PROGNOSTIC AND BIOLOGIC SIGNIFICANCE OF CIRCULAR RNA PROFILING IN YOUNGER ADULT PATIENTS WITH CYTOGENETICALLY NORMAL ACUTE MYELOID LEUKEMIA (CN-AML)
Author(s): ,
Dimitrios Papaioannou
Affiliations:
Comprehensive Cancer Center,The Ohio State University,Columbus,United States
,
Stefano Volinia
Affiliations:
Department of Morphology, Surgery and Experimental Medicine,University of Ferrara,Ferrara,Italy
,
Deedra Nicolet
Affiliations:
Comprehensive Cancer Center,The Ohio State University,Columbus,United States;Alliance Statistics and Data Center,The Ohio State University,Columbus,United States
,
Michal Swierniak
Affiliations:
Centre of new Technologies,University of Warsaw,Warsaw,Poland
,
Andreas Petri
Affiliations:
Center for RNA Medicine, Department of Clinical Medicine,Aalborg University,Copenhagen,Denmark
,
Krzysztof Mrózek
Affiliations:
Comprehensive Cancer Center,The Ohio State University,Columbus,United States
,
Andrew J. Carroll
Affiliations:
Department of Genetics,University of Alabama at Birmingham,Birmingham,United States
,
Jessica Kohlschmidt
Affiliations:
Comprehensive Cancer Center,The Ohio State University,Columbus,United States;Alliance Statistics and Data Center,The Ohio State University,Columbus,United States
,
Bayard L. Powell
Affiliations:
The Comprehensive Cancer Center of Wake Forest University,Winston-Salem,United States
,
Geoffrey L. Uy
Affiliations:
Siteman Cancer Center ,Washington University School of Medicine,St. Louis,United States
,
Jonathan E. Kolitz
Affiliations:
Monter Cancer Center,Hofstra Northwell School of Medicine,Lake Success,United States
,
Eunice S. Wang
Affiliations:
Roswell Park Comprehensive Cancer Center ,Buffalo,United States
,
Sakari Kauppinen
Affiliations:
Center for RNA Medicine, Department of Clinical Medicine,Aalborg University,Copenhagen,Denmark
,
Richard M. Stone
Affiliations:
Dana-Farber Cancer Institute,Harvard University,Boston,United States
,
Ramiro Garzon
Affiliations:
Comprehensive Cancer Center,The Ohio State University,Columbus,United States
Clara D. Bloomfield
Affiliations:
Comprehensive Cancer Center,The Ohio State University,Columbus,United States
(Abstract release date: 05/17/18) EHA Library. PAPAIOANNOU D. 06/15/18; 214594; S120
Dr. Dimitrios PAPAIOANNOU
Dr. Dimitrios PAPAIOANNOU
Contributions
Abstract

Abstract: S120

Type: Oral Presentation

Presentation during EHA23: On Friday, June 15, 2018 from 11:30 - 11:45

Location: Room K1

Background
Circular RNAs (circRNAs) constitute a novel class of non-coding RNAs characterized by an out-of-order arrangement of exons called “back-splicing”. While individual circRNAs have been linked to cancer pathogenesis, the prognostic value and biologic implications of circRNA expression in CN-AML pts have not been studied.

Aims

The aims of our study were to determine whether circRNA expression associates with clinical outcome of CN-AML pts, and to gain biological insights into circRNA function in CN-AML.

Methods
We conducted whole transcriptome profiling (RNAseq) in a training (n=254) and a validation (n=111) set of younger adults (aged <60 years) with de novo CN-AML. We applied a novel algorithm (MScircRNA) to quantify circRNA expression in both datasets. All pts were treated on frontline Cancer and Leukemia Group B (CALGB)/Alliance trials.

Results

We first performed RNAseq in RNAseR (a linear RNA-degrading exonuclease) v mock-treated samples of 7 AML pts and 3 AML cell lines (EOL-1, K562, OCI-AML3). We found 85% of the MScircRNA algorithm-predicted circRNAs to represent true circularized transcripts, as shown by enrichment upon RNAseR treatment. We next studied 180 circRNAs in the training set, and identified 4 (circCFLAR, circFCHO2, circKHLH8, circSMC1A) that associated with disease-free (DFS), overall (OS), and event-free (EFS) survival in both the training and validation sets. circKHLH8 was most strongly associated with clinical outcome. Using median expression values, we dichotomized the pts into high and low circKHLH8 expressers. In the validation set, high circKHLH8 expressers had longer 5-year DFS (50% v 21%, P<.001), OS (53% v 29%, P<.001) and EFS (45% v 18%, P<.001) than low circKHLH8 expressers. Regarding pretreatment clinical and molecular features, pts with high circKHLH8 expression had higher platelet counts (P=.05) and lower % of blasts in blood (P=.002) and bone marrow (P=.05). High circKHLH8 expressers also had more often FLT3-TKD (P=.05) and less often FLT3-ITD (P<.001) or high expression of miR-155 (P<.001) and ERG (P<.001) than low circKHLH8 expressers. In multivariable analyses, high circKHLH8 expression independently associated with longer DFS (P=.02; HR: 0.53), OS (P=.03; HR: 0.54) and EFS (P=.02; HR: 0.54) after adjusting for other co-variates. To ensure that our findings on circKHLH8 were not merely reflecting the prognostic significance of the linear KHLH8, we confirmed that the correlation between the transcripts was weak (Pearson’s r: 0.29) and that linear KHLH8 expression was not prognostic for clinical outcome in either of the 2 datasets.

To study the biologic significance of circRNA expression, we performed knock-down (KD)-based screening of candidate circRNAs associated with outcome or ELN prognostic gene mutations in KG1a and OCI-AML3 cells. We used back-splice site-targeting locked nucleic acid-modified oligonucleotides, which depleted circRNAs without affecting the corresponding linear transcripts. Of the circRNAs tested, circFBXW7 KD increased the proliferative capacity of KG1a and OCI-AML3 cells, as measured by WST1 reagent degradation (P=.001 and P<.001, respectively) and bromodeoxyuridine-based cell cycle analysis (P=.02 and P=.01, respectively). Colony forming unit assays with blasts of 3 AML pts revealed a consistent increase in the number of colonies formed upon circFBXW7 KD (P=.03, P=.04 and P=.02, respectively). Altogether, our data suggest that circFBXW7 has a tumor suppressor function in CN-AML.

Conclusion

We conclude that circRNA expression has prognostic and biologic significance in CN-AML.

Session topic: 3. Acute myeloid leukemia - Biology & Translational Research

Keyword(s): AML, prognosis, Transcription

Abstract: S120

Type: Oral Presentation

Presentation during EHA23: On Friday, June 15, 2018 from 11:30 - 11:45

Location: Room K1

Background
Circular RNAs (circRNAs) constitute a novel class of non-coding RNAs characterized by an out-of-order arrangement of exons called “back-splicing”. While individual circRNAs have been linked to cancer pathogenesis, the prognostic value and biologic implications of circRNA expression in CN-AML pts have not been studied.

Aims

The aims of our study were to determine whether circRNA expression associates with clinical outcome of CN-AML pts, and to gain biological insights into circRNA function in CN-AML.

Methods
We conducted whole transcriptome profiling (RNAseq) in a training (n=254) and a validation (n=111) set of younger adults (aged <60 years) with de novo CN-AML. We applied a novel algorithm (MScircRNA) to quantify circRNA expression in both datasets. All pts were treated on frontline Cancer and Leukemia Group B (CALGB)/Alliance trials.

Results

We first performed RNAseq in RNAseR (a linear RNA-degrading exonuclease) v mock-treated samples of 7 AML pts and 3 AML cell lines (EOL-1, K562, OCI-AML3). We found 85% of the MScircRNA algorithm-predicted circRNAs to represent true circularized transcripts, as shown by enrichment upon RNAseR treatment. We next studied 180 circRNAs in the training set, and identified 4 (circCFLAR, circFCHO2, circKHLH8, circSMC1A) that associated with disease-free (DFS), overall (OS), and event-free (EFS) survival in both the training and validation sets. circKHLH8 was most strongly associated with clinical outcome. Using median expression values, we dichotomized the pts into high and low circKHLH8 expressers. In the validation set, high circKHLH8 expressers had longer 5-year DFS (50% v 21%, P<.001), OS (53% v 29%, P<.001) and EFS (45% v 18%, P<.001) than low circKHLH8 expressers. Regarding pretreatment clinical and molecular features, pts with high circKHLH8 expression had higher platelet counts (P=.05) and lower % of blasts in blood (P=.002) and bone marrow (P=.05). High circKHLH8 expressers also had more often FLT3-TKD (P=.05) and less often FLT3-ITD (P<.001) or high expression of miR-155 (P<.001) and ERG (P<.001) than low circKHLH8 expressers. In multivariable analyses, high circKHLH8 expression independently associated with longer DFS (P=.02; HR: 0.53), OS (P=.03; HR: 0.54) and EFS (P=.02; HR: 0.54) after adjusting for other co-variates. To ensure that our findings on circKHLH8 were not merely reflecting the prognostic significance of the linear KHLH8, we confirmed that the correlation between the transcripts was weak (Pearson’s r: 0.29) and that linear KHLH8 expression was not prognostic for clinical outcome in either of the 2 datasets.

To study the biologic significance of circRNA expression, we performed knock-down (KD)-based screening of candidate circRNAs associated with outcome or ELN prognostic gene mutations in KG1a and OCI-AML3 cells. We used back-splice site-targeting locked nucleic acid-modified oligonucleotides, which depleted circRNAs without affecting the corresponding linear transcripts. Of the circRNAs tested, circFBXW7 KD increased the proliferative capacity of KG1a and OCI-AML3 cells, as measured by WST1 reagent degradation (P=.001 and P<.001, respectively) and bromodeoxyuridine-based cell cycle analysis (P=.02 and P=.01, respectively). Colony forming unit assays with blasts of 3 AML pts revealed a consistent increase in the number of colonies formed upon circFBXW7 KD (P=.03, P=.04 and P=.02, respectively). Altogether, our data suggest that circFBXW7 has a tumor suppressor function in CN-AML.

Conclusion

We conclude that circRNA expression has prognostic and biologic significance in CN-AML.

Session topic: 3. Acute myeloid leukemia - Biology & Translational Research

Keyword(s): AML, prognosis, Transcription

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