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NFATC3-PLA2G15 IS A NOVEL INTERGENICALLY SPLICED CHIMERA THAT IS ASSOCIATED WITH AGGRESSIVE T-ACUTE LYMPHOBLASTIC LEUKAEMIA BIOLOGY.
Author(s):
Jonathan Bond
,
Jonathan Bond
Affiliations:
Université Paris Descartes Sorbonne Cité, Institut Necker-Enfants Malades (INEM), Institut national de recherche médicale (INSERM) U1151,Paris,France;Laboratory of Onco-Haematology, Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Necker Enfants-Malades,Paris,France
Christine Trang-Quang
,
Christine Trang-Quang
Affiliations:
Institut Curie, PSL Research University, CNRS UMR 3348,Orsay,France;Université Paris Sud, Université Paris-Saclay,Orsay,France
Aurélie Bergon
,
Aurélie Bergon
Affiliations:
Technological Advances for Genomics and Clinics (TAGC), INSERM U1090, Aix-Marseille University UMR-S 1090,Marseille,France
Mohamed Belhocine
,
Mohamed Belhocine
Affiliations:
Technological Advances for Genomics and Clinics (TAGC), INSERM U1090, Aix-Marseille University UMR-S 1090,Marseille,France
Guillaume Hypolite
,
Guillaume Hypolite
Affiliations:
Université Paris Descartes Sorbonne Cité, Institut Necker-Enfants Malades (INEM), Institut national de recherche médicale (INSERM) U1151,Paris,France;Laboratory of Onco-Haematology, Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Necker Enfants-Malades,Paris,France
Wilson Goma
,
Wilson Goma
Affiliations:
Technological Advances for Genomics and Clinics (TAGC), INSERM U1090, Aix-Marseille University UMR-S 1090,Marseille,France
Jacques Ghysdael
,
Jacques Ghysdael
Affiliations:
Institut Curie, PSL Research University, CNRS UMR 3348,Orsay,France;Université Paris Sud, Université Paris-Saclay,Orsay,France
Elizabeth Macintyre
,
Elizabeth Macintyre
Affiliations:
Université Paris Descartes Sorbonne Cité, Institut Necker-Enfants Malades (INEM), Institut national de recherche médicale (INSERM) U1151,Paris,France;Laboratory of Onco-Haematology, Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Necker Enfants-Malades,Paris,France
Nicolas Boissel
,
Nicolas Boissel
Affiliations:
Université Paris Diderot, Institut Universitaire d'Hématologie, EA-3518, Assistance Publique-Hôpitaux de Paris, University Hospital Saint-Louis,Paris,France
Salvatore Spicuglia
,
Salvatore Spicuglia
Affiliations:
Technological Advances for Genomics and Clinics (TAGC), INSERM U1090, Aix-Marseille University UMR-S 1090,Marseille,France
Vahid Asnafi
Vahid Asnafi
Affiliations:
Université Paris Descartes Sorbonne Cité, Institut Necker-Enfants Malades (INEM), Institut national de recherche médicale (INSERM) U1151,Paris,France;Laboratory of Onco-Haematology, Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Necker Enfants-Malades,Paris,France
(Abstract release date: 05/18/17) EHA Library. Bond J. 06/24/17; 181727; S440
Jonathan Bond
Jonathan Bond
Contributions
PDF Abstract
Abstract

Abstract: S440

Type: Oral Presentation

Presentation during EHA22: On Saturday, June 24, 2017 from 12:30 - 12:45

Location: Room N105

Background
Transcriptional read-through of a single mRNA between contiguous loci, or cis-splicing of adjacent genes (cis-SAGe), results in transcription of intergenically-spliced chimeric RNAs (ISCs) in the absence of structural genomic changes. Recent advances in high-throughput RNA-sequencing analysis have permitted identification of aberrant ISC expression as a potential cancer driver, but knowledge of leukaemia-related ISCs is lacking.

Aims
To examine whether cis-SAGe generates biologically important ISCs in T-acute lymphoblastic leukemia (T-ALL).

Methods
We performed RNA-sequencing of 12 cases of T-ALL and normal thymic RNA, and used targeted analysis pipelines to detect T-ALL-specific fusion chimeras.

Results

We identified 140 T-ALL-specific fusions, of which 55 involved genes located within 30kb of each other, in the same transcriptional orientation. This distance is consistent with that previously observed for cis-SAGE, suggesting that ISC expression is common in T-ALL. In total, putative ISCs were detected in 10/12 samples, with a median of 4 (range 0-15) per patient.
We performed further analysis on the candidate ISC NFATC3-PLA2G15, which includes the Nuclear Factor of Activated T-cells (NFAT) family member NFATC3, a critical regulator of normal thymopoiesis and known modulator of T-ALL biology. We found that primary T-ALLs exhibited a wide range of NFATC3-PLA2G15 expression, while levels in normal tissue were either very low or undetectable. 5’ RACE PCR analysis of leukemic cDNA revealed that fusion transcription was initiated in exon 1 of NFATC3. We also performed array competitive genomic hybridization of 115 diagnostic T-ALL samples, and found no evidence of microdeletions that would result in NFATC3-PLA2G15 expression, providing strong evidence that NFATC3-PLA2G15 is a true ISC that is generated by cis-SAGe.
We found that the NFATC3-PLA2G15 fusion had lower activity than wild-type NFATC3 in both luciferase reporter experiments and proliferation and survival complementation assays in NFAT-null ALL cell lines in vitro. Gene set enrichment analysis revealed that primary T-ALL blasts with elevated NFATC3-PLA2G15 levels had reduced transcription of canonical NFAT target genes in vivo, suggesting that these cases may have lower activity of normal physiological NFAT pathways.
Strikingly, we found that higher NFATC3-PLA2G15 levels strongly correlated with both shorter time to leukemia development (p = 0.01) and survival (p = 0.003) in patient-derived T-ALL xenografts in immunodeficient mice. These findings were corroborated by survival analyses of human T-ALL patients treated as part of the Francophone multinational GRAALL-2003 and -2005 studies, as cases with the highest quartile of NFATC3-PLA2G15 expression had significantly reduced 5 year overall survival (52.6%, 95% CI 33.3% - 68.7%) compared with NFATC3-PLA2G15 low cases (69.8%, 95% CI 58.8% - 78.3%, p = 0.047).

Conclusion
Our results suggest that ISC expression is common in T-ALL, and that high expression of the NFATC3-PLA2G15 ISC correlates with reduced canonical NFAT pathway activity and poor patient outcome.

Session topic: 1. Acute lymphoblastic leukemia - Biology

Keyword(s): Transcriptional regulation, T-ALL, Fusion

Abstract: S440

Type: Oral Presentation

Presentation during EHA22: On Saturday, June 24, 2017 from 12:30 - 12:45

Location: Room N105

Background
Transcriptional read-through of a single mRNA between contiguous loci, or cis-splicing of adjacent genes (cis-SAGe), results in transcription of intergenically-spliced chimeric RNAs (ISCs) in the absence of structural genomic changes. Recent advances in high-throughput RNA-sequencing analysis have permitted identification of aberrant ISC expression as a potential cancer driver, but knowledge of leukaemia-related ISCs is lacking.

Aims
To examine whether cis-SAGe generates biologically important ISCs in T-acute lymphoblastic leukemia (T-ALL).

Methods
We performed RNA-sequencing of 12 cases of T-ALL and normal thymic RNA, and used targeted analysis pipelines to detect T-ALL-specific fusion chimeras.

Results

We identified 140 T-ALL-specific fusions, of which 55 involved genes located within 30kb of each other, in the same transcriptional orientation. This distance is consistent with that previously observed for cis-SAGE, suggesting that ISC expression is common in T-ALL. In total, putative ISCs were detected in 10/12 samples, with a median of 4 (range 0-15) per patient.
We performed further analysis on the candidate ISC NFATC3-PLA2G15, which includes the Nuclear Factor of Activated T-cells (NFAT) family member NFATC3, a critical regulator of normal thymopoiesis and known modulator of T-ALL biology. We found that primary T-ALLs exhibited a wide range of NFATC3-PLA2G15 expression, while levels in normal tissue were either very low or undetectable. 5’ RACE PCR analysis of leukemic cDNA revealed that fusion transcription was initiated in exon 1 of NFATC3. We also performed array competitive genomic hybridization of 115 diagnostic T-ALL samples, and found no evidence of microdeletions that would result in NFATC3-PLA2G15 expression, providing strong evidence that NFATC3-PLA2G15 is a true ISC that is generated by cis-SAGe.
We found that the NFATC3-PLA2G15 fusion had lower activity than wild-type NFATC3 in both luciferase reporter experiments and proliferation and survival complementation assays in NFAT-null ALL cell lines in vitro. Gene set enrichment analysis revealed that primary T-ALL blasts with elevated NFATC3-PLA2G15 levels had reduced transcription of canonical NFAT target genes in vivo, suggesting that these cases may have lower activity of normal physiological NFAT pathways.
Strikingly, we found that higher NFATC3-PLA2G15 levels strongly correlated with both shorter time to leukemia development (p = 0.01) and survival (p = 0.003) in patient-derived T-ALL xenografts in immunodeficient mice. These findings were corroborated by survival analyses of human T-ALL patients treated as part of the Francophone multinational GRAALL-2003 and -2005 studies, as cases with the highest quartile of NFATC3-PLA2G15 expression had significantly reduced 5 year overall survival (52.6%, 95% CI 33.3% - 68.7%) compared with NFATC3-PLA2G15 low cases (69.8%, 95% CI 58.8% - 78.3%, p = 0.047).

Conclusion
Our results suggest that ISC expression is common in T-ALL, and that high expression of the NFATC3-PLA2G15 ISC correlates with reduced canonical NFAT pathway activity and poor patient outcome.

Session topic: 1. Acute lymphoblastic leukemia - Biology

Keyword(s): Transcriptional regulation, T-ALL, Fusion

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