Molecular Pathogenesis Of Multiple Myeloma Research Articles

Abstract B032: Genome-wide 5-hydroxymethylation mapping in cell-free DNA to characterize the epigenetic differences in minimal residual disease of multiple myeloma

Abstract Background: Multiple myeloma (MM), a B-cell neoplasm characterized by the infiltration of malignant plasma cells into the bone marrow, is the second most common blood malignancy in the United States. The molecular pathogenesis of MM involves both genetic and epigenetic alterations. While 60-75% of newly diagnosed patients achieve a complete response or better with current modern combination therapies, almost all patients eventually progress largely due to the persistence or re-emergence of minimal residual disease (MRD). Monitoring for MRD has become essential for evaluating treatment efficacy and predicting long-term outcomes in MM patients. We aimed to investigate whether epigenetic changes, particularly 5- hydroxymethylcytosines (5hmC), at diagnosis are associated with MRD status following treatment. Methods: Utilizing 5hmC-Seal, a highly sensitive chemical labeling technique, we profiled genome-wide 5hmC in circulating cell-free DNA (cfDNA) and bone marrow genomic DNA (gDNA) at baseline and after 8 cycles of treatment (C8) from newly diagnosed MM patients (n=25) enrolled in a clinical trial (NCT01816971). MRD testing was performed on bone marrow samples using the clonoSEQ next generation sequencing assay (limit of detection 10-6). Results: The 5hmC modification levels were summarized across various genomic features, revealing enrichment in gene bodies and enhancer regions, consistent with the known regulatory role of 5hmC. We ranked 5hmC-modified genes at baseline by their variability, and found that the number of overlapping genes between cfDNA and gDNA was significantly higher than expected by permutation analysis. Subsequently, we identified the top 100 most variable 5hmC- modified genes in gDNA, which exhibited a higher Pearson correlation with cfDNA within individuals (mean r=0.90) compared to between individuals (mean r=0.82; p<0.05), suggesting that cfDNA has the potential to reflect gDNA in capturing some MM-related variations. Among the 11 patients with MRD+ at C8, a genome-wide scan of 5hmC in cfDNA comparing baseline and C8 identified 1,614 differentially modified genes (p<0.05) after adjusting for age and sex. [BC1] Furthermore, we identified 459 differentially modified genes comparing baseline 5hmC levels between patients who were MRD+ (n=11) and MRD- (n= 14) at C8 (p<0.05), after adjusting for age and sex. These results highlight the differential epigenetic landscape associated with MRD status. Conclusions: Genome-wide 5hmC profiling of cfDNA from blood revealed a similar epigenetic landscape to gDNA from bone marrow in patients with MM, suggesting that cfDNA may offer a less invasive alternative for assessing epigenetic modifications. Moreover, this profiling identified distinct epigenetic changes associated with the achievement of MRD- negativity. These findings provide a foundation for further investigation into the molecular alterations linked to MM disease biology that could potentially lead to MRD negativity. Future directions will focus on identifying biomarkers that could inform personalized treatment strategies. Citation Format: Zhou Zhang, Bei Wang, Krissana Kowitwanich, John Cursio, Daniel Appelbaum, Chuan He, Wei Zhang, Benjamin Derman, Brian Chiu, Andrzej Jakubowiak. Genome-wide 5-hydroxymethylation mapping in cell-free DNA to characterize the epigenetic differences in minimal residual disease of multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B032.

Genome-wide 5-hydroxymethylation mapping and epigenetic pathways in multiple myeloma.

7566 Background: Multiple myeloma (MM), a B-cell neoplasm characterized by bone marrow infiltration of malignant plasma cells, is the second most common blood malignancy, with an estimated number of ~35,000 new patients annually in the United States. The molecular pathogenesis of MM is complex and involves various genetic and epigenetic alterations. Key molecular mechanisms, including chromosomal abnormalities such as translocations involving the immunoglobulin heavy chain locus and various oncogenes (e.g., MMSET, FGFR3, CCND1, MAF), alterations in epigenetic regulators (e.g., EZH2, DNMT3A), dysregulation of cell cycle control, and aberrant activation of signaling pathways (e.g., NF-kB, PI3K/AKT, and JAK/STAT), play critical roles in MM pathogenesis. However, epigenetic pathways implicated in MM has not been comprehensively investigated, partly due to technical limitations that cannot distinguish major cytosine modification types. Methods: Using the 5hmC-Seal, a highly sensitive chemical labeling technique, we profiled genome-wide 5-hydroxymethylcytosines (5hmC) in circulating cell-free DNA (cfDNA) from a population-based case-control study of MM (cases, n = 313; controls, n = 317) conducted in Canada. Results: The 5hmC modification levels were summarized for various genomic features, showing an enrichment in gene bodies and enhancer markers, consistent with the putative role of gene regulation for 5hmC modification. A genome-wide scan of gene bodies identified 771 differential features between cases and controls, adjusting for age, sex, smoking status, education, and first two principal components, at a permutation-based empirical p-value cutoff of 10-4. For instance, IL1RAP, a component of the interleukin-1 signaling cascade, may impact tumor progression and immune system evasion. Furthermore, functional analysis indicated canonical pathways associated with MM pathology and treatment, such as calcium signaling, and synthesis and secretion of cortisol/aldosterone, were enriched in the differential 5hmC features between cases and controls. Notably, the calcium signaling pathway, integral to Ca2+ transport and involved in various physiological and pathological processes, plays a critical role in MM pathogenesis. Within this pathway, the CAMK1D gene, which has been identified as a crucial regulator of tumor-intrinsic immune resistance, showed differential 5hmC level between cases and controls, highlighting a vital connection between epigenetic modifications and immune evasion mechanisms in MM. Conclusions: Leveraging a state-of-the-art technique, we identified novel epigenetic modifications and pathways in the risk of MM. This approach establishes a solid foundation for further investigating etiology of MM, deepening our understanding of the disease, and advancing the discovery of biomarkers, which could potentially guide preventive strategies.

Use of daratumumab drug formulation for subcutaneous administration in multiple myeloma therapy: Literature review and clinical observation

Study of the molecular pathogenesis of multiple myeloma led to development and introduction of antitumor agents with new mechanisms of action and their combinations into clinical practice. According to several trials, use of anti-CD38 monoclonal antibody daratumumab as part of triplet therapies at an early stage leads to deep and long-term antitumor response, achievement of MRD-negative status, and, as a result, increased survival and improved prognosis in all patient subgroups. High effectiveness of daratumumab subcutaneous formulation close to intravenous formulation has favorable safety profile, short administration time and low rate of infusion reactions which improves patients’ quality of life, affects their treatment compliance, and decreases healthcare costs.We present a clinical observation of a 60-year-old patient with newly diagnosed multiple myeloma who previously underwent treatment for another lymphoproliferative disorder which included autologous hematopoietic stem cell transplantation leading to complete and long-term remission. After 9 intravenous daratumumab infusions (with recommended dose of 16 mg/kg) in accordance to the D-Rd regimen, evaluation of antitumor effect and consideration of socially active lifestyle of the patient, daratumumab formulation for intravenous administration was replaced with subcutaneous formulation of fixed dose 1800 mg. Further deepening of the antitumor response was observed along with manageable safety profile and absence of significant adverse events which allowed to maintain intercourse intervals and improve the patient’s quality of life.

Molecular features and outcomes for patients receiving elranatamab for relapsed or refractory multiple myeloma in MagnetisMM-1.

e20021 Background: Multiple myeloma (MM) is a plasma cell dyscrasia that expresses B-cell maturation antigen (BCMA). Elranatamab (PF-06863135), a BCMA x CD3 bispecific antibody, redirects the T-cell mediated immune response against MM. MagnetisMM-1 (NCT03269136) is the Phase 1 study evaluating safety, pharmacokinetics (PK), pharmacodynamics, and efficacy of elranatamab for patients (pts) with relapsed or refractory MM. Methods: Elranatamab was given subcutaneously at doses 80–1000 µg/kg weekly or every 2 weeks. Treatment-emergent adverse events (TEAEs) were graded by National Cancer Institute Common Terminology Criteria for Adverse Events (v4.03), whereas cytokine release syndrome (CRS) was assessed by criteria of American Society for Transplantation and Cellular Therapy. PK and cytokines were analyzed over time. Bone marrow aspirates were used for cytogenetic analysis, whole exome sequencing, and minimal residual disease (MRD). Clinical response and MRD (with sensitivity of 1×10-5 by next generation sequencing) were assessed by International Myeloma Working Group criteria. Results: 55 pts received elranatamab at efficacious doses ≥215 μg/kg as of September 30, 2022. Pts had median age of 64 years (range 42-80), median of 5 prior regimens (range 2-14), 91% were triple-class refractory, 29% had high cytogenetic risk, and 24% received prior BCMA-targeted therapy. The most common TEAEs included CRS, injection site reaction, neutropenia, anemia, lymphopenia, and thrombocytopenia. Premedication and 1-step priming mitigated CRS. Elranatamab showed linear PK. Genetic alterations potentially relevant to molecular pathogenesis of MM were identified and included functional mutations in KRAS, NRAS, and TP53. With median follow-up of 12.0 months (range 0.3-32.3), objective response rate (ORR) was 64% (95% CI 50-75%) with 56% of pts (31/55) achieving very good partial response (VGPR) or better and 38% of pts (21/55) achieving complete response (CR) or better. Among 13 pts with prior BCMA-targeted therapy, 54% (7/13) achieved response including 46% (6/13) with VGPR or better. All 13 MRD-evaluable pts with confirmed CR or better and dominant clone sequence at baseline achieved MRD negativity, including 2 patients with ongoing stringent CR beyond 2 years. Median duration of response (DOR) was 17.1 months (n = 35; 95% CI 11.1-NE). Median progression-free survival (PFS) was 11.8 months (n = 55; 95% CI 6.0-19.1), and median overall survival (OS) was 21.2 months (n = 55; 95% CI 10.9-NE). Conclusions: Elranatamab induced durable clinical and molecular responses with an acceptable safety profile for pts with relapsed or refractory MM. The ORR was 64% with more than half of these pts (38%) achieving CR or better, and 100% of evaluable pts achieved MRD negativity. These results, along with emerging evidence for both PFS and OS, support the clinical activity of elranatamab for pts with MM. Clinical trial information: NCT03269136 .

Identification of FCER1G as a key gene in multiple myeloma based on weighted gene co-expression network analysis

ABSTRACTPurpose:Although the prognosis of multiple myeloma (MM) has remarkably improved with the emerge of novel agents, it remains incurable and relapses inevitably. The molecular mechanisms of MM have not been well-studied. Herein, this study aimed to identify key genes in MM.Materials and Methods:The GSE39754 dataset was used to screen differentially expressed genes (DEGs) and construct a co-expression network. Hub nodes were identified in the protein and protein interaction (PPI) network. Datasets GSE13591 and GSE2658 were used to validate hub genes. Moreover, function and gene set enrichment analyses were performed to elucidate the molecular pathogenesis of MM.Results:In this study, 11 genes were found to be hub genes in the co-expression network, among which four genes (CD68, FCER1G, PLAUR and LCP2) were also identified as hub nodes. In the test dataset GSE13591, CD68 and FCER1G were significantly downregulated in MM. Besides, the areas under the curve (AUCs) of CD68 and FCER1G were greater than 0.8 in both the training dataset and the test dataset. Our results also confirmed that FCER1G highly expressed patients had remarkably longer survival times in MM. Function and pathway enrichment analyses suggested that hub genes were associated with epithelial mesenchymal transition, TNF-α signaling via NF-κB and inflammatory response. GSEA in our study indicated that FCER1G participated in NK cell mediated cytotoxicity and the NOD-like receptor signaling pathway.Conclusion:Our study identified FCER1G as a key gene in MM, providing a novel biomarker and potential molecular mechanisms of MM for further studies.

The molecular pathogenesis of multiple myeloma.

Multiple Myeloma (MM) is characterized by uncontrolled proliferation and accumulation of clonal plasma cells within the bone marrow. However, the cell of origin is a B-lymphocyte acquiring aberrant genomic events in the germinal center of a lymph node as off-target events during somatichypermutation and class-switch recombination driven by activation-induced-deaminase. Whether pre-germinal center events are also required for transformation, and which additional events are required for disease progression is still matter of debate. As early treatment in asymptomatic phases is gaining traction in the clinic, a better understanding of the molecular pathogenesis of myeloma progression would allow stratification of patients based on their risk of progression, thus rationalizing efficacy and cost of clinical interventions. In this review, we will discuss the development of MM, from the cell of origin through asymptomatic stages such as monoclonal gammopathy of undetermined significance and smoldering MM, to the development of symptomatic disease. We will explain the genetic heterogeneity of MM, one of the major drivers of disease recurrence. In this context, moreover, we will propose how this knowledge may influence future diagnostic and therapeutic interventions.

A Journey Through Myeloma Evolution: From the Normal Plasma Cell to Disease Complexity.

The knowledge of cancer origin and the subsequent tracking of disease evolution represent unmet needs that will soon be within clinical reach. This will provide the opportunity to improve patient's stratification and to personalize treatments based on cancer biology along its life history. In this review, we focus on the molecular pathogenesis of multiple myeloma (MM), a hematologic malignancy with a well-known multi-stage disease course, where such approach can sooner translate into a clinical benefit. We describe novel insights into modes and timing of disease initiation. We dissect the biology of the preclinical and pre-malignant phases, elucidating how knowledge of the genomics of the disease and the composition of the microenvironment allow stratification of patients based on risk of disease progression. Then, we explore cell-intrinsic and cell-extrinsic drivers of MM evolution to symptomatic disease. Finally, we discuss how this may relate to the development of refractory disease after treatment. By integrating an evolutionary view of myeloma biology with the recent acquisitions on its clonal heterogeneity, we envision a way to drive the clinical management of the disease based on its detailed biological features more than surrogates of disease burden.

Super-Enhancer Profiling Identifies Novel Oncogenes and Therapeutic Targets in Multiple Myeloma

Background: Multiple myeloma (MM) is an aggressive neoplastic plasma cell cancer characterized by diversely cytogenetic abnormalities. MM can be divided into subtypes with immunoglobulin heavy chain (IGH) gene translocations involving CCND1-3, FGFR3/MMSET, MAFs and hyperdiploid myeloma containing trisomies of several odd numbered chromosomes 3, 5, 7, 9, 11, 15, 19, and 21. Although several new drugs have been introduced into clinic, treatment for MM patients remains challenge and refractory/resistant to therapy is often seen. Thus, a better understanding of the molecular pathogenesis of MM can lead to generate new prognostic classification and identify new therapeutic targets. Super-enhancers (SEs) are defined as large clusters of cis-acting enhancers, marked by high level bindings of acetylation of histone H3 lysine 27 (H3K27ac) and mediator complex. SEs have been shown to control genes for maintaining cellular identity and also key tumor drivers in various malignancies. Methods: H3K27Ac ChIP-seq and RNA-seq were performed on primary MM patient samples, MM cell lines. Normal plasma cells and lymphoma cell lines were served as controls. We systematically compared SEs and their associated genes of normal and cancerous tissue. THZ1, a CDK7 inhibitor, was used to efficiently down-regulate SE-associated genes. Combinatory analysis of THZ1-sensitive and SE-associated gene revealed a number of promising MM oncogenes. CRISPR/Cas9 technology and ectopic expression experiments in conjunction with cellular functional assays were performed to determine the effects of candidate SE-genes on MM cells. Circularized chromatin conformation capture followed by sequencing (4C-seq) was applied to explore the direct contact of SE and promoter. Results: SE analysis uncovered some cell lineage-specific transcription factors (TFs) and known oncogenes in MM. Several key TFs, including IRF4, PRDM1, MYC and XBP1, were identified in most MM samples, confirming the origin of MM cells. These data reinforce the concept that SE establishment is a key component of MM biology. The acquisition of SEs around oncogene drivers is widely observed during tumorigenesis. ST3GAL6 and ADM were two known oncogenic drivers in myeloma cells, which were associated with super-enhancers in all MM samples but not in normal plasma cell and lymphoma cells. We also found SE constituents for multiple subtype-specific key oncogenes such as CCND1 in t(11;14) cells, C-MAF in t(14;16) cells, and NSD2 and FGFR3 in t(4;14) cells. Furthermore, THZ1 showed prominent anti-neoplastic effect against MM cells. SE-associated genes were more sensitive to THZ1 compared with those genes associated with typical enhancers (TEs). By overlapping THZ1-sensitve gene with SE-associated genes, we identified a number of novel MM oncogenes, including MAGI2, EDEM3, HJURP, LAMP5, MBD1 and UCK2 as a potential druggable kinase. The expression level of MAGI2 and HJURP confers poor prognosis in several MM datasets. MAGI2 silencing in MM cells decreased cell proliferation and induced apoptosis. qRT-PCR and Western blot analysis confirmed the overexpression of HJURP in t(4;14) cells relative to non-t(4;14) MM cells. Furthermore, 4C-seq analysis revealed the physical interaction between HJURP-SE and promoter and THZ1 treatment diminished this interaction. Motif search at SE constituents revealed a highly significant enrichment of NSD2 recognition. Significant reduction of NSD2 binding at HJURP-SE region was observed in KMS11 infected with NSD2-specific shRNAs. Interestingly, blocking SE sites by CRISPR/Cas9i or silencing HJURP by shRNA led to decreased HJURP expression and cell apoptosis, whereas overexpression of this gene promoted cell growth. Taken together, our data demonstrated that HJURP is a novel SE-associated oncogene in t(4;14) MM. Conclusions: Our integrative approaches by combing H3K27Ac ChIP-seq, RNA-seq and THZ1-sensitive transcript defined the landscape of SE and identified SE-associated novel oncogenes, as well as lineage-specific TFs in MM. Furthermore, we also revealed subtype-specific SE-driving oncogenic program in MM. Taken together, these results not provide novel insight into the MM pathology, but also offer novel, potential therapeutic targets, such as MAGI2, and HJURP for the treatment of MM patients. Disclosures No relevant conflicts of interest to declare.

PS1367 COMPREHENSIVE GENETIC ANALYSIS OF MULTIPLE MYELOMA IN JAPAN

Background:Next generation sequencing (NGS) technology has enabled comprehensive analyses of genomic abnormalities occurring in cancer. Previous genetic studies using large cohorts of patients with multiple myeloma (MM) have revealed the genomic landscape of MM. Although some of these genomic abnormalities are believed to be associated with the molecular pathogenesis of MM and/or clinical outcome, these relationships are not fully understood. In addition, most previous studies have been performed in European and American cohorts. Thus, the genomic profiles of Asian or Japanese MM patients remains unclear.Aims:We aimed to clarify the genomic profiles of Japanese patients with MM and to explore genomic alterations correlated to the clinical or pathological features of MM.Methods:The cohort in this study included 138 Japanese patients, newly diagnosed with MM between Dec. 2013 and Jan. 2018. The median age at diagnosis was 69 years (36–87), and the male to female ratio was 1:1. Tumor DNA was isolated from CD138‐positive bone marrow plasma cells, and germline control DNA was derived from peripheral blood mononuclear cells. A total of 340 genes related to MM and immunoglobulin (Ig) gene rearrangement regions were analyzed for somatic mutations and clonal Ig rearrangements by targeted capture sequencing. Single nucleotide polymorphism‐baits were also included to detect copy number alterations (CNA) by sequencing. IgH‐CCND1 translocations were detected, and expression levels of CCND1 mRNA were measured using FISH and RQ/RT‐PCR, respectively.Results:Clonal Ig gene rearrangements in myeloma‐derived cells were identified in all patients. In total, 136/138 patients (98.6%) harbored at least one single‐nucleotide variant or indel, with a median of 4 (0–12). KRAS was the most commonly mutated gene (48/138, 34.8%), and abnormalities in RAS/MAPK pathway genes were present in 85/138 patients (61.6%). In copy number analysis, 130 /138 patients (94.2%) had some kind of CNA and 52/138 (37.7%) were considered to be hyperdiploid subtypes according to WHO classification. Analysis of relationships between somatic mutations and CNA revealed MAX gene mutations in 9 patients (6.5%). All of these were accompanied by loss of heterozygosity (LOH) at chr14q23. An expression analysis was performed in 92 (66.7%) patients. Overexpression of CCND1 was observed in all 24 patients with t(11;14) as previously reported. Moreover, patients with a gain of CCND1 and uniparental disomy (UPD) of chr11 showed CCND1 overexpression. Del(17p) was observed in 13/138 patients (9.4%) and was predictive of shorter overall survival (OS) (p < 0.001).In cases with t(11;14), the presence of a KRAS mutation also tended to predict shorter OS (p = 0.0379).Figure:Frequency of mutated genes in this cohort.Summary/Conclusion:To the best of our knowledge, this is the first report of a comprehensive genetic analysis of Japanese MM patients. Although most of our results were compatible with those of the previous reports, hyperdiploid cases were less frequent, and KRAS mutations were more frequent than in other cohorts. These characteristics, although based on small numbers, may represent racial differences. In addition, all of the patients who had MAX mutations displayed LOH in this cohort. Because the function of MAX is not fully understood in MM, this correlation may add to our understanding of the molecular pathogenesis of myeloma.image

Abstract A32: Comprehensive analysis of molecular pathogenesis of multiple myeloma by genetic ancestry

Abstract Introduction: Multiple Myeloma (MM) is a complex malignancy of plasma cells triggered by immunoglobulin gene rearrangements and well-described hyperdyploidy, accounting for slightly more than 10% of all hematologic cancers. MM is the most common hematologic malignancy in African American population with conspicuous racial disparities in both, mortality rates and incidence in cancer compared to European American. This observation evolved into a central hypothesis that MM has distinct biological differences across different ethnicities with yet unidentified race specific markers of tumor heterogeneity. Clear understanding of these molecular differences among ethnic minorities with MM will fulfill a major unmet medical need and eliminate racial disparity. Methods: We acquired data from the Multiple Myeloma Research Foundation (MMRF) initiated comprehensive longitudinal study (CoMMpass) with an overall goal to profile 1,000 multiple myeloma patients at diagnosis, with multiple follow-up points throughout the course of the disease. To generate population subgroups based on genetic ancestry, we used a population stratification principle component analysis (PCA) and STRUCTURE to stratify myeloma patients by Ancestry Informative Markers. These well-established methods have allowed us to avoid confounders associated with self-reporting, and thus stratify the myeloma samples by genetic ancestry mapped along with anchor populations developed by 1000 genome project. We then assessed mutational frequencies as a function of PCA for each ancestry group using complex bioinformatics algorithms. Results: We confirmed known commonly mutated genes in MM including KRAS, NRAS, FAM46C, and DIS3. Among the most striking and novel observations in our preliminary analysis of CoMMpass data using genetic ancestry and PCA was a significant difference in the frequency of nonsilent mutations in TP53, with a frequency of 7.1% (33/464) in patients clustering within the European ancestry compared to none (0/142) in African ancestry populations. Further analysis of enrichment of differentially mutated key factors within the TP53 pathway showed ATM as another gene with a significantly (p= 0.019) higher mutation frequency in EA PCA 4.7% (22/464) compared to AA PCA 0.7% (1/142). Analysis of clinical outcomes data showed poorer overall survival in patients harboring TP53 alterations. Furthermore, a comprehensive mutation analysis across samples identified a novel candidate PTCHD3 (p = 7.07E-06) with a significantly higher mutation occurrence in patients of African ancestry. Moreover, the frequency of copy number alterations known to be associated with poor prognosis revealed notable, but not significant (p=0.259) lower frequency of 1q gain in tumors from African compared to European descent. Lastly, we also observed a significant (p=0.0157) two-fold increase in early age of onset of MM in patients of African descent compared to those of European descent. Conclusion: CoMMpass has constructed a fruitful discovery environment at nexus of high-resolution next generation deep sequencing with detailed clinical data allowing to elucidate potential ancestral drivers of MM paving the way to personalized treatments. Ultimately, these data may help us further delineate the influence of percent admixture on biological factors that drive differences in incidence and outcomes among multi-ethnic MM patients. Citation Format: Zarko Manojlovic, Austin Christofferson, Gil Speyer, Seungchan Kim, Winnie Liang, Mary Derome, Daniel Auclair, David Craig, Jonathan Keats, John Carpten. Comprehensive analysis of molecular pathogenesis of multiple myeloma by genetic ancestry [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A32.

Small interfering RNA-mediated silencing of nicotinamide phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) induce growth inhibition and apoptosis in human multiple myeloma cells: A preliminary study.

Multiple myeloma (MM) is a malignancy of B lymphocytes or plasma cells. Our array-based comparative genomic hybridization findings revealed chromosomal gains at 7q22.3 and 1q42.3, where nicotinamide (NAM) phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) genes are localized, respectively. This led us to further study the functions of these genes in myeloma cells. NAMPT is a key enzyme involved in nicotinamide adenine dinucleotide salvage pathway, and it is frequently overexpressed in human cancers. In contrast, little is known about the function of LYST in cancer. The expression of LYST is shown to affect lysosomal size, granule size, and autophagy in human cells. In this study, the effects of small interfering RNA (siRNA)-mediated silencing of NAMPT and LYST on cell proliferation and apoptosis were evaluated in RPMI 8226 myeloma cells. Transfection efficiencies were determined by quantitative real time reverse transcriptase PCR. Cell proliferation was determined using MTT assay, while apoptosis was analyzed with flow cytometry using Annexin V-fluorescein isothiocyanate/propidium iodide assay. The NAMPT protein expression in siRNA-treated cells was estimated by enzyme-linked immunosorbent assay. Our results showed that NAMPT and LYST were successfully knockdown by siRNA transfection (p < 0.05). NAMPT or LYST gene silencing significantly inhibited cell proliferation and induced apoptosis in RPMI 8226 cells (p < 0.05). Silencing of NAMPT gene also decreased NAMPT protein levels (p < 0.01). Our study demonstrated that NAMPT and LYST play pivotal roles in the molecular pathogenesis of MM. This is the first report describing the possible functions of LYST in myelomagenesis and its potential role as a therapeutic target in MM.

Distinct lncRNA transcriptional fingerprints characterize progressive stages of multiple myeloma.

Although many efforts have recently contributed to improve our knowledge of molecular pathogenesis of multiple myeloma (MM), the role and significance of long non-coding RNAs (lncRNAs) in plasma cells (PC) malignancies remains virtually absent. To this aim, we developed a custom annotation pipeline of microarray data investigating lncRNA expression in PCs from 20 monoclonal gammopathies of undetermined significance, 33 smoldering MM, 170 MM, and 36 extra-medullary MMs/plasma cell leukemia patients, and 9 healthy donors. Our study identified 31 lncRNAs deregulated in tumor samples compared to normal controls; among these, the upregulation of MALAT1 appeared associated in MM patients with molecular pathways involving cell cycle regulation, p53-mediated DNA damage response, and mRNA maturation processes. Furthermore, we found 21 lncRNAs whose expression were progressively deregulated trough the more aggressive stages of PC dyscrasia, suggesting a possible role in the progression of the disease. Finally, in the context of molecular heterogeneity of MM, we identified a transcriptional fingerprint in hyperdiploid patients, characterized by the upregulation of lncRNAs/pseudogenes related to ribosomal protein genes, known to be upregulated in this molecular group. Overall, the data provides an important resource for future studies on the functions of lncRNAs in the pathology.

Molecular pathogenesis of multiple myeloma.

Multiple myeloma (MM), one of the most intractable malignancies, is characterized by the infiltration and growth of plasma cells, the most differentiated cells in the B-cell lineage, in the bone marrow. Despite the introduction of novel therapeutic agents, including proteasome inhibitors and immunomodulatory drugs, the prognosis of patients with MM is still worse than that of most hematological malignancies. A better understanding of the molecular pathogenesis of the disease is essential to achieve any improvement of treatment outcome of MM patients. All MM cases pass through the phase of asymptomatic expansion of clonal plasma cells, referred to as monoclonal gammopathy of undetermined significance (MGUS). It has long been believed that MM evolves linearly from MGUS to terminal phases, such as extramedullary tumors and plasma cell leukemia via the accumulation of novel mutations. However, recent studies using next-generation sequencing have disclosed the complex genomic architecture of the disease. At each step of progression, the acquisition of novel mutations is accompanied by subclonal evolution from reservoir clones with branching patterns. Each subclone may carry novel mutations and distinct phenotypes, including drug sensitivity. In addition, minor clones already exist at the MGUS stage, which could expand later in the clinical course, resulting in relapse and/or leukemic conversion. The ultimate goal of treatment is to eradicate all clones, including subclonal populations with distinct biological characteristics. This goal could be achieved by further improving treatment strategies that reflect the genomic landscape of the disease.

Anti-Galectin-3 Therapy: A New Chance for Multiple Myeloma and Ovarian Cancer?

Here we review the role of Galectins in the molecular pathogenesis of multiple myeloma and ovarian cancer, with a special focus on Glectin-3. Multiple myeloma is the second most common hematologic malignancy worldwide. Because the pathogenesis of multiple myeloma is still incompletely understood, there is no ultimately effective cure, and this cancer results fatal. Ovarian cancer is the most lethal gynecologic malignancy worldwide. Due to the lack of screening techniques for early detection, patients are mostly diagnosed with advanced disease, which results ultimately fatal. Multiple myeloma and ovarian cancer have different biologies, but they share a strong dependence on adhesion with extracellular matrix and other cells. Galectin-3 plays a key role in regulating such adhesive abilities of tumor cells. Here we discuss the outcomes and possible mechanism of action of a truncated, dominant negative form of Galectin-3, Galectin-3C, in these malignancies. Overall, we report that Galectin-3C is a promising new compound for effective adjuvant therapies in advanced, refractory multiple myeloma and ovarian cancer.

Understanding Myeloma Cancer Stem Cells

Multiple myeloma (MM) is a neoplasm of mature plasma cells [1], characterized by the accumulation of malignant cells in the bone marrow that are dedicated to the production of a monoclonal immunoglobulin [1]. It is also char­ acterized by osteolytic lesions [2], anemia, hyper­ calcemia and renal failure [3]. MM is responsible for approximately 10% of all hematological can­ cers [4] and its incidence is increasing, in part because of an aging trend in the world popula­ tion. Approximately half of all MM patients are older than 70 years and 20% of them are older than 80 years at diagnosis [5]. A major change in the treatment of MM occurred in the last decade, with the discovery of new therapeutic agents, such as immuno­ modulatory drugs (thalidomide and lenalido­ mide) and proteasome inhibitors (bortezomib) [6]. The use of these drugs, in different combi­ nations, improved the complete response rate in patients eligible for autologous hematopoietic stem cell transplantation, and has contributed to prolong progression­free survival and to improve the quality of life of MM patients [7]. However, after more than 30 years of autologous hematopoietic stem cell transplantation use in MM treatment, this procedure is not consid­ ered curative [2]. Even with multiple approaches and promising therapeutic agents, MM remains an incurable disease [3,8]. Thus, a better under­ standing of the molecular pathogenesis of MM is essential for the development of novel drugs capable of overcoming relapses and improving survival in MM.

The Comprehensive Genomic Characterization Of All Commercially and Non-Commercially Available Multiple Myeloma Cell Lines

Human myeloma cell lines (HMCL) provide both a discovery and validation platform to improve our understanding of the molecular pathogenesis of multiple myeloma. We have completed a project to characterize the underlying genetics of all commonly used HMCL with a primary goal of identifying appropriate model systems for findings from large scale patient studies like the multiple myeloma genomics initiative (MMGI). We first purchased all 37 commercially available HMCL from DSMZ, JCRB, ECACC, and ATCC. An additional, 32 non-commercial cell lines were obtained from Diane Jelinek and Leif Bergsagel. Subsequently each HMCL was thawed and cultured under strict parameters, which yielded cells for analysis, by comparative genomic hybridization, whole exome sequencing (Agilent SureSelect V4+UTR) and mRNA sequencing. The combination of these two assays provides a detailed map of the genetic complexity underlying this deadly disease.For variant discovery, alignment was done using BWA followed by realignment, quality recalibration and duplicate removal. High quality calls were identified using Mutect by requiring a minor allele frequency of 0.25 and a minimum depth of coverage of 10x. This identified a median of 81999 mutations per sample with 25th and 75thpercentile values of 76621 and 87440 respectively. To identify likely somatic mutations, we filtered out variants found in dbSNP, 1000 genomes project and the NHLBI Exome Sequencing Project when any of the SNPs within each database had a global minor allele frequency of greater than 0.1%. In addition, we included only those variants in gene space defined by RefSeq or GENCODE and targeted by Agilent exome capture V4+UTR. After these filtering steps a median of 1389 putative somatic mutations remained. To identify key events responsible for mutagenesis, we extracted non-synonymous variants in genes, which show substantially expression in RNASeq(FPKM>5). This identified a median of 371 and 135 variants per sample respectively. Overall, across all the 66 samples, there were at total of 9074 non-synonymous mutations in 4453 genes, which are significantly expressed.To identify potential oncogenes we focused on mutations that occurred at the same position in the genome or altered the same amino acid in expressed genes. This identified 63 genes including KRAS(n=16) and NRAS(n=11) as expected but also BRD9, CCNE1 and MAP2K3. To identify putative tumor suppressors, we looked for genes with homozygous mutations or genes with more than one mutation in the same sample. This identified 112 genes including TP53, TRAF3, FAM46C and DIS3 as expected along with LTB and KDM6B. Independently, to attempt to identify genes involved in MM pathogenesis, genes with mutations having a SIFT score of less than 0.05 which occurred in at least 5%(n>3) of the cell-lines were identified. This identified 185 genes of which 25 are in our putative oncogene list and 37 are in the tumor suppressor list.These study results provide the myeloma community with the unique resource of a fully characterized series of models that can be used for laboratory-based tests. To increase the utility of the dataset to the community all these data are freely available at www.keatslab.org or by request. Disclosures:No relevant conflicts of interest to declare.

Molecular pathogenesis of multiple myeloma: basic and clinical updates

Multiple myeloma is divided into two distinct genetic subtypes based on chromosome content. Hyperdiploid myeloma is characterized by multiple trisomies of chromosomes 3, 5, 7, 9 11, 15, 19 and 21, and lacks recurrent immunoglobulin gene translocations. Non-hyperdiploid myeloma in contrast is characterized by chromosome translocations t(4;14), t(14;16), t(14;20), t(6;14) and t(11;14). A unifying event in the pathogenesis of multiple myeloma is the dysregulated expression of a cyclin D gene, either directly by juxtaposition to an immunoglobulin enhancer, as a result of ectopic expression of a MAF family transcription factor, or indirectly by as yet unidentified mechanisms. Secondary genetic events include rearrangements of MYC, activating mutations of NRAS, KRAS or BRAF, a promiscuous array of mutations that activate NFkB and deletions of 17p. Among the poor-risk genetic features are t(4;14), t(14;16), t(14;20), del 17p and gains of 1q. Available evidence supports the use of a risk-stratified approach to the treatment of patients with multiple myeloma, with the early and prolonged use of bortezomib particularly in patients with t(4;14) and del 17p.

Molecular pathogenesis of multiple myeloma

Molecular pathogenesis of multiple myeloma

The Comprehensive Genomic Characterization of All Commercially Available Multiple Myeloma Cell Lines.

AbstractAbstract 2920Human myeloma cell lines (HMCL) provide both a discovery and validation platform to improve our understanding of the molecular pathogenesis of multiple myeloma. We have completed a project to characterize the underlying genetics of all commercially available HMCL with a primary goal of identifying appropriate model systems for findings from large scale patient studies like the multiple myeloma genomics initiative (MMGI). We first purchased all 33 commercially available HMCL from DSMZ, JCRB, ECACC, and ATCC. Subsequently each HMCL was thawed and cultured under strict parameters, which yielded cells for analysis, by Agilent 400k CGH, whole exome sequencing (Agilent 70Mb Exon+UTR), and mRNA sequencing. The combination of these three assays provides a detailed map of the genetic complexity underlying this deadly disease.For variant discovery, alignment was done using BWA followed by indel realignment, quality recalibration and duplicate removal. High quality calls were identified from the intersection of variants called by both Samtools and GATK. This identified a median of 32691 high confidence variants per sample with upper and lower quartile values of 34307.75 and 32241.25, respectively. To identify likely somatic mutations, we removed variants found in the 1000 genomes project and the NHLBI Exome Sequencing project. In addition, we removed variants present in dbSNP unless these mutations were also present in the COSMIC database. After these filtering steps a median of 702 potential mutations remained. From these lists we identified a median of 209.5 non-synonymous variants per sample and in genes which are typically expressed in the cohort, a median of 91 variants were found. Overall, these steps identified 2678 variants in 1978 genes.The primary goal was to identify appropriate models for novel findings from studies like the MMGI. For instance we identified HMCL with mutations in FAM46C and DIS3 among others. Secondarily, we focused on attempting to identify potential oncogenes and tumor suppressors through the integration of our three data types and data from published studies (Chapman et al. and Walker et al.). To identify potential oncogenes we focused on mutations that occurred at the same position in the genome or altered the same amino acid at a minimum. This identified 23 genes; including expected genes like KRAS (n=11) and, NRAS (n=7) but it also identified potentially activating mutations in IKBKB, SOX2, KDM4C, CD81, OSBP, NOTCH2, WDR92 and UBR2. To identify potential tumor suppressors we focused on genes that are typically expressed, which showed bi-allelic inactivation in two or more samples by either a homozygous deletion event, a deletion plus mutation, or two independent mutations. This identified 116 genes; including expected genes like TP53, CDKN2C, RB1, BIRC2/3, TRAF3, KDM6A, CDKN1B, FAM46C, and DIS3. Outside of the expected genes we identified recurrent inactivation in ANKRD11, ATP6AP1, ATXN1, BCL2L11, CDK8, RNF7, STS, TSPAN7, and TBL1XR1.These studies have highlighted the value in studying HMCL as most novel genes reported from recent studies were independently identified in this small cohort of samples. This is in large part because HMCL provide an unlimited DNA and RNA resource that allowed for multiple independent assays to be performed on each sample. Ultimately this study will provide the myeloma community with a detailed resource from which they can acquire appropriate model systems for their research goals from the various cell line repositories around the world. Disclosures:Keats:Tgen: Employment.

Molecular pathogenesis of multiple myeloma and its premalignant precursor

Multiple myeloma is a monoclonal tumor of plasma cells, and its development is preceded by a premalignant tumor with which it shares genetic abnormalities, including universal dysregulation of the cyclin D/retinoblastoma (cyclin D/RB) pathway. A complex interaction with the BM microenvironment, characterized by activation of osteoclasts and suppression of osteoblasts, leads to lytic bone disease. Intratumor genetic heterogeneity, which occurs in addition to intertumor heterogeneity, contributes to the rapid emergence of drug resistance in high-risk disease. Despite recent therapeutic advances, which have doubled the median survival time, myeloma continues to be a mostly incurable disease. Here we review the current understanding of myeloma pathogenesis and insight into new therapeutic strategies provided by animal models and genetic screens.