Over-expression of ADAR1 in mice does not initiate or accelerate cancer formation in vivo.

Adenosine to inosine editing (A-to-I) in regions of double stranded RNA (dsRNA) is mediated by adenosine deaminase acting on RNA 1 (ADAR1) or ADAR2. ADAR1 and A-to-I editing levels are increased in many human cancers. Inhibition of ADAR1 has emerged as a high priority oncology target, however, whether ADAR1 overexpression enables cancer initiation or progression has not been directly tested. We established a series of in vivo models to allow overexpression of full-length ADAR1, or its individual isoforms, to test if increased ADAR1 expression was oncogenic. Widespread over-expression of ADAR1 or the p110 or p150 isoforms individually as sole lesions was well tolerated and did not result in cancer initiation. Therefore, ADAR1 overexpression alone is not sufficient to initiate cancer. We demonstrate that endogenous ADAR1 and A-to-I editing increased upon immortalization in murine cells, consistent with the observations from human cancers. We tested if ADAR1 over-expression could co-operate with cancer initiated by loss of tumour suppressors using a model of osteosarcoma. We did not see a disease potentiating or modifying effect of overexpressing ADAR1 or its isoforms in the models assessed. We conclude that increased ADAR1 expression and A-to-I editing in cancers is most likely a consequence of tumor formation.

The phenotype of the most common human ADAR1p150 Zα mutation P193A in mice is partially penetrant.

ADAR1 -mediated A-to-I RNA editing is a self-/non-self-discrimination mechanism for cellular double-stranded RNAs. ADAR mutations are one cause of Aicardi-Goutières Syndrome, an inherited paediatric encephalopathy, classed as a "Type I interferonopathy." The most common ADAR1 mutation is a proline 193 alanine (p.P193A) mutation, mapping to the ADAR1p150 isoform-specific Zα domain. Here, we report the development of an independent murine P195A knock-in mouse, homologous to human P193A. The Adar1P195A/P195A mice are largely normal and the mutation is well tolerated. When the P195A mutation is compounded with an Adar1 null allele (Adar1P195A/- ), approximately half the animals are runted with a shortened lifespan while the remaining Adar1P195A/- animals are normal, contrasting with previous reports. The phenotype of the Adar1P195A/- animals is both associated with the parental genotype and partly non-genetic/environmental. Complementation with an editing-deficient ADAR1 (Adar1P195A/E861A ), or the loss of MDA5, rescues phenotypes in the Adar1P195A/- mice.

Srsf2P95H/+ co-operates with loss of TET2 to promote myeloid bias and initiate a chronic myelomonocytic leukemia-like disease in mice.

Recurrent mutations in RNA splicing proteins and epigenetic regulators contribute to the development of myelodysplastic syndrome (MDS) and related myeloid neoplasms. In chronic myelomonocytic leukemia (CMML), SRSF2 mutations occur in ~50% of patients and TET2 mutations in ~60%. Clonal analysis indicates that either mutation can arise as the founder lesion. Based on human cancer genetics we crossed an inducible Srsf2P95H/+ mutant model with Tet2fl/fl mice to mutate both concomitantly in hematopoietic stem cells. At 20-24 weeks post mutation induction, we observed subtle differences in the Srsf2/Tet2 mutants compared to either single mutant. Under conditions of native hematopoiesis with aging, we see a distinct myeloid bias and monocytosis in the Srsf2/Tet2 mutants. A subset of the compound Srsf2/Tet2 mutants display an increased granulocytic and distinctive monocytic proliferation (myelomonocytic hyperplasia), with increased immature promonocytes and monoblasts and binucleate promonocytes. Exome analysis of progressed disease demonstrated mutations in genes and pathways similar to those reported in human CMML. Upon transplantation, recipients developed leukocytosis, monocytosis, and splenomegaly. We reproduce Srsf2/Tet2 co-operativity in vivo, yielding a disease with core characteristics of CMML, unlike single Srsf2 or Tet2 mutation. This model represents a significant step toward building high fidelity and genetically tractable models of CMML.

Genome-wide screening identifies cell-cycle control as a synthetic lethal pathway with SRSF2P95H mutation.

Current strategies to target RNA splicing mutant myeloid cancers proposes targeting the remaining splicing apparatus. This approach has only been modestly sensitizing and is also toxic to non-mutant-bearing wild-type cells. To explore potentially exploitable genetic interactions with spliceosome mutations, we combined data mining and functional screening for synthetic lethal interactions with an Srsf2P95H/+ mutation. Analysis of missplicing events in a series of both human and murine SRSF2P95H mutant samples across multiple myeloid diseases (acute myeloid leukemia, myelodysplastic syndromes, chronic myelomonocytic leukemia) was performed to identify conserved missplicing events. From this analysis, we identified that the cell-cycle and DNA repair pathways were overrepresented within the conserved misspliced transcript sets. In parallel, to functionally define pathways essential for survival and proliferation of Srsf2P95H/+ cells, we performed a genome-wide Clustered regularly interspaced short palindromic repeat loss-of-function screen using Hoxb8 immortalized R26-CreERki/+Srsf2P95H/+ and R26-CreERki/+Srsf2+/+ cell lines. We assessed loss of single guide RNA representation at 3 timepoints: immediately after Srsf2P95H/+ activation, and at 1 week and 2 weeks after Srsf2P95H/+ mutation. Pathway analysis demonstrated that the cell-cycle and DNA damage response pathways were among the top synthetic lethal pathways with Srsf2P95H/+ mutation. Based on the loss of guide RNAs targeting Cdk6, we identified that palbociclib, a CDK6 inhibitor, showed preferential sensitivity in Srsf2P95H/+ cell lines and in primary nonimmortalized lin-cKIT+Sca-1+ cells compared with wild-type controls. Our data strongly suggest that the cell-cycle and DNA damage response pathways are required for Srsf2P95H/+ cell survival, and that palbociclib could be an alternative therapeutic option for targeting SRSF2 mutant cancers.

The characterization of distinct populations of murine skeletal cells that have different roles in B lymphopoiesis.

Hematopoiesis is extrinsically controlled by cells of the bone marrow microenvironment, including skeletal lineage cells. The identification and subsequent studies of distinct subpopulations of maturing skeletal cells is currently limited because of a lack of methods to isolate these cells. We found that murine Lin-CD31-Sca-1-CD51+ cells can be divided into 4 subpopulations by using flow cytometry based on their expression of the platelet-derived growth factor receptors ⍺ and ß (PDGFR⍺ and PDGFRß). The use of different skeletal lineage reporters confirmed the skeletal origin of the 4 populations. Multiplex immunohistochemistry studies revealed that all 4 populations were localized near the growth plate and trabecular bone and were rarely found near cortical bone regions or in central bone marrow. Functional studies revealed differences in their abundance, colony-forming unit-fibroblast capacity, and potential to differentiate into mineralized osteoblasts or adipocytes in vitro. Furthermore, the 4 populations had distinct gene expression profiles and differential cell surface expression of leptin receptor (LEPR) and vascular cell adhesion molecule 1 (VCAM-1). Interestingly, we discovered that 1 of these 4 different skeletal populations showed the highest expression of genes involved in the extrinsic regulation of B lymphopoiesis. This cell population varied in abundance between distinct hematopoietically active skeletal sites, and significant differences in the proportions of B-lymphocyte precursors were also observed in these distinct skeletal sites. This cell population also supported pre-B lymphopoiesis in culture. Our method of isolating 4 distinct maturing skeletal populations will help elucidate the roles of distinct skeletal niche cells in regulating hematopoiesis and bone.

Hematopoietic stem and progenitor cell-restricted Cdx2 expression induces transformation to myelodysplasia and acute leukemia.

The caudal-related homeobox transcription factor CDX2 is expressed in leukemic cells but not during normal blood formation. Retroviral overexpression of Cdx2 induces AML in mice, however the developmental stage at which CDX2 exerts its effect is unknown. We developed a conditionally inducible Cdx2 mouse model to determine the effects of in vivo, inducible Cdx2 expression in hematopoietic stem and progenitor cells (HSPCs). Cdx2-transgenic mice develop myelodysplastic syndrome with progression to acute leukemia associated with acquisition of additional driver mutations. Cdx2-expressing HSPCs demonstrate enrichment of hematopoietic-specific enhancers associated with pro-differentiation transcription factors. Furthermore, treatment of Cdx2 AML with azacitidine decreases leukemic burden. Extended scheduling of low-dose azacitidine shows greater efficacy in comparison to intermittent higher-dose azacitidine, linked to more specific epigenetic modulation. Conditional Cdx2 expression in HSPCs is an inducible model of de novo leukemic transformation and can be used to optimize treatment in high-risk AML.

The majority of A-to-I RNA editing is not required for mammalian homeostasis.

BACKGROUND: Adenosine-to-inosine (A-to-I) RNA editing, mediated by ADAR1 and ADAR2, occurs at tens of thousands to millions of sites across mammalian transcriptomes. A-to-I editing can change the protein coding potential of a transcript and alter RNA splicing, miRNA biology, RNA secondary structure and formation of other RNA species. In vivo, the editing-dependent protein recoding of GRIA2 is the essential function of ADAR2, while ADAR1 editing prevents innate immune sensing of endogenous RNAs by MDA5 in both human and mouse. However, a significant proportion of A-to-I editing sites can be edited by both ADAR1 and ADAR2, particularly within the brain where both are highly expressed. The physiological function(s) of these shared sites, including those evolutionarily conserved, is largely unknown. RESULTS: To generate completely A-to-I editing-deficient mammals, we crossed the viable rescued ADAR1-editing-deficient animals (Adar1E861A/E861AIfih1-/-) with rescued ADAR2-deficient (Adarb1-/-Gria2R/R) animals. Unexpectedly, the global absence of editing was well tolerated. Adar1E861A/E861AIfih1-/-Adarb1-/-Gria2R/R were recovered at Mendelian ratios and age normally. Detailed transcriptome analysis demonstrated that editing was absent in the brains of the compound mutants and that ADAR1 and ADAR2 have similar editing site preferences and patterns. CONCLUSIONS: We conclude that ADAR1 and ADAR2 are non-redundant and do not compensate for each other's essential functions in vivo. Physiologically essential A-to-I editing comprises a small subset of the editome, and the majority of editing is dispensable for mammalian homeostasis. Moreover, in vivo biologically essential protein recoding mediated by A-to-I editing is an exception in mammals.

Defining the functions of adenosine-to-inosine RNA editing through hematology.

PURPOSE OF REVIEW: The direct modification of RNA is now understood to be widespread, evolutionarily conserved and of consequence to cellular and organismal homeostasis. adenosine-to-inosine (A-to-I) RNA editing is one of the most common mammalian RNA modifications. Transcriptome-wide maps of the A-to-I editing exist, yet functions for the majority of editing sites remain opaque. Herein we discuss how hematology has been applied to determine physiological and malignant functions of A-to-I editing. RECENT FINDINGS: Functional studies have established that A-to-I editing and ADAR1, responsible for the majority of editing in blood cells, are essential for normal blood cell homeostasis. ADAR1 edits endogenous RNA and reshapes its secondary structure, preventing MDA5 from perceiving the cells own RNA as pathogenic. Roles for ADAR1 in human leukaemia, and most recently, cancer cell intrinsic and extrinsic functions of ADAR1 have been identified that highlight ADAR1 as a therapeutic target in cancer. SUMMARY: The studies reviewed have identified the key physiological function of ADAR1 and mechanistic basis for A-to-I editing in normal physiology and have now been extended to cancer. As our understanding of the biology and consequences of A-to-I editing evolve, it may be possible to target ADAR1 function advantageously in a number of settings.

Srsf2P95H initiates myeloid bias and myelodysplastic/myeloproliferative syndrome from hemopoietic stem cells.

Mutations in SRSF2 occur in myelodysplastic syndromes (MDS) and MDS/myeloproliferative neoplasms (MPN). SRSF2 mutations cluster at proline 95, with the most frequent mutation being a histidine (P95H) substitution. They undergo positive selection, arise early in the course of disease, and have been identified in age-related clonal hemopoiesis. It is not clear how mutation of SRSF2 modifies hemopoiesis or contributes to the development of myeloid bias or MDS/MPN. Two prior mouse models of Srsf2P95H mutation have been reported; however, these models do not recapitulate many of the clinical features of SRSF2-mutant disease and relied on bone marrow (BM) transplantation stress to elicit the reported phenotypes. We describe a new conditional murine Srsf2P95H mutation model, where the P95H mutation is expressed physiologically and heterozygously from its endogenous locus after Cre activation. Using multiple Cre lines, we demonstrate that during native hemopoiesis (ie, no BM transplantation), the Srsf2P95H mutation needs to occur within the hemopoietic stem-cell-containing populations to promote myelomonocytic bias and expansion with corresponding transcriptional and RNA splicing changes. With age, nontransplanted Srsf2P95H animals developed a progressive, transplantable disease characterized by myeloid bias, morphological dysplasia, and monocytosis, hallmarks of MDS/MPN in humans. Analysis of cooccurring mutations within the BM demonstrated the acquisition of additional mutations that are recurrent in humans with SRSF2 mutations. The tractable Srsf2P95H/+ knock-in model we have generated is highly relevant to human disease and will serve to elucidate the effect of SRSF2 mutations on initiation and maintenance of MDS/MPN.

Protein recoding by ADAR1-mediated RNA editing is not essential for normal development and homeostasis.

BACKGROUND: Adenosine-to-inosine (A-to-I) editing of dsRNA by ADAR proteins is a pervasive epitranscriptome feature. Tens of thousands of A-to-I editing events are defined in the mouse, yet the functional impact of most is unknown. Editing causing protein recoding is the essential function of ADAR2, but an essential role for recoding by ADAR1 has not been demonstrated. ADAR1 has been proposed to have editing-dependent and editing-independent functions. The relative contribution of these in vivo has not been clearly defined. A critical function of ADAR1 is editing of endogenous RNA to prevent activation of the dsRNA sensor MDA5 (Ifih1). Outside of this, how ADAR1 editing contributes to normal development and homeostasis is uncertain. RESULTS: We describe the consequences of ADAR1 editing deficiency on murine homeostasis. Adar1 E861A/E861A Ifih1 -/- mice are strikingly normal, including their lifespan. There is a mild, non-pathogenic innate immune activation signature in the Adar1 E861A/E861A Ifih1 -/- mice. Assessing A-to-I editing across adult tissues demonstrates that outside of the brain, ADAR1 performs the majority of editing and that ADAR2 cannot compensate in its absence. Direct comparison of the Adar1 -/- and Adar1 E861A/E861A alleles demonstrates a high degree of concordance on both Ifih1 +/+ and Ifih1 -/- backgrounds, suggesting no substantial contribution from ADAR1 editing-independent functions. CONCLUSIONS: These analyses demonstrate that the lifetime absence of ADAR1-editing is well tolerated in the absence of MDA5. We conclude that protein recoding arising from ADAR1-mediated editing is not essential for organismal homeostasis. Additionally, the phenotypes associated with loss of ADAR1 are the result of RNA editing and MDA5-dependent functions.

Adenosine-to-inosine RNA editing by ADAR1 is essential for normal murine erythropoiesis.

Adenosine deaminases that act on RNA (ADARs) convert adenosine residues to inosine in double-stranded RNA. In vivo, ADAR1 is essential for the maintenance of hematopoietic stem/progenitors. Whether other hematopoietic cell types also require ADAR1 has not been assessed. Using erythroid- and myeloid-restricted deletion of Adar1, we demonstrate that ADAR1 is dispensable for myelopoiesis but is essential for normal erythropoiesis. Adar1-deficient erythroid cells display a profound activation of innate immune signaling and high levels of cell death. No changes in microRNA levels were found in ADAR1-deficient erythroid cells. Using an editing-deficient allele, we demonstrate that RNA editing is the essential function of ADAR1 during erythropoiesis. Mapping of adenosine-to-inosine editing in purified erythroid cells identified clusters of hyperedited adenosines located in long 3'-untranslated regions of erythroid-specific transcripts and these are ADAR1-specific editing events. ADAR1-mediated RNA editing is essential for normal erythropoiesis.

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.

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.

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

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 (Adar1(E861A), where E861A denotes Glu(861)→Ala(861)). Adar1(E861A/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 Adar1(E861A/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.

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.

Wnt inhibitory factor 1 (WIF1) is a marker of osteoblastic differentiation stage and is not silenced by DNA methylation in osteosarcoma.

Wnt pathway targeting is of high clinical interest for treating bone loss disorders such as osteoporosis. These therapies inhibit the action of negative regulators of osteoblastic Wnt signaling. The report that Wnt inhibitory factor 1 (WIF1) was epigenetically silenced via promoter DNA methylation in osteosarcoma (OS) raised potential concerns for such treatment approaches. Here we confirm that Wif1 expression is frequently reduced in OS. However, we demonstrate that silencing is not driven by DNA methylation. Treatment of mouse and human OS cells showed that Wif1 expression was robustly induced by HDAC inhibition but not by methylation inhibition. Consistent with HDAC dependent silencing, the Wif1 locus in OS was characterized by low acetylation levels and a bivalent H3K4/H3K27-trimethylation state. Wif1 expression marked late stages of normal osteoblast maturation and stratified OS tumors based on differentiation stage across species. Culture of OS cells under differentiation inductive conditions increased expression of Wif1. Together these results demonstrate that Wif1 is not targeted for silencing by DNA methylation in OS. Instead, the reduced expression of Wif1 in OS cells is in context with their stage in differentiation.

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.

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.

Gene expression profiling to define the cell intrinsic role of the SKI proto-oncogene in hematopoiesis and myeloid neoplasms.

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 gene signatures associated with hematopoietic stem cells and myeloid differentiation. Here we provide detailed experimental methods and analysis for the gene expression profiling described in our recently published study of Singbrant et al. (2014) in Haematologica. Our data sets (available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE39457) provide a resource for exploring the underlying molecular mechanisms of the involvement of the proto-oncogene SKI in hematopoietic stem cell function and development of myeloid neoplasms.