Alistair M. Chalk

RNA editing by ADAR1 prevents MDA5 sensing of endogenous dsRNA as nonself

RNA editing helps identify cellular RNAs Adenosine bases in messenger RNA (mRNAs) can be enzymatically modified and changed into inosine bases. This RNA “editing” is mediated by adenosine deaminase acting on RNA (ADAR) enzymes. Liddicoat et al. show that the in vivo targets of the principal editing enzyme, ADAR1, are long double-stranded RNA (dsRNA) structures in noncoding portions of cellular mRNAs. ADAR1-directed editing of these cellular targets is critical to avoid activation of an immune response to dsRNA in the cytoplasm, because dsRNA is also a marker of viral infection. Science, this issue p. 1115 The principal RNA-editing enzyme modifies cellular RNAs to prevent their erroneous identification as foreign RNA. Adenosine-to-inosine (A-to-I) editing is a highly prevalent posttranscriptional modification of RNA, mediated by ADAR (adenosine deaminase acting on RNA) enzymes. In addition to RNA editing, additional functions have been proposed for ADAR1. To determine the specific role of RNA editing by ADAR1, we generated mice with an editing-deficient knock-in mutation (Adar1E861A, where E861A denotes Glu861→Ala861). Adar1E861A/E861A embryos died at ~E13.5 (embryonic day 13.5), with activated interferon and double-stranded RNA (dsRNA)–sensing pathways. Genome-wide analysis of the in vivo substrates of ADAR1 identified clustered hyperediting within long dsRNA stem loops within 3′ untranslated regions of endogenous transcripts. Finally, embryonic death and phenotypes of Adar1E861A/E861A were rescued by concurrent deletion of the cytosolic sensor of dsRNA, MDA5. A-to-I editing of endogenous dsRNA is the essential function of ADAR1, preventing the activation of the cytosolic dsRNA response by endogenous transcripts.

BET inhibitors induce apoptosis through a MYC independent mechanism and synergise with CDK inhibitors to kill osteosarcoma cells

Osteosarcoma (OS) survival rates have plateaued in part due to a lack of new therapeutic options. Here we demonstrate that bromodomain inhibitors (BETi), JQ1, I-BET151, I-BET762, exert potent anti-tumour activity against primary and established OS cell lines, mediated by inhibition of BRD4. Strikingly, unlike previous observations in long-term established human OS cell lines, the antiproliferative activity of JQ1 in primary OS cells was driven by the induction of apoptosis, not cell cycle arrest. In further contrast, JQ1 activity in OS was mediated independently of MYC downregulation. We identified that JQ1 suppresses the transcription factor FOSL1 by displacement of BRD4 from its locus. Loss of FOSL1 phenocopied the antiproliferative effects of JQ1, identifying FOSL1 suppression as a potential novel therapeutic approach for OS. As a monotherapy JQ1 demonstrated significant anti-tumour activity in vivo in an OS graft model. Further, combinatorial treatment approaches showed that JQ1 increased the sensitivity of OS cells to doxorubicin and induced potent synergistic activity when rationally combined with CDK inhibitors. The greater level of activity achieved with the combination of BETi with CDK inhibitors demonstrates the efficacy of this combination therapy. Taken together, our studies show that BET inhibitors are a promising new therapeutic for OS.

Modeling distinct osteosarcoma subtypes in vivo using Cre:lox and lineage-restricted transgenic shRNA

Osteosarcoma is the most common primary cancer of bone and one that predominantly affects children and adolescents. Osteoblastic osteosarcoma represents the major subtype of this tumor, with approximately equal representation of fibroblastic and chondroblastic subtypes. We and others have previously described murine models of osteosarcoma based on osteoblast-restricted Cre:lox deletion of Trp53 (p53) and Rb1 (Rb), resulting in a phenotype most similar to fibroblastic osteosarcoma in humans. We now report a model of the most prevalent form of human osteosarcoma, the osteoblastic subtype. In contrast to other osteosarcoma models that have used Cre:lox mediated gene deletion, this model was generated through shRNA-based knockdown of p53. As is the case with the human disease the shRNA tumors most frequently present in the long bones and preferentially disseminate to the lungs; feature less consistently modeled using Cre:lox approaches. Our approach allowed direct comparison of the in vivo consequences of targeting the same genetic drivers using two different technologies, Cre:lox and shRNA. This demonstrated that the effects of Cre:lox and shRNA mediated knock-down are qualitatively different, at least in the context of osteosarcoma, and yielded distinct subtypes of osteosarcoma. Through the use of complementary genetic modification strategies we have established a model of the most common clinical subtype of osteosarcoma that was not previously represented and more fully recapitulated the clinical spectrum of this cancer.

Systematic Screening Identifies Dual PI3K and mTOR Inhibition as a Conserved Therapeutic Vulnerability in Osteosarcoma

Purpose: Osteosarcoma is the most common cancer of bone occurring mostly in teenagers. Despite rapid advances in our knowledge of the genetics and cell biology of osteosarcoma, significant improvements in patient survival have not been observed. The identification of effective therapeutics has been largely empirically based. The identification of new therapies and therapeutic targets are urgently needed to enable improved outcomes for osteosarcoma patients. Experimental Design: We have used genetically engineered murine models of human osteosarcoma in a systematic, genome-wide screen to identify new candidate therapeutic targets. We performed a genome-wide siRNA screen, with or without doxorubicin. In parallel, a screen of therapeutically relevant small molecules was conducted on primary murine– and primary human osteosarcoma–derived cell cultures. All results were validated across independent cell cultures and across human and mouse osteosarcoma. Results: The results from the genetic and chemical screens significantly overlapped, with a profound enrichment of pathways regulated by PI3K and mTOR pathways. Drugs that concurrently target both PI3K and mTOR were effective at inducing apoptosis in primary osteosarcoma cell cultures in vitro in both human and mouse osteosarcoma, whereas specific PI3K or mTOR inhibitors were not effective. The results were confirmed with siRNA and small molecule approaches. Rationale combinations of specific PI3K and mTOR inhibitors could recapitulate the effect on osteosarcoma cell cultures. Conclusions: The approaches described here have identified dual inhibition of the PI3K–mTOR pathway as a sensitive, druggable target in osteosarcoma, and provide rationale for translational studies with these agents. Clin Cancer Res; 21(14); 3216–29. ©2015 AACR.

Knockdown of PTHR1 in osteosarcoma cells decreases invasion and growth and increases tumor differentiation in vivo

Osteosarcoma (OS) is the most common cancer of bone. Parathyroid hormone (PTH) regulates calcium homeostasis and bone development, while the paracrine/autocrine PTH-related protein (PTHrP) has central roles in endochondral bone formation and bone remodeling. Using a murine OS model, we found that OS cells express PTHrP and the common PTH/PTHrP receptor (PTHR1). To investigate the role of PTHR1 signaling in OS cell behavior, we used shRNA to reduce PTHR1 expression. This only mildly inhibited proliferation in vitro, but markedly reduced invasion through collagen and reduced expression of RANK ligand (RANKL). Administration of PTH(1–34) did not stimulate OS proliferation in vivo but, strikingly, PTHR1 knockdown resulted in a profound growth inhibition and increased differentiation/mineralization of the tumors. Treatment with neutralizing antibody to PTHrP did not recapitulate the knockdown of PTHR1. Consistent with this lack of activity, PTHrP was predominantly intracellular in OS cells. Knockdown of PTHR1 resulted in increased expression of late osteoblast differentiation genes and upregulation of Wnt antagonists. RANKL production was reduced in knockdown tumors, providing for reduced homotypic signaling through the receptor, RANK. Loss of PTHR1 resulted in the coordinated loss of gene signatures associated with the polycomb repressive complex 2 (PRC2). Using Ezh2 inhibitors, we demonstrate that the increased expression of osteoblast maturation markers is in part mediated by the loss of PRC2 activity. Collectively these results demonstrate that PTHR1 signaling is important in maintaining OS proliferation and undifferentiated state. This is in part mediated by intracellular PTHrP and through regulation of the OS epigenome.

ADAR1, inosine and the immune sensing system: distinguishing self from non‐self

The conversion of genomically encoded adenosine to inosine in dsRNA is termed as A‐to‐I RNA editing. This process is catalyzed by two of the three mammalian ADAR proteins (ADAR1 and ADAR2) both of which have essential functions for normal organismal homeostasis. The phenotype of ADAR2 deficiency can be primarily ascribed to a lack of site‐selective editing of a single transcript in the brain. In contrast, the biology and substrates responsible for the Adar1−/− phenotype have remained more elusive. Several recent studies have identified that a feature of absence or reductions of ADAR1 activity, conserved across human and mouse models, is a profound activation of interferon‐stimulated gene signatures and innate immune responses. Further analysis of this observation has lead to the conclusion that editing by ADAR1 is required to prevent activation of the cytosolic innate immune system, primarily focused on the dsRNA sensor MDA5 and leading to downstream signaling via MAVS. The delineation of this mechanism places ADAR1 at the interface between the cells ability to differentiate self‐ from non‐self dsRNA. Based on MDA5 dsRNA recognition requisites, the mechanism indicates that the type of dsRNA must fulfil a particular structural characteristic, rather than a sequence‐specific requirement. While additional studies are required to molecularly verify the genetic model, the observations to date collectively identify A‐to‐I editing by ADAR1 as a key modifier of the cellular response to endogenous dsRNA. WIREs RNA 2016, 7:157–172. doi: 10.1002/wrna.1322

The Rothmund-Thomson syndrome helicase RECQL4 is essential for hematopoiesis

Mutations within the gene encoding the DNA helicase RECQL4 underlie the autosomal recessive cancer-predisposition disorder Rothmund-Thomson syndrome, though it is unclear how these mutations lead to disease. Here, we demonstrated that somatic deletion of Recql4 causes a rapid bone marrow failure in mice that involves cells from across the myeloid, lymphoid, and, most profoundly, erythroid lineages. Apoptosis was markedly elevated in multipotent progenitors lacking RECQL4 compared with WT cells. While the stem cell compartment was relatively spared in RECQL4-deficent mice, HSCs from these animals were not transplantable and even selected against. The requirement for RECQL4 was intrinsic in hematopoietic cells, and loss of RECQL4 in these cells was associated with increased replicative DNA damage and failed cell-cycle progression. Concurrent deletion of p53, which rescues loss of function in animals lacking the related helicase BLM, did not rescue BM phenotypes in RECQL4-deficient animals. In contrast, hematopoietic defects in cells from Recql4Δ/Δ mice were fully rescued by a RECQL4 variant without RecQ helicase activity, demonstrating that RECQL4 maintains hematopoiesis independently of helicase activity. Together, our data indicate that RECQL4 participates in DNA replication rather than genome stability and identify RECQL4 as a regulator of hematopoiesis with a nonredundant role compared with other RecQ helicases.

Increased miR-155-5p and reduced miR-148a-3p contribute to the suppression of osteosarcoma cell death

Osteosarcoma (OS) is the most common cancer of bone and the 5th leading cause of cancer-related death in young adults. Currently, 5-year survival rates have plateaued at ~70% for patients with localized disease. Those with disseminated disease have an ~20% 5-year survival. An improved understanding of the molecular genetics of OS may yield new approaches to improve outcomes for OS patients. To this end, we applied murine models that replicate human OS to identify and understand dysregulated microRNAs (miRNAs) in OS. miRNA expression patterns were profiled in murine primary osteoblasts, osteoblast cultures and primary OS cell cultures (from primary and paired metastatic locations) isolated from two genetically engineered murine models of OS. The differentially expressed miRNA were further assessed by a cross-species comparison with human osteoblasts and OS cultures. We identified miR-155-5p and miR-148a-3p as deregulated in OS. miR-155-5p suppression or miR-148a-3p overexpression potently reduced proliferation and induced apoptosis in OS cells, yet strikingly, did not impact normal osteoblasts. To define how these miRNAs regulated OS cell fate, we used an integrated computational approach to identify putative candidate targets and then correlated these with the cell biological impact. Although we could not resolve the mechanism through which miR-148a-3p impacts OS, we identified that miR-155-5p overexpression suppressed its target Ripk1 (receptor (TNFRSF)-interacting serine–threonine kinase 1) expression, and miR-155-5p inhibition elevated Ripk1 levels. Ripk1 is directly involved in apoptosis/necroptosis. In OS cells, small interfering RNA against Ripk1 prevented cell death induced by the sequestration of miR-155-5p. Collectively, we show that miR-148a-3p and miR-155-5p are species-conserved deregulated miRNA in OS. Modulation of these miRNAs was specifically toxic to tumor cells but not normal osteoblasts, raising the possibility that these may be tractable targets for miRNA-based therapies for OS.

Activation of PTHrP-cAMP-CREB1 signaling following p53 loss is essential for osteosarcoma initiation and maintenance

Mutations in the P53 pathway are a hallmark of human cancer. The identification of pathways upon which p53-deficient cells depend could reveal therapeutic targets that may spare normal cells with intact p53. In contrast to P53 point mutations in other cancer, complete loss of P53 is a frequent event in osteosarcoma (OS), the most common cancer of bone. The consequences of p53 loss for osteoblastic cells and OS development are poorly understood. Here we use murine OS models to demonstrate that elevated Pthlh (Pthrp), cAMP levels and signalling via CREB1 are characteristic of both p53-deficient osteoblasts and OS. Normal osteoblasts survive depletion of both PTHrP and CREB1. In contrast, p53-deficient osteoblasts and OS depend upon continuous activation of this pathway and undergo proliferation arrest and apoptosis in the absence of PTHrP or CREB1. Our results identify the PTHrP-cAMP-CREB1 axis as an attractive pathway for therapeutic inhibition in OS. DOI: http://dx.doi.org/10.7554/eLife.13446.001

The SKI proto-oncogene enhances the in vivo repopulation of hematopoietic stem cells and causes myeloproliferative disease

The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced a gene signature associated with hematopoietic stem cells and myeloid differentiation, as well as hepatocyte growth factor signaling. Here we demonstrate that, in contrast to what has generally been assumed, the significant impact of SKI on hematopoiesis is independent of its ability to inhibit TGF-beta signaling. Instead, myeloid progenitors expressing SKI are partially dependent on functional hepatocyte growth factor signaling. Collectively our results demonstrate that SKI is an important regulator of hematopoietic stem cell activity and its overexpression leads to myeloproliferative disease.