Six neoplastically-derived cell lines and three cell lines derived from normal tissues were compared for their sensitivity to isoprenoid trans-trans farnesol. Assays of cell numbers and of protein ...concentrations per culture revealed greater sensitivity of neoplastic cells than of the normal cells. Similar differences were obtained from the comparison of incorporation of methyl-3Hcholine into cellular lipids, with neoplastic cells showing greater inhibition than normal cells.
Abstract only
Chromosomal translocations of the mixed‐lineage leukemia (
MLL
) gene with various partner genes result in aggressive MLL leukemia with dismal outcomes. Despite relatively equal ...contributions of the rearranged allele and the remaining wild‐type allele at the mRNA level, the MLL chimeric protein is much more stable than the wild‐type MLL protein. Here, we used Multidimensional Protein Identification Technology (MudPIT) and established a pooled genome‐wide shRNA library screen to identify the factors and pathways involved in regulating the stability of the wild‐type MLL protein. Targeting wild‐type MLL degradation, through blocking of these pathways and factors, preferentially impedes MLL leukemia cell proliferation and downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the Super Elongation Complex (SEC), as revealed by RNA sequencing and Chromatin Immunoprecipitation Sequencing (ChIP‐Seq). Stabilizing wild‐type MLL protein by inhibition of these pathways displaces the MLL chimera from some of its target genes and therefore relieves the cellular oncogenic addiction to MLL chimeras. Furthermore, pharmacologically inhibition of these pathways with small molecules substantially delays progression and improves survival of murine MLL‐AF9 leukemia in vivo. Therefore, disrupting the balance between wild‐type MLL and MLL chimeras by stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers driven by chromosomal translocations.
Support or Funding Information
This study was supported by the Samuel Waxman Cancer Research Foundation to John D. Crispino and National Institute of Health grants, CA211428 to Edwin R. Smith, CA117907 to Joaquin M. Espinosa, CA101774 to John D. Crispino. and R35CA197569 to Ali Shilatifard
Acute leukemia cells of the established line CEM-C1 were treated during growth in serum-free medium with various concentrations of trans-trans farnesol. At concentrations ranging from 9.0 to 31.5 ...microM, farnesol inhibited growth of these cells without causing cell lysis. This effect was preceded by very rapid inhibition of choline incorporation in cellular lipid fraction. The growth inhibitory effect was prevented to a large extent by incubation with phosphatidylcholine or diacylglycerol.
The epigenetic regulation of imprinted genes by monoallelic DNA methylation of either maternal or paternal alleles is critical for embryonic growth and development. Imprinted genes were recently ...shown to be expressed in mammalian adult stem cells to support self-renewal of neural and lung stem cells; however, a role for imprinting per se in adult stem cells remains elusive. Here we show upregulation of growth-restricting imprinted genes, including in the H19-Igf2 locus, in long-term haematopoietic stem cells and their downregulation upon haematopoietic stem cell activation and proliferation. A differentially methylated region upstream of H19 (H19-DMR), serving as the imprinting control region, determines the reciprocal expression of H19 from the maternal allele and Igf2 from the paternal allele. In addition, H19 serves as a source of miR-675, which restricts Igflr expression. We demonstrate that conditional deletion of the maternal but not the paternal H19-DMR reduces adult haematopoietic stem cell quiescence, a state required for long-term maintenance of haematopoietic stem cells, and compromises haematopoietic stem cell function. Maternal-specific H19-DMR deletion results in activation of the Igf2-Igfrl pathway, as shown by the translocation of phosphorylated Fox03 (an inactive form) from nucleus to cytoplasm and the release of Fox03-mediated cell cycle arrest, thus leading to increased activation, proliferation and eventual exhaustion of haematopoietic stem cells. Mechanistically, maternal-specific H19-DMR deletion leads to Igf2 upregulation and increased translation of Igflr, which is normally suppressed by H19-derived miR-675. Similarly, genetic inactivation of Igflr partly rescues the H19-DMR deletion phenotype. Our work establishes a new role for this unique form of epigenetic control at the H19-Igf2 locus in maintaining adult stem cells. PUBLICATION ABSTRACT
The epigenetic regulation of imprinted genes via monoallelic DNA methylation of either maternal or paternal alleles is critical for embryonic growth and development
1
. Imprinted genes were recently ...shown to be expressed in mammalian adult stem cells to support self-renewal of neural and lung stem cells
2
,
3
,
4
; however, a role for imprinting
per se
in adult stem cells remains elusive. Here we show up-regulation of growth-restricting imprinted genes, including within the
H19
-
Igf2
locus
5
, in long-term hematopoietic stem cells (LT-HSCs) and their down-regulation upon HSC activation and proliferation. A differentially methylated region (DMR) upstream of
H19
(H19-DMR), serving as the imprinting control region, determines the reciprocal expression of
H19
from the maternal allele and
Igf2
from the paternal allele
1
. In addition,
H19
also serves as a source of miR-675, which restricts
Igf1r
expression
6
. We demonstrated that conditional deletion of the maternal but not the paternal H19-DMR reduced adult HSC quiescence, a state required for long-term maintenance of HSCs, and compromised HSC function. Maternal-specific H19-DMR deletion resulted in activation of the Igf2-Igfr1 pathway as revealed by the translocation of phosphorylated Foxo3 (an inactive form) from nucleus to cytoplasm and the release of Foxo3-mediated cell-cycle arrest, thus leading to increased activation, proliferation, and eventual exhaustion of HSCs. Mechanistically, maternal-specific H19-DMR deletion led to
Igf2
up-regulation and increased translation of Igf1r, which is normally suppressed by
H19
-derived miR-675. Similarly, genetic inactivation of
Igf1r
partially rescued the H19-DMR deletion phenotype. Our work establishes a novel role for this unique form of epigenetic control at the
H19
-
Igf2
locus in maintaining adult stem cells.
In the initial published version of this article, there was an inadvertent omission from the Acknowledgements that this work was supported by Stowers Institute for Medical Research (SIMR-1004) and ...NIH National Cancer Institute grant to University of Kansas Cancer Center (P30 CA168524). This omission does not affect the description of the results or the conclusions of this work.
Transplantation of hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB) holds great promise for treating a broad spectrum of hematological disorders including cancer. However, the ...limited number of HSCs in a single hUCB unit restricts its widespread use. Although extensive efforts have led to multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for stem cell self-renewal. Recent studies indicate that N
-methyladenosine (m
A) modulates the expression of a group of mRNAs critical for stem cell-fate determination by influencing their stability. Among several m
A readers, YTHDF2 is recognized as promoting targeted mRNA decay. However, the physiological functions of YTHDF2 in adult stem cells are unknown. Here we show that following the conditional knockout (KO) of mouse Ythdf2 the numbers of functional HSC were increased without skewing lineage differentiation or leading to hematopoietic malignancies. Furthermore, knockdown (KD) of human YTHDF2 led to more than a 10-fold increase in the ex vivo expansion of hUCB HSCs, a fivefold increase in colony-forming units (CFUs), and more than an eightfold increase in functional hUCB HSCs in the secondary serial of a limiting dilution transplantation assay. Mapping of m
A in RNAs from mouse hematopoietic stem and progenitor cells (HSPCs) as well as from hUCB HSCs revealed its enrichment in mRNAs encoding transcription factors critical for stem cell self-renewal. These m
A-marked mRNAs were recognized by Ythdf2 and underwent decay. In Ythdf2 KO HSPCs and YTHDF2 KD hUCB HSCs, these mRNAs were stabilized, facilitating HSC expansion. Knocking down one of YTHDF2's key targets, Tal1 mRNA, partially rescued the phenotype. Our study provides the first demonstration of the function of YTHDF2 in adult stem cell maintenance and identifies its important role in regulating HSC ex vivo expansion by regulating the stability of multiple mRNAs critical for HSC self-renewal, thus identifying potential for future clinical applications.
This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy).
This article has been retracted at the request of the Authors.
Our ...paper reported the identification of a nuclear protein complex comprising DNA-PKcs, PIDD, and caspase-2 and characterization of its role in G2/M checkpoint maintenance, thereby providing insight into the functional significance of nuclear caspase-2. We recently identified errors affecting several figure panels where original data were processed inappropriately such that the figure panels do not accurately report the original data. We believe that the most responsible course of action is to retract the paper. We sincerely apologize to the scientific community for any inconvenience this might cause.
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