Multiple myeloma (MM) remains a largely incurable, genetically heterogeneous plasma-cell malignancy that contains - just like many other cancers - a small fraction of clonogenic stem cell-like cells ...that exhibit pronounced self-renewal and differentiation capacities, but also pronounced drug resistance. These MM stem cells (MMSCs) are a controversial but highly significant issue in myeloma research because, in our opinion, they are at the root of the failure of anti-neoplastic chemotherapies to transform myeloma to a manageable chronic disease. Several markers including CD138-, ALDH1+ and SP have been used to identify MMSCs; however, no single marker is reliable for the isolation of MMSC. Nonetheless, it is now known that MMSCs depend on self-renewal and pro-survival pathways, such as AKT, Wnt/β-catenin, Notch and Hedgehog, which can be targeted with novel drugs that have shown promise in pre-clinical and clinical trials. Here, we review the pathways of myeloma "stemness", the interactions with the bone marrow microenvironment that promote drug resistance, and the obstacles that must be overcome to eradicate MMSCs and make myeloma a curable disease.
MicroRNAs (miRNAs) are small RNAs that regulate gene expression pathways. Previous studies have shown interactions between hepatitis C virus (HCV) and host miRNAs. We measured miR-122 and miR-21 ...levels in HCV-infected human liver biopsies relative to uninfected human livers and correlated these with clinical patient data. miR-122 is required for HCV replication in vitro, and miR-21 is involved in cellular proliferation and tumorigenesis. We found that miR-21 expression correlated with viral load, fibrosis and serum liver transaminase levels. miR-122 expression inversely correlated with fibrosis, liver transaminase levels and patient age. miR-21 was induced ∼twofold, and miR-122 was downregulated on infection of cultured cells with the HCV J6/JFH infectious clone, thus establishing a link to HCV. To further examine the relationship between fibrosis and the levels of miR-21 and miR-122, we measured their expression levels in a mouse carbon tetrachloride fibrosis model. As in the HCV-infected patient samples, fibrotic stage positively correlated with miR-21 and negatively correlated with miR-122 levels. Transforming growth factor β (TGF-β) is a critical mediator of fibrogenesis. We identified SMAD7 as a novel miR-21 target. SMAD7 is a negative regulator of TGF-β signaling, and its expression is induced by TGF-β. To confirm the relationship between miR-21 and the TGF-β signaling pathway, we measured the effect of miR-21 on a TGF-β-responsive reporter. We found that miR-21 enhanced TGF-β signaling, further supporting a relationship between miR-21 and fibrosis. We suggest a model in which miR-21 targeting of SMAD7 could increase TGF-β signaling, leading to increased fibrogenesis.
Recognition of microbial products by members of the Toll-like receptor (TLR) family initiates intracellular signaling cascades that result in NF-κB activation and subsequent production of ...inflammatory cytokines. We explored the potential roles of microRNAs (miRNAs) in regulating TLR pathways. A target analysis approach to the TLR4 pathway adaptor molecules identified several putative targets of miR-200a, miR-200b and miR-200c. miRNA mimics were co-transfected with a NF-κB activity reporter plasmid into HEK293 cells stably expressing TLR4 (HEK293-TLR4). Mimics of both miR-200b and miR-200c, but not miR-200a, decreased NF-κB reporter activity in either untreated cells or in cells treated with endotoxin:MD2 as a TLR4 agonist. Transfection of HEK293-TLR4 cells with miR-200b or miR-200c significantly decreased expression of MyD88, whereas TLR4, IRAK-1 and TRAF-6 mRNAs were unaffected. When miR-200b or miR-200c mimics were transfected into the differentiated monocytic THP-1 cell line, the abundance of MyD88 transcripts, as well as LPS-induced expression of the pro-inflammatory molecules IL-6, CXCL9 and TNF-α were diminished. These data define miRNAs miR-200b and miR-200c as factors that modify the efficiency of TLR4 signaling through the MyD88-dependent pathway and can thus affect host innate defenses against microbial pathogens.
Multiple myeloma (MM) is a genetically heterogeneous disease characterized by genomic chaos making it difficult to distinguish driver from passenger mutations. In this study, we integrated data from ...whole genome gene expression profiling (GEP) microarrays and CytoScan HD high-resolution genomic arrays to integrate GEP with copy number variations (CNV) to more precisely define molecular alterations in MM important for disease initiation, progression and poor clinical outcome. We utilized gene expression arrays from 351 MM samples and CytoScan HD arrays from 97 MM samples to identify eight CNV events that represent possible MM drivers. By integrating GEP and CNV data we divided the MM into eight unique subgroups and demonstrated that patients within one of the eight distinct subgroups exhibited common and unique protein network signatures that can be utilized to identify new therapeutic interventions based on pathway dysregulation. Data also point to the central role of 1q gains and the upregulated expression of ANP32E, DTL, IFI16, UBE2Q1, and UBE2T as potential drivers of MM aggressiveness. The data presented here utilized a novel approach to identify potential driver CNV events in MM, the creation of an improved definition of the molecular basis of MM and the identification of potential new points of therapeutic intervention.
Multiple myeloma (MM) is an incurable B-cell malignancy. The proteasome inhibitor bortezomib (BTZ) is a frontline MM drug; however, intrinsic or acquired resistance to BTZ remains a clinical hurdle. ...As BTZ induces oxidative stress in MM cells, we queried if altered redox homeostasis promotes BTZ resistance. In primary human MM samples, increased gene expression of copper-zinc superoxide dismutase (CuZnSOD or SOD1) correlated with cancer progression, high-risk disease, and adverse overall and event-free survival outcomes. As an in vitro model, human MM cell lines (MM.1S, 8226, U266) and the BTZ-resistant (BR) lines (MM.1SBR, 8226BR) were utilized to determine the role of antioxidants in intrinsic or acquired BTZ-resistance. An up-regulation of CuZnSOD, glutathione peroxidase-1 (GPx-1), and glutathione (GSH) were associated with BTZ resistance and attenuated prooxidant production by BTZ. Enforced overexpression of SOD1 induced BTZ resistance and pharmacological inhibition of CuZnSOD with disulfiram (DSF) augmented BTZ cytotoxicity in both BTZ-sensitive and BTZ-resistant cell lines. Our data validates CuZnSOD as a novel therapeutic target in MM. We propose DSF as an adjuvant to BTZ in MM that is expected to overcome intrinsic and acquired BTZ resistance as well as augment BTZ cytotoxicity.
Many SARS-CoV-2 variants have emerged during the course of the COVID-19 pandemic. These variants have acquired mutations conferring phenotypes such as increased transmissibility or virulence, or ...causing diagnostic, therapeutic, or immune escape. Detection of Alpha and the majority of Omicron sublineages by PCR relied on the so-called S gene target failure due to the deletion of six nucleotides coding for amino acids 69-70 in the spike (S) protein. Detection of hallmark mutations in other variants present in samples relied on whole genome sequencing. However, whole genome sequencing as a diagnostic tool is still in its infancy due to geographic inequities in sequencing capabilities, higher cost compared to other molecular assays, longer turnaround time from sample to result, and technical challenges associated with producing complete genome sequences from samples that have low viral load and/or high background. Hence, there is a need for rapid genotyping assays. In order to rapidly generate information on the presence of a variant in a given sample, we have created a panel of four triplex RT-qPCR assays targeting 12 mutations to detect and differentiate all five variants of concern: Alpha, Beta, Gamma, Delta, and Omicron. We also developed an expanded pentaplex assay that can reliably distinguish among the major sublineages (BA.1-BA.5) of Omicron.
, analytical and clinical testing of the variant panel indicate that the assays exhibit high sensitivity and specificity. This panel can help fulfill the need for rapid identification of variants in samples, leading to quick decision making with respect to public health measures, as well as treatment options for individuals. Compared to sequencing, these genotyping PCR assays allow much faster turn-around time from sample to results-just a couple hours instead of days or weeks.
Ibrutinib (Imbruvica), a small-drug inhibitor of Bruton tyrosine kinase (BTK), is currently undergoing clinical testing in patients with multiple myeloma, yet important questions on the role of BTK ...in myeloma biology and treatment are outstanding. Using flow-sorted side population cells from human myeloma cell lines and multiple myeloma primary samples as surrogate for the elusive multiple myeloma stem cell, we found that elevated expression of BTK in myeloma cells leads to AKT/WNT/β-catenin-dependent upregulation of key stemness genes (OCT4, SOX2, NANOG, and MYC) and enhanced self-renewal. Enforced transgenic expression of BTK in myeloma cells increased features of cancer stemness, including clonogenicity and resistance to widely used myeloma drugs, whereas inducible knockdown of BTK abolished them. Furthermore, overexpression of BTK in myeloma cells promoted tumor growth in laboratory mice and rendered side population-derived tumors that contained high levels of BTK more sensitive to the selective, second-generation BTK inhibitor, CGI1746, than side population-derived tumors that harbored low levels of BTK. Taken together, these findings implicate BTK as a positive regulator of myeloma stemness and provide additional support for the clinical testing of BTK-targeted therapies in patients with myeloma.
We reported previously that increased expression of aldehyde dehydrogenase 1 (ALDH1) in multiple myeloma (MM) is a marker of tumor-initiating cells (TICs) that is further associated with chromosomal ...instability (CIN). Here we demonstrate that member A1 of the ALDH1 family of proteins, ALDH1A1, is most abundantly expressed in myeloma. Enforced expression of ALDH1A1 in myeloma cells led to increased clonogenicity, tumor formation in mice, and resistance to myeloma drugs in vitro and in vivo. The mechanism underlying these phenotypes included the ALDH1A1-dependent activation of drug-efflux pump, ABCB1, and survival proteins, AKT and BCL2. Over expression of ALDH1A1 in myeloma cells led to increased mRNA and protein levels of NIMA-related kinase 2 (NEK2), whereas shRNA-mediated knock down of NEK2 decreased drug efflux pump activity and drug resistance. The activation of NEK2 in myeloma cells relied on the ALDH1A1-dependent generation of the retinoid X receptor α (RXRα) ligand, 9-cis retinoic acid (9CRA) - not the retinoic acid receptor α (RARα) ligand, all-trans retinoic acid (ATRA). These findings implicate the ALDH1A1-RXRα-NEK2 pathway in drug resistance and disease relapse in myeloma and suggest that specific inhibitors of ALDH1A1 are worthy of consideration for clinical development of new approaches to overcome drug resistance in myeloma.
The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible in vaccinated and unvaccinated populations. The dynamics that govern its establishment and ...propensity toward fixation (reaching 100% frequency in the SARS-CoV-2 population) in communities remain unknown. Here, we describe the dynamics of Omicron at 3 institutions of higher education (IHEs) in the greater Boston area.
We use diagnostic and variant-specifying molecular assays and epidemiological analytical approaches to describe the rapid dominance of Omicron following its introduction into 3 IHEs with asymptomatic surveillance programs.
We show that the establishment of Omicron at IHEs precedes that of the state and region and that the time to fixation is shorter at IHEs (9.5-12.5 days) than in the state (14.8 days) or region. We show that the trajectory of Omicron fixation among university employees resembles that of students, with a 2- to 3-day delay. Finally, we compare cycle threshold values in Omicron vs Delta variant cases on college campuses and identify lower viral loads among college affiliates who harbor Omicron infections.
We document the rapid takeover of the Omicron variant at IHEs, reaching near-fixation within the span of 9.5-12.5 days despite lower viral loads, on average, than the previously dominant Delta variant. These findings highlight the transmissibility of Omicron, its propensity to rapidly dominate small populations, and the ability of robust asymptomatic surveillance programs to offer early insights into the dynamics of pathogen arrival and spread.
Background: Multiple myeloma is the second most common blood borne neoplasia, accounting for nearly 10% of all diagnosed hematologic malignancies and has a disproportionately high incidence in ...elderly populations. Here we explored copy number variations using the high fidelity CytoScan HD arrays to develop a detailed map of copy number variations and identify novel mediators of disease progression. The results from CytoScan HD microarrays provide a detailed view of the entire genome with a resolution up to 25kb. Furthermore, 750,000 single-nucleotide polymorphisms are included and the array provides information about loss of heterozygosity and uniparental disomy.
Materials and methods: CytoScan HD arrays were performed on 97 myeloma patient samples to identify cytogenetic regions important to the development and progression of the disease. Gene expression profiles from 351 patients were analyzed to identify genes with a change in gene expression of 1.5 fold or more. Data from CytoScan and gene expression arrays was combined to perform chromosomal positional enrichment analysis to identify cytogenetic driver lesions, or lesions that provide a small, but significant growth and survival advantage to the cell. Furthermore, Kaplan-Meier, log-rank test and Hazard ratio analyses were performed to identify gene within the driver lesions that have a significant impact on survival when dysregulated.
Results: The results from the CytoScan HD analysis closely mirrored what has been shown by FISH and SNP arrays, with gains to the odd numbered chromosomes, specifically 3, 5, 7, 9, 11, 15 and 17 as well as losses to chromosomes 1p and 13. Interestingly, we identified gains to a small region within chromosome 8p, contrary to published reports demonstrating a large scale loss of this region. We identified numerous genes within this region that are important for survival and their overexpression resulted in a decreased progression free survival. For example, Cathepsin B (CTSB) is encoded for in chromosome 8p22-p21 with an increased gene expression of at least 1.5 fold over normal controls, among others. Furthermore, Cathepsin B, a cysteine protease, has been linked to cancer of the ileum, suggesting that a similar role may be present within myeloma.
We then integrated the 97 copy number profiles results with 351 myeloma gene expression profiles to identify cytogenetic driver lesions in myeloma important for disease development, progression and poor clinical outcome. Chromosomal positional enrichment analysis was employed to identify global myeloma cytogenetic driver aneuploidies as well as develop unique cytogenetic copy number profiles. Our results identified portions of chromosomes 1q, 3, 8p, 9, 13q and 16q, among others, as important driver lesions with changes to these regions providing growth advantages to the cell. Furthermore, our analysis identified five unique cytogenetic classifications based on common cytogenetic lesions. We continue to explore these driver regions to identify lesions important for the oncogenic properties of the larger regions.
Conclusion: The data presented here represents a novel and highly sensitive approach for the identification of novel copy number variations and driver lesions. Furthermore, correlations between copy number variations and gene expression arrays identified novel targets important for disease progression and patient survival. CytoScan HD arrays in conjunction with gene expression analysis provided a high resolution image of important cytogenetic lesions in myeloma and identified potentially important therapeutic targets for drug development. Further work is needed to validate our findings and determine the therapeutic efficacy of the identified targets.
No relevant conflicts of interest to declare.
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