Supplementary Figures 1-6 from The <i>miR-17-92</i> Cluster of MicroRNAs Confers Tumorigenicity by Inhibiting Oncogene-Induced Senescence

Autoreactive B cells are major contributors to autoimmune disease pathogenesis, but the molecular pathways responsible for the break of B cell tolerance in these patients remain largely unknown. In this study, we performed an in vivo functional screen of a lymphocyte-expressed miRNA library and identified miR-148a as a key regulator of this process. Elevated miR-148a expression impaired B cell tolerance by protecting immature B cells from BCR engagement-induced apoptosis, through downregulation of its targets Gadd45a, Bcl2l11, and Pten. Moreover, increased expression of miR-148a, which has been consistently found in lupus patients and lupus-prone mice, accelerated the development of lethal autoimmunity in a lupus mouse model. These results demonstrate that miR-148a functions as an important regulator of B cell tolerance and autoimmunity.

The molecular mechanisms that regulate B-cell development and tolerance remain incompletely understood. In this study, we identify a critical role for the miR-17∼92 microRNA cluster in regulating B-cell central tolerance and demonstrate that these miRNAs control early B-cell development in a cell-intrinsic manner. While the cluster member miR-19 suppresses the expression of Pten and plays a key role in regulating B-cell tolerance, miR-17 controls early B-cell development through other molecular pathways. These findings demonstrate differential control of two closely linked B-cell developmental stages by different members of a single microRNA cluster through distinct molecular pathways.

Aberrant activation of the PI3K and NF-κB pathways occurs frequently in human B-cell lymphomas.1, 2 Recent studies suggested reciprocal molecular interactions between these two pathways in lymphomagenesis. For example, PI3K inhibition suppresses NF-κB activity in human Burkitt's lymphoma and diffuse large B-cell lymphoma,3, 4 while blockade of NF-κB causes suppression of PI3K activity in primary effusion lymphoma cell lines.5 Despite frequent alterations and molecular interactions of these two pathways in human lymphomas, genetic activation of anyone of these two pathways was not sufficient to initiate lymphoma development in mice.6, 7, 8

Transient transfection of chemically synthesized microRNA (miRNA) mimics is being used extensively to study the functions and mechanisms of endogenous miRNAs. However, it remains unclear whether transfected miRNAs behave similarly to endogenous miRNAs. Here we show that transient transfection of miRNA mimics into HeLa cells by a commonly used method led to the accumulation of high molecular weight RNA species and a few hundred fold increase in mature miRNA levels. In contrast, expression of the same miRNAs through lentiviral infection or plasmid transfection of HeLa cells, transgenic expression in primary lymphocytes, and endogenous overexpression in lymphoma and leukemia cell lines did not lead to the appearance of high molecular weight RNA species. The increase of mature miRNA levels in these cells was below 10-fold, which was sufficient to suppress target gene expression and to drive lymphoma development in mice. Moreover, transient transfection of miRNA mimics at high concentrations caused non-specific alterations in gene expression, while at low concentrations achieved expression levels comparable to other methods but failed to efficiently suppress target gene expression. Small RNA deep sequencing analysis revealed that the guide strands of miRNA mimics were frequently mutated, while unnatural passenger strands of some miRNA mimics accumulated to high levels. The high molecular weight RNA species were a heterogeneous mixture of several classes of RNA species generated by concatemerization, 5'- and 3'-end tailing of miRNA mimics. We speculate that the supraphysiological levels of mature miRNAs and these artifactual RNA species led to non-specific changes in gene expression. Our results have important implications for the design and interpretation of experiments primarily employing transient transfection of miRNA mimics.

The miR-17–92 cluster is a prototypical example of a polycistronic miRNA gene. Recently, miR-17–92 has emerged as a pleiotropic regulator in immune system. Its loss or deregulation leads to defects in lymphocyte development and response, and lymphoma development. Although the six individual miRNAs of the cluster are expressed together from the same primary transcript, their relative abundance, functional contributions and interactions vary in different cellular contexts.

Autoreactive B cells have critical roles in a large diversity of autoimmune diseases, but the molecular pathways that control these cells remain poorly understood. We performed an in vivo functional screen of a lymphocyte-expressed microRNA library and identified miR-148a as a potent regulator of B cell tolerance. Elevated miR-148a expression impaired B cell tolerance by promoting the survival of immature B cells after engagement of the B cell antigen receptor by suppressing the expression of the autoimmune suppressor Gadd45α, the tumor suppressor PTEN and the pro-apoptotic protein Bim. Furthermore, increased expression of miR-148a, which occurs frequently in patients with lupus and lupus-prone mice, facilitated the development of lethal autoimmune disease in a mouse model of lupus. Our studies demonstrate a function for miR-148a as a regulator of B cell tolerance and autoimmunity.

Conception and design: ML, HYJ, CX; Collection and assembly of data: ML, HYJ, CX Data analysis and interpretation: ML, HYJ, CX Manuscript writing: ML, CX; Final approval of manuscript: ML, CX

microRNAs (miRNAs) are a class of small non-coding RNAs of ~22 nucleotides in length that bind to their target mRNAs (mRNAs) and regulate their expression by translation repression and mRNA degradation. Over the past decade, several miRNA genes have been implicated in human cancers, but their exact function and underlying molecular mechanism in carcinogenesis remain poorly understood. This is a key obstacle in the development of miRNA-based cancer therapeutics. miR-17~92, a cluster of 6 miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92), was the first miRNA gene implicated in cancer. Its overexpression occurs in a broad spectrum of human cancers, including lymphoma, leukemia, and solid tissue cancers. miR17~92 overexpression is mainly driven by gene amplification at the human chromosome 13q31 region, which harbors the miR-17~92 gene (termed MIR17HG), and by Myc-mediated transcriptional upregulation. A recent RNA sequencing analysis of patient biopsies showed that miR-17~92 expression was upregulated by 2~36-fold in 14 out of 79 (18%) cases of diffuse large B-cell lymphomas and in all the 28 cases of Burkitt lymphomas analyzed. As Myc overexpression is the defining feature of Burkitt lymphoma, these results confirmed that the Myc-miR-17~92 axis is operating in all patients with this malignancy. Previous studies showed that miR17~92 overexpression contributes to carcinogenesis. Thus, retroviral overexpression of miR-17~92 accelerated Myc-driven lymphomagenesis and Notch-driven leukemogenesis, while a miR-17~92 transgene promoted retinoblastoma initiated by inactivation of the Rb pathway. Another study showed that miR-17~92 played important roles in sustaining the optimal growth of Myc-driven lymphoma cell lines in tissue culture and in immunodeficient host. However, it was unclear whether elevated miR-17~92 expression, per se, is sufficient to drive carcinogenesis, and exactly what role miR-17~92 plays in Myc-driven carcinogenesis. A recent study from our lab provided some answers to these important questions. First, we created transgenic mice specifically overexpressing miR-17~92 in B cells. Those mice developed B-cell lymphomas with high penetrance, establishing miR-17~92 as a powerful cancer driver and validating its encoded miRNAs and downstream pathways as therapeutic targets (see below). Second, we used CD19-Cre to conditionally delete miR17~92 in B cells of λ-Myc mice, a commonly used model of Burkitt lymphoma. λ-Myc;CD19-Cre;miR-17~92 l mice exhibited a delay in lymphomagenesis. Strikingly, all lymphomas arising from those mice contained 2 intact miR-17~92 alleles. They escaped CD19-Cre-mediated deletion of miR-17~92 by restricting Myc-driven malignant transformation in CD19-negative early B-cell precursors or in a small fraction of CD19-positive cells with low Cre expression. These results show that Myc-driven lymphomagenesis stringently requires 2 intact alleles of miR17~92. This, together with a recent study demonstrating that miR-17~92 is essential for the development of retinoblastoma driven by the double deletion of Rb and p53, prompts us to hypothesize that the requirement of miR-17~92 is a common denominator of cancers. As miR-17~92 overexpression is found in many human cancers, future studies are warranted to investigate the effect of miR-17~92 deletion on carcinogenesis in other mouse models. We next explored the molecular mechanisms through which miR-17~92 drives lymphomagenesis. We performed PAR– CLIP analysis of human B cells, identified 868 protein coding genes containing miR17~92-binding sites that are conserved between human and mouse, and validated a select group of target genes in miR-17~92 transgenic B cells. Our analyses showed that miR-17~92 suppresses the expression of multiple inhibitors of the PI3K (Pten and Phlpp2) and NFκB (Cyld, A20, Itch, Rnf11, and Tax1BP1) pathways, and that both pathways are constitutively active in miR-17~92-driven lymphoma cells. Furthermore, chemical inhibition of either pathway significantly controlled tumor growth and prolonged survival of mice bearing miR-17~92-driven lymphomas. These results suggest that activation of the PI3K and NFκB pathways plays important roles in both the development and maintenance of miR-17~92-driven lymphomas, and that dual targeting of PI3K and NFκB pathways is a potential strategy to treat miR-17~92-driven lymphomas (Fig. 1). An alternative strategy to treat miR17~92-driven lymphomas is to sequester or knockdown miR-17~92 miRNAs directly. As miR-17~92 encodes for 6 distinct miRNAs, which may cooperate or even antagonize each other in driving lymphomagenesis, it will be essential to

MicroRNAs (miRNAs) have been broadly implicated in cancer, but their exact function and mechanism in carcinogenesis remain poorly understood. Elevated miR‐17∼92 expression is frequently found in human cancers, mainly due to gene amplification and Myc‐mediated transcriptional upregulation. Here we show that B cell‐specific miR‐17∼92 transgenic mice developed lymphomas with high penetrance and that, conversely, Myc‐driven lymphomagenesis stringently requires two intact alleles of miR‐17∼92. We experimentally identified miR‐17∼92 target genes by PAR‐CLIP and validated select target genes in miR‐17∼92 transgenic mice. These analyses demonstrate that miR‐17∼92 drives lymphomagenesis by suppressing the expression of multiple negative regulators of the PI3K and NFκB pathways and by inhibiting the mitochondrial apoptosis pathway. Accordingly, miR‐17∼92‐driven lymphoma cells exhibited constitutive activation of the PI3K and NFκB pathways and chemical inhibition of either pathway reduced tumour size and prolonged the survival of lymphoma‐bearing mice. These findings establish miR‐17∼92 as a powerful cancer driver that coordinates the activation of multiple oncogenic pathways, and demonstrate for the first time that chemical inhibition of miRNA downstream pathways has therapeutic value in treating cancers caused by miRNA dysregulation. B cell‐specific knockout of the miRNA 17∼92 cluster corroborates its oncogenic properties. Application of PAR‐CLIP establishes unique and novel molecular targets of this first‐ever described oncogenic miRNA

The p38 mitogen-activated protein kinase (MAPK) pathway regulates multiple physiologic and pathologic processes, including cancer development. PRAK, a p38 substrate protein kinase, has previously been implicated in the suppression of skin carcinogenesis. In the current study, we show that PRAK deletion accelerates hematopoietic cancer development in a mouse model harboring an oncogenic ras allele, Eμ-N-Ras(G12D), specifically expressed in hematopoietic cells. Further investigation reveals that enhanced hematopoietic tumorigenesis by PRAK deficiency is associated with hyperactivation of the c-jun-NH(2)-kinase (JNK) pathway both in vivo and in primary hematopoietic cells isolated from spleens. In primary splenocytes, PRAK deficiency further enhanced oncogenic ras-induced cell proliferation and promoted ras-mediated colony formation on semisolid medium in a JNK-dependent manner. In addition, deletion of PRAK leads to abrogation of ras-induced accumulation of senescence markers. These findings indicate that PRAK suppresses hematopoietic cancer formation in this mouse model by antagonizing oncogenic ras-induced activation of the JNK pathway. Our results suggest that PRAK may function as a tumor suppressor in multiple types of cancers.

Downlo ammalian cells, activation of oncogenes usually triggers innate tumor-suppressing defense mechanisms, ing apoptosis and senescence, which are compromised by additional mutations before cancers are develThe miR-17-92 gene cluster, a polycistron encoding six microRNAs (miRNA), is frequently overexpressed an cancers and has been shown to promote several aspects of oncogenic transformation, including evaf apoptosis. In the current study, we show a new role of miR-17-92 in inhibiting oncogenic ras-induced ence. Further dissection of the miRNA components in this cluster reveals that the miR-17/20a seed family nts for this antisenescence activity. miR-17 and miR-20a are both necessary and sufficient for conferring nce to ras-induced senescence by directly targeting p21, a key effector of senescence. By contrast, components are not essential for the ability of miR-17-92 to evade Myc-induced apoptosis. Moreover, tion of senescence by miR-17-92 or its miR-17/20a components leads to enhanced oncogenic transforn by activated ras in primary human cells. Taken together with previous reports that miR-17-92 inhibits sis by suppressing Pten via the miR-19 components, our results indicate that this miRNA cluster proapopto motes tumorigenesis by antagonizing both tumor-suppressing mechanisms, apoptosis, and senescence, through the activities of different miRNA components encoded in this cluster. Cancer Res; 70(21); 8547–57. ©2010 AACR.

Abstract In mammalian cells, activation of oncogenes usually triggers innate tumor-suppressing defense mechanisms, including apoptosis and senescence, which are compromised by additional mutations before cancers are developed. The miR-17-92 gene cluster, a polycistron encoding six microRNAs (miRNA), is frequently overexpressed in human cancers and has been shown to promote several aspects of oncogenic transformation, including evasion of apoptosis. In the current study, we show a new role of miR-17-92 in inhibiting oncogenic ras-induced senescence. Further dissection of the miRNA components in this cluster reveals that the miR-17/20a seed family accounts for this antisenescence activity. miR-17 and miR-20a are both necessary and sufficient for conferring resistance to ras-induced senescence by directly targeting p21WAF1, a key effector of senescence. By contrast, these components are not essential for the ability of miR-17-92 to evade Myc-induced apoptosis. Moreover, disruption of senescence by miR-17-92 or its miR-17/20a components leads to enhanced oncogenic transformation by activated ras in primary human cells. Taken together with previous reports that miR-17-92 inhibits apoptosis by suppressing Pten via the miR-19 components, our results indicate that this miRNA cluster promotes tumorigenesis by antagonizing both tumor-suppressing mechanisms, apoptosis, and senescence, through the activities of different miRNA components encoded in this cluster. Cancer Res; 70(21); 8547–57. ©2010 AACR.

Vpr ( viral protein R) is a vital HIV-1 accessory protein with multiple functions in the viral life cycle, including nuclear import of preintegration complex, induction of apoptosis and G(2) cell cycle arrest. The cell cycle perturbation activity of Vpr requires activation of the ATR ( Ataxia-Telangiectasia and Rad3-related) pathway and the integrity of Vpr C-terminal motif that is crucial for chromatin binding. Recent studies also demonstrated Vpr as one of the viral factors that influence HIV disease progression, as mutations in Vpr were overrepresented in some cohorts of long-term nonprogressors (LTNP). The LTNP-associated mutations of Vpr are frequently observed in the C-terminal domain. This raises the question whether the LTNP phenotype of Vpr is the result of the loss its ability to induce G(2) arrest. Here we report that the LTNP-associated mutants of Vpr function normally in the induction of G(2) arrest. No defects in ATR activation and direct binding to chromatin are observed. These mutants also show similar levels of apoptosis induction as wild-type Vpr. These data differentiate the LTNP-associated mutations of Vpr with those defective in inducing G(2) arrest. We propose that the G(2) arrest function of Vpr is separated from the LTNP phenotype, and the role of Vpr in HIV disease progression may involve other functions of Vpr.

BRCA1 (Breast Cancer Susceptibility Gene 1) possesses an N-terminal Ring domain and tandem C-terminal BRCT motifs. While the Ring domain has E3 ubiquitin ligase activity, the BRCA1 BRCT domains specifically recognize phospho-serine motifs. Here, we demonstrate that BRCA1 Ring domain catalyzes CtIP ubiquitination in a manner that depends on a phosphorylation-mediated interaction between CtIP and BRCA1 BRCT domains. The BRCA1-dependent ubiquitination of CtIP does not target CtIP for degradation. Instead, ubiquitinated CtIP associates with chromatin following DNA damage and participates in G2/M checkpoint control. Thus, we propose that BRCA1 can regulate the functions of its substrates through nonproteasomal pathways that do not involve substrate degradation.

Nat. Struct. Mol. Biol. 12, 663–653 (2005). The manuscript contained an error in the name of one of the authors. Maciej Gajec was misspelled Maciez Gajek. The correct author list should read: Barbara A L Owen, Zungyoon Zang, Maoyi Lai, Maciej Gajec, John D Badger II, Jeffrey J Hayes, Winfried Edelmann, Raju Kucherlapati, Teresa M Wilson & Cynthia T McMurray.

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) protein Vpr (viral protein R) arrests cells in the G2 phase of the cell cycle, a process that requires activation of the ATR (ataxia-telangiectasia and Rad3-related) pathway. In this study we demonstrate that the expression of Vpr does not cause DNA double-strand breaks but rather induces ATR activation, as indicated by induction of Chk1 phosphorylation and the formation of γ-H2AX and 53BP1 nuclear foci. We define a C-terminal domain containing repeated H(F/S)RIG sequences required for Vpr-induced activation of ATR. Further investigation of the mechanism by which Vpr activates the ATR pathway reveals an increase in chromatin binding of replication protein A (RPA) upon Vpr expression. Immunostaining shows that RPA localizes to nuclear foci in Vpr-expressing cells. Furthermore, we demonstrate direct binding of Vpr to chromatin in vivo, whereas Vpr C-terminal domain mutants lose this chromatin-binding activity. These data support a mechanism whereby HIV-1 Vpr induces ATR activation by targeting the host cell DNA and probably interfering with normal DNA replication.

We describe a simple technology used to cure an established metastatic disease. Intradermal injection of plasmid DNA encoding a transcriptionally targeted cytotoxic gene, along with hsp70, not only promoted tissue-specific, inflammatory killing of normal melanocytes, but also induced a CD8 + T-cell–dependent, antigen-specific response in mice that eradicated systemically established B16 tumors. This CD8 + T cell response was subsequently suppressed in vivo within a few days. The data demonstrate that deliberate destruction of normal tissue can be exploited to generate immunity against a malignant disease originating from that tissue. This approach obviates the need to identify tumor antigens and does not require complex isolation of tumor cells or their derivatives. In addition, it provides a model system for studying the mechanisms underlying the etiology and control of autoimmune diseases. Finally, despite targeting normal tissue, therapy could be separated from development of overt autoimmune symptoms, suggesting that the strategy may be valuable against tumors derived from both non-essential and essential tissue types. *Note: In the version of this article originally published online, the name of one of the authors was spelled incorrectly. Mayoi Lai should be Maoyi Lai. This mistake has been corrected in the HTML version and will appear correctly in print.