to mitosis), CDK4, CDK6, CHK, CDC36, CDC34, and cyclins E and H. Consistent with these data, knockdown (KD) of XRCC5 was associated with reduced HR as well as reduced proliferation rate followed by a complete cell death over a period of two to three weeks in different experiments, in all 3 myeloma cell lines tested. Moreover, the investigation in U266 cells showed that XRCC5-KD is associated with 3-fold increase in the fraction of cells in G2 phase of cell cycle. Importantly, the elevated expression of XRCC5 was associated with shorter event free (p < 0.013) as well as poor overall survival (p < 0.008) in 170 patient dataset. We evaluted the expression and clinical correlation of XRCC5 in RNA-seq data from 311 newly-diagnosed MM patients and observed that the elevated expression of XRCC5 also correlated with event free survival (p = 0.03). In summary, we report that XRCC5, besides its known role in NHEJ, has important roles in HR, cell cycle and may be involved in the crosstalk among these DNA repair pathways. Elevated XRCC5 expression is associated with dysregulation of HR with consequent impact on survival of myeloma patients. Elevated XRCC5 is, therefore, a promising new target to inhibit/reduce genomic evolution as well as MM cell growth. Disclosures Avet-Loiseau: celgene: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; onyx: 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; jansen: Membership on an entity's Board of Directors or advisory committees; BMS: 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.

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Chromothripsis and Chromoplexy Are Associated with DNA Instability and Adverse Clinical Outcome in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-117359 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 408-408 ◽

Cited By ~ 1

Author(s):

Cody Ashby ◽

Michael A Bauer ◽

Yan Wang ◽

Christopher P. Wardell ◽

Ruslana G. Tytarenko ◽

...

Keyword(s):

Dna Repair ◽

Board Of Directors ◽

Copy Number ◽

Structural Rearrangements ◽

Employment Equity ◽

Advisory Committees ◽

Oxford Nanopore ◽

Long Read ◽

Equity Ownership ◽

Median Expression

Abstract Introduction: Chromothripsis and chromoplexy are gross structural events that deregulate multiple genes simultaneously and may help explain rapid changes in clinical behavior. Previous screening studies in multiple myeloma (MM) using copy number arrays have identified chromothripsis at a low frequency (1.3%) and suggested it adversely impacts prognosis. Here, using whole genome sequencing (WGS) data we have identified a higher frequency of these events, suggesting they are more common than previously thought. Methods: 10X ChromiumWGS (10XWGS) from 76 newly diagnosed MM (NDMM) patients were analyzed for structural rearrangements using Longranger. Oxford Nanopore long read sequencing was performed on 2 samples. Long insert WGS data from 813 NDMM patient samples from the Myeloma Genome Project (MGP) were analyzed for structural rearrangements using Manta. Whole exome sequencing was available for 712 samples. RNA-seq was available for 643 samples. Chromothripsis was determined by manual curation of breakpoint and copy number data. Chromoplexy was defined as rearrangements within 1 Mb of one another involving 3 or more chromosomes. Results: Chromoplexy was detected in 33/76 (46%) cases using 10XWGS data, and cross validated in the MGP WGS dataset being found in 30% (247/813) of samples and was most frequent on chromosomes 8 (11.7% of samples), 14 (10.6%), 11 (9.6%), 1 (9.5%), 6 (8.0%), 22 (7.6%), 12 (6.7%), and 17 (6.7%). The gene regions most involved in chromoplexy events were MYC (chr8; 7.3%), IGH (chr14, 8.8%), IGL (chr22; 4.6%), CCND1 (chr11; 3.9%), TXNDC5 (chr6; 1.7%), FCHSD2 (chr11; 1.4%), FAM46C (chr1; 1.2%), MMSET (chr4; 1.2%), and MAP3K14 (chr17; 0.7%). Chromoplexy samples involved pairings of super-enhancer donors (IGH, IGL, FAM46C, TXNDC5) and oncogenic receptors (CCND1, MMSET, MAP3K14, MYC) implicating transcriptional deregulation. To confirm, RNASeq showed an elevation of expression over median in the oncogenic receptors when paired with a donor: CCND1 (median expression = 12.0 vs. median expression with donor = 17.9), MAP3K14 (10.8 vs. 14.7), MYC (12.7 vs. 14.1) and MMSET (11.9 vs. 16.7). We also identified elevated expression of PAX5 (8.23 vs. 13.79) and two cases where BCL2 (13.32 vs. 14.68) partnered with MYC, one involved IGH similar to follicular lymphoma. To determine if chromoplexy events were happening on the same allele, we performed long read sequencing using Oxford Nanopore on a sample with a t(2;6;8;11) event. We observed a read mapped to chromosome 2, with secondary alignment to chromosomes 6 and 8. This single 32 kb read was a continuous t(2;6;8) event, proving these events occurred on the same allele. However, despite close proximity, the data did not put the t(8;11) in the same read meaning this event occurred on a different allele or sub-clone, suggesting ongoing genomic instability. Chromothripsis was detected in 16/76 (21%) cases using 10XWGS, and was consistent in MGP data, (170/813; 21%). Chromothripsis occurred on all chromosomes but at different frequencies where chromosome 1 had most events (5.1%), followed by 14 (2.4%), 11 (2.3%), 12 (2.2%), 20 (1.9%), 17 (1.9%), and 8 (1.9%). We hypothesized the presence of both chromoplexy and chromothripsis could be associated with ineffective DNA repair and indeed, using WES data, patients with both events show more mutations in TP53 (19% vs. 5%) and ATM (10% vs. 4%) implicating homologous recombination deficiency as an etiologic mechanism. Gene set enrichment analysis showed significant enrichment and positive normalized enrichment score (NES) for the DNA Repair (P = 0.01; NES = 1.7) and MYC pathways (P = 0.01; NES = 3.2) consistent with previous results. In relation to prognosis, chromoplexy and chromothripsis have a negative impact on progression free survival (28.6 months vs. 42.8 months, P=0.03 and 28.6 months vs. 40.7 months P=0.01, respectively). When patients with both chromoplexy and chromothripsis (9%) were examined there was a pronounced effect on PFS (40.7 months vs. 22.7 months, P<0.001). Conclusion: Complex structural events are seen frequently in MM and could help explain disease progression. Severe cases with both chromoplexy and chromothripsis are associated with acquired genomic instability and an adverse impact on prognosis either directly or due to their association with DNA repair abnormalities. This opens the possibility of specifically therapeutically targeting the underlying DNA abnormalities. Disclosures Flynt: Celgene Corporation: Employment, Equity Ownership. Ortiz:Celgene Research SL (Spain), part of Celgene Corporation: Employment, Equity Ownership. Dervan:Celgene Corporation: Employment, Equity Ownership. Gockley:Celgene Corporation: Employment. Davies:Janssen: Consultancy, Honoraria; TRM Oncology: Honoraria; Abbvie: Consultancy; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; ASH: Honoraria; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; MMRF: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Thakurta:Celgene Corporation: Employment, Equity Ownership. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Research Funding.

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Longitudinal Genomic Analyses In Chronic Lymphocytic Leukemia (CLL) Patients Reveal Clonal Relationship and Genomic Evolution In Disease Progression and After Therapy

Blood ◽

10.1182/blood.v116.21.3605.3605 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3605-3605

Author(s):

Esteban Braggio ◽

Neil E. Kay ◽

Scott Van Wier ◽

Stephanie Smoley ◽

Jeanette Eckel-Passow ◽

...

Keyword(s):

Disease Progression ◽

Board Of Directors ◽

Copy Number ◽

Research Funding ◽

Clonal Evolution ◽

The Other ◽

Genomic Evolution ◽

Advisory Committees ◽

Time Points ◽

Time Point

Abstract Abstract 3605 CLL is a malignant B-cell disorder characterized by the accumulation of small B lymphocytes with a mature appearance in blood, marrow and lymph nodes. Despite effective treatment options, all patients with CLL will eventually relapse after therapy. This could be due in part to the presence of subclones of the CLL cell population that harbor genetic abnormalities, which confer resistance to treatment. The aims of this study were to investigate the clonal evolution in longitudinal samples of CLL patients and to identify genetic alterations associated with disease progression and resistance to therapy. Sequential analyses were performed in 51 samples from 23 patients who were included in a previously reported clinical trial of pentostatin, cyclophosphamide and rituximab (PCR) given every 3 weeks for 6 cycles in previously untreated CLL (Blood 109:2007). In all cases the first sample analyzed was prior to therapy. In 5 of 23 patients, three time points were analyzed: >6 months prior to entry onto PCR trial (time point A), just before starting with the PCR regimen (time point B), and the time of relapse after PCR trial (time point C). Seven patients were analyzed at time points A and B; 9 at time points B and C and 2 at time points A and C. The median time between points A and B was 17.5 months (range 8–48 months) and between points B and C was 20.5 months (7–60 m). All samples were examined by array-based comparative genomic hybridization (aCGH) using the Agilent Sureprint G3 (1 million probe) array. aCGH findings were confirmed by interphase FISH using probes for D13S319 (MIR16–1/MIR15A), RB1, MDM2, CEP12, CEP6, MYB, TP53, NFKBIA, PERP and FGFR1 loci. Overall, we observed a small increase in the number of copy-number abnormalities (CNA) with disease progression. Twenty-two of the 23 patients with paired samples harbored at least one CNA that persisted in all samples, indicating clonal relationship between the sequential samples. In 15 of the 23 patients the tumor clone was stable and no CNA differences between time points were identified. Conversely, genomic evolution was found in 8 patients. In 3 cases the genetic differences were observed pre treatment (between time points A and B) and in the other 5 cases, the observed changes were found after therapy (between time points A and C or between B and C). One remarkable case with genome evolution exhibited two subclones sharing trisomies 12 and 19, but with several unique CNA confined to each subclone. The first subclone was characterized by deletions of 6q, RB1, MIR16-1/MIR15A and 3 other losses, while the second subclone showed homozygous deletion of MIR16–1/MIR15A and 5 other monoallelic deletions. The first subclone was predominant at time points A and B (60–70% of cells), but was present in only 10–20% of cells at time point C as confirmed by FISH. Conversely, the second subclone was observed in ~20% of cells at time points A and B and became predominant after therapy, found in ~80% of cells at time point C. Another case was characterized by deletion 11q32 (including ATM and others) as the sole abnormality at time point B. Significant genomic complexity was observed at time point C, including deletions of 11q32, 9p21 (CDKN2A), 9q12-q33, 14q13.2 (NFKBIA) and 17p (TP53), and gains of 2p16 (REL) and 9q34. Interestingly, the deletion 11q32 from both time points arose independently at each time point, as they exhibited different chromosomal breakpoints and copy number variants. Moreover, the other CNA found at relapse were not identified at diagnosis (confirmed by aCGH and FISH). For evolution of specific CNA, trisomy 12 was found in 5 cases at the first sample analyzed and was stable with no changes between time points. The frequency of deletions 13q14.3 (MIR16-1/MIR15A) and 17p increased at the later time points. Conversely, –6q decreased in frequency across time points (3 cases in time points A–B and 1 case in time point C). In summary, at least 35% of CLL patients exhibited clonal evolution and at least 9% showed evidence of multiple subclones. This subgroup of CLL patients provides an exceptional framework for comprehensive analysis of genome evolution during disease progression before and after therapy. Our observations also support the hypothesis of a common CLL progenitor cell can give rise to clonally related, but genetically evolving subpopulations of tumor cells. Finally, this study may bring novel information regarding the drug resistance pathways utilized by CLL B cell clones post therapy. Disclosures: Kipps: GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding; Memgen: Research Funding; Igenica: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Research Funding; Abbott Laboratories: Research Funding. Fonseca:Genzyme: Consultancy; Medtronic: Consultancy; BMS: Consultancy; AMGEN: Consultancy; Otsuka: Consultancy; Celgene: Consultancy, Research Funding; Intellikine: Consultancy; Cylene: Research Funding; Onyx: Research Funding; FISH probes prognostication in myeloma: Patents & Royalties.

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Aberrant a-to-I RNA Editing and Prognostic Impact of Adar in Multiple Myeloma Patients with 1q Amplification

Blood ◽

10.1182/blood.v128.22.357.357 ◽

2016 ◽

Vol 128 (22) ◽

pp. 357-357

Author(s):

Alessandro Lagana ◽

Deepak Perumal ◽

Violetta V Leshchenko ◽

Pei-Yu Kuo ◽

Brian Kidd ◽

...

Keyword(s):

Overall Survival ◽

Board Of Directors ◽

Rna Editing ◽

Copy Number ◽

Research Funding ◽

Cox Regression ◽

Advisory Committees ◽

Cox Regression Analysis ◽

Kaplan Meier ◽

Equity Ownership

Abstract Amplification of 1q is observed in 40% of Multiple Myeloma (MM) patients and is associated with a more aggressive clinical course of the disease. The frequency of 1q21 amplifications has been shown to increase significantly in the transition from monoclonal gammopathy of undetermined significance (MGUS) to overt myeloma and to relapse. Previous studies have reported genes on 1q such as ANP32E and CSK1B that have significant impact on survival. However, the biological mechanisms underlying disease aggressiveness associated to 1q amplification still remain unclear. ADAR (Adenosine Deaminase Acting on RNA) is an enzyme responsible for A-to-I editing, a post-transcriptional modification of double stranded RNA consisting in the conversion of specific Adenosines (A) into Inosines (I) by deamination. As Inosine is structurally similar to Guanosine (G), editing events can result in functional consequences in RNA and protein structure, including non-synonymous changes in protein coding sequences and creation/disruption of miRNA binding sites on UTRs. Dysregulation of A-to-I editing by ADAR has been recently linked to cancer. Since the ADAR gene is located in 1q21.3 (the critical minimally amplified region in MM), we asked whether 1q amplification affected ADAR expression, RNA editing and overall prognosis in MM patients. We identified 44 patients with 1q amplification from the IA6 release of the MMRF CoMMpass dataset. As a control group (wt), we selected an equal number of patients from CoMMpass without any 1q alteration. Gene expression analysis showed significantly higher expression of ADAR in 1q-amp patients compared to wt (q = 3.64e-7) (Fig. 1) and significant correlation between ADAR copy number and its expression (Spearman ρ=0.69, p = 4.52e-14). To evaluate the functional impact of ADAR up-regulation, we applied a computational pipeline based on the tool REDItools and our in-house scripts to detect A-to-I edited sites in RNA-Seq samples. The pipeline identified candidate A-to-G mutations in RNA sequences using corresponding Whole-Exome Sequencing data to filter out actual DNA mutations. We calculated sample-wise mean editing frequency across all edited sites and found significantly increased editing in 1q-amp patients compared to wt (p = 4.3e-5) (Fig. 2). Mean editing frequency was significantly correlated with ADAR expression (ρ = 0.62, p < 2e-16) and ADAR copy number (ρ= 0.5, p= 4.32e-7). Our analysis identified 3,286 sites residing in Alu sequences and 1,303 in non-Alu regions. A-to-I editing has been shown to occur predominantly in Alu elements, repetitive sequences abundantly interspersed throughout the human genome, mostly within introns and untranslated regions (UTRs). As expected, most sites were reported within 3' UTRs (66%) and introns (12%). Overall, at the site level, we observed increased editing in 1q-amp vs wt (p < 2e-16). We found that 2,173 sites (47%) had significant differential editing frequency between 1q-amp and wt patients (FDR < 20%). Next, we sought to assess the prognostic implications of ADAR activity. Cox regression analysis revealed a trend toward higher risk in terms of EFS (Event Free Survival) for 1q-amp vs wt (HR = 1.7, 95% CI = 0.83-3.59, p = 0.13), as well as for patients with higher expression of ADAR (HR = 2.4, 95% CI = 0.79-7.15, p = 0.11) and higher mean editing frequency (HR = 2, 95% CI = 0.72-5.59, p = 0.17). Since survival data in the CoMMpass dataset is not yet mature, we evaluated the effects of ADAR expression on survival on an independent dataset consisting of 559 samples from newly diagnosed patients pre-TT2 and -TT3 treatments (GSE2658, Shaughnessy et al, Blood 2007; 109:2276-84). Cox regression analysis showed a significant difference in terms of overall survival between patients with low and high ADAR expression, the latter being correlated with higher risk (HR = 2, 95% CI = 1.18-3.66, p = 0.01) (Fig. 3). In conclusion, we found a significant increase in ADAR expression and aberrant A-to-I RNA editing in MM patients with amplification of 1q. These results demonstrate a novel mechanism by which 1q amplification can contribute to MM pathogenesis via induction of A-to-I RNA editing by ADAR. Figure 1 ADAR expression in 1q-amp vs wt patients. Figure 1. ADAR expression in 1q-amp vs wt patients. Figure 2 Difference in mean RNA editing frequency between 1q-amp and wt patients. Figure 2. Difference in mean RNA editing frequency between 1q-amp and wt patients. Figure 3 Kaplan-Meier curves of overall survival in the Shaughnessy cohort stratified by ADAR expression (GSE2658) Figure 3. Kaplan-Meier curves of overall survival in the Shaughnessy cohort stratified by ADAR expression (GSE2658) Disclosures Chari: Janssen: Consultancy, Research Funding; Pharmacyclics: Research Funding; Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Amgen Inc.: Honoraria, Research Funding; Array Biopharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Cho:Ludwig Institute for Cancer Research: Membership on an entity's Board of Directors or advisory committees; Agenus, Inc.: Research Funding; Genentech Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding. Barlogie:Signal Genetics: Patents & Royalties. Jagannath:Janssen: Consultancy; Celgene: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Dudley:NuMedii, Inc.: Patents & Royalties; AstraZeneca: Speakers Bureau; Ontomics, Inc.: Equity Ownership; NuMedii, Inc.: Equity Ownership; Ecoeos, Inc.: Equity Ownership; Ayasdi, Inc.: Equity Ownership; Janssen Pharmaceuticals, Inc.: Consultancy; GlaxoSmithKline: Consultancy; Personalis: Patents & Royalties.

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Deciphering the Chronology of Copy Number Alterations in Multiple Myeloma (MM): What Comes First?

Blood ◽

10.1182/blood-2018-99-120101 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3171-3171

Author(s):

Anil Aktas Samur ◽

Mehmet Kemal Samur ◽

Stephane Minvielle ◽

Florence Magrangeas ◽

Masood A. Shammas ◽

...

Keyword(s):

Board Of Directors ◽

Copy Number ◽

Early Stage ◽

Chromosome 21 ◽

Chromosome 15 ◽

Newly Diagnosed ◽

Copy Number Alterations ◽

Advisory Committees ◽

Equity Ownership ◽

1Q Gain

Abstract Multiple Myeloma (MM) is characterized by genomic heterogeneity with copy number alterations (CNA) as one of the most prominent genomic perturbation. Hyperdiploidy involving chromosomes 3,5,7,9,11,15,19, and 21 is observed in nearly half of the patients, however the chronological sequence of their occurrence during MM development remains unknown. Here, we have acquired one of the largest genomic datasets from 647 patients that combines data from monoclonal gammopathy of undermined significance (MGUS) to newly diagnosed MM (336 newly diagnosed MM, 147 smoldering MM (SMM) and 164 MGUS) to characterize when and in what sequence CNA occurs in MM development. We deduce the order of CNA events by identifying their pattern of clonality basically inferring that clonal genomic change suggest its origin at an earlier stage of the disease. In hyperdiploid MM (HMM), gains in chromosome 19 (95%), 15 (90%) and 9 (90%) are the most frequent events, followed by gains in 5,11,3,7 and 21. Based on the clonality assessment the gain of chromosome 15 is the first and most frequent clonal/near clonal event, observed in 95% of HMM patients followed by Chromosome 9. Surprisingly, although chromosome 19 gain is the most frequent event overall, its clonal occurrence was lower than clonal chromosome 15 gain. More than 96% of HMM samples had concurrent gains in at least 2 of the 3 most frequent chromosomes (9,15 and 19). Moreover, less frequent events such as chromosome 21 gain, 18p gain, and 1q gain showed higher frequency of clonal/near clonal occurrence compared to other events indicating that when these events occur they are early events. Majority of the deletions occur as late subclonal events with few or none observed as clonal events. In the nonhyperdiploid MM (NHMM), del13, gain of 1q and gain of 11 had the highest frequency of clonal occurrence. Most were clonal events signifying its importance in the early stages of the disease. As all MM originates from its precursor conditions, MGUS and SMM, clonal and likely early CNAs in MM, must also exist in MGUS and SMM. So, we next investigated genomic data from SMM and MGUS for the occurrence of clonal events observed in MM. We confirmed same patterns for top MM-related CNA events in SMM and MGUS and observed no significant difference (p=0.1) between the number of events in hyperdiploid groups in MM (median=10, IQR= [8-12]) and SMM (median=9, IQR= [7-11]).To further confirm the analysis, we calculated an average clonality score for each chromosomal alteration using a 1 to 5 clonality index (1 being clonal 5 being low subclonal) in MM, SMM and MGUS and observed that similar clonal trisomies median=5, IQR=[4-6] are observed in both HMM and hyperdiploid MGUS; and that not all trisomies are required or occur at the same time. With occurrence in over 96% of cases trisomy involving chromosome 15 is central to the development of MGUS and later on MM. This is closely followed by trisomy of 9, and 19. Gain of chromosome 21 is also an early event. Major events like deletion 13 and 1q gain occur relatively later than first hyperdiploid events. NHMM on the other hand is well known to have clonal IgH-associated translocation as an initiating feature which is also observed in SMM and MGUS. However, different from HMM, it shows only few CNAs at an early stage and does not accumulate frequent additional alterations. The only exception to this rule is a deletion group observed in HMM, NHMM and SMM but not MGUS. In this deletion in over 10 whole chromosome or its p or q arm are involved as subclonal events. Its absence in MGUS suggests them to be a later event in MM development. On the other hand, number of deletions are observed at the same locations in both hyperdiploid and non-hyperdiploid groups with similar frequency. Moreover, similarity of events in this deletion groups strongly suggest that in sub group of both HMM and NHMM a similar process may be operative to induce such deletions. Our results also highlight that for both HMM and NHMM groups the major copy number events are not adequate for eventual malignant transformation since only a small fraction of MGUS patients progress to MM. Here, we describe the time line of initial copy number alterations observed in MM and confirm their early occurrence using data from a unique early stage plasma cell cases. Similarities between stages show that large scale DNA alterations happen early however some copy number hotspots are enriched over the time which could be important for disease progression. Disclosures Moreau: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: 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; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Anderson:Bristol Myers Squibb: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Gilead: Membership on an entity's Board of Directors or advisory committees; OncoPep: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy; Celgene: Consultancy. Munshi:OncoPep: Other: Board of director.

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Perturbation of Genomic Instability by Wortmannin in Myeloma.

Blood ◽

10.1182/blood.v114.22.1105.1105 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1105-1105

Author(s):

Jagannath Pal ◽

Dheeraj Pelluru ◽

Mariateresa Fulciniti ◽

Samir B Amin ◽

Leutz Buon ◽

...

Keyword(s):

Drug Resistance ◽

Tumor Growth ◽

Board Of Directors ◽

Gene Product ◽

Genomic Instability ◽

Research Funding ◽

Advisory Committees ◽

Myeloma Cells ◽

Over Time

Abstract Abstract 1105 Poster Board I-127 Genetic recombination plays a critical role in telomere maintenance, chromosomal translocation, and gene amplification, and may therefore underlie the chromosomal aberrations observed with high frequency in number of malignancies. The molecular mechanism/s inducing genomic instability remains ill-defined and their elucidation may provide methods to prevent tumor progression and development of drug resistance. Our earlier work has demonstrated that homologous recombination (HR) activity is elevated in multiple myeloma (MM) cells and leads to increased rate of mutation and progressive accumulation of genetic variation over time. We have further demonstrated that the inhibition of HR activity in MM cells by siRNAs targeting recombinase leads to significant reduction in the acquisition of new genetic changes in the genome; and conversely, induction of HR activity leads to significant elevation in the number of new mutations over time, and development of drug resistance in MM cells. Here we have evaluated a PI3K inhibitor Wortmaninin which has significant inhibitory activity against both HR and non-HR (nHR) pathways. Exposure of MM cells (OPM1, ARP and RPMI 8226) to wortmannin (WM) led to reduced expression of recombinase (hsRAD51) and nearly complete inhibition of HR activity, within 24 hrs as determined by a plasmid based assay in which generation of active gene product by recombination is measured. Similarly nHR was evaluated by measuring generation of intact gene product from a linearized plasmid. We evaluated effect of WM on nHR by 3 hours preincubation before transfecting the plasmid followed by cell culture for 72 hrs at 37° C. Cells were harvested and analyzed for nHR as previously described. Treatment with WM led to >40% reduction in nHR, indicating that WM affects both HR and NHR pathways. Downregulation of these pathways by wortmannin was also associated with a reduced growth rate of myeloma cells in culture by 20-25% at 48 hours. Importantly, WM treatment markedly decreased the acquisition of new genomic changes in MM as measured by genome-wide loss of heterozygosity assay as an indicator of genomic stability. To evaluate the impact of WM on in vivo tumor growth, OPM1 cells were injected subcutaneously in SCID mice and following appearance of palpable tumors, mice were treated with WM at 0.75 mg/kg, injecting daily intraperitoneally. Treatment with WM was associated with almost complete inhibition of tumor growth in vivo. Long term exposure of myeloma cells to WM was consistently associated with reduced telomere length, probably by blocking HR dependent ALT pathway. These data identifies dysregulated recombination activity as a key mediator of DNA instability and progression of MM, and WM as a potential therapeutic agent for prevention of myeloma progression and possibly drug resistance. Disclosures Anderson: Millenium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Munshi:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis : Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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Whole Genome Sequencing Defines the Clonal Architecture and Genomic Evolution in Myeloma: Tumor Heterogeneity with Continued Acquisition of New Mutational Change

Blood ◽

10.1182/blood.v118.21.297.297 ◽

2011 ◽

Vol 118 (21) ◽

pp. 297-297

Author(s):

Nikhil C. Munshi ◽

Hervé Avet-Loiseau ◽

Philip J. Stephens ◽

Graham R Bignell ◽

Yu-Tzu Tai ◽

...

Keyword(s):

Board Of Directors ◽

Copy Number ◽

Genomic Evolution ◽

Copy Number Alterations ◽

Advisory Committees ◽

Genomic Changes ◽

Time Points ◽

Progression Of Disease ◽

Serial Samples ◽

Generation Sequencing

Abstract Abstract 297 Genetic instability, a central feature of malignant cells, plays an important role in oncogenesis by perturbing critical cell signaling pathways, including activation of oncogene and/or deletion of tumor suppressor genes; moreover, ongoing genomic changes are associated with tumor progression, invasiveness, and drug resistance. We hypothesized that the inherent genomic instability in tumors would lead to a heterogeneous tumor cell population at diagnosis, thereby providing a substantial substrate for ongoing selection during progression of the disease. We have here investigated serial samples from patients with multiple myeloma (MM) using a variety of methodologies to study the genomic evolution. Purified MM cells, as well as matching normal samples from the same patients, were collected at 2 time points at least 4 months apart and subjected to genomic analyses. To compare the changes between matching normal and MM cells collected at two time points (range 5–18 months apart), we utilized SNP 6.0 array to identify copy number alterations (CAN); identified genome-wide rearrangements utilizing a low-coverage whole genome shotgun approach generated via next-generation sequencing; and, importantly, for the first time in 13 patients performed whole exome sequencing based on a solution phase capture and next generation sequencing. Variants identified in both the rearrangement and exome screens were validated on orthogonal platform. Our analysis demonstrates: 1) a significant intratumoural heterogeneity at the initial time of evaluation, suggesting that even at diagnosis multiple sub-clones may be co-existing; 2) discernable shifts in the clonal structure of disease at the time of progression (2nd sample) that indicates appearance of previously undetected sub-clones. We have observed frequent mutational changes (3 or more samples) involving CCND1, DTX1, KRAS genes. The changes are irrespective of intervention and disease status. We have also observed appearance of new copy number alterations and heterozygosity between 2 serial samples, ranging from 0.021 – 2.674 % (i.e. per 100 informative loci investigated), as well as insertion/deletion changes. These data therefore confirm evolution of genomic changes in MM patients over time and identify molecular alterations associated with progression of disease and development of drug resistance. This study begins to define the clonal architecture of MM and will provide insights into the impact of this structure and heterogeneity on pathogenesis and progression of disease. Disclosures: Munshi: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees.

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Whole Exome Sequencing Of Multiple Myeloma Reveals An Heterogeneous Clonal Architecture and Genomic Evolution

Blood ◽

10.1182/blood.v122.21.399.399 ◽

2013 ◽

Vol 122 (21) ◽

pp. 399-399

Author(s):

Niccolo Bolli ◽

Herve Avet-Loiseau ◽

David Wedge ◽

Peter Van Loo ◽

Ludmil Alexandrov ◽

...

Keyword(s):

Board Of Directors ◽

Copy Number ◽

Clonal Structure ◽

Genomic Evolution ◽

Advisory Committees ◽

Whole Exome ◽

Chromosomal Copy Number ◽

Copy Number Changes ◽

Mutational Processes ◽

Over Time

Abstract Multiple myeloma (MM) is a malignancy of post-germinal centre B-cells whose pathogenesis is only partially understood. Chromosomal hyperdiploidy and recurrent immunoglobulin gene locus rearrangements are frequent, but are insufficient for malignant transformation, which is associated with additional events such as somatic mutations, epigenomic aberrations, and chromosomal copy-number changes. To investigate genomic event underlying MM pathogenesis and evolution, we used whole exome sequencing, copy number profiling and cytogenetics in 67 patients and 84 samples. For 15 patients, 2 or 3 serial samples (median 299 days apart) were available. Exome reads were used to call substitutions and indels. We used the Genome-Wide SNP Array 6.0 or exome reads to estimate the allele-specific copy number of the tumor. To cluster variants and estimate the clonal architecture of each sample and its evolution over time, we used the mutation burden, corrected for copy number and normal cell contamination. Analysis of the clonal structure of the tumors showed at least one subclone in 94% of patients at diagnosis, suggesting that myeloma is a heterogeneous disease at presentation. Interestingly, many mutations of known MM driver genes (KRAS, NRAS, BRAF, TP53, FAM46C) were subclonal at diagnosis. In 5/67 patients, BRAF and KRAS/NRAS mutations co-existed in the same sample, raising therapeutic implications given the paradoxical ERK-activating effect of BRAF inhibitors in RAS-mutated cells. Furthermore, only 3/10 BRAF variants were V600E, the current target of most inhibitors. Altogether, only the 5 previously known genes were significantly enriched in our cohort, highlighting marked heterogeneity of the spectrum of candidate driver gene mutations across MM patients. Nevertheless, we identified several new recurrent gene lesions: inactivating mutations of SP140 (7.5%), a gene previously linked to germline susceptibility to CLL, and in ROBO1 (7.5%), a gene recently implicated in pancreatic cancer; clustered missense substitutions in EGR1 (6%), a gene previously implicated in plasma cell apoptosis; clustered truncating mutations in LTB (4.5%), a TNF-family protein implicated in lymphoid development. The subclonal structure of the sample changed over time in 72% paired samples, highlighting genomic evolution at relapse. We described 4 different scenarios with striking concordance between mutations and chromosomal copy number changes: no change, linear evolution (a new clone appears in the later sample), differential clonal response (the relative proportions of the subclones change over time), and branching evolution (new clones emerge, while others decline in frequency or disappear). All subclonal variants in known driver myeloma genes increased their clonal fraction at the later time-point, consistent with the expected positive selection for the subclones harboring them. To investigate mutational processes responsible for the generation of the mutational repertoire in MM, we extracted the variant context and analyzed the mutational signatures. We found two signatures in our samples. The most represented one is enriched for spontaneous deamination of methylated cytosines, a common process in cancer and aged cells. The second signature was more represented in samples showing extremely high numbers of variants, sometimes clustered in small regions of ∼200 bp (kataegis). We hypothesize that it results from aberrant activity of the APOBEC family of cytosine deaminases, recently described in breast cancer. Interestingly, cases of extramedulary relapse were always associated with branching evolution and showed increased contribution from this APOBEC signature. In conclusion, in our cohort of MM samples we show: 1) evidence of tumor heterogeneity at the time of diagnosis; 2) discernable genetic changes and shifts in the clonal structure of disease at the time of progression; 3) different mutational processes responsible for an heterogeneous mutational repertoire across patients, and over time in the same patient; 4) a comprehensive list of recurrent variants, many of which are previously unreported. Our study provides new insights into the genomic architecture of MM, and will help identify molecular alterations associated with progression of disease and development of drug resistance. Disclosures: Tai: Onyx: Consultancy. Richardson:Millennium: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees. Moreau:Celgene: Honoraria, Speakers Bureau. Attal:CELGENE: Honoraria, Speakers Bureau; JANSSEN: Honoraria, Speakers Bureau. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity’s Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder , Scientific Founder Other.

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Prospective Investigation of Genetic Alterations in Osteolytic Lesions Compared to Paired Random Aspirates

Blood ◽

10.1182/blood.v128.22.4419.4419 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4419-4419

Author(s):

Sandra Sauer ◽

Jens Hillengass ◽

Barbara Wagner ◽

Daniel Spira ◽

Marc Andre Weber ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Copy Number ◽

Research Funding ◽

Iliac Crest ◽

Genetic Alterations ◽

Advisory Committees ◽

Single Nucleotide ◽

Osteolytic Lesions ◽

Myeloma Cells

Abstract Background: Bone disease is the most frequent clinical manifestation of multiple myeloma. In this prospective study we ask whether osteolytic lesions (OL) are driven by myeloma cells showing a different background of genetic alterations in terms of chromosomal aberrations and expressed single nucleotide variants (SNVs) compared to random aspirates (RA) from diffuse myeloma cell infiltration at the iliac crest (spatial genetic heterogeneity). Material and Methods: Consecutive sample-pairs (n=41) were prospectively obtained by CT-guided biopsies of OLs as well as simultaneous random bone marrow aspirates of the iliac crest, the latter undergoing CD138-purification of myeloma cells, in transplant eligible patients with previously untreated symptomatic multiple myeloma, after written informed consent. Peripheral blood mononuclear cells were used as germline control. Plasma cell infiltration in biopsies was quantified histologically. Samples pairs (n=8) were subjected to RNA-sequencing (Illumina HiSeq2000), gene expression profiling using DNA-microarrays (Affymetrix U133 2.0), whole exome sequencing (Illumina NextSeq 500), and arrayCGH (Affymetrix cytoscan array). Results and Discussion: Expressed single nucleotide variants.The spectrum of mutated genes in our samples comprises two of the most frequently mutated in symptomatic myeloma, i.e. KRAS and FAM46C, alongside those implicated in myeloma pathophysiology, e.g. mutations in IRF4, FGFR3, and CD200. In total, 1-10 clonal expressed non-synonymous SNVs were exclusively found in OL compared to RA, comprising e.g. WHSC1, FAM46C, and ROCK1P1. In 2/8 patients (25%), no expressed clonal differences between RA and OL were present. Single nucleotide variants.In investigated samples, 77-1569 non-synonymous SNVs appear with an allele frequency of ≥10% in OL and RA, clustering in 4-5 groups. The clonal constitution can vary, but subclones are detectable in both. Subclonal complexity is maintained (subclones remain present) in OL compared to RA, and the vast majority of subclonal changes is present in both, especially for expressed non-synonymous SNVs, incompatible with an "osteolytic clonal variant" driving OL in the majority of patients. Copy number alterations and loss of heterozygosity.Subtle differences in copy number between OL and RA are present. However, only 1/8 patients (12.5%) showed further "gained" aberrations in OL compared to RA, i.e. deletions on chromosome 7p, 8p, and 11p as well as 19p gain. Loss of heterozygosity was observed in 3/8 patients (37.5%) with a shared pattern between OL and RA in all of them. Conclusions: In our prospective study, the majority of alterations is shared between RA and OL. Spatial heterogeneity is present, but nature and frequency of alterations detectable exclusively in OL make them unlikely candidates in most myeloma patients for being causative for generation of OL. Disclosures Hillengass: Novartis: Research Funding; Sanofi: Research Funding; BMS: Honoraria; Celgene: Honoraria; Amgen: Consultancy, Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Goldschmidt:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Durie:Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy. Hose:EngMab: Research Funding; Takeda: Other: Travel grant; Sanofi: Research Funding.

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MYC Rearrangements in Multiple Myeloma Are Complex, Can Involve More Than Five Different Chromosomes, and Correlate with Increased Expression of MYC and a Distinct Downstream Gene Expression Pattern

Blood ◽

10.1182/blood.v130.suppl_1.65.65 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 65-65

Author(s):

Aneta Mikulasova ◽

Cody Cody Ashby ◽

Ruslana G. Tytarenko ◽

Michael Bauer ◽

Konstantimos Mavrommatis ◽

...

Keyword(s):

Gene Expression ◽

Board Of Directors ◽

Copy Number ◽

Chromosomal Rearrangement ◽

Hot Spot ◽

Chromosomal Rearrangements ◽

Advisory Committees ◽

Equity Ownership ◽

Downstream Gene Expression

Abstract Introduction: The proto-oncogene MYC (locus 8q24.21) is a key transcription factor in multiple myeloma (MM) resulting in significant gene deregulation and impacting on many biological functions, including cell growth, proliferation, apoptosis, differentiation, and transformation. Chromosomal rearrangement and copy number change at the MYC locus are secondary events involved in MM progression, which are thought to lead to aggressive disease. Current analyses of the MYC locus have not been large and have reported rearrangements in 15% of new-diagnosed MM. However, more recent studies using advanced genomic techniques suggest that the frequency of MYC rearrangements may be much higher, and that a full reassessment of the role of MYC in MM pathogenesis may be critical. In this study, we analyzed 1280 MM patients to provide a better understanding of the role of this important genomic driver in MM pathogenesis. Methods: In total, 1280 tumor normal pairs of CD138 sorted bone marrow plasma cells and their germline control samples were analyzed by: 1. Targeted sequencing of 131 genes and 27 chromosome regions (n=100) with 4.5 Mb captured region surrounding MYC ; 2. Exome sequencing (n=461) with 2.3 Mb captured region surrounding MYC ; 3. Whole genome sequencing (n=719). Normalized tumor/germline depth ratio in targeted-sequencing cases and MANTA were used for detection of somatic copy number and structural variants. Expression analysis was performed using RNA-seq or microarrays. Results: MYC translocations were found in 25% (323/1280) of patients and occurred most frequently as inter-chromosomal translocations involving 2-5 chromosomes (90%, 291/323). Of the remaining cases, 5% (17/323) of the translocations involved inversion of chromosome 8 and 5% (15/323) were complex, affecting more than 5 chromosomal loci. The proportion of MYC translocations involving 2, 3, 4, and 5 loci was 62% (200/323), 23% (74/323), 8% (26/323) and 3% (8/323), respectively. Using abnormal rearranged cases (29/100), we found copy number imbalances >14.2 kb in size associated with a MYC translocation in 76% (22/29). Another 7% (2/29) of cases with translocations showed complex intra-chromosomal rearrangement. A region of 2.0 Mb surrounding MYC was identified as a translocation breakpoint hot-spot incorporating 96% of breakpoints. This region also contained two hotspots for chromosomal gain and tandem duplications. MYC rearrangements were not randomly distributed across the spectrum of MM with an excess being seen in hyperdiploidy (76% of rearranged samples, P <0.0001). Importantly, 67% (207/308) of cases with a MYC translocation involving 5 or less chromosomes had one of the commonly known super-enhancers involved in the translocation. Gene expression analysis was used to explore the impact of these events on downstream gene expression patterns. The results showed that inter- and/or intra-chromosomal rearrangements were associated with a significantly (P <0.0001) higher MYC expression (4.1-fold). In patients where rearrangements were associated with additional copies of MYC there was higher expression of MYC in comparison to cases with a translocation but lacking copy number gain (P=0.04). To identify downstream genes deregulated by MYC rearrangements we compared gene expression between those with and without a translocation, independently of hyperdiploidy. Genes that showed >2-fold change in expression (P <0.01) included MYC and the non-protein coding oncogene PVT1 that is located next to MYC . Genes with significantly lower levels of expression were involved in B-cell biology including CD79A and AHR, or were associated with cell proliferation, migration, adhesion, apoptosis and/or angiogenesis (FGF16, ADAMTS1, FBXL7, HRK, PDGFD, and PRKD1) . Conclusions: This study confirms the central role of MYC in the pathogenesis of clinical cases of MM, and as such defining it as a critical therapeutic target. We will be able to target MYC better if we understand how it is deregulated and in this respect we show that the MYC locus rearrangements are complex and it is a hot-spot for heterogeneous inter- as well intra-chromosomal rearrangements, including complex rearrangements involving >5 chromosomes. These events lead to increased MYC expression consistent with it being a driver of disease progression, particularly in the hyperdiploid subset of MM. Disclosures Mavrommatis: Celgene Corporation: Employment. Trotter: Celgene Corporation: Equity Ownership; Celgene Institute for Translational Research Europe: Employment. Davies: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria. Thakurta: Celgene Corporation: Employment, Equity Ownership. Morgan: Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Myers: Consultancy, Honoraria.

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