Abstract
Chromothripsis is detectable in 20–30% of newly diagnosed multiple myeloma (NDMM) patients and is emerging as a new independent adverse prognostic factor. In this study we interrogate 752 ...NDMM patients using whole genome sequencing (WGS) to investigate the relationship of copy number (CN) signatures to chromothripsis and show they are highly associated. CN signatures are highly predictive of the presence of chromothripsis (AUC = 0.90) and can be used identify its adverse prognostic impact. The ability of CN signatures to predict the presence of chromothripsis is confirmed in a validation series of WGS comprised of 235 hematological cancers (AUC = 0.97) and an independent series of 34 NDMM (AUC = 0.87). We show that CN signatures can also be derived from whole exome data (WES) and using 677 cases from the same series of NDMM, we are able to predict both the presence of chromothripsis (AUC = 0.82) and its adverse prognostic impact. CN signatures constitute a flexible tool to identify the presence of chromothripsis and is applicable to WES and WGS data.
Multiple Myeloma is an incurable plasma cell malignancy with a poor survival rate that is usually treated with immunomodulatory drugs (iMiDs) and proteosome inhibitors (PIs). The malignant plasma ...cells quickly become resistant to these agents causing relapse and uncontrolled growth of resistant clones. From whole genome sequencing (WGS) and RNA sequencing (RNA-seq) studies, different high-risk translocation, copy number, mutational, and transcriptional markers can be identified. One of these markers, PHF19, epigenetically regulates cell cycle and other processes and is already studied using RNA-seq. In this study, we generate a large (325,025 cells and 49 patients) single cell multi-omic dataset and jointly quantify ATAC- and RNA-seq for each cell and matched genomic profiles for each patient. We identify an association between one plasma cell subtype with myeloma progression that we call relapsed/refractory plasma cells (RRPCs). These cells are associated with chromosome 1q alterations, TP53 mutations, and higher expression of PHF19. We also identify downstream regulation of cell cycle inhibitors in these cells, possible regulation by the transcription factor (TF) PBX1 on chromosome 1q, and determine that PHF19 may be acting primarily through this subset of cells.
Multiple myeloma (MM) progression is characterized by the seeding of cancer cells in different anatomic sites. To characterize this evolutionary process, we interrogated, by whole genome sequencing, ...25 samples collected at autopsy from 4 patients with relapsed MM and an additional set of 125 whole exomes collected from 51 patients. Mutational signatures analysis showed how cytotoxic agents introduce hundreds of unique mutations in each surviving cancer cell, detectable by bulk sequencing only in cases of clonal expansion of a single cancer cell bearing the mutational signature. Thus, a unique, single-cell genomic barcode can link chemotherapy exposure to a discrete time window in a patient's life. We leveraged this concept to show that MM systemic seeding is accelerated at relapse and appears to be driven by the survival and subsequent expansion of a single myeloma cell following treatment with high-dose melphalan therapy and autologous stem cell transplant.
The landscape of structural variants (SVs) in multiple myeloma remains poorly understood. Here, we performed comprehensive analysis of SVs in a large cohort of 752 multiple myeloma patients by low ...coverage long-insert whole genome sequencing. We identified 68 SV hotspots involving 17 new candidate driver genes, including the therapeutic targets BCMA (
),
and
Catastrophic complex rearrangements termed chromothripsis were present in 24% of patients and independently associated with poor clinical outcomes. Templated insertions were the second most frequent complex event (19%), mostly involved in super-enhancer hijacking and activation of oncogenes such as
and
. Importantly, in 31% of patients two or more seemingly independent putative driver events were caused by a single structural event, demonstrating that the complex genomic landscape of multiple myeloma can be acquired through few key events during tumor evolutionary history. Overall, this study reveals the critical role of SVs in multiple myeloma pathogenesis.
Despite improvements in outcome, 15-25% of newly diagnosed multiple myeloma (MM) patients have treatment resistant high-risk (HR) disease with a poor survival. The lack of a genetic basis for HR has ...focused attention on the role played by epigenetic changes. Aberrant expression and somatic mutations affecting genes involved in the regulation of tri-methylation of the lysine (K) 27 on histone 3 H3 (H3K27me3) are common in cancer. H3K27me3 is catalyzed by EZH2, the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2). The deregulation of H3K27me3 has been shown to be involved in oncogenic transformation and tumor progression in a variety of hematological malignancies including MM. Recently we have shown that aberrant overexpression of the PRC2 subunit PHD Finger Protein 19 (PHF19) is the most significant overall contributor to HR status further focusing attention on the role played by epigenetic change in MM. By modulating both the PRC2/EZH2 catalytic activity and recruitment, PHF19 regulates the expression of key genes involved in cell growth and differentiation. Here we review the expression, regulation and function of PHF19 both in normal and the pathological contexts of solid cancers and MM. We present evidence that strongly implicates PHF19 in the regulation of genes important in cell cycle and the genetic stability of MM cells making it highly relevant to HR MM behavior. A detailed understanding of the normal and pathological functions of PHF19 will allow us to design therapeutic strategies able to target aggressive subsets of MM.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
New therapeutic targets for advanced colorectal cancer (CRC) are critically needed. Our laboratory recently performed an insertional mutagenesis screen in mice to identify novel CRC driver genes and, ...thus, potential drug targets. Here, we define Transmembrane 9 Superfamily 2 (TM9SF2) as a novel CRC oncogene. TM9SF2 is an understudied protein, belonging to a well conserved protein family characterized by their nine putative transmembrane domains. Based on our transposon screen we found that TM9SF2 is a candidate progression driver in digestive tract tumors. Analysis of The Cancer Genome Atlas (TCGA) data revealed that approximately 35% of CRC patients have elevated levels of TM9SF2 mRNA, data we validated using an independent set of CRC samples. RNAi silencing of TM9SF2 reduced CRC cell growth in an anchorage-independent manner, a hallmark of cancer. Furthermore, CRISPR/Cas9 knockout of TM9SF2 substantially diminished CRC tumor fitness in vitro and in vivo. Transcriptome analysis of TM9SF2 knockout cells revealed a potential role for TM9SF2 in cell cycle progression, oxidative phosphorylation, and ceramide signaling. Lastly, we report that increased TM9SF2 expression correlates with disease stage and low TM9SF2 expression correlate with a more favorable relapse-free survival. Taken together, this study provides evidence that TM9SF2 is a novel CRC oncogene.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Deciphering genomic architecture is key to identifying novel disease drivers and understanding the mechanisms underlying myeloma initiation and progression. In this work, using the CoMMpass dataset, ...we show that structural variants (SV) occur in a nonrandom fashion throughout the genome with an increased frequency in the t(4;14), RB1, or TP53 mutated cases and reduced frequency in t(11;14) cases. By mapping sites of chromosomal rearrangements to topologically associated domains and identifying significantly upregulated genes by RNAseq we identify both predicted and novel putative driver genes. These data highlight the heterogeneity of transcriptional dysregulation occurring as a consequence of both the canonical and novel structural variants. Further, it shows that the complex rearrangements chromoplexy, chromothripsis and templated insertions are common in MM with each variant having its own distinct frequency and impact on clinical outcome. Chromothripsis is associated with a significant independent negative impact on clinical outcome in newly diagnosed cases consistent with its use alongside other clinical and genetic risk factors to identify prognosis.
INTRODUCTION: Many key genomic events leading to relapse in multiple myeloma (MM) occur outside of coding regions and can only be identified using whole-genome sequencing (WGS). Studies of MM ...progression examining single nucleotide variants (SNVs) and insertions/deletions (InDels) have identified a distinct pattern of mutations associated with resistance, however these do not fully account for drivers of progression. Studies of relapsed patients provide hints about the molecular mechanisms driving progression based on identification of homozygous loss of tumor suppressor genes, aberrant copy-number variants (CNVs), and rare examples of complex structural variants (SV). We have analyzed a set of presentation and relapsed MM samples, including paired data, derived from WGS of plasma cells and focused on SVs as key drivers of progression. METHODS: WGS was performed on 338 MM patient samples using CD138-sorted plasma cells, among which 237 samples were paired including 101 patients (30%) at presentation and at relapse, 35 of whom had more than one sample at relapse. A bioinformatics pipeline employing a consensus mechanism for determining the final set of somatic events was used based on Mutect2, Strelka2, and VarScan2 for SNVs; Mutect2, Strelka2, VarScan2, and SvABA for InDels; Battenberg and FACETS for CNVs; Manta, SvABA, DELLY2, and IgCaller for SVs. Additionally, an admixture workflow was used to estimate each individual's ancestral lineage using continentally-distinct references, comprising 23 regional populations within 5 super-populations from the 1000 Genomes Project (https://github.com/pblaney/mgp1000). Complex rearranged genomes were reconstructed using the graph-based R package JaBbA (https://github.com/mskilab-org/JaBbA). Additionally, the python package Pairtree (https://github.com/morrislab/pairtree) was used to describe the evolutionary history of acquired mutations in these patients. RESULTS: Themedian age of the cases studied was 67, and 42% were female. Racial admixture correlated with self-identification, including 74% of European ancestry with the remainder being predominantly of African ancestry. High-risk cytogenetic features were found in 10% and 13% at diagnosis and relapse, respectively. As expected, the tumor mutational burden of the relapse cases was higher than that at presentation. The patterns of SNV mutational drivers were similar at relapse in comparison to presentation with again evidence for a role of biallelic tumor suppressor gene inactivation as being a key mechanism. We also found that the number of SVs increased at relapse. Complex SVs including templated insertions, chromoplexy, and chromothripsis were detected and occurred exclusively at relapse in some cases as well as earlier in the natural history in others, suggesting they can occur as both early and late driver events. Characterizing complex SVs, further we identified them at lower levels in cases at presentation compared to relapse, consistent with these being at a higher clonal fraction and providing further evidence for their role as driver events. We did not see the emergence of additional rearrangements at relapse in either the templated insertions or chromoplexy events, suggesting that these occur at a single timepoint and remain structurally stable overtime. CONCLUSIONS: Complex SVs provide a novel mechanism driving relapse in MM which can deregulate multiple genes simultaneously providing new potential markers of aggressive disease behavior and disease evolution.
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IJS, IMTLJ, KILJ, NLZOH, NUK, SAZU, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Whole-genome sequencing (WGS) of patients with newly diagnosed multiple myeloma (NDMM) has shown recurrent structural variant (SV) involvement in distinct regions of the genome (i.e., hotspots) and ...causing recurrent copy-number alterations. Together with canonical immunoglobulin translocations, these SVs are recognized as "recurrent SVs." More than half of SVs were not involved in recurrent events. The significance of these "rare SVs" has not been previously examined.
In this study, we utilize 752 WGS and 591 RNA sequencing data from patients with NDMM to determine the role of rare SVs in myeloma pathogenesis.
Ninety-four percent of patients harbored at least one rare SV event. Rare SVs showed an SV class-specific enrichment within genes and superenhancers associated with outlier gene expression. Furthermore, known myeloma driver genes recurrently impacted by point mutations were dysregulated by rare SVs.
Overall, we demonstrate the association of rare SVs with aberrant gene expression supporting a potential driver role in myeloma pathogenesis.
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