A retroviral mutagenesis screen reveals strong cooperation between Bcl11a overexpression and loss of the Nf1 tumor suppressor gene

Yin, Bin; Delwel, Ruud; Valk, Peter J.M.; Wallace, Margaret R.; Loh, Mignon L.; Shannon, Kevin; Largaespada, David A.

doi:10.1182/blood-2008-03-144436

Cited by 51 publications

(55 citation statements)

References 67 publications

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“…These alterations could contribute to the leukemogenic potential of the NKM cells. In light of a recent study showing that overexpression of Bcl11a cooperates with Nf1 deficiency in leukemogenesis, 5 we assessed Bcl11a expression in NKM cells. However, Bcl11a was not increased in NKM cells.…”

Section: Resultsmentioning

confidence: 99%

“…3,4 Both models exhibit growth factor hypersensitivity of hematopoietic cells, splenomegaly, and infiltration of myeloid cells in the liver-but they do not progress to acute leukemia, unless accompanied by other genetic alterations. 5,6 NF1 encodes neurofibromin, a GTPase-activating protein for the RAS proteins. 7 Thus, NF1-deficient cells have increased levels of RAS-GTP that result in hyperactive RAS signaling.…”

Section: Introductionmentioning

confidence: 99%

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Nf1 deficiency cooperates with oncogenic K-RAS to induce acute myeloid leukemia in mice

Cutts

Sjögren

Andersson

et al. 2009

Blood

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Hyperactive RAS signaling is caused by mutations in RAS genes or a deficiency of the neurofibromatosis gene (NF1) and is common in myeloid malignancies. In mice, expression of oncogenic K-RAS or inactivation of Nf1 in hematopoietic cells results in myeloproliferative disorders (MPDs) that do not progress to acute myeloid leukemia (AML IntroductionHyperactive RAS signaling is associated with the development of myeloid malignancies and can be caused by a deficiency in the neurofibromatosis type 1 gene (NF1) or by oncogenic mutations in RAS genes. Children with NF1 deficiency have an increased risk of developing juvenile myelomonocytic leukemia (JMML) and oncogenic mutations in RAS proteins are common in JMML and chronic myelomonocytic leukemia. 1 JMML and chronic myelomonocytic leukemia are aggressive myeloproliferative disorders (MPDs) that can progress to acute myeloid leukemia (AML) but usually only together with other genetic alterations. Thus, hyperactive signaling through the RAS proteins is likely an initiating event in the development of MPD.This reasoning draws support from mouse models of hyperactive RAS signaling: Conditional inactivation of Nf1 in hematopoietic cells produces a fatal MPD with a long latency 2 ; similarly, conditional expression of oncogenic K-RAS in hematopoietic cells produces a fatal MPD, but with a shorter latency. 3,4 Both models exhibit growth factor hypersensitivity of hematopoietic cells, splenomegaly, and infiltration of myeloid cells in the liver-but they do not progress to acute leukemia, unless accompanied by other genetic alterations. 5,6 NF1 encodes neurofibromin, a GTPase-activating protein for the RAS proteins. 7 Thus, NF1-deficient cells have increased levels of RAS-GTP that result in hyperactive RAS signaling. Consequently, NF1 deficiency has been viewed as functionally equivalent to an oncogenic RAS mutation. This is supported by the fact that NF1 and RAS mutations are not found in the same patient. 8 But NF1 may also be involved in other signaling pathways. 9-11 At this point, the impact of Nf1 deficiency in the setting of an oncogenic RAS in bone marrow cells is unknown.The MPD induced by Nf1 deficiency in mice is less severe than the K-RAS-induced MPD. We argued that if Nf1 deficiency was functionally equivalent to an oncogenic RAS, then inactivating Nf1 should have a limited effect on the development of K-RASinduced MPD. To approach this issue, we bred Nf1 conditional knockout mice on a background of the latent oncogenic K-RAS allele and the Mx1-Cre transgene. After Cre recombination in the triple-transgenic mice, we could define the impact of simultaneously inactivating Nf1 and expressing K-RAS G12D in hematopoietic cells. Methods Mouse breeding and in vivo experimentsMice with conditional Nf1 fl alleles 12 (designated N) were bred with Kras2 LSL mice 13 (designated K) to generate NK mice. NK mice were bred with mice harboring the interferon-inducible Mx1-Cre transgene 14 (designated M) to generate NKM mice. NKM mice were compared with littermate NM and KM mice ...

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Nf1 deficiency cooperates with oncogenic K-RAS to induce acute myeloid leukemia in mice

Cutts

Sjögren

Andersson

et al. 2009

Blood

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“…The number of recovered PISs is not only affected by the capacity of APE-PCR, but also determined by the abundance of proviral insertions present in the genomic DNA of leukemia cells derived from BXH-2 strain of mice. This strain of mouse represents a unique animal model to study the mechanisms underlying the development of acute myeloid leukemia (Yin et al 2009). However, one of the key elements, the sufficient isolation of PISs, used to be a challenge.…”

Section: Resultsmentioning

confidence: 99%

“…These PCR methods have been adopted to map a large amount of PISs present in leukemia cells derived from BXH-2 mice (Yin et al 2009). BXH-2 recombinant inbred mice spontaneously produce a B-tropic murine leukemia virus (MuLV) beginning early in life.…”

Section: Introductionmentioning

confidence: 99%

“…The development of methods for the identification of insertion sites of a given mutagen under study has been at the core of the strategy. Among various methods developed, ligation-mediated polymerase chain reaction (PCR)-based approaches, genome walking strategies, have been successful for the investigation of different insertional mutagens, such as retroviruses and transposons Yin et al 2009;Leoni et al 2011;Vassiliou et al 2011;Pérez-Mancera et al 2012).…”

Section: Introductionmentioning

confidence: 99%

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The development of APE-PCR for the cloning of genomic insertion sites of DNA elements

Xu¹,

Li²,

Mao

et al. 2013

Biologia

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Insertional mutagenesis is a productive strategy for the exploration of genetic regulation of important biological and pathological processes, such as tumorigenesis. Successful implementation of this strategy depends heavily on an efficient approach to the identification of insertion sites present in the host genome. Here, we have introduced an easy and efficient protocol, called Adenosine-ended Primer Extension Polymerase Chain Reaction (APE-PCR), which represents several advantages, including the Addition technique we previously developed, primer extension approach coupled with biotin-streptavidin based purification, introduction of nano-scale magnetic particles, and digestion of DNA with a combination of enzymes. We have demonstrated that APE-PCR is able to amplify more and larger specific proviral insertion site (PIS)-derived fragments, with a lower non-specific background produced, fewer steps and less DNA samples required, flexibility in choice of restriction enzymes applied, at a lower cost. Replacement of regular magnetic beads with nano-scale ones in the protocol can further increase its power. Moreover, even with small amount of sample DNA, PISs can be recovered and analyzed. Thus, based on the results provided from this study, we believe that APE-PCR represents an efficient method in mapping of PISs and likely, the insertion sites of other types of DNA elements as well.

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Gene expression profile analysis of SUDHL6 cells with siRNA‐mediated BCL11A downregulation

Gao

Ding

et al. 2014

Cell Biology International

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Our previous study has shown that downregulation of B-cell chronic lymphocytic leukemia (CLL)/lymphoma11A (BCL11A) gene by small interfering RNA (siRNA) resulted in the growth inhibition and apoptosis of B cell lymphoma cell line SUDHL6. To gain further insight into the molecular mechanisms of this process and identify the differentially expressed genes in SUDHL6 cells after BCL11A downregulation, the global gene expression profile was identified and analyzed using the Affymetrix HG-U133 Plus 2.0 array. Twenty-one differentially expressed genes were validated and analyzed from the BCL11A siRNA-treated SUDHL6 cells. There was a significant dysregulation in the global gene expression of the BCL11A-suppressed SUDHL6 cells. There were 1903 genes differentially expressed with >2-fold changes between the BCL11A siRNA- and negative control-transfected cells. Of these, there were 916 upregulated genes and 987 downregulated genes. The differential genes are involved in various molecular functions and signaling pathways. QRT-PCR validation of the selected differentially expressed genes demonstrated there was a good correlation with the microarray analysis. There was a significant deregulation of expression in the apoptosis-related genes such as BCL-2, BCL2L11 and involved in TGFβ, MAPK, WNT signaling pathways after BCL11A was downregulated in SUDHL6 cells. Our results show that the suppression of BCL11A by RNA interference altered gene expression profile of SUDHL6 cells. The apoptosis-related genes BCL-2, BCL2L11 and the gene alterations in TGFβ, MAPK, WNT signaling pathways might be important in BCL11A siRNA-induced apoptosis of SUDHL6 cells, suggesting BCL11A is involved in gene networks associated with apoptosis.

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A retroviral mutagenesis screen reveals strong cooperation between Bcl11a overexpression and loss of the Nf1 tumor suppressor gene

Cited by 51 publications

References 67 publications

Nf1 deficiency cooperates with oncogenic K-RAS to induce acute myeloid leukemia in mice

Nf1 deficiency cooperates with oncogenic K-RAS to induce acute myeloid leukemia in mice

The development of APE-PCR for the cloning of genomic insertion sites of DNA elements

Gene expression profile analysis of SUDHL6 cells with siRNA‐mediated BCL11A downregulation

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