Potential Role of CD9 and Implication of Motility Process in Pathogenesis of TEL/ALM1-positive ALL Relapses (LAL TEL/ALM1 and CD9).

• The potential discriminating state of CD9. - To determine the functional impact of CD9 on motility assays in TEL/AML1-positive blasts - To explore the regulation of the expression of the CD9 transcript inTEL/AML1-positive blasts [ Time Frame: 3 years ] [ Designated as safety issue: No ]

  Due to the importance of the motility process in malignant cells and the role of CD9 in cell motility regulation, we considered the potential discriminating state of CD9.

  • To determine the functional impact of CD9 on motility assays in TEL/AML1-positive blasts
  • To explore the regulation of the expression of the CD9 transcript inTEL/AML1-positive blasts
  
  

• - Migratory potential of blasts according to CD9 expression [ Time Frame: 3 years ] [ Designated as safety issue: No ]
  - Migratory potential of blasts according to CD9 expression
  
  
• - Adhesion properties of blasts according to CD9 expression [ Time Frame: 3 years ] [ Designated as safety issue: No ]
  - Adhesion properties of blasts according to CD9 expression
  
  
• - Level of miRNA, that could affect CD9 transcript levels inTEL/AML1-positive blasts versus TEL/AML1-negative ones [ Time Frame: 3 years ] [ Designated as safety issue: No ]
  - Level of miRNA, that could affect CD9 transcript levels inTEL/AML1-positive blasts versus TEL/AML1-negative ones
  
  

Other: Impact of CD9 expression level on motility assays

1) Assess of the impact of CD9 expression level on motility assays (migration and adhesion) We have initiated motility assays (fibronectin adhesion experiments and CXCL12 chemoattracted migration tests with modified Boyden chamber technique) using the CD9 positive TEL/AML1-positive cell line REH and the CD9 negative cell line RAJI (wild or transfected with CD9 cDNA). Data will be analyzed in combination with blocking antibodies and chemical antagonist according to the level of CD9 (transcript and protein) and of CXCR4. Protein quantifications will be performed by flow cytometry and Western Blot. Interactions will be explored by confocal microscopy and biological pathways by immunoblot.

Adhesion results will be validated on patient samples of B-ALL.

Other Name: Not Apllicable
Other: Post-transcriptional regulation of CD9

2) Post-transcriptional regulation of CD9 in TEL/AML1-positive ALL To identify miRNAs that are potentially deregulated in TEL/AML1-positive acute lymphoblastic leukaemia and especially to screen for CD9 -targeted miRNAs, we will use a TaqMan ®MicroRNA Arrays approach allowing the simultaneous measurement of about 760 human miRNA.

Small RNA will be extracted from bone marrow samples of twenty childhood B-ALL to screen miRNAs which are differentially expressed between CD9-positive and CD9-negative ALL and further compared with miRNAs which were predicted to target CD9 in databases. Validation of the selection will be performed by single Q-PCR for selected miRNAs using a novel cohort of ten bone marrow samples.

Other Name: Not Apllicable

1. Assess of the impact of CD9 expression level on motility assays (migration and adhesion) We have initiated motility assays (fibronectin adhesion experiments and CXCL12 chemoattracted migration tests with modified Boyden chamber technique) using the CD9 positive TEL/AML1-positive cell line REH and the CD9 negative cell line RAJI (wild or transfected with CD9 cDNA). Data will be analyzed in combination with blocking antibodies and chemical antagonist according to the level of CD9 (transcript and protein) and of CXCR4. Protein quantifications will be performed by flow cytometry and Western Blot. Interactions will be explored by confocal microscopy and biological pathways by immunoblot.

   Adhesion results will be validated on patient samples of B-ALL.

2. Post-transcriptional regulation of CD9 in TEL/AML1-positive ALL To identify miRNAs that are potentially deregulated in TEL/AML1-positive acute lymphoblastic leukaemia and especially to screen for CD9 -targeted miRNAs, we will use a TaqMan ®MicroRNA Arrays approach allowing the simultaneous measurement of about 760 human miRNA.

Small RNA will be extracted from bone marrow samples of twenty childhood B-ALL to screen miRNAs which are differentially expressed between CD9-positive and CD9-negative ALL and further compared with miRNAs which were predicted to target CD9 in databases. Validation of the selection will be performed by single Q-PCR for selected miRNAs using a novel cohort of ten bone marrow samples. Transfection assays and luciferase assays will be further realized to confirm that the differential miRNAs really target and affect CD9 expression .