Fine-needle aspiration biopsy and flow cytometry immunophenotyping of lymphoid and myeloproliferative disorders of the spleen.

BACKGROUND: Flow cytometry (FC) is a useful adjunct to fine-needle aspiration biopsy (FNAB) in the evaluation of lymphoproliferative disorders. The application of FC to FNAB of the spleen (sFNAB) is reported. METHODS: Flow cytometry was performed on 18 sFNAB collected over 3 years. The series comprised 10 cases of non- Hodgkin lymphomas (NHL), 2 cases insufficient for diagnosis, 2 cases of reactive hyperplasia (RH), and 4 cases of myeloid metaplasia (MM). FNAB was performed under ultrasound guidance using a 22-gauge needle. One or two passes were sufficient to prepare a conventional smear that was immediately evaluated to select the cases studied and to prepare a cell suspension for FC. The following fluoresceinated antibodies were used: CD3, CD19/kappa/lambda, FMC7/CD23/CD19, Bcl-2, and CD13/HLA-DR. In six cases, cytospins were also prepared for immunocytochemistry and were tested for CD20 (L26), CD45Ro, and kappa and lambda light chain expression. RESULTS: Flow cytometry contributed to the diagnosis of all cases of NHL by assessing light chain restriction. The specific subtype was also diagnosed by CD19/CD5 and CD 19/CD10 coexpression in two cases. Flow cytometry quantified the percentage of myeloid cells in MM cases and contributed to the cytologic diagnosis showing a polyclonal light chain expression in RH cases.Immunocytochemistry was effective and concordant in four cases. Patients tolerated the sFNAB well and no complications were reported. Cytologic and FC diagnoses were confirmed by follow-up and by histologic evaluation in cases in which splenectomy was performed for therapeutic purposes. CONCLUSION: Flow cytometry applied to sFNAB corroborates the cytologic diagnosis in lymphoid and myeloproliferative disorders of the spleen and allows therapeutic decisions avoiding splenectomy

miR-29b sensitizes multiple myeloma cells to bortezomib-induced apoptosis through the activation of a feedback loop with the transcription factor Sp1

MicroRNAs (miRNAs) with tumor-suppressor potential might have therapeutic applications in multiple myeloma (MM) through the modulation of still undiscovered molecular pathways. Here, we investigated the effects of enforced expression of miR-29b on the apoptotic occurrence in MM and highlighted its role in the context of a new transcriptional loop that is finely tuned by the proteasome inhibitor bortezomib. In details, in vitro growth inhibition and apoptosis of MM cells was induced by either transient expression of synthetic miR-29b or its stable lentivirus-enforced expression. We identified Sp1, a transcription factor endowed with oncogenic activity, as a negative regulator of miR-29b expression in MM cells. Since Sp1 expression and functions are regulated via the 26S proteasome, we investigated the effects of bortezomib on miR-29b-Sp1 loop, showing that miR-29b levels were indeed upregulated by the drug. At the same time, the bortezomib/miR-29b combination produced significant pro-apoptotic effects. We also demonstrated that the PI3K/AKT pathway plays a major role in the regulation of miR-29b-Sp1 loop and induction of apoptosis in MM cells. Finally, MM xenografts constitutively expressing miR-29b showed significant reduction of their tumorigenic potential. Our findings indicate that miR-29b is involved in a regulatory loop amenable of pharmacologic intervention and modulates the anti-MM activity of bortezomib in MM cells

Therapeutic vulnerability of multiple myeloma to MIR17PTi, a first-in-class inhibitor of pri-mir-17-92

The microRNA cluster miR-17-92 is oncogenic and represents a valuable therapeutic target in c-MYC (MYC)-driven malignancies. Here, we developed novel LNA gapmeR antisense oligonucleotides (ASOs) to induce RNase H-mediated degradation of MIR17HG primary transcripts and, consequently, to prevent biogenesis of miR-17-92 microRNAs (miR-17-92s). The leading LNA-ASO, named MIR17PTi, impaired proliferation of several cancer cell lines (n=48) established from both solid and hematologic tumors by on-target antisense activity, and more effectively as compared to miR-17-92s inhibitors. By focusing on multiple myeloma (MM), we found that MIR17PTi triggers apoptosis via impairment of homeostatic MYC/miR-17-92 feed-forward loops (FFLs) in patient-derived MM cells; and induced MYC-dependent synthetic lethality. We show that alteration of a BIM-centered FFL is instrumental for MIR17PTi to induce cytotoxicity in MM cells. MIR17PTi exerts strong in vivo anti-tumor activity in NOD-SCID mice bearing clinically relevant models of MM, with advantageous safety and pharmacokinetics profiles in non-human primates. Altogether, MIR17PTi is a novel pharmacological tool to be tested in early-phase clinical trials against MM and other MYC-driven malignancies

A targeted sequencing approach in multiple myeloma reveals a complex landscape of genomic lesions that has implications for prognosis

Background: Next-generation sequencing (NGS) studies have shown that mul- tiple myeloma is a heterogeneous disease with a complex subclonal architecture and few recurrently mutated genes. The analysis of smaller regions of interest in the genome (\u201ctargeted studies\u201d) allows interrogation of recurrent genomic events with reduces complexity of downstream analysis at a lower price. Aims: Here, we performed the largest targeted study to date in multiple myelo- ma to analyze gene mutations, deletions and amplifications, chromosomal copy number changes and immunoglobulin heavy chain locus (IGH) translo- cations and correlate results with biological and clinical features. Methods: We used Agilent SureSelect cRNA pull down baits to target: 246 genes implicated in myeloma or cancer in general in a mixed gene discovery/confirmation effort; 2538 single nucleotide polymorphisms to detect amplifications and deletions at the single-gene and chromosome level; the IGH locus to detect translocations. We sequenced unmatched DNA from CD138- purified plasma cells from 418 patients with multiple myeloma at diagnosis, with a median follow-up of 5.3 years. We sequenced at an average depth of 337x using Hiseq2000 machines (Illumina Inc.). We applied algorithms developed in- house to call genomic events, filtering out potential artifacts and germline vari- ants. We then ranker each event on its likelihood of being \u201concogenic\u201d based on clustering, recurrence and cross-reference with the COSMIC database. Results: We identified 2270 gene mutations in 412/418 patients, and of those 688 were oncogenic. 342 patients harbored at least one oncogenic mutation. 215/246 genes showed at lease one likely somatic mutation, but only 106 showed at least one oncogenic mutation. 63% of oncogenic mutations were accounted for by the top 9 driver genes previously identified (KRAS, NRAS, TP53, FAM46C, BRAF, DIS3, TRAF3, SP140, IRF4), implying our gene discov- ery effort did not identify novel mutated genes. We included deletion of tumor suppressors, amplification of oncogenes, chromosomal copy number changes and IGH translocations for a total of 76 variables, so that 413/418 patients showed at least one informative driver genomic event, (median 4/patient). We investigated pairwise associations between events and found significant corre- lations, such as TP53 mutations and del(17p), CYLD mutations and del(16), FAM46C mutations and del(1p), SF3B1 mutations and t(11;14). Hotspots muta- tions of IRF4 lysine p.123 showed an inverse correlation with a hyperdiploid karyotype and del(16) as opposed to other missense mutations scattered along the gene, which has pathogenic implications. Survival was negatively affected by the cumulative burden of lesions in an almost linear fashion, with median survival of 10.97 and 4.07 years in patients with =7 lesions respectively, and this was independent of the nature of the genomic events. Given the het- erogeneity and complex interplay of the variables we fitted a cox-proportional hazard model to predict survival. We found that mutations in TP53, amplifications of MYC, deletions of CYLD, amp(1q), del12p13.31 and del17p13 where the only significant events, all promoting shorter survival. In particular, TP53 muta- tions and deletions, often co-occurring, had an additive effect so that carriers of both showed a dismal survival of 17 months (Figure 1).Summary/Conclusions: Due to the complex genomic landscape in MM, a discovery effort still requires large studies to derive significant associations. We conclude that a targeted sequencing approach may provide prognostic models and give insights into myeloma biology

Drugging the lncRNA MALAT1 via LNA gapmeR ASO inhibits gene expression of proteasome subunits and triggers anti-multiple myeloma activity

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) are still to be investigated. Here, we studied the functional significance and the druggability of the oncogenic lncRNA MALAT1 in MM. Targeting MALAT1 by novel LNA-gapmeR antisense oligonucleotide antagonized MM cell proliferation and triggered apoptosis both in vitro and in vivo in a murine xenograft model of human MM. Of note, antagonism of MALAT1 downmodulated the two major transcriptional activators of proteasome subunit genes, namely NRF1 and NRF2, and resulted in reduced trypsin, chymotrypsin and caspase-like proteasome activities and in accumulation of polyubiquitinated proteins. NRF1 and NRF2 decrease upon MALAT1 targeting was due to transcriptional activation of their negative regulator KEAP1, and resulted in reduced expression of anti-oxidant genes and increased ROS levels. In turn, NRF1 promoted MALAT1 expression thus establishing a positive feedback loop. Our findings demonstrate a crucial role of MALAT1 in the regulation of the proteasome machinery, and provide proof-of-concept that its targeting is a novel powerful option for the treatment of MM

Nucleotide excision repair is a potential therapeutic target in multiple myeloma

Despite the development of novel drugs, alkylating agents remain an important component of therapy in multiple myeloma (MM). DNA repair processes contribute towards sensitivity to alkylating agents and therefore we here evaluate the role of nucleotide excision repair (NER), which is involved in the removal of bulky adducts and DNA crosslinks in MM. We first evaluated NER activity using a novel functional assay and observed a heterogeneous NER efficiency in MM cell lines and patient samples. Using next-generation sequencing data, we identified that expression of the canonical NER gene, excision repair cross-complementation group 3 (ERCC3), significantly impacted the outcome in newly diagnosed MM patients treated with alkylating agents. Next, using small RNA interference, stable knockdown and overexpression, and small-molecule inhibitors targeting xeroderma pigmentosum complementation group B (XPB), the DNA helicase encoded by ERCC3, we demonstrate that NER inhibition significantly increases sensitivity and overcomes resistance to alkylating agents in MM. Moreover, inhibiting XPB leads to the dual inhibition of NER and transcription and is particularly efficient in myeloma cells. Altogether, we show that NER impacts alkylating agents sensitivity in myeloma cells and identify ERCC3 as a potential therapeutic target in MM

CDK-Mediated Regulation of Cell Functions via c-Jun Phosphorylation and AP-1 Activation

Cyclin-dependent kinases (CDKs) and their targets have been primarily associated with regulation of cell-cycle progression. Here we identify c-Jun, a transcription factor involved in the regulation of a broad spectrum of cellular functions, as a newly recognized CDK substrate. Using immune cells from mouse and human, and several complementary in vitro and in vivo approaches including dominant negative protein expression, pharmacologic inhibitors, kinase assays and CDK4 deficient cells, we demonstrate the ability of CDK4 to phosphorylate c-Jun. Additionally, the activity of AP-1, a ubiquitous transcription factor containing phosphorylated c-Jun as a subunit, was inhibited by abrogating CDK4. Surprisingly, the regulation of c-Jun phosphorylation by CDK4 occurred in non-dividing cells, indicating that this pathway is utilized for cell functions that are independent of proliferation. Our studies identify a new substrate for CDK4 and suggest a mechanism by which CDKs can regulate multiple cellular activation functions, not all of which are directly associated with cell cycle progression. These findings point to additional roles of CDKs in cell signaling and reveal potential implications for therapeutic manipulations of this kinase pathway

A high-risk, Double-Hit, group of newly diagnosed myeloma identified by genomic analysis

Patients with newly diagnosed multiple myeloma (NDMM) with high-risk disease are in need of new treatment strategies to improve the outcomes. Multiple clinical, cytogenetic, or gene expression features have been used to identify high-risk patients, each of which has significant weaknesses. Inclusion of molecular features into risk stratification could resolve the current challenges. In a genome-wide analysis of the largest set of molecular and clinical data established to date from NDMM, as part of the Myeloma Genome Project, we have defined DNA drivers of aggressive clinical behavior. Whole-genome and exome data from 1273 NDMM patients identified genetic factors that contribute significantly to progression free survival (PFS) and overall survival (OS) (cumulative R2 = 18.4% and 25.2%, respectively). Integrating DNA drivers and clinical data into a Cox model using 784 patients with ISS, age, PFS, OS, and genomic data, the model has a cumlative R2 of 34.3% for PFS and 46.5% for OS. A high-risk subgroup was defined by recursive partitioning using either a) bi-allelic TP53 inactivation or b) amplification (≥4 copies) of CKS1B (1q21) on the background of International Staging System III, comprising 6.1% of the population (median PFS = 15.4 months; OS = 20.7 months) that was validated in an independent dataset. Double-Hit patients have a dire prognosis despite modern therapies and should be considered for novel therapeutic approaches

miR-23b/SP1/c-myc forms a feed-forward loop supporting multiple myeloma cell growth

Deregulated microRNA (miR)/transcription factor (TF)-based networks represent a hallmark of cancer. We report here a novel c-Myc/miR-23b/Sp1 feed-forward loop with a critical role in multiple myeloma (MM) and Waldenstrom's macroglobulinemia (WM) cell growth and survival. We have found miR-23b to be downregulated in MM and WM cells especially in the presence of components of the tumor bone marrow milieu. Promoter methylation is one mechanism of miR-23b suppression in myeloma. In gain-of-function studies using miR-23b mimics-transfected or in miR-23b-stably expressing MM and WM cell lines, we observed a significant decrease in cell proliferation and survival, along with induction of caspase-3/7 activity over time, thus supporting a tumor suppressor role for miR-23b. At the molecular level, miR-23b targeted Sp1 3'UTR and significantly reduced Sp1-driven nuclear factor-kappa B activity. Finally, c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, transcriptionally repressed miR-23b. Thus MYC-dependent miR-23b repression in myeloma cells may promote activation of oncogenic Sp1-mediated signaling, representing the first feed-forward loop with critical growth and survival role in myeloma