Yuan-Xiao Zhu

Genome-wide studies in multiple myeloma identify XPO1/CRM1 as a critical target validated using the selective nuclear export inhibitor KPT-276

RNA interference screening identified XPO1 (exportin 1) among the 55 most vulnerable targets in multiple myeloma (MM). XPO1 encodes CRM1, a nuclear export protein. XPO1 expression increases with MM disease progression. Patients with MM have a higher expression of XPO1 compared with normal plasma cells (P<0.04) and to patients with monoclonal gammopathy of undetermined significance/smoldering MM (P<0.0001). The highest XPO1 level was found in human MM cell lines (HMCLs). A selective inhibitor of nuclear export compound KPT-276 specifically and irreversibly inhibits the nuclear export function of XPO1. The viability of 12 HMCLs treated with KTP-276 was significantly reduced. KPT-276 also actively induced apoptosis in primary MM patient samples. In gene expression analyses, two genes of probable relevance were dysregulated by KPT-276: cell division cycle 25 homolog A (CDC25A) and bromodomain-containing protein 4 (BRD4), both of which are associated with c-MYC pathway. Western blotting and reverse transcription-PCR confirm that c-MYC, CDC25A and BRD4 are all downregulated after treatment with KPT-276. KPT-276 reduced monoclonal spikes in the Vk*MYC transgenic MM mouse model, and inhibited tumor growth in a xenograft MM mouse model. A phase I clinical trial of an analog of KPT-276 is ongoing in hematological malignancies including MM.

Cyproheptadine displays preclinical activity in myeloma and leukemia

D-cyclins are regulators of cell division that act in a complex with cyclin-dependent kinases to commit cells to a program of DNA replication. D-cyclins are overexpressed in many tumors, including multiple myeloma and leukemia, and contribute to disease progression and chemoresistance. To better understand the role and impact of D-cyclins in hematologic malignancies, we conducted a high throughput screen for inhibitors of the cyclin D2 promoter and identified the drug cyproheptadine. In myeloma and leukemia cells, cyproheptadine decreased expression of cyclins D1, D2, and D3 and arrested these cells in the G(0)/G(1) phase. After D-cyclin suppression, cyproheptadine induced apoptosis in myeloma and leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. In mouse models of myeloma and leukemia, cyproheptadine inhibited tumor growth without significant toxicity. Cyproheptadine-induced apoptosis was preceded by activation of the mitochondrial pathway of caspase activation and was independent of the drugs known activity as an H1 histamine and serotonin receptor antagonist. Thus, cyproheptadine represents a lead for a novel therapeutic agent for the treatment of malignancy. Because the drug is well tolerated and already approved in multiple countries for clinical use as an antihistamine and appetite stimulant, it could be moved directly into clinical trials for cancer.

Mechanism of immunomodulatory drugs' action in the treatment of multiple myeloma

Although immunomodulatory drugs (IMiDs), such as thalidomide, lenalidomide, and pomalidomide, are widely used in the treatment of multiple myeloma (MM), the molecular mechanism of IMiDs action is largely unknown. In this review, we will summarize recent advances in the application of IMiDs in MM cancer treatment as well as their effects on immunomodulatory activities, anti-angiogenic activities, intervention of cell surface adhesion molecules between myeloma cells and bone marrow stromal cells, anti-inflammatory activities, anti-proliferation, pro-apoptotic effects, cell cycle arrest, and inhibition of cell migration and metastasis. In addition, the potential IMiDs target protein, IMiDs target proteins functional role, and the potential molecular mechanisms of IMiDs resistance will be discussed. We wish, by presentation of our naive discussion, that this review article will facilitate further investigation in these fields.

Enhanced adaptive immunity in mice lacking the immunoinhibitory adaptor Hacs1

Hacs1, a SH3 and SAM domain‐containing adaptor protein, is up‐regulated by IL‐4 in activated B cells and strongly expressed in dendritic cells. To elucidate the function of Hacs1 in immune regulation, we generated Hacs1−/− mice by deletion of the SH3 and SAM domains. Hacs1−/− mice were viable and fertile and had normal bone marrow B‐cell development and normal splenic T‐ and B‐cell populations. However, adult Hacs1−/− mice had increased peritoneal Bla cells (IgM+CD5+). On immunization with T‐cell‐independent antigen TNP‐Ficoll, Hacs1−/− mice had increased production of anti‐TNP IgM and IgG3. Purified splenic B cells from Hacs1−/− mice showed increased cell proliferation on BCR (B‐cell receptor) stimulation. We further demonstrate that the Hacs1−/− B cells had increased global tyrosine phosphorylation, including tyrosine kinases Lyn and Akt. Both T‐helper type 1 (Th1) and T‐helper type 2 (Th2) humoral responses were enhanced in Hacs1−/− mice. In vitro bone marrow‐derived Hacs1−/− dendritic cells showed increased IL‐12 production on stimulation with ovalbumin (OVA). This study suggests that Hacs1 is an immunoinhibitory adaptor that might be a useful target for immune suppression therapy.—Wang, D., Stewart, A. K., Zhuang, L., Zhu, Y., Wang, Y., Shi, C., Keating, A., Slutsky, A., Zhang, H., Wen, X.‐Y. Enhanced adaptive immunity in mice lacking the immunoinhibitory adaptor Hacs1. FASEBJ. 24, 947–956 (20l0). www.fasebj.org

Panel sequencing for clinically oriented variant screening and copy number detection in 142 untreated multiple myeloma patients

We employed a customized Multiple Myeloma (MM)-specific Mutation Panel (M3P) to screen a homogenous cohort of 142 untreated MM patients for relevant mutations in a selection of disease-specific genes. M3Pv2.0 includes 77 genes selected for being either actionable targets, potentially related to drug–response or part of known key pathways in MM biology. We identified mutations in potentially actionable genes in 49% of patients and provided prognostic evidence of STAT3 mutations. This panel may serve as a practical alternative to more comprehensive sequencing approaches, providing genomic information in a timely and cost-effective manner, thus allowing clinically oriented variant screening in MM.

Cereblon binding molecules in multiple myeloma.

Immunomodulation is an established treatment strategy in multiple myeloma with thalidomide and its derivatives lenalidomide and pomalidomide as its FDA approved representatives. Just recently the method of action of these cereblon binding molecules was deciphered and results from large phase 3 trials confirmed the backbone function of this drug family in various combination therapies. This review details the to-date knowledge concerning mechanism of IMiD action, clinical applications and plausible escape mechanisms in which cells may become resistant/refractory to cereblon binding molecule based treatment.

Longitudinal analysis of 25 sequential sample-pairs using a custom multiple myeloma mutation sequencing panel (M 3 P)

Recent advances in genomic sequencing technologies now allow results from deep next-generation sequencing to be obtained within clinically meaningful timeframes, making this an attractive approach to better guide personalized treatment strategies. No multiple myeloma-specific gene panel has been established so far; we therefore designed a 47-gene-targeting gene panel, containing 39 genes known to be mutated in ≥3xa0% of multiple myeloma cases and eight genes in pathways therapeutically targeted in multiple myeloma (MM). We performed targeted sequencing on tumor/germline DNA of 25 MM patients in which we also had a sequential sample post treatment. Mutation analysis revealed KRAS as the most commonly mutated gene (36xa0% in each time point), followed by NRAS (20 and 16xa0%), TP53 (16 and 16xa0%), DIS3 (16 and 16xa0%), FAM46C (12 and 16xa0%), and SP140 (12 and 12xa0%). We successfully tracked clonal evolution and identified mutation acquisition and/or loss in FAM46C, FAT1, KRAS, NRAS, SPEN, PRDM1, NEB, and TP53 as well as two mutations in XBP1, a gene associated with bortezomib resistance. Thus, we present the first longitudinal analysis of a MM-specific targeted sequencing gene panel that can be used for individual tumor characterization and for tracking clonal evolution over time.

Expression of the cereblon binding protein argonaute 2 plays an important role for multiple myeloma cell growth and survival

BackgroundImmunomodulatory drugs (IMiDs), such as lenalidomide, are therapeutically active compounds that bind and modulate the E3 ubiquitin ligase substrate recruiter cereblon, thereby affect steady-state levels of cereblon and cereblon binding partners, such as ikaros and aiolos, and induce many cellular responses, including cytotoxicity to multiple myeloma (MM) cells. Nevertheless, it takes many days for MM cells to die after IMiD induced depletion of ikaros and aiolos and thus we searched for other cereblon binding partners that participate in IMiD cytotoxicity.MethodsCereblon binding partners were identified from a MM cell line expressing histidine-tagged cereblon by pulling down cereblon and its binding partners and verified by co-immunoprecipitation. IMiD effects were determined by western blot analysis, cell viability assay, microRNA array and apoptosis analysis.ResultsWe identified argonaute 2 (AGO2) as a cereblon binding partner and found that the steady-state levels of AGO2 were regulated by cereblon. Upon treatment of IMiD-sensitive MM cells with lenalidomide, the steady-state levels of cereblon were significantly increased, whereas levels of AGO2 were significantly decreased. It has been reported that AGO2 plays a pivotal role in microRNA maturation and function. Interestingly, upon treatment of MM cells with lenalidomide, the steady-state levels of microRNAs were significantly altered. In addition, silencing of AGO2 in MM cells, regardless of sensitivity to IMiDs, significantly decreased the levels of AGO2 and microRNAs and massively induced cell death.ConclusionThese results support the notion that the cereblon binding partner AGO2 plays an important role in regulating MM cell growth and survival and AGO2 could be considered as a novel drug target for overcoming IMiD resistance in MM cells.

Additional file 1: Figure S1. of Expression of the cereblon binding protein argonaute 2 plays an important role for multiple myeloma cell growth and survival

Sequence of human AGO2 small hairpin RNA (shRNA). Table S2. Proteins detected by tandem mass spectrometry analyses. Table S3. The miRNA up- or down-regulated by AGO2 shRNA. Table S4. The miRNA up- or down-regulated by lenalidomide treatment. (DOCX 159Âxa0kb)

Bioinformatics Strategies for Identifying Regions of Epigenetic Deregulation Associated with Aberrant Transcript Splicing and RNA-editing

Epigenetic modifications are associated with the regulation of co/post-transcriptional processing and differential transcript isoforms are known to be important during cancer progression. It remains unclear how disruptions of chromatin-based modifications contribute to tumorigenesis and how this knowledge can be leveraged to develop more potent treatment strategies that target specific isoforms or other products of the co/post-transcriptional regulation pathway. Rapid developments in all areas of next-generation sequencing (DNA, RNA-seq, ChIP-seq, Methyl-CpG, etc.) have provided new opportunities to develop novel integration and data-mining approaches, and also allows for exciting hypothesis driven bioinformatics and computational studies. Here, we present a program that we developed and summarize the results of applying our methods to analyze datasets from patient matched tumor or normal (T/N) paired samples, as well as cell lines that were either sensitive or resistant (S/R) to treatment with an anti-cancer drug, 5-Azacytidine (http://sourceforge.net/projects/chiprnaseqpro/). We discuss additional options for user-defined approaches and general guidelines for simultaneously analyzing and annotating epigenetic and RNA-seq datasets in order to identify and rank significant regions of epigenetic deregulation associated with aberrant splicing and RNA-editing.