Sukhinder K. Sandhu

Src homology 2 domain–containing inositol-5-phosphatase and CCAAT enhancer-binding protein β are targeted by miR-155 in B cells of Eμ-MiR-155 transgenic mice

We showed that Emicro-MiR-155 transgenic mice develop acute lymphoblastic leukemia/high-grade lymphoma. Most of these leukemias start at approximately 9 months irrespective of the mouse strain. They are preceded by a polyclonal pre-B-cell proliferation, have variable clinical presentation, are transplantable, and develop oligo/monoclonal expansion. In this study, we show that in these transgenic mice the B-cell precursors have the highest MiR-155 transgene expression and are at the origin of the leukemias. We determine that Src homology 2 domain-containing inositol-5-phosphatase (SHIP) and CCAAT enhancer-binding protein beta (C/EBPbeta), 2 important regulators of the interleukin-6 signaling pathway, are direct targets of MiR-155 and become gradually more down-regulated in the leukemic than in the preleukemic mice. We hypothesize that miR-155, by down-modulating Ship and C/EBPbeta, initiates a chain of events that leads to the accumulation of large pre-B cells and acute lymphoblastic leukemia/high-grade lymphoma.

Modulation of mismatch repair and genomic stability by miR-155

Inactivation of mismatch repair (MMR) is the cause of the common cancer predisposition disorder Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer (HNPCC), as well as 10–40% of sporadic colorectal, endometrial, ovarian, gastric, and urothelial cancers. Elevated mutation rates (mutator phenotype), including simple repeat instability [microsatellite instability (MSI)] are a signature of MMR defects. MicroRNAs (miRs) have been implicated in the control of critical cellular pathways involved in development and cancer. Here we show that overexpression of miR-155 significantly down-regulates the core MMR proteins, hMSH2, hMSH6, and hMLH1, inducing a mutator phenotype and MSI. An inverse correlation between the expression of miR-155 and the expression of MLH1 or MSH2 proteins was found in human colorectal cancer. Finally, a number of MSI tumors with unknown cause of MMR inactivation displayed miR-155 overexpression. These data provide support for miR-155 modulation of MMR as a mechanism of cancer pathogenesis.

Regulation of acute graft-versus-host disease by microRNA-155

Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic hematopoietic stem cell transplant (alloHSCT), underscoring the need to further elucidate its mechanisms and develop novel treatments. Based on recent observations that microRNA-155 (miR-155) is up-regulated during T-cell activation, we hypothesized that miR-155 is involved in the modulation of aGVHD. Here we show that miR-155 expression was up-regulated in T cells from mice developing aGVHD after alloHSCT. Mice receiving miR-155-deficient donor lymphocytes had markedly reduced lethal aGVHD, whereas lethal aGVHD developed rapidly in mice recipients of miR-155 overexpressing T cells. Blocking miR-155 expression using a synthetic anti-miR-155 after alloHSCT decreased aGVHD severity and prolonged survival in mice. Finally, miR-155 up-regulation was shown in specimens from patients with pathologic evidence of intestinal aGVHD. Altogether, our data indicate a role for miR-155 in the regulation of GVHD and point to miR-155 as a novel target for therapeutic intervention in this disease.

miR-155 targets histone deacetylase 4 (HDAC4) and impairs transcriptional activity of B-cell lymphoma 6 (BCL6) in the Eμ-miR-155 transgenic mouse model.

Multiple studies have established that microRNAs (miRNAs) are involved in the initiation and progression of cancer. Notably, miR-155 is one of the most overexpressed miRNAs in several solid and hematological malignancies. Ectopic miR-155 expression in mice B cells (Eμ-miR-155 transgenic mice) has been shown to induce pre–B-cell proliferation followed by high-grade lymphoma/leukemia. Loss of miR-155 in mice resulted in impaired immunity due to defective T-cell–mediated immune response. Here we provide a mechanistic insight into miR-155–induced leukemogenesis in the Eμ-miR-155 mouse model through genome-wide transcriptome analysis of naïve B cells and target studies. We found that a key transcriptional repressor and proto-oncogene, Bcl6 is significantly down-regulated in Eμ-miR-155 mice. The reduction of Bcl6 subsequently leads to de-repression of some of the known Bcl6 targets like inhibitor of differentiation (Id2), interleukin-6 (IL6), cMyc, Cyclin D1, and Mip1α/ccl3, all of which promote cell survival and proliferation. We show that Bcl6 is indirectly regulated by miR-155 through Mxd1/Mad1 up-regulation. Interestingly, we found that miR-155 directly targets HDAC4, a corepressor partner of BCL6. Furthermore, ectopic expression of HDAC4 in human-activated B-cell–type diffuse large B-cell lymphoma (DLBCL) cells results in reduced miR-155–induced proliferation, clonogenic potential, and increased apoptosis. Meta-analysis of the diffuse large B-cell lymphoma patient microarray data showed that miR-155 expression is inversely correlated with Bcl6 and Hdac4. Hence this study provides a better understanding of how miR-155 causes disruption of the BCL6 transcriptional machinery that leads to up-regulation of the survival and proliferation genes in miR-155–induced leukemias.

Potential Applications of MicroRNAs in Cancer Diagnosis, Prognosis, and Treatment

Early studies have established that microRNAs (miRNAs) are widely deregulated in cancer and play a critical role in cancer pathogenesis. Recent research efforts are directed now towards translating these basic discoveries into novel tests or treatments that could improve the diagnosis and outcome of cancer patients. In this review, we will summarize the potential applications of miRNAs for cancer diagnosis, prognosis, and treatment. In addition, we will discuss current pitfalls and future directions.

MicroRNA expression signatures in solid malignancies.

AbstractAn ongoing challenge in cancer research is represented by the identification of new specific clinical molecular markers and pharmacological targets. During the last 10 years, microRNAs (miRNAs) have become one of the hottest subjects in the area of cancer genomics. MicroRNAs are single-stranded RNAs of 19 to 24 nucleotides in length generated through a complex maturation process. Recent studies have demonstrated that microRNAs can have an oncogene or tumor suppressor role by regulating the expression of target genes. Therefore, microRNAs are highly related to cancer processes, including initiation, growth, apoptosis, invasion, and metastasis. In this panorama, several high-through put technologies studies have revealed miRNA roles in classifying tumors and predicting patient outcome with high accuracy. We provide a review highlighting recent progress on the understanding of the cellular function of human microRNAs and their expression in solid tumors.

Micro-RNA Expression and Function in Lymphomas

The recent discovery of microRNAs (miRNAs) has introduced a new layer of complexity to the process of gene regulation. MiRNAs are essential for cellular function, and their dysregulation often results in disease. Study of miRNA expression and function in animal models and human lymphomas has improved our knowledge of the pathogenesis of this heterogeneous disease. In this paper, we attempt to describe the expression of miRNAs and their function in lymphomas and discuss potential miRNA-based therapies in the diagnosis and treatment of lymphomas.

A large scale expression study associates uc.283-plus lncRNA with pluripotent stem cells and human glioma

BackgroundThere are 481 ultra-conserved regions (UCRs) longer than 200 bases in the genomes of human, mouse and rat. These DNA sequences are absolutely conserved and show 100% identity with no insertions or deletions. About half of these UCRs are reported as transcribed and many correspond to long non-coding RNAs (lncRNAs).MethodsWe used custom microarrays with 962 probes representing sense and antisense sequences for the 481 UCRs to examine their expression across 374 normal samples from 46 different tissues and 510 samples representing 10 different types of cancer. The expression in embryonic stem cells of selected UCRs was validated by real time PCR.ResultsWe identified tissue selective UCRs and studied UCRs in embryonic and induced pluripotent stem cells. Among the normal tissues, the uc.283 lncRNA was highly specific for pluripotent stem cells. Intriguingly, the uc.283-plus lncRNA was highly expressed in some solid cancers, particularly in one of the most untreatable types, glioma.ConclusionOur results suggest that uc.283-plus lncRNA might have a role in pluripotency of stem cells and in the biology of glioma.

Abstract 3: OncomiR-155 targets oncogenes HDAC4 and BCL6 in a murine B cell leukemia model: A paradigm shift in the oncogenic mechanisms of microRNAs

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC miR-155 belongs to a family of non-coding RNA, namely microRNAs (miRNAs) and is over-expressed in several lymphoid malignancies including chronic lymphocytic leukemia and diffuse large B-cell lymphomas. Transgenic over-expression of miR-155 in mouse B cells has been shown to induce pre-B cell proliferation followed by high-grade B-cell lymphoma. miR-155 exerts its oncogenic activity by targeting directly or indirectly tumor suppressor genes-SHIP-1 and C/EBPβ in B cells. To further characterize the mechanisms by which miR-155 contributes to leukemogenesis we did mRNA profiling of the B cells from the transgenic (TG) miR-155 mice spleens. Interestingly, we found that an oncogene Bcl-6 (B cell leukemia 6) was down regulated in the miR-155 TG B cells. Bcl-6 is a transcriptional repressor and a key regulator of germinal center B-cell development where antigen activated B cells differentiate to memory B cells or antibody producing plasma cells. Bcl6 is also involved in repression of cell cycle arrest and apoptosis associated genes by recruiting corepressors like histone deacetylases (HDACs). Further analysis of miR155 targets indicated another oncogene hdac4 to be a potential target. Luciferase reporter assay validated hdac4 as a direct target of miR155 which was further confirmed by 50% reduction in hdac4 mRNA and protein levels in TG mice by RT-PCR and immunoblotting respectively. To further investigate the effect of downegulation of hdac4 and Bcl6, we hypothesized that Bcl6 is functionally inactivated due to increased acetylation in TG mice and is targeted for ubiquitin mediated proteasome degradation due to downregulation of hdac4. Increased levels of acetylated Bcl6 in immunoprecipitated Bcl6 from total splenocytes of TG mice compared to WT control mice confirmed our hypothesis. This partly explains the post-translational regulation of Bcl6 indirectly by miR-155 through HDAC4. We further confirmed these findings in vitro in cultured cells lines with high and low expression of miR-155 and observed similar results of down regulation of HDAC4 and BCL6. In addition, we also observed significantly decreased proliferation of cells with high expression of miR-155 compared to cell lines with low expression of mir-155. mRNA analysis also showed significantly upregulation of genes involved in cell cycle arrest such as Cyclin D2/ccnd2, Cdkn1a and Inhibitor of differentiation 2/Id2) in TG mice compared to WT. These findings enlighten the complex interplay between tumor suppressor and oncogene pathways affected by a single gene, that when over-expressed results in leukemia. This indicates a shift in the paradigm of typical oncogenic miRNAs mechanisms, whereby they can act, not only by targeting tumor suppressors but also oncogenes. 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 3.

Abstract LB-276: Characterizing tumorigenesis of ovarian cancer across metastatic tumors and circulating, cell-free DNA

Tumor heterogeneity creates significant challenges for the generation and monitoring of treatment strategies. Circulating, cell-free DNA (cfDNA) can facilitate these processes by providing a noninvasive method to detect and track mutations without the need for multiple biopsies. To study both tumor heterogeneity and its relationship to cfDNA, we used samples taken from multiple positions on two metastatic tumors from a stage 3B ovarian carcinosarcoma patient as well as a cfDNA sample that was collected at the time of surgery. To maximize low frequency variant detection from next-generation sequencing (NGS) data, molecular identifiers (MIDs) were employed, allowing accurate distinction of true variants from PCR-induced errors along with sequencing artifacts. The use of MIDs to uniquely label input DNA generates tagged library molecules for the detection and removal of PCR duplicates while simultaneously preserving fragmentation and strand duplicates. By over-sequencing the MID tagged library, sequencing reads from PCR duplicates can be grouped based on their shared MID tag, generating a consensus sequence to identify and subsequently remove errors. To validate this technology, we performed low frequency spike-in experiments to 0.5% and 1% by combining Coriell NA12878 and HG005 genomic DNA as well as different cfDNA samples. Libraries were sequenced to 8000x coverage and a consensus sequence was generated with BMFtools (ARUP labs). All known variants present at 1% and 0.5% allele frequencies were maintained in the resulting data set. Further, true variants were preserved while sequencing and PCR-induced errors were removed, demonstrating improved sensitivity but also improved specificity using MIDs. After validation of the technology, libraries with MIDs were prepared from the ovarian carcinosarcoma FFPE and cfDNA samples. Libraries were sequenced on an Illumina® HiSeq® to a minimum of 13,000x coverage, and we determined data retention after de-duplication with and without the use of MIDs. We observed an increase in data retention for both Covaris-sheared FFPE and cfDNA libraries that led to a 2 to 3-fold increase in coverage using MIDs. Variant calling depicted significant inter-tumor heterogeneity in these samples as well as low frequency variants that represent intra-tumor heterogeneity. Specifically, we identified a pathogenic TP53 mutation present in one tumor sample and the cfDNA but absent from the second tumor. Additionally, we observed a significant loss of heterozygosity for only one of the tumors. We also identified low frequency, pathogenic mutations (as low as 1%) that are either unique to one tumor or unique to one sample from a tumor. This study highlights the use of MID technology to enable low frequency variant calling during disease diagnosis and tracking to facilitate precise, personalized treatment of complex cancers. Citation Format: Ashley Wood, Sukhinder Sandhu, Matthew Dashkoff, Olga Camacho-Vanegas, Laurie Kurihara, Timothy Harkins, John Martignetti, Vladimir Makarov, Peter Dottino. Characterizing tumorigenesis of ovarian cancer across metastatic tumors and circulating, cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-276. doi:10.1158/1538-7445.AM2017-LB-276