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Daniel M. Knowles - One of the best experts on this subject based on the ideXlab platform.

  • microrna mediated down regulation of prdm1 blimp 1 in hodgkin reed sternberg cells a potential pathogenetic Lesion in hodgkin lymphomas
    American Journal of Pathology, 2008
    Co-Authors: Mario Gomez, Pablo Landgraf, Thomas Tuschl, Amy Chadburn, José Francisco García, Daniel M. Knowles
    Abstract:

    PRDM1/Blimp-1, a master regulator in terminal B-cell differentiation, has been recently identified as a tumor suppressor target for mutational inactivation in diffuse large B-cell lymphomas of the activated B-cell type. Our studies here demonstrate that PRDM1/blimp-1 is also a target for microRNA (miRNA)-mediated down-regulation by miR-9 and let-7a in Hodgkin/Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL). MiRNA expression profiling by direct miRNA cloning demonstrated that both of these miRNAs are among the most highly expressed in cultured HRS cells. These miRNAs functionally targeted specific binding sites in the 3′ untranslated region of PRDM1/blimp-1 mRNA and repressed luciferase reporter activities through repression of translation. In addition, high levels of miR-9 and let-7a in HL cell lines correlated with low levels of PRDM1/Blimp-1. Similar to their in vitro counterparts, the majority of HRS cells in primary HL cases showed weak or no PRDM1/Blimp-1 expression. Over-expression of miR-9 or let-7a reduced PRDM1/Blimp-1 levels in U266 cells by 30% to 50%, whereas simultaneous inhibition of their activities in L428 cells resulted in an approximately 2.6-fold induction in PRDM1/Blimp-1. MiRNA-mediated down-regulation of PRDM1/Blimp-1 may contribute to the phenotype maintenance and pathogenesis of HRS cells by interfering with normal B-cell terminal differentiation, thus representing a novel Molecular Lesion, as well as a potential therapeutic target in HL.

  • microrna mediated translation repression of prdm1 in hodgkin reed sternberg cells a potential pathogenetic Lesion in hodgkin lymphoma
    Blood, 2006
    Co-Authors: Mario Gomez, Pablo Landgraf, Thomas Tuschl, Amy Chadburn, José Francisco García, Daniel M. Knowles
    Abstract:

    PR (PRDI-BF1-RIZ-homology) domain zinc finger protein 1 (PRDM1) is a master regulator in plasma cell differentiation recently identified as a tumor suppressor target for inactivation in diffuse large B-cell lymphomas of the activated B-cell type (Tam et al ., Blood107: 4090–4100, 2006). Since Hodgkin/Reed-Sternberg (HRS) cells in Hodgkin lymphomas (HL) show resemblance to post-germinal center B cells, we investigated whether PRDM1 inactivation might play a role in HL pathogenesis. Western blotting and immunohistochemistry on HL cell lines and primary clinical HL samples demonstrated that HRS cells, regardless of Epstein-Barr virus (EBV) status, frequently lacked or expressed low levels of PRDM1. Despite similar levels of PRDM1 transcripts, expression of PRDM1 is 4 to 10 fold lower in HL cell lines compared to the myeloma cell line U266. We provide evidence that PRDM1 down-regulation in HRS cells is determined by a post-transcriptional mechanism mediated by microRNAs (miRNAs). Several lines of experimental evidence support PRDM1 mRNA as a target for at least three miRNAs: miR-9, let-7a and EBV-encoded miR-BHRF1. First, sequence analysis predicted binding sites for these miRNAs in the 3′ untranslated region of PRDM1 mRNA: three for miR-9, and one each for let-7a and miR-BHRF1. Second, all three miRNAs repressed luciferase activities in reporter assays by at least 50% via translation inhibition relative to non-targeting RNA oligonucleotide. This inhibition is dependent upon specific interactions of the miRNAs with their predicted binding sites, since point mutations within the “seed” sequences of the binding sites relieve the repression. In addition, cooperativity exists between the three miR-9 binding sites, as well as between let-7a and miR-9 or between let-7a and miR-BHRF1. Transfection of both let-7a and miR-9 or let-7a and miR-BHRF1 diminished luciferase activities by at least 70%. Third, levels of these miRNAs inversely correlate with endogenous PRDM1 protein expression between the HL cell lines and U266. miR-9 or let-7a levels in HL lines are 2 to 4 times that in U266. Though miR-9 is expressed at low levels in the EBV(+) HL line L591, miR-BHRF1 is abundant, accounting for ~9% of the total miRNA population in this cell line. Lastly, Western blotting showed that EBV(−) HL line L428 transfected with anti-sense miR-9 or let-7a RNA oligonucleotides resulted in PRDM1 induction of approximately 1.5 fold. A higher level of induction (~3 fold) was seen when both miR-9 and let-7a were inhibited, suggesting co-operativity between miR-9 and let-7a in regulation of PRDM1 in HRS cells at physiological levels. Over-expression of miR-9 in U266 cells reduced endogenous PRDM1 expression by about 50%. In summary, our studies show that miR-9, let-7a and miR-BHRF1 can target PRDM1 mRNA and repress translation of PRDM1 in HRS cells, implicating interference of terminal differentiation in HL pathogenesis. MiRNA-mediated translation repression of PRDM1 is likely to be an alternative, epigenetic mechanism of PRDM1 inactivation, and represents a novel Molecular Lesion and potential therapeutic target in HL.

Edward H. Schuchman - One of the best experts on this subject based on the ideXlab platform.

  • identification and characterization of the Molecular Lesion causing mucopolysaccharidosis type i in cats
    Molecular Genetics and Metabolism, 1999
    Co-Authors: Calogera M Simonaro, Robert J Desnick, Qi Wan, Mark E Haskins, Edward H. Schuchman
    Abstract:

    Mucopolysaccharidosis Type I (MPS I) is the lysosomal storage disease caused by the deficient activity of alpha-L-iduronidase (IDUA). In man, MPS I can occur in severe, mild, or intermediate forms known as the Hurler, Scheie, or Hurler/Scheie syndromes, respectively. MPS I also has been described in cats, dogs, and mice. This manuscript reports the identification and characterization of the mutation causing MPS I in cats. To obtain wild-type feline IDUA cDNAs, two PCR-based strategies were used. PCR primers were constructed from a conserved region of the published human and dog sequences and used to amplify a 224-bp IDUA fragment from normal cat genomic DNA. This fragment was then used to screen a feline uterus cDNA library. PCR also was used to directly amplify IDUA fragments from the same cDNA library. Two overlapping feline IDUA cDNAs encoding 466 amino acid residues of the feline IDUA polypeptide ( approximately 85% of the mature protein based on comparison to the human, dog, and mouse sequences) were obtained by these strategies. To identify the mutation causing MPS I in cats, DNA sequencing was carried out on the corresponding IDUA region from several affected animals. A 3-bp deletion was found on both IDUA alleles in each of the MPS I animals, predicting the deletion of a single aspartate residue from the feline IDUA polypeptide. To confirm the authenticity of this mutation, heteroduplex, SSCP, and transient expression studies were carried out. Over 100 animals from the MPS I colony were screened for the presence of the mutation by heteroduplex and SSCP analyses-in all cases the presence of the 3-bp deletion was 100% concordant with the disease phenotype. For transient expression studies, the two partial, overlapping feline cDNAs were combined and joined in-frame to the 5' end of the canine IDUA cDNA. This wild-type, hybrid cDNA expressed IDUA activity up to sixfold over endogenous levels after transfection into COS-1 cells. A modified full-length IDUA cDNA containing the 3-bp deletion did not express IDUA activity in a transient expression system, providing proof that this Lesion was the cause of feline MPS I.

  • Molecular cloning and characterization of a full length complementary dna encoding human acid ceramidase identification of the first Molecular Lesion causing farber disease
    Journal of Biological Chemistry, 1996
    Co-Authors: Jurgen Koch, Sabine Gartner, Lothar E Quintern, Katussevani Bernardo, Orna Levran, Doris Schnabel, Robert J Desnick, Edward H. Schuchman
    Abstract:

    Abstract Human acid ceramidase ((AC) N-acylsphingosine amidohydrolase, EC 3.5.1.23) hydrolyzes the sphingolipid ceramide into sphingosine and free fatty acid. Ceramide is an essential component of all sphingolipids and an important cell-signaling molecule. Moreover, an inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease. Human AC was purified from urine, and 117 amino acid residues were determined by microsequencing. Degenerative oligonucleotide probes were then constructed and used to screen for human fibroblast and pituitary cDNA libraries. Several partial cDNA clones were obtained, and two of these were combined to construct a full-length cDNA containing a 17-base pair (bp) 5′-untranslated sequence, a 1185-bp open reading frame encoding 395 amino acids, a 1110-bp 3′-untranslated sequence, and an 18-bp poly(A) tail. Transient expression of the full-length cDNA in COS-1 cells led to a 10-fold increase in AC activity. In addition, biosynthetic studies carried out in the transfected cells demonstrated that 13-kDa (α) and 40-kDa (β) AC subunits were derived from a common 55-kDa precursor encoded by the full-length cDNA. This protein pattern was identical to that seen in normal human skin fibroblasts. A homoallelic point mutation (T222K) was also identified in the AC gene of a patient suffering from Farber disease, further confirming the authenticity of the full-length cDNA.

  • mucopolysaccharidosis type vi in rats isolation of cdnas encoding arylsulfatase b chromosomal localization of the gene and identification of the mutation
    Genomics, 1995
    Co-Authors: T Kunieda, Hiromi Ikadai, Robert J Desnick, Calogera M Simonaro, Midori Yoshida, Goran Levan, Edward H. Schuchman
    Abstract:

    Mucopolysaccharidosis (MPS) type VI, the lysosomal storage disorder caused by the deficiency of arylsulfatase B (ARSB) activity, occurs in humans, cats, and rats. To characterize the Molecular Lesion(s) causing MPS VI in rats, cDNAs encoding rat ARSB were isolated from a rat liver cDNA library. The nucleotide and deduced amino acid sequences of rat ARSB had {approximately}80 and 85% identity with the human ARSB sequences, respectively. The chromosomal location of the rat ARSB gene was determined by PCR analysis of rat-mouse somatic cell hybrid panel. The ARSB gene was assigned to rat chromosome 2, where the locus for the MPS VI phenotype in rats has been localized by linkage analysis. To identify the mutations within the ARSB gene causing MPS VI in rats, the ARSB sequence were amplified from affected animals and completely sequenced. Notably, a homoallelic one-base insertion at nucleotide 507 (507insC) was identified, resulting in a frame shift mutation and premature termination at codon 258. The presence of the insertion completely correlated with the occurrence of the MPS VI phenotype among 66 members of the MPR rat colony. Thus, we conclude that 507insC is the causative mutation in these animals and that the MPS VI rats are anmore » authentic model of human MPS VI. 27 refs., 3 figs., 1 tab.« less

Robert J Desnick - One of the best experts on this subject based on the ideXlab platform.

  • Rapid Publication Schindler Disease: The Molecular Lesion in the a-N-Acetylgalactosaminidase Gene that Causes an Infantile Neuroaxonal Dystrophy
    2016
    Co-Authors: Anne M Wang, Detlev Schindler, Robert J Desnick
    Abstract:

    Schindler disease is a recently recognized infantile neuroax-onal dystrophy resulting from the deficient activity of the lyso-somal hydrolase, a-N-acetylgalctosaminidase (a-GalNAc). The recent isolation and expression of the full-length cDNA encoding a-GalNAc facilitated the identification of the molec-ular Lesions in the affected brothers from family D, the first cases described with this autosomal recessive disease. South-ern and Northern hybridization analyses of DNA and RNA from the affected homozygotes revealed a grossly normal a-GalNAc gene structure and normal transcript sizes and amounts. Therefore, the a-GalNAc transcript from an affected homozygote was reverse-transcribed, amplified by the poly-merase chain reaction (PCR), and sequenced. A single G to A transition at nucleotide 973 was detected in multiple subclones containing the PCR products. This point mutation resulted in a glutamic acid to lysine substitution in residue 325 (E325K) of the a-GalNAc polypeptide. The base substitution was con-firmed by dot blot hybridization analyses of PCR-amplified genomic DNA from family members with allele-specific oligo-nucleotides. Furthermore, transient expression of an a-Gal-NAc construct containing the E325K mutation resulted in the expression of an immunoreactive polypeptide which had no detectable a-GalNAc activity. (J. Clin. Invest. 1990.86:1752

  • identification and characterization of the Molecular Lesion causing mucopolysaccharidosis type i in cats
    Molecular Genetics and Metabolism, 1999
    Co-Authors: Calogera M Simonaro, Robert J Desnick, Qi Wan, Mark E Haskins, Edward H. Schuchman
    Abstract:

    Mucopolysaccharidosis Type I (MPS I) is the lysosomal storage disease caused by the deficient activity of alpha-L-iduronidase (IDUA). In man, MPS I can occur in severe, mild, or intermediate forms known as the Hurler, Scheie, or Hurler/Scheie syndromes, respectively. MPS I also has been described in cats, dogs, and mice. This manuscript reports the identification and characterization of the mutation causing MPS I in cats. To obtain wild-type feline IDUA cDNAs, two PCR-based strategies were used. PCR primers were constructed from a conserved region of the published human and dog sequences and used to amplify a 224-bp IDUA fragment from normal cat genomic DNA. This fragment was then used to screen a feline uterus cDNA library. PCR also was used to directly amplify IDUA fragments from the same cDNA library. Two overlapping feline IDUA cDNAs encoding 466 amino acid residues of the feline IDUA polypeptide ( approximately 85% of the mature protein based on comparison to the human, dog, and mouse sequences) were obtained by these strategies. To identify the mutation causing MPS I in cats, DNA sequencing was carried out on the corresponding IDUA region from several affected animals. A 3-bp deletion was found on both IDUA alleles in each of the MPS I animals, predicting the deletion of a single aspartate residue from the feline IDUA polypeptide. To confirm the authenticity of this mutation, heteroduplex, SSCP, and transient expression studies were carried out. Over 100 animals from the MPS I colony were screened for the presence of the mutation by heteroduplex and SSCP analyses-in all cases the presence of the 3-bp deletion was 100% concordant with the disease phenotype. For transient expression studies, the two partial, overlapping feline cDNAs were combined and joined in-frame to the 5' end of the canine IDUA cDNA. This wild-type, hybrid cDNA expressed IDUA activity up to sixfold over endogenous levels after transfection into COS-1 cells. A modified full-length IDUA cDNA containing the 3-bp deletion did not express IDUA activity in a transient expression system, providing proof that this Lesion was the cause of feline MPS I.

  • Molecular cloning and characterization of a full length complementary dna encoding human acid ceramidase identification of the first Molecular Lesion causing farber disease
    Journal of Biological Chemistry, 1996
    Co-Authors: Jurgen Koch, Sabine Gartner, Lothar E Quintern, Katussevani Bernardo, Orna Levran, Doris Schnabel, Robert J Desnick, Edward H. Schuchman
    Abstract:

    Abstract Human acid ceramidase ((AC) N-acylsphingosine amidohydrolase, EC 3.5.1.23) hydrolyzes the sphingolipid ceramide into sphingosine and free fatty acid. Ceramide is an essential component of all sphingolipids and an important cell-signaling molecule. Moreover, an inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease. Human AC was purified from urine, and 117 amino acid residues were determined by microsequencing. Degenerative oligonucleotide probes were then constructed and used to screen for human fibroblast and pituitary cDNA libraries. Several partial cDNA clones were obtained, and two of these were combined to construct a full-length cDNA containing a 17-base pair (bp) 5′-untranslated sequence, a 1185-bp open reading frame encoding 395 amino acids, a 1110-bp 3′-untranslated sequence, and an 18-bp poly(A) tail. Transient expression of the full-length cDNA in COS-1 cells led to a 10-fold increase in AC activity. In addition, biosynthetic studies carried out in the transfected cells demonstrated that 13-kDa (α) and 40-kDa (β) AC subunits were derived from a common 55-kDa precursor encoded by the full-length cDNA. This protein pattern was identical to that seen in normal human skin fibroblasts. A homoallelic point mutation (T222K) was also identified in the AC gene of a patient suffering from Farber disease, further confirming the authenticity of the full-length cDNA.

  • mucopolysaccharidosis type vi in rats isolation of cdnas encoding arylsulfatase b chromosomal localization of the gene and identification of the mutation
    Genomics, 1995
    Co-Authors: T Kunieda, Hiromi Ikadai, Robert J Desnick, Calogera M Simonaro, Midori Yoshida, Goran Levan, Edward H. Schuchman
    Abstract:

    Mucopolysaccharidosis (MPS) type VI, the lysosomal storage disorder caused by the deficiency of arylsulfatase B (ARSB) activity, occurs in humans, cats, and rats. To characterize the Molecular Lesion(s) causing MPS VI in rats, cDNAs encoding rat ARSB were isolated from a rat liver cDNA library. The nucleotide and deduced amino acid sequences of rat ARSB had {approximately}80 and 85% identity with the human ARSB sequences, respectively. The chromosomal location of the rat ARSB gene was determined by PCR analysis of rat-mouse somatic cell hybrid panel. The ARSB gene was assigned to rat chromosome 2, where the locus for the MPS VI phenotype in rats has been localized by linkage analysis. To identify the mutations within the ARSB gene causing MPS VI in rats, the ARSB sequence were amplified from affected animals and completely sequenced. Notably, a homoallelic one-base insertion at nucleotide 507 (507insC) was identified, resulting in a frame shift mutation and premature termination at codon 258. The presence of the insertion completely correlated with the occurrence of the MPS VI phenotype among 66 members of the MPR rat colony. Thus, we conclude that 507insC is the causative mutation in these animals and that the MPS VI rats are anmore » authentic model of human MPS VI. 27 refs., 3 figs., 1 tab.« less

  • the Molecular Lesion in the alpha n acetylgalactosaminidase gene that causes angiokeratoma corporis diffusum with glycopeptiduria
    Journal of Clinical Investigation, 1994
    Co-Authors: Anne M Wang, Tamotsu Kanzaki, Robert J Desnick
    Abstract:

    Angiokeratoma corporis diffusum with glycopeptiduria is a recently recognized inborn error of glycoprotein catabolism resulting from the deficient activity of human alpha-N-acetylgalactosaminidase (E.C. 3.2.1.49; alpha-GalNAc). The first patient with this autosomal recessive disorder, a 46-yr-old consanguineous Japanese woman, presented with diffuse angiokeratoma, mild intellectual impairment, and peripheral neuroaxonal degeneration. Deficient alpha-GalNAc activity also has been reported in consanguineous brothers with an infantile-onset form of neuroaxonal dystrophy resulting from a missense mutation (designated E325K) in the alpha-GalNAc gene. To identify the mutation causing the phenotypically distinct adult-onset disorder, Southern and Northern hybridization analyses of DNA and RNA from the affected homozygote were performed which revealed a grossly normal alpha-GalNAc gene structure and normal transcript size and abundancy. Reverse transcription, amplification, and sequencing of the alpha-GalNAc transcript identified a single C to T transition at nucleotide (nt) 985 that predicted an arginine to tryptophan substitution in residue 329 (designated R329W) of the alpha-GalNAc polypeptide. This base substitution was confirmed by hybridization of PCR-amplified genomic DNA from family members with allele-specific oligonucleotides. Transient expression of an alpha-GalNAc construct containing the R329W mutation resulted in the expression of an immunoreactive polypeptide which had no detectable alpha-GalNAc activity. Comparison of the biosynthesis and stabilities of the transiently expressed and radiolabeled normal, E325K (infantile-onset) and R329W (adult-onset) alpha-GalNAc polypeptides in COS-1 cells indicated that both the mutant precursors were processed to the mature form; however, the E325K mutant polypeptide was more rapidly degraded than the R329W subunit, thereby providing a basis for the distinctly different infantile- and adult-onset phenotypes.

Mario Gomez - One of the best experts on this subject based on the ideXlab platform.

  • microrna mediated down regulation of prdm1 blimp 1 in hodgkin reed sternberg cells a potential pathogenetic Lesion in hodgkin lymphomas
    American Journal of Pathology, 2008
    Co-Authors: Mario Gomez, Pablo Landgraf, Thomas Tuschl, Amy Chadburn, José Francisco García, Daniel M. Knowles
    Abstract:

    PRDM1/Blimp-1, a master regulator in terminal B-cell differentiation, has been recently identified as a tumor suppressor target for mutational inactivation in diffuse large B-cell lymphomas of the activated B-cell type. Our studies here demonstrate that PRDM1/blimp-1 is also a target for microRNA (miRNA)-mediated down-regulation by miR-9 and let-7a in Hodgkin/Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL). MiRNA expression profiling by direct miRNA cloning demonstrated that both of these miRNAs are among the most highly expressed in cultured HRS cells. These miRNAs functionally targeted specific binding sites in the 3′ untranslated region of PRDM1/blimp-1 mRNA and repressed luciferase reporter activities through repression of translation. In addition, high levels of miR-9 and let-7a in HL cell lines correlated with low levels of PRDM1/Blimp-1. Similar to their in vitro counterparts, the majority of HRS cells in primary HL cases showed weak or no PRDM1/Blimp-1 expression. Over-expression of miR-9 or let-7a reduced PRDM1/Blimp-1 levels in U266 cells by 30% to 50%, whereas simultaneous inhibition of their activities in L428 cells resulted in an approximately 2.6-fold induction in PRDM1/Blimp-1. MiRNA-mediated down-regulation of PRDM1/Blimp-1 may contribute to the phenotype maintenance and pathogenesis of HRS cells by interfering with normal B-cell terminal differentiation, thus representing a novel Molecular Lesion, as well as a potential therapeutic target in HL.

  • microrna mediated translation repression of prdm1 in hodgkin reed sternberg cells a potential pathogenetic Lesion in hodgkin lymphoma
    Blood, 2006
    Co-Authors: Mario Gomez, Pablo Landgraf, Thomas Tuschl, Amy Chadburn, José Francisco García, Daniel M. Knowles
    Abstract:

    PR (PRDI-BF1-RIZ-homology) domain zinc finger protein 1 (PRDM1) is a master regulator in plasma cell differentiation recently identified as a tumor suppressor target for inactivation in diffuse large B-cell lymphomas of the activated B-cell type (Tam et al ., Blood107: 4090–4100, 2006). Since Hodgkin/Reed-Sternberg (HRS) cells in Hodgkin lymphomas (HL) show resemblance to post-germinal center B cells, we investigated whether PRDM1 inactivation might play a role in HL pathogenesis. Western blotting and immunohistochemistry on HL cell lines and primary clinical HL samples demonstrated that HRS cells, regardless of Epstein-Barr virus (EBV) status, frequently lacked or expressed low levels of PRDM1. Despite similar levels of PRDM1 transcripts, expression of PRDM1 is 4 to 10 fold lower in HL cell lines compared to the myeloma cell line U266. We provide evidence that PRDM1 down-regulation in HRS cells is determined by a post-transcriptional mechanism mediated by microRNAs (miRNAs). Several lines of experimental evidence support PRDM1 mRNA as a target for at least three miRNAs: miR-9, let-7a and EBV-encoded miR-BHRF1. First, sequence analysis predicted binding sites for these miRNAs in the 3′ untranslated region of PRDM1 mRNA: three for miR-9, and one each for let-7a and miR-BHRF1. Second, all three miRNAs repressed luciferase activities in reporter assays by at least 50% via translation inhibition relative to non-targeting RNA oligonucleotide. This inhibition is dependent upon specific interactions of the miRNAs with their predicted binding sites, since point mutations within the “seed” sequences of the binding sites relieve the repression. In addition, cooperativity exists between the three miR-9 binding sites, as well as between let-7a and miR-9 or between let-7a and miR-BHRF1. Transfection of both let-7a and miR-9 or let-7a and miR-BHRF1 diminished luciferase activities by at least 70%. Third, levels of these miRNAs inversely correlate with endogenous PRDM1 protein expression between the HL cell lines and U266. miR-9 or let-7a levels in HL lines are 2 to 4 times that in U266. Though miR-9 is expressed at low levels in the EBV(+) HL line L591, miR-BHRF1 is abundant, accounting for ~9% of the total miRNA population in this cell line. Lastly, Western blotting showed that EBV(−) HL line L428 transfected with anti-sense miR-9 or let-7a RNA oligonucleotides resulted in PRDM1 induction of approximately 1.5 fold. A higher level of induction (~3 fold) was seen when both miR-9 and let-7a were inhibited, suggesting co-operativity between miR-9 and let-7a in regulation of PRDM1 in HRS cells at physiological levels. Over-expression of miR-9 in U266 cells reduced endogenous PRDM1 expression by about 50%. In summary, our studies show that miR-9, let-7a and miR-BHRF1 can target PRDM1 mRNA and repress translation of PRDM1 in HRS cells, implicating interference of terminal differentiation in HL pathogenesis. MiRNA-mediated translation repression of PRDM1 is likely to be an alternative, epigenetic mechanism of PRDM1 inactivation, and represents a novel Molecular Lesion and potential therapeutic target in HL.

Jelena V Jovanovic - One of the best experts on this subject based on the ideXlab platform.

  • strikingly different Molecular relapse kinetics in npm1c pml rara runx1 runx1t1 and cbfb myh11 acute myeloid leukemias
    Blood, 2010
    Co-Authors: Hans Beier Ommen, Susanne Schnittger, M Ostergaard, Jelena V Jovanovic, Ingrid Beier Ommen, David Grimwade, Henrik Hasle, Peter Hokland
    Abstract:

    Early relapse detection in acute myeloid leukemia is possible using standardized real-time quantitative polymerase chain reaction (RQ-PCR) protocols. However, optimal sampling intervals have not been defined and are likely to vary according to the underlying Molecular Lesion. In 74 patients experiencing hematologic relapse and harboring aberrations amenable to RQ-PCR (mutated NPM1 [designated NPM1c ], PML-RARA , RUNX1-RUNX1T1 , and CBFB-MYH11 ), we observed strikingly different relapse kinetics. The median doubling time of the CBFB-MYH11 leukemic clone was significantly longer (36 days) than that of clones harboring other markers ( RUNX1-RUNX1T1 , 14 days; PML-RARA , 12 days; and NPM1c , 11 days; P < .001). Furthermore, we used a mathematical model to determine frequency of relapse detection and median time from detection of minimal residual disease to hematologic relapse as a function of sampling interval length. For example, to obtain a relapse detection fraction of 90% and a median time of 60 days, blood sampling every sixth month should be performed for CBFB-MYH11 leukemias. By contrast, in NPM1c +/ FLT3 -ITD−, NPM1c + /FLT3 -ITD+, RUNX1-RUNX1T1 , and PML-RARA leukemias, bone marrow sampling is necessary every sixth, fourth, and fourth and second month, respectively. These data carry important implications for the development of optimal RQ-PCR monitoring schedules suitable for evaluation of minimal residual disease–directed therapies in future clinical trials.

  • low dose imatinib mesylate leads to rapid induction of major Molecular responses and achievement of complete Molecular remission in fip1l1 pdgfra positive chronic eosinophilic leukemia
    Blood, 2007
    Co-Authors: Jelena V Jovanovic, Daniela Cilloni, Enrico Gottardi, J. Score, Katherine Waghorn, Helena Popp, Andreas Hochhaus
    Abstract:

    The FIP1L1-PDGFRA fusion gene is a recurrent Molecular Lesion in eosinophilia-associated myeloproliferative disorders, predicting a favorable response to imatinib mesylate. To investigate its prevalence, 376 patients with persistent unexplained hypereosinophilia were screened by the United Kingdom reference laboratory, revealing 40 positive cases (11%). To determine response kinetics following imatinib, real-time quantitative-polymerase chain reaction (RQ-PCR) assays were developed and evaluated in samples accrued from across the European LeukemiaNet. The FIP1L1-PDGFRA fusion transcript was detected at a sensitivity of 1 in 10(5) in serial dilution of the EOL-1 cell line. Normalized FIP1L1-PDGFRA transcript levels in patient samples prior to imatinib varied by almost 3 logs. Serial monitoring was undertaken in patients with a high level of FIP1L1-PDGFRA expression prior to initiation of imatinib (100 mg/d-400 mg/d). Overall, 11 of 11 evaluable patients achieved at least a 3-log reduction in FIP1L1-PDGFRA fusion transcripts relative to the pretreatment level within 12 months, with achievement of Molecular remission in 9 of 11 (assay sensitivities 1 in 10(3)-10(5)). In 2 patients, withdrawal of imatinib was followed by a rapid rise in FIP1L1-PDGFRA transcript levels. Overall, these data are consistent with the exquisite sensitivity of the FIP1L1-PDGFRalpha fusion to imatinib, as compared with BCR-ABL, and underline the importance of RQ-PCR monitoring to guide management using Molecularly targeted therapies.

  • Sensitive Detection of FIP1L1-PDGFRA Fusion Transcripts by Real-Time Quantitative RT-PCR (RQ-PCR) Reveals Achievement of Molecular Remission in Chronic Eosinophilic Leukemia Treated with Low-Dose Imatinib Therapy.
    Blood, 2005
    Co-Authors: Jelena V Jovanovic, Georgia Metzgeroth, Andreas Reiter, Daniela Cilloni, Enrico Gottardi, J. Score, Henning D. Popp, C Walz, A. Hochhaus, Catherine Roche
    Abstract:

    The FIP1L1-PDGFRA fusion gene generated by a cryptic interstitial deletion at 4q12 is a recurrent Molecular Lesion in idiopathic hypereosinophilic syndrome (HES), that forms a basis for diagnosis of chronic eosinophilic leukemia (CEL). This disease entity is particularly important to recognize, since presence of the FIP1L1-PDGFRA fusion predicts a favorable response to Molecularly targeted therapy in the form of imatinib, with clinical remissions being achieved with lower doses than are required in BCR-ABL+ chronic myeloid leukemia (CML). In order to improve our understanding of the biology of CEL and to provide a tool to improve the management of patients with this disorder we have developed real-time quantitative reverse transcriptase PCR (RQ-PCR) assays for the FIP1L1-PDGFRA fusion. Taking into account the marked heterogeneity in upstream breakpoints within the FIP1L1 gene, RQ-PCR assays were designed for cases with FIP1L1 breakpoints leading to fusion of exon 9, 10, 11, 12 or 13 to PDGFRA exon 12. FIP1L1-PDGFRA expression was compared to that of validated Europe Against Cancer endogenous control genes - s2microglobulin (B2M) and ABL. Serial dilution of the FIP1L1-PDGFRA+ EOL1 cell-line in fusion gene negative filler cells (HL60) revealed an assay sensitivity of 1 in 105. While identification of the FIP1L1-PDGFRA fusion using conventional RT-PCR approaches can be problematic, RQ-PCR analysis undertaken in diagnostic samples from 31 patients with FIP1L1-PDGFRA+ CEL (median age 53, 31–64 years) revealed that, in the majority, the fusion transcript was expressed at relatively high level (median deltaCt FIP1L1-PDGFRA - B2M, 12.2; median deltaCt FIP1L1-PDGFRA - ABL, 2.3). The FIP1L1-PDGFRA fusion was expressed at comparable level in blood and marrow at diagnosis of CEL. Serial monitoring was undertaken in 17 patients following initiation of imatinib 100mg/d. Overall, 8/8 evaluable patients achieved at least a 3-log reduction in FIP1L1-PDGFRA fusion transcript level within the first year of therapy. In follow-up samples affording a sensitivity of at least 1 in 1000, PCR negativity by quantitative and conventional nested RT-PCR was documented in 8/17 patients following a median of 21 weeks of imatinib (4–64 weeks); in two cases profound PCR negativity (i.e. at a sensitivity level of at least 1 in 105) was documented following 13 weeks and 2 years of imatinib, respectively. Overall, these data demonstrate that CEL with the FIP1L1-PDGFRA fusion is uniquely sensitive to imatinib therapy. This contrasts with BCR-ABL+ CML, in which Molecular remission is generally not achieved - a phenomenon that has been postulated to reflect the persistence of a primitive quiescent stem cell population that is resistant to this agent. Understanding the biological basis for the differences in Molecular response to imatinib in CML and CEL, may yield further improvements in Molecularly-targeted therapeutic approaches.