Jian-Jun Zhao

MicroRNAs 15a/16-1 function as tumor suppressor genes in multiple myeloma

Multiple myeloma is characterized by frequent chromosomal alterations. Deletion of chr 13, especially band 13q14, is commonly observed in early stages of MM, suggesting the presence of tumor suppressor genes within this region. Here, we functionally validate the role of the microRNAs-15a/16-1 cluster, centered at the deleted region, as TSGs and delineate their downstream target genes in MM. Using sponge lentiviral vectors to competitive stably inhibit mature microRNAs in vitro and in vivo, we have documented enhanced proliferative and invasive capacity of cells with stably inhibition of miR-16. Importantly, miR-16 inhibition decreased animal survival in a xenograft model of MM by increasing tumor load and host angiogenesis. Expression profiling analysis of miR-16-deficient cells identified a large number of downstream target genes including FGFR1, PI3KCa, MDM4, VEGFa, as well as secondary affected genes such as JUN and Jag1. We validated designated genes showing binding sites within the conserved 3-UTR and also within the mRNA coding region as direct miR-16 targets, thus indicating that the miRNAs may have many more targets than anticipated by conventional prediction methods. This loss-of-function system, which mimics the 13q chromosomal deletion, provides a valuable tool to investigate their function in MM pathogenesis and their potential use as therapeutic targets.

Abstract 2934: Targeting Bruton's tyrosine kinase with PCI-32765 blocks growth and survival of multiple myeloma and Waldenström macroglobulinemia via potent inhibition of osteoclastogenesis, cytokines/chemokine secretion, and myeloma stem-like cells in the bone marrow microenvironment

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Specific expression of Brutons tyrosine kinase (Btk) in osteoclasts (OC), but not osteoblasts (OB), suggests its role in osteoclastogenesis. Since Btk has not been characterized in multiple myeloma (MM) and Waldenstrom Macroglobulinemia (WM), we investigate effects of PCI-32765, an oral, potent, and selective Btk inhibitor with promising clinical activity in B-cell malignancies, on OC differentiation and function within MM bone marrow (BM) milieu, as well as on MM and WM cancer cells. In CD14+ OC precursor cells, PCI-32765 abrogated RANKL/M-CSF-induced Btk activation and downstream PLCγ2, resulting in decreased number of multinucleated OC (>3 nuclei) by tartrate-resistant acid phosphatase (TRAP) staining and inhibition of TRAP5b (ED50 = 17 nM), a specific mature OC marker. It induced defective bone resorption activity, as evidenced by diminished pit formation on dentine slices. Lack of effect of Dexamethasone on osteoclastic activity was overcome by combination of Dexamethasone with PCI-32765. PCI-32765 potently downregulates cytokine and chemokine secretion from OC cultures, i.e., MIP1α, MIP1α, IL-8, TGFβ1, RANTES, APRIL, SDF-1, and activin A (ED50 = 0.1-0.48 nM). It significantly decreased IL-6, SDF-1, MIP1α, MIP1α, and M-CSF in 2-week cultures of CD138-negative cells from active MM patients, associated with decreased TRAP staining. In MM and WM cells, immunoblotting analysis showed Btk expression in higher percentage of CD138+ myeloma cells from patients (4 out of 5 samples) than MM cell lines (5 out of 9 cell lines), whereas microarray analysis demonstrated increased expression of Btk and its downstream signaling components in WM cells than in CD19+ normal BM cells (p<0.001). Importantly, PCI-32765 blocked SDF-1-induced adhesion and migration of MM cell lines and patient MM cells via blockade of Btk signaling cascade. Quantigene analysis further showed PCI-32765-inhibited MIP1α mRNA in MM cells and many NF-κB-targeted transcripts in OC-lineages. PCI-32765 mitigated MM cell growth and survival triggered by IL-6 and coculture with BM stromal cells (BMSCs) or OCs. It blocked WM cell proliferation and induced apoptosis. Furthermore, myeloma stem-like cells from MM patients expressed Btk and PCI-32765 (10-100 nM) specifically blocked their abilities to form colonies in methylcellulose (n=5). In contrast, no toxicity was observed in Btk-negative BMSCs and OB. Oral administration of PCI-32765 (12 mg/kg) in mice significantly suppressed MM cell growth (p< 0.03) and MM cell-induced osteolysis on implanted human bone chips in a humanized myeloma (SCID-hu) model. Together, these results strongly support clinical trials of targeting Btk by PCI-32765 in the BM microenvironment to improve patient outcome in MM and WM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2934. doi:1538-7445.AM2012-2934

Abstract 3054: MicroRNA expression profile in cutaneous melanoma: miR-17-92 cluster is a progression and prognostic marker

MicroRNAs (miRNA) represent a novel class of RNAs that play important roles in cell survival, proliferation, differentiation and angiogenesis through negative regulation of protein-coding genes. Deregulation of miRNAs has been detected in various types of human malignancies. Some miRNAs are implicated in diagnosis, prognosis, treatment response and pathogenesis. However, aberrant miRNA expression and its pathologic significance in human melanoma have not been well documented. Here, we report on the miRNA expression profile of human cutaneous melanoma as well as melanoma cell lines. Of 650 miRNAs examined, at least 28 miRNAs were significantly deregulated in melanoma compared to normal skin and primary melanocytes, with the most common aberrantly expressed miRNAs being miR-17-92 cluster, miR-21, miR-146a/b. miR-148a/b, miR-126, miR-606, and miR-655. Notably, miRNA expression signatures could stratify patients into 2 groups (e.g., good and poor prognosis). Further analysis revealed that elevated levels of the miR17-92 cluster are associated with tumor progression and shorter overall survival. Our results suggest that miRNAs may be a useful new biomarker for and be a therapeutic target in cutaneous melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3054.