The bone marrow (BM) microenvironment of multiple myeloma (MM) is reported to play a role in the biology of disease. In this study, we found that the extracellular BM microenvironment in MM contains a unique miRNA signature detectable by miRNA microarray and quantitative real-time PCR, which is partially represented in the peripheral blood. Eleven miRNAs were significantly decreased in both BM and serum of MM patients in comparison with controls. Evaluation of these miRNAs in plasma of a separate cohort of MM patients and controls confirmed significantly aberrant levels of let-7a, let-7b, let-7i, miR15b, miR-16, and miR-20a in both serum and plasma. We then studied the myeloma precursor diseases and found that a subset of the MM miRNAs exhibited aberrant expression in monoclonal gammopathy of undetermined significance and smoldering myeloma. miRNA analysis of enriched CD138 þ plasma cells from MM and monoclonal gammopathy of undetermined significance found that most of the validated MM BM signature miRNAs were significantly decreased in MM plasma cells. Gene expression profiling indicated that multiple targets of the decreased miRNAs found increased expression in MM plasma cells, including ATF2, HRAS, HDAC4, TGFB1, TGFBR1, and mitogen-activated protein kinases. The findings suggest that these miRNAs are detectable in aberrant levels in the peripheral blood of patients with plasma cell proliferation and may play a role in aberrant plasma cell proliferation and disease progression. Multiple myeloma (MM) is a malignant plasma cell (PC) neoplasm that evolves from an underlying asymptomatic precursor clonal PC proliferation designated monoclonal gammopathy of undetermined significance (MGUS). MGUS is present in >3% of the population aged >50 years and progresses to myeloma at a rate of nearly 1% per year. 1Smoldering myeloma (SMM) represents an intermediate entity with increased bone marrow (BM) clonal PCs without symptomatic disease and carries an increased rate of progression to myeloma of nearly 10% per year.2 Currently, no single factor can predict patients with MGUS that are likely to progress to MM. A biomarker of disease progression in the peripheral blood (PB) could assist in the early identification of patients evolving to MM. Recent data suggest that serum miRNAs are altered in MM and MGUS and may serve as diagnostic and prognostic biomarkers. 3,4 miRNAs use a post-transcriptional gene regulation mechanism that was shown to play a role in development, differentiation, and tumorigenesis. 5e7 miRNAs are evolutionarily conserved small non-coding RNAs, which regulate gene
The lymph node (LN) is the site of chronic lymphocytic leukemia (CLL) cell activation and proliferation. Aberrant microRNA (miRNA) expression has been shown to have a role in CLL pathogenesis; however, a comparison of miRNA expression between CLL cells in the LN and the peripheral blood (PB) has previously not been reported. On the basis of the analysis of 17 paired LN and PB samples from CLL patients, we identify a panel of miRNAs that are increased in LN CLL cells correlating with an activation phenotype. When evaluated in CLL cells from 38 patients pre and post treatment with ibrutinib, a subset of these miRNAs (miR-22, miR-34a, miR-146b and miR-181b) was significantly decreased in response to ibrutinib. A concomitant increase in putative miRNA target transcripts (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4) was also observed. Functional studies confirmed targets of ibrutinib-responsive miRNAs to include messenger RNA transcripts of multiple tumor suppressors. Knockdown of endogenous miR-34a and miR146b resulted in increased transcription of tumor suppressors and inhibition of cell proliferation. These findings demonstrate that ibrutinib downregulates the expression of a subset of miRNAs related to B-cell activation leading to increased expression of miRNA targets including tumor suppressors and a reduction in cell proliferation.
Currently, no reliable biomarkers are available to predict transformation from smoldering myeloma (SMM) to multiple myeloma (MM). Using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) we assessed the levels of a broad range of cytokines and chemokines in the peripheral blood (PB) and bone marrow (BM) supernatant collected from 14 SMM and 38 MM patients and compared to healthy donors. We found significantly increased levels of key cytokines, in particular CXCL8 (IL-8), associated with progressive disease state (controls→SMM→MM). Cytokine profiles were found similar in PB and BM. Five of fourteen SMM patients (36%) progressed to MM. Our findings, although based on a limited number of patients, suggest that serum-based cytokines may have a future role as biomarkers for disease progression and could potentially be assessed as novel targets for treatment.
GATA2 is a transcription factor critical for hematopoiesis. Germline mutations in GATA binding protein 2 (GATA2) led to haploinsufficiency, severe cytopenias of multiple cell lineages, susceptibility to infections and strong propensity to develop myelodysplastic syndrome, and acute myeloid leukemia. Mechanisms of progressive cytopenias remain unclear. MicroRNA (miRNA) represents a unique mechanism of post-transcriptional gene regulation. In this study, miRNA profiles were evaluated and eight miRNAs were found to be differentially expressed (≥2-fold, P ≤ 0.05) in patient-derived cell lines (N = 13) in comparison to controls (N = 10). miR-9, miR-181a-2-3p, miR-181c, miR-181c-3p, miR-486-3p, and miR-582 showed increased expression, whereas miR-223 and miR-424-3p showed decreased expression. Cell death assays indicated that miR-181c potently induces cell death in lymphoid (Ly-8 and SP-53) and myeloid (HL-60) cell lines. miR-181c was predicted to target myeloid cell leukemia (MCL)1, which was confirmed by transfection assays, resulting in significantly reduced MCL1 mRNA and decreased live cell numbers. Bone marrow analysis of 34 GATA2 patients showed significantly decreased cellularity, CD34-positive cells, monocytes, dendritic cells, NK cells, B cells, and B cell precursors in comparison to healthy controls (N = 29; P < 0.001 for each), which was accompanied by decreased levels of MCL1 (P < 0.05). GATA2 expression led to significant repression of miR-181c expression in transfection experiments. Conversely, knockdown of GATA2 led to increased miR-181c expression. These findings indicate that miR-181c expression is increased and MCL1 levels decreased in GATA2 deficiency cells, and that GATA2 represses miR-181c transcription. Increased miR-181c may contribute to elevated cell death and cytopenia in GATA2 deficiency potentially through down-regulation of MCL1.
570 Background. MicroRNAs (miRNAs) play a pivotal role in tumorigenesis, due to their ability to target mRNAs involved in the regulation of cell proliferation, survival and differentiation. In particular, the Let-7 miRNA family members have been described to act as tumor suppressors, as demonstrated both in solid cancer and hematologic malignancies. However, the role of Let-7 in multiple myeloma (MM) has not been studied. Method. Circulating miRNA profiling has been performed in MM patients compared to healthy individuals using TaqMan human miRNA profiling, and validated by qRT-PCR. Exosomes were collected from both normal and MM peripheral blood, using ultracentrifugation; and further studied by using electron microscopy and immunogold labeling for the detection of CD63 and CD81. Exosomes were then evaluated for their miRNA content, by qRT-PCR. Gain- and loss-of functions studies were performed on MM cell lines (MM.1S; U266), using Let-7-mimic and Lin28B siRNA, respectively. Scramble probe-transfected cells were used as control. Cell proliferation and cell survival have been evaluated by using BrdU assay and MTT assay, respectively. Effects of Let-7 and Lin28B on signaling cascades have been evaluated by western blot. Results. We identified a MM specific signature characterized by down-regulation of miRNA-15a, −19b, −21, let-7b, let-7c and over-expression of miR-720 (P < 0.001). Circulating exosomes were studied at ultrastructural level showing positivity for CD81 and CD63, as demonstrated by immunogold labeling and electron microscopy. The same miRNA signature was found in the circulating exosomes, suggesting that circulating miRNAs may be transported by exosomes. The Let-7 family members were significantly decreased in peripheral blood of MM patient compared to healthy individuals, suggesting that Let-7 family could be down-regulated in MM cells. We then performed qRT-PCR in MM primary cells; and found that the Let-7 family is significantly down-regulated in MM primary cells, especially for Let-7b and c (5 fold change, P< 0.05). Over-expression of Let-7b and Let-7c in MM cells (U266; MM1S) transfected decreased cell proliferation. The RNA binding protein Lin28B is known to regulate the Let-7 family: we therefore performed Lin28-loss of function studies which led to up-regulation of the Let-7 family members, in MM transfected cells; and found that Lin28B-knockdown cells presented with reduced cell proliferation, supported by down-regulation of c-Myc and K-Ras, known to be Let-7-related targets. Conclusion. This data indicate that Let-7 miRNA family members play an important role in modulating MM biology, thus providing the basis for the development of new miRNA-based target therapies and biomarker in this disease. Disclosures: Ghobrial: Novartis: advisory board, advisory board Other; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: advisory board, advisory board Other.
Background: MicroRNAs (miRNAs, miRs) are small (~22nt) noncoding RNAs that are involved in post-transcriptional regulation of specific mRNA targets. Aberrant miRNA expression has been shown to play a role in chronic lymphocytic leukemia (CLL) pathogenesis and to have prognostic implications. The lymph node (LN) has been demonstrated to be the site of CLL cell proliferation and activation. In particular, CLL cells in the LN display significantly increased BCR and NF-kB signaling in comparison to CLL cells in the peripheral blood (PB). Ibrutinib is a clinically approved BTK inhibitor that disrupts BCR and NF-kB signaling; and has been shown to be effective in relapsed or refractory CLL. We hypothesized that 1) miRNAs would be differentially expressed between activated CLL cells in the LN and CLL cells in the PB; and 2) ibrutinib would alter miRNA expression in CLL, which may affect therapeutic response by regulating the expression of miR target genes. Methods: RNA was isolated from CD19 positive selected cells from two separate cohorts of CLL patients. The first group included 17 treatment naïve patients donating paired LN and PB CD19 positive cells (10 IGHV un-mutated [59%], 4 IGHV mutated [23.5%] and 3 IGHV unknown [17.5%]); the second group contained 38 pairs of PB CD19 positive cells (22 IGHV un-mutated [58%] and 16 IGHV mutated [42%]) before and after ibrutinib treatment on day 28. A panel of 22 miRs previously reported to play a role in CLL pathogenesis, prognosis, or BCR signaling was profiled by qPCR. The expression level of a custom panel of genes encoding putative miR targets was quantified using the nanoString nCounter assay and verified by qPCR. Functional studies were performed in MEC-1 and SP-53 cell lines, which were treated separately with 1 μM ibrutinib or 1 μM fludarabine for 48 hours. Live cells were identified by trypan blue staining; miR and mRNA expression was quantified by qPCR. Results: Nineteen of 22 (86.4%) miRs were significantly increased (p < 0.05) in CLL cells from the LN in comparison to paired CLL cells from the PB, correlating with an activated B cell phenotype. Examination of mRNA transcripts of putative miR targets in 13 paired LN and PB samples showed that multiple tumor suppressor transcripts including CYLD, ARID1B and FOXP1 were significantly decreased in LN-derived CLL cells. Assay of 22 miRs in PB CLL cells from 38 patients before and after ibrutinib treatment indicated that a subset of miRs (miR-22, miR-34a, miR-146b and miR-181b) were significantly decreased (p < 0.05) in response to ibrutinib. Quantification of mRNA transcripts of putative targets of the ibrutinib responsive miRs by the nanoString nCounter assay showed that several tumor suppressor transcripts (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4) were significantly increased. Functional studies in MEC-1 and SP-53 cell lines confirmed that miR-22, miR-34a, miR-146b and miR-181b were down-regulated and the tumor suppressor targets of these miRs (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, PTEN and SMAD4) were significantly increased after ibrutinib treatment. The alteration of miR and gene expression was specific to the effects of ibrutinib as similar changes were not observed post treatment with the chemotherapeutic agent fludarabine. Conclusions: miRNA expression patterns are significantly altered between CLL cells in the LN and those in the PB. An overall increase in the expression of miRs and down-regulation of mRNAs of tumor suppressor genes (ARID1B, CYLD and FOXP1) was observed in CLL cells in the LN, correlating with an activated B-cell phenotype. Ibrutinib treatment and inhibition of BCR signaling resulted in down-regulation of miR-22, miR-34a, miR-146b and miR-181b and concomitant up-regulation of multiple tumor suppressor targets including ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4 in PB CLL cells. Similar findings were also observed in ibrutinib treated MEC-1 and SP-53 cell lines. Additional studies are needed to address how much the down-regulation of miRs and consequent up-regulation of tumor suppressors in response to ibrutinib contributes to the overall disease response and whether this effect could sensitize CLL cells to treatment with cytotoxic agents. Disclosures Wiestner: Pharmacyclics: Research Funding.
8597 Background: Presently, there is no reliable biomarker for predicting clinical progression from smoldering (SMM) to symptomatic MM for individual patients. To improve our understanding on the pathogenesis from SMM to MM, we conducted a screening study of circulating cytokines using peripheral blood (PB) and bone marrow supernatant (BM) collected from treatment naïve SMM and MM patients as well as healthy donors as controls. Methods: PB samples from 14 SMM and 38 MM patients and 7 controls and BM obtained from 17 MM patients and 9 controls were assayed in duplicates using ultra-sensitive Human TH1/TH2 10-plex multi-spot plate and multi-array plate for interleukin-(IL)-6. Two-tailed Mann-Whitney test was used for statistical analysis. Results: PB of SMM patients (vs controls) had increased levels of several cytokines, including IL-8 (p=0.008) and IFN-gamma (p=0.002). In PB, MM patients (compared to SMM patient and controls) had increased levels of IL-6 (p=0.006 and 0.001, respectively), IL-8 (p=0.0001 and 0.008), IL-10 (p=0.02 and 0.02), TNF-alpha (p=0.01 and 0.009), and IFN-gamma (p=0.01 and 0.02). Analysis of BM revealed a similar profile with increased levels of IL-2, IL-6, IL-8, and TNF-alpha in MM patients compared with controls (p=0.007, p=0.0003, p=0.0001 and p=0.0008). Conclusions: We found significantly increased levels of key cytokines associated with progressive disease state (controls→SMM→MM). Patterns of cytokines were similar in BM and PB, suggesting that serum based cytokines may have a future role as biomarkers for disease progression, and could potentially be assessed as novel targets for treatment.
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