The molecular dynamics of long noncoding RNA control of transcription in PTEN and its pseudogene.

RNA has been found to interact with chromatin and modulate gene transcription. In human cells, little is known about how long noncoding RNAs (lncRNAs) interact with target loci in the context of chromatin. We find here, using the phosphatase and tensin homolog (PTEN) pseudogene as a model system, that antisense lncRNAs interact first with a 5' UTR-containing promoter-spanning transcript, which is then followed by the recruitment of DNA methyltransferase 3a (DNMT3a), ultimately resulting in the transcriptional and epigenetic control of gene expression. Moreover, we find that the lncRNA and promoter-spanning transcript interaction are based on a combination of structural and sequence components of the antisense lncRNA. These observations suggest, on the basis of this one example, that evolutionary pressures may be placed on RNA structure more so than sequence conservation. Collectively, the observations presented here suggest a much more complex and vibrant RNA regulatory world may be operative in the regulation of gene expression.

ClusterSignificance: a bioconductor package facilitating statistical analysis of class cluster separations in dimensionality reduced data.

SUMMARY: Multi-dimensional data generated via high-throughput experiments is increasingly used in conjunction with dimensionality reduction methods to ascertain if resulting separations of the data correspond with known classes. This is particularly useful to determine if a subset of the variables, e.g. genes in a specific pathway, alone can separate samples into these established classes. Despite this, the evaluation of class separations is often subjective and performed via visualization. Here we present the ClusterSignificance package; a set of tools designed to assess the statistical significance of class separations downstream of dimensionality reduction algorithms. In addition, we demonstrate the design and utility of the ClusterSignificance package and utilize it to determine the importance of long non-coding RNA expression in the identity of multiple hematological malignancies. AVAILABILITY AND IMPLEMENTATION: ClusterSignificance is an R package available via Bioconductor (https://bioconductor.org/packages/ClusterSignificance) under GPL-3. CONTACT: dan.grander@ki.se. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Pseudogene-Expressed RNAs: Emerging Roles in Gene Regulation and Disease.

Pseudogenes have for long been considered as non-functional relics littering the human genome. Only now, it is becoming apparent that many pseudogenes are transcribed into long noncoding RNAs, some with proven biological functions. Here, we review the current knowledge of pseudogenes and their widespread functional properties with an emphasis on pseudogenes that have been functionally investigated in greater detail. Pseudogenes are emerging as a novel class of long noncoding RNAs functioning, for example, through microRNA sponging and chromatin remodeling. The examples discussed herein underline that pseudogene-encoded RNAs are important regulatory molecules involved in diseases such as cancer.

The mutational landscape in pediatric acute lymphoblastic leukemia deciphered by whole genome sequencing.

Genomic characterization of pediatric acute lymphoblastic leukemia (ALL) has identified distinct patterns of genes and pathways altered in patients with well-defined genetic aberrations. To extend the spectrum of known somatic variants in ALL, we performed whole genome and transcriptome sequencing of three B-cell precursor patients, of which one carried the t(12;21)ETV6-RUNX1 translocation and two lacked a known primary genetic aberration, and one T-ALL patient. We found that each patient had a unique genome, with a combination of well-known and previously undetected genomic aberrations. By targeted sequencing in 168 patients, we identified KMT2D and KIF1B as novel putative driver genes. We also identified a putative regulatory non-coding variant that coincided with overexpression of the growth factor MDK. Our results contribute to an increased understanding of the biological mechanisms that lead to ALL and suggest that regulatory variants may be more important for cancer development than recognized to date. The heterogeneity of the genetic aberrations in ALL renders whole genome sequencing particularly well suited for analysis of somatic variants in both research and diagnostic applications.

Evolutionary conservation of long non-coding RNAs; sequence, structure, function.

BACKGROUND: Recent advances in genomewide studies have revealed the abundance of long non-coding RNAs (lncRNAs) in mammalian transcriptomes. The ENCODE Consortium has elucidated the prevalence of human lncRNA genes, which are as numerous as protein-coding genes. Surprisingly, many lncRNAs do not show the same pattern of high interspecies conservation as protein-coding genes. The absence of functional studies and the frequent lack of sequence conservation therefore make functional interpretation of these newly discovered transcripts challenging. Many investigators have suggested the presence and importance of secondary structural elements within lncRNAs, but mammalian lncRNA secondary structure remains poorly understood. It is intriguing to speculate that in this group of genes, RNA secondary structures might be preserved throughout evolution and that this might explain the lack of sequence conservation among many lncRNAs. SCOPE OF REVIEW: Here, we review the extent of interspecies conservation among different lncRNAs, with a focus on a subset of lncRNAs that have been functionally investigated. The function of lncRNAs is widespread and we investigate whether different forms of functionalities may be conserved. MAJOR CONCLUSIONS: Lack of conservation does not imbue a lack of function. We highlight several examples of lncRNAs where RNA structure appears to be the main functional unit and evolutionary constraint. We survey existing genomewide studies of mammalian lncRNA conservation and summarize their limitations. We further review specific human lncRNAs which lack evolutionary conservation beyond primates but have proven to be both functional and therapeutically relevant. GENERAL SIGNIFICANCE: Pioneering studies highlight a role in lncRNAs for secondary structures, and possibly the presence of functional "modules", which are interspersed with longer and less conserved stretches of nucleotide sequences. Taken together, high-throughput analysis of conservation and functional composition of the still-mysterious lncRNA genes is only now becoming feasible.

Cell crowding induces interferon regulatory factor 9, which confers resistance to chemotherapeutic drugs.

The mechanism of multicellular drug resistance, defined as the reduced efficacy of chemotherapeutic drugs in solid tumors is incompletely understood. Here we report that colon carcinoma cells cultured as 3D microtissues (spheroids) display dramatic increases in the expression of a subset of type I interferon-(IFN)-stimulated genes (ISGs). A similar gene signature was associated previously with resistance to radiation and chemotherapy, prompting us to examine the underlying biological mechanisms. Analysis of spheroids formed by different tumor cell lines and studies using knock-down of gene expression showed that cell crowding leads to the induction of IFN regulatory factor-9 (IRF9) which together with STAT2 and independently of IFNs, is necessary for ISG upregulation. Increased expression of IRF9 alone was sufficient to induce the ISG subset in monolayer cells and to confer increased resistance to clinically used cytotoxic drugs. Our data reveal a novel mechanism of regulation of a subset of ISGs, leading to drug resistance in solid tumors.

An activated JAK/STAT3 pathway and CD45 expression are associated with sensitivity to Hsp90 inhibitors in multiple myeloma.

The molecular chaperone Hsp90 is required to maintain the activity of many signaling proteins, including members of the JAK/STAT and the PI3K pathways. Inhibitors of Hsp90 (Hsp90-Is) demonstrated varying activity against multiple myeloma (MM) in clinical trials. We aimed to determine which signaling pathways that account for the differential sensitivity to the Hsp90-I 17DMAG on a panel of MM cell lines and freshly obtained MM cells. Three CD45(+) cell lines with an activated JAK/STAT3 pathway were sensitive to 17DMAG and underwent prominent apoptosis upon treatment, while the majority of CD45(-) cell lines, that were dependent on the activated PI3K pathway, were more resistant to the drug. Culturing the most resistant cell line, LP1, in the presence of IL-6 resulted in up-regulation of CD45 and pSTAT3, and sensitized to 17DMAG-induced apoptosis, primarily in the induced CD45(+) sub-population of cells. The high CD45 expressers among primary myeloma cells also expressed significantly higher levels of pSTAT3, as compared to the low CD45 expressers. Ex vivo treatment of primary myeloma cells with 17DMAG resulted in a stronger caspase3 activation in tumor samples with the prevalence of high CD45 expressers. STAT3 activity was efficiently inhibited by Hsp90-Is in both cell lines and primary cells suggesting an importance of STAT3 inactivation for the pro-apoptotic effects of HSP90-Is. Indeed, over-expression of STAT3C, a variant with an increased DNA binding activity, in U266 cells protected them from 17DMAG-induced cell death. The down-regulation of the STAT3 target gene Mcl-1 at both the mRNA and protein levels following 17DMAG treatment was significantly attenuated in STAT3C-expressing cells, and transient over-expression of Mcl-1 protected U266 cells from 17DMAG-induced cell death. The finding that CD45(+) MM cells with an IL-6-activated JAK/STAT3 pathway are particularly sensitive to Hsp90-Is as compared to the low CD45 expressers may provide a rational basis for selection of MM patients amenable to Hsp90-I treatment.

Requirement of apoptotic protease-activating factor-1 for bortezomib-induced apoptosis but not for Fas-mediated apoptosis in human leukemic cells.

Bortezomib is a highly selective inhibitor of the 26S proteasome and has been approved for clinical use in the treatment of relapsing and refractory multiple myeloma and mantle cell lymphoma. Clinical trials are also underway to assess the role of bortezomib in several other human malignancies, including leukemia. However, the mechanism(s) by which bortezomib acts remain to be fully understood. Here, we studied the molecular requirements of bortezomib-induced apoptosis using the human T-cell leukemic Jurkat cells stably transfected with or without shRNA against apoptotic protease-activating factor-1 (Apaf-1). The Apaf-1-deficient Jurkat T cells were resistant to bortezomib-induced apoptosis, as assessed by caspase-3 activity, poly(ADP-ribose) polymerase cleavage, phosphatidylserine externalization, and hypodiploid DNA content. In contrast, Apaf-1-deficient cells were sensitive to Fas-induced apoptosis. Bortezomib induced an upregulation of the pro-apoptotic protein Noxa, loss of mitochondrial transmembrane potential, and release of cytochrome c in cells expressing or not expressing Apaf-1. Transient silencing of Apaf-1 expression in RPMI 8402 T-cell leukemic cells also diminished bortezomib-induced apoptosis. Fas-associated death domain (FADD)-deficient Jurkat cells were resistant to Fas-mediated apoptosis yet remained sensitive to bortezomib. Our results show that bortezomib induces apoptosis by regulating pathways that are mechanistically different from those activated upon death receptor ligation. Furthermore, in silico analyses of public transcriptomics databases indicated elevated Apaf-1 expression in several hematologic malignancies, including acute lymphoblastic and myeloid leukemia. We also noted variable Apaf-1 expression in a panel of samples from patients with acute lymphoblastic leukemia. Our results suggest that the expression of Apaf-1 may be predictive of the response to proteasome inhibition.

MicroRNA-125b down-regulates matrix metallopeptidase 13 and inhibits cutaneous squamous cell carcinoma cell proliferation, migration, and invasion.

Cutaneous squamous cell carcinoma (cSCC) is the second most common human cancer. Although dysregulation of microRNAs (miRNAs) is known to be involved in a variety of cancers, the role of miRNAs in cSCC is unclear. In this study, we aimed to identify tumor suppressive and oncogenic miRNAs involved in the pathogenesis of cSCC. MiRNA expression profiles in healthy skins (n = 4) and cSCCs (n = 4) were analyzed using MicroRNA Low Density Array. MiR-125b expression was analyzed by quantitative real-time PCR and in situ hybridization in skin biopsies from 40 healthy donors, 13 actinic keratosis, and 74 cSCC patients. The effect of miR-125b was analyzed in wound closure, colony formation, migration, and invasion assays in two cSCC cell lines, UT-SCC-7 and A431. The genes regulated by miR-125b in cSCC were identified by microarray analysis and its direct target was validated by luciferase reporter assay. Comparing cSCC with healthy skin, we identified four up-regulated miRNAs (miR-31, miR-135b, miR-21, and miR-223) and 54 down-regulated miRNAs, including miR-125b, whose function was further examined. We found that miR-125b suppressed proliferation, colony formation, migratory, and invasive capacity of cSCC cells. Matrix metallopeptidase 13 (MMP13) was identified as a direct target suppressed by miR-125b, and there was an inverse relationship between the expression of miR-125b and MMP13 in cSCC. Knockdown of MMP13 expression phenocopied the effects of miR-125b overexpression. These findings provide a novel molecular mechanism by which MMP13 is up-regulated in cSCCs and indicate that miR-125b plays a tumor suppressive role in cSCC.

TRIM13 (RFP2) downregulation decreases tumour cell growth in multiple myeloma through inhibition of NF Kappa B pathway and proteasome activity.

Multiple myeloma (MM) is an incurable neoplasm caused by proliferation of malignant plasma cells in the bone marrow (BM). MM is characterized frequently by a complete or partial deletion of chromosome 13q14, seen in more than 50% of patients at diagnosis. Within this deleted region the tripartite motif containing 13 (TRIM13, also termed RFP2) gene product has been proposed to be a tumour suppressor gene (TSG). Here, we show that low expression levels of TRIM13 in MM are associated with chromosome 13q deletion and poor clinical outcome. We present a functional analysis of TRIM13 using a loss-of-function approach, and demonstrate that TRIM13 downregulation decreases tumour cell survival as well as cell cycle progression and proliferation of MM cells. In addition, we provide evidence for the involvement of TRIM13 downregulation in inhibiting the NF kappa B pathway and the activity of the 20S proteasome. Although this data does not support a role of TRIM13 as a TSG, it substantiates important roles of TRIM13 in MM tumour survival and proliferation, underscoring its potential role as a novel target for therapeutic intervention.

Activation of STAT1 is required for interferon-alpha-mediated cell death.

Interferon-alpha (IFNα)-induced cell death of tumor cells is likely mediated through several signaling pathways. We previously demonstrated that blocking the activation of phosphoinositide-3-kinase, PI3K, or mammalian target of rapamycin, mTOR, partially inhibited apoptosis induced by IFNα. Here, we postulate using pharmacological inhibition and dominant negative mutants that activation of signal transducer and activator of transcription-1, STAT1, is also required for the cell death induced by IFNα. Inhibition of STAT1 tyrosine phosphorylation and DNA binding by a naturally occurring rotenoid deguelin also rescued U266 myeloma cell lines from IFNα-induced apoptosis. Deguelin had no effect on upstream Jak kinases or STAT2 phosphorylation suggesting the involvement of a yet unknown mechanism. Inhibition of STAT1 tyrosine phosphorylation and activity was independent of the known effects of deguelin on PI3K, Akt or mTOR as shown using selective pharmacological inhibitors against these kinases. The combination of deguelin and PI3K or mTOR antagonists further inhibited apoptosis suggesting that both the Jak-STAT and the PI3K/mTOR pathways contribute to the induction of apoptosis by IFNα in these cells. Over-expression of STAT1-Y701A or K410/413A mutants in Rhek-1 keratinocytes largely inhibited apoptosis further supporting the importance of STAT1 phosphorylation and activity for IFNα-induced cell death. Thus, at least two signaling pathways, one of which requires STAT1 activation, cooperate to mediate IFNα-induced apoptosis.

The p53 target Wig-1 regulates p53 mRNA stability through an AU-rich element.

The p53 target gene Wig-1 encodes a double-stranded-RNA-binding zinc finger protein. We show here that Wig-1 binds to p53 mRNA and stabilizes it through an AU-rich element (ARE) in the 3' UTR of the p53 mRNA. This effect is mirrored by enhanced p53 protein levels in both unstressed cells and cells exposed to p53-activating stress agents. Thus, the p53 target Wig-1 is a previously undescribed ARE-regulating protein that acts as a positive feedback regulator of p53, with implications both for the steady-state levels of p53 and for the p53 stress response. Our data reveal a previously undescribed link between the tumor suppressor p53 and posttranscriptional gene regulation via AREs in mRNA.

PTENP1-AS contributes to BRAF inhibitor resistance and is associated with adverse clinical outcome in stage III melanoma.

BRAF inhibitors (BRAFi) selectively target oncogenic BRAFV600E/K and are effective in 80% of advanced cutaneous malignant melanoma cases carrying the V600 mutation. However, the development of drug resistance limits their clinical efficacy. Better characterization of the underlying molecular processes is needed to further improve treatments. We previously demonstrated that transcription of PTEN is negatively regulated by the PTEN pseudogene antisense RNA, PTENP1-AS, and here we investigated the impact of this transcript on clinical outcome and BRAFi resistance in melanoma. We observed that increased expression levels of PTENP1-AS in BRAFi resistant cells associated with enrichment of EZH2 and H3K27me3 at the PTEN promoter, consequently reducing the expression levels of PTEN. Further, we showed that targeting of the PTENP1-AS transcript sensitized resistant cells to BRAFi treatment and that high expression of PTENP1-AS in stage III melanoma correlated with poor survival. Collectively, the data presented here show that PTENP1-AS is a promising target for re-sensitizing cells to BRAFi and also a possible prognostic marker for clinical outcome in stage III melanoma.

Inactivation of FBXW7/hCDC4-ß expression by promoter hypermethylation is associated with favorable prognosis in primary breast cancer.

INTRODUCTION: Mutational inactivation of the FBXW7/hCDC4 tumor suppressor gene (TSG) is common in many cancer types, but infrequent in breast cancers. This study investigates the presence and impact of FBXW7/hCDC4 promoter methylation in breast cancer. METHODS: FBXW7/hCDC4-ß expression and promoter methylation was assessed in 161 tumors from two independent breast cancer cohorts. Associations between methylation status and clinicopathologic characteristics were assessed by Fisher's exact test. Survival was analyzed using the Kaplan-Meier method in addition to modeling the risk by use of a multivariate proportional hazard (Cox) model adjusting for possible confounders of survival. RESULTS: Methylation of the promoter and loss of mRNA expression was found both in cell lines and primary tumors (43% and 51%, respectively). Using Cox modeling, a trend was found towards decreased hazard ratio (HR) for death in women with methylation of FBXW7/hCDC4-ß in both cohorts (HR 0.53 (95% CI 0.23 to 1.23) and HR 0.50 (95% CI 0.23 to 1.08), respectively), despite an association between methylation and high-grade tumors (P = 0.017). Interestingly, in subgroups of patients whose tumors are p53 mutated or lymph-node positive, promoter methylation identified patients with significantly improved survival (P = 0.048 and P = 0.017, respectively). CONCLUSIONS: We demonstrate an alternative mechanism for inactivation of the TSG FBXW7/hCDC4, namely promoter specific methylation. Importantly, in breast cancer, methylation of FBXW7/hCDC4-ß is related to favorable prognosis despite its association with poorly differentiated tumors. Future work may define whether FBXW7/hCDC4 methylation is a biomarker of the response to chemotherapy and a target for epigenetic modulation therapy.

Characterisation of hairy cell leukaemia by tiling resolution array-based comparative genome hybridisation: a series of 13 cases and review of the literature.

Little is known about the cytogenetic features and molecular mechanisms behind hairy cell leukaemia (HCL), despite the advances in diagnosis and treatment. Therefore, we used high-resolution genome-wide array-based comparative genomic hybridisation (array-CGH) and multiplex ligation-dependent probe amplification (MLPA) to characterise copy number alterations (CNAs) in DNA from 13 cases of HCL. We also summarise CNAs and cytogenetic features in 109 HCL cases comprising our 13 cases and 96 cases from the literature. Genomic array-CGH revealed imbalances in two out of 13 cases in addition to previously described copy number variants (CNVs) found in healthy individuals. In one case, a 700 kb deletion of 20q11.22 was detected encompassing ten characterised genes, among them the TP53INP2, DNCL2A and ITCH genes. In the second case, trisomy 5, and a deletion of 5p15.2 encompassing a non-characterised gene AY328033 was found. Altogether only 20/81 (25%) of all cases studied by CGH or gene dose array revealed CNAs. The most common recurrent deletions and breakpoints were 14q22-32 (33%), 6q25 (16%), 2p12 (10%), 22q11 (10%), 17p11-13 (10%), 7q32-36 (9%), 18q11-13 (7%), 1q32-44 (6%), 8p22-23 (6%) and 7q11 (6%). Trisomy 5 occurred in 15%. In addition, several other recurrent breakpoints were identified. Although a number of genomic imbalances were identified in the HCL samples, the genome appeared remarkably stable.

DLEU2, frequently deleted in malignancy, functions as a critical host gene of the cell cycle inhibitory microRNAs miR-15a and miR-16-1.

The microRNAs miR-15a and miR-16-1 are downregulated in multiple tumor types and are frequently deleted in chronic lymphocytic leukemia (CLL), myeloma and mantle cell lymphoma. Despite their abundance in most cells the transcriptional regulation of miR-15a/16-1 remains unclear. Here we demonstrate that the putative tumor suppressor DLEU2 acts as a host gene of these microRNAs. Mature miR-15a/miR-16-1 are produced in a Drosha-dependent process from DLEU2 and binding of the Myc oncoprotein to two alterative DLEU2 promoters represses both the host gene transcript and levels of mature miR-15a/miR-16-1. In line with a functional role for DLEU2 in the expression of the microRNAs, the miR-15a/miR-16-1 locus is retained in four CLL cases that delete both promoters of this gene and expression analysis indicates that this leads to functional loss of mature miR-15a/16-1. We additionally show that DLEU2 negatively regulates the G1 Cyclins E1 and D1 through miR-15a/miR-16-1 and provide evidence that these oncoproteins are subject to miR-15a/miR-16-1-mediated repression under normal conditions. We also demonstrate that DLEU2 overexpression blocks cellular proliferation and inhibits the colony-forming ability of tumor cell lines in a miR-15a/miR-16-1-dependent way. Together the data illuminate how inactivation of DLEU2 promotes cell proliferation and tumor progression through functional loss of miR-15a/miR-16-1.

The RBCC gene RFP2 (Leu5) encodes a novel transmembrane E3 ubiquitin ligase involved in ERAD.

RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-delta, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.

Silencing of CEBPB-AS1 modulates CEBPB expression and resensitizes BRAF-inhibitor resistant melanoma cells to vemurafenib.

Introduction of targeted therapy in the treatment of metastatic cutaneous malignant melanoma (CMM) has improved clinical outcome during the last years. However, only in a subset of the CMM patients, this will lead to long-term effects. CEBPB is a transcription factor that has been implicated in various physiological and pathological processes, including cancer development. We have investigated its prognostic impact on CMM and unexpectedly found that higher CEBPB mRNA levels correlated with a longer overall survival. Furthermore, in a small cohort of patients with metastatic CMM treated with BRAF-inhibitors, higher levels of CEBPB mRNA expression in the tumor cells prior treatment correlated to a longer progression-free survival. We have characterized an overlapping antisense transcript, CEBPB-AS1, with the aim to investigate the regulation of CEBPB expression in CMM and its impact on BRAF-inhibitor sensitivity. We demonstrated that silencing of CEBPB-AS1 resulted in epigenetic modifications in the CEBPB promoter and in increased CEBPB mRNA and protein levels, inhibited proliferation and partially resensitized BRAF-inhibitor resistant CMM cells to this drug-induced apoptosis. Our data suggest that targeting CEBPB-AS1 may represent a valuable tool to sensitize CMM cells to the BRAF-inhibitor-based therapies.

Ribonucleotide reductase inhibitors suppress SAMHD1 ara-CTPase activity enhancing cytarabine efficacy.

The deoxycytidine analogue cytarabine (ara-C) remains the backbone treatment of acute myeloid leukaemia (AML) as well as other haematological and lymphoid malignancies, but must be combined with other chemotherapeutics to achieve cure. Yet, the underlying mechanism dictating synergistic efficacy of combination chemotherapy remains largely unknown. The dNTPase SAMHD1, which regulates dNTP homoeostasis antagonistically to ribonucleotide reductase (RNR), limits ara-C efficacy by hydrolysing the active triphosphate metabolite ara-CTP. Here, we report that clinically used inhibitors of RNR, such as gemcitabine and hydroxyurea, overcome the SAMHD1-mediated barrier to ara-C efficacy in primary blasts and mouse models of AML, displaying SAMHD1-dependent synergy with ara-C. We present evidence that this is mediated by dNTP pool imbalances leading to allosteric reduction of SAMHD1 ara-CTPase activity. Thus, SAMHD1 constitutes a novel biomarker for combination therapies of ara-C and RNR inhibitors with immediate consequences for clinical practice to improve treatment of AML.

The tumor suppressor gene hCDC4 is frequently mutated in human T-cell acute lymphoblastic leukemia with functional consequences for Notch signaling.

Notch signaling is of crucial importance in normal T-cell development and Notch 1 is frequently mutated in T-cell acute lymphoblastic leukemias (T-ALL), leading to aberrantly high Notch signaling. In this report, we determine whether T-ALL mutations occur not only in Notch1 but also in the F-box protein hCdc4 (Sel-10, Ago, or Fbxw7), a negative regulator of Notch1. We show that the hCDC4 gene is mutated in leukemic cells from more than 30% of patients with pediatric T-ALL and derived cell lines. Most hCDC4 mutations found were missense substitutions at critical arginine residues (Arg(465), Arg(479), and Arg(505)) localized in the substrate-binding region of hCdc4. Cells inactivated for hCdc4 and T-ALL cells containing hCDC4 mutations exhibited an increased Notch1 protein half-life, consistent with the proposed role of hCdc4 in ubiquitin-dependent proteolysis of Notch1. Furthermore, restoration of wild-type but not mutant hCdc4 in HCT 116 hCDC4-negative cells led to an increased Notch1 ubiquitylation and decreased Notch1 signaling. These results show that hCdc4 mutations interfere with normal Notch1 regulation in vivo. Finally, we found that mutations in hCDC4 and NOTCH1 can occur in the same cancers and that patients carrying hCDC4 and/or NOTCH1 mutations have a favorable overall survival. Collectively, these data show that mutation of hCDC4 is a frequent event in T-ALL and suggest that hCDC4 mutations and gain-of-function mutations in NOTCH1 might synergize in contributing to the development of pediatric T-ALL leukemogenesis.