Contributions
Abstract: PB1677
Type: Publication Only
Background
Non-coding RNA (ncRNAs) genes are at least as frequent as protein-coding genes in the human genome; however, our knowledge on their function is still preliminary. Their emerging role as new players in cancer development and therapy response is widely supported by several scientific reports.
Aims
Here, we aim to investigate BALR-2 involvement in pediatric acute myeloid leukemia (AML).
Methods
We retrospectively analyzed by RQ-PCR bone marrow samples of 132 children with de novo AML harboring different genetic abnormalities (CBF,MLL, and NUP98 rearrangements, FLT3-ITD, other rare translocations and patients without any recurrent molecular abnormality) diagnosed between 2002-2014 in one of the AIEOP centers and treated according to AML-2002/01 protocol. We compared patients’ gene expression signatures (n=58, HTA affymetrix 2.0) with either high (4th quartile) or low expression (1st+2nd+3rd quartiles) of BALR-2, and used gene set and single sample enrichment analysis (GSEA, ssGSEA) to search biological differences. AML cell lines with different BALR-2 expression (high and low) were used to perform in vitro experiments.
Results
The expression of BALR-2 was found higher in all AML samples as compared with those collected from healthy volunteers. Moreover, we did not find any correlation between BALR-2 expression and any specific genetic subtype. We subdivided patients by using BALR-2 expression quartiles and found that patients with higher BALR-2 expression (4th quartile, n=32) had worse, although not statistically significant, EFS when compared to that of patients allocated to the 1st+2nd+3rd quartiles (n=100), butwe observed that the 4th quartile was enriched for patients who did not reach complete remission (CR) after induction therapy (28% vs 12%, p=0.03). Supervised clustering analyses showed that cases belonging to the 4th quartile (n=18) clustered separately from the remaining quartiles (n=40), irrespectively of the genetics. In particular, we found that patients with high BALR-2 (4th quartile) had 57 coding and 12 non-coding RNAs significantly differentially expressed (Fold Change|2|, p<0.01), and an upregulation of processes regulating mitochondrial mass and activity was found (p<0.05). We silenced BALR-2 in SHI-1 AML cell line (high BALR-2 expression) and revealed a decrease of mitochondrial mass by TOM20 staining (p<0.001), an enhanced mitochondrial depolarizion by JC1 staining, and an higher sensibility to FCCP after 6 and 24h of BALR-2 knockdown. We further investigated CDK6, being chromosomally adjacent to BALR-2, and demonstrated a positive correlation of these genes expression levels (Pearson correlation>0.7, p<0.05). Silencing of BALR-2 in vitro reduced CDK6 mRNA and protein levels, as well as phospho-RB, its direct target. Noteworthy, we observed BALR-2 depletion increasing myelomonocytic differentiation, with CD11 and PU.1, both Runt-related transcription factor (RUNX1) targets, being upregulated. In agreement with these findings we showed that additional 81 RUNX1 target genes were down-regulated in patients with high BALR-2 (4th quartile) and-CDK6 expression.
Conclusion
Taken together, our data suggest that pediatric AML may have a broad heterogeneity in metabolic requirements and capacities as well as mitochondrial energetic through BALR-2 expression. We also highlight a cis-regulatory transcriptional relationship between BALR-2 and CDK6 linked to myeloid differentiation to be further dissected.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): AML, Differentiation, Gene expression, Mitochondria
Abstract: PB1677
Type: Publication Only
Background
Non-coding RNA (ncRNAs) genes are at least as frequent as protein-coding genes in the human genome; however, our knowledge on their function is still preliminary. Their emerging role as new players in cancer development and therapy response is widely supported by several scientific reports.
Aims
Here, we aim to investigate BALR-2 involvement in pediatric acute myeloid leukemia (AML).
Methods
We retrospectively analyzed by RQ-PCR bone marrow samples of 132 children with de novo AML harboring different genetic abnormalities (CBF,MLL, and NUP98 rearrangements, FLT3-ITD, other rare translocations and patients without any recurrent molecular abnormality) diagnosed between 2002-2014 in one of the AIEOP centers and treated according to AML-2002/01 protocol. We compared patients’ gene expression signatures (n=58, HTA affymetrix 2.0) with either high (4th quartile) or low expression (1st+2nd+3rd quartiles) of BALR-2, and used gene set and single sample enrichment analysis (GSEA, ssGSEA) to search biological differences. AML cell lines with different BALR-2 expression (high and low) were used to perform in vitro experiments.
Results
The expression of BALR-2 was found higher in all AML samples as compared with those collected from healthy volunteers. Moreover, we did not find any correlation between BALR-2 expression and any specific genetic subtype. We subdivided patients by using BALR-2 expression quartiles and found that patients with higher BALR-2 expression (4th quartile, n=32) had worse, although not statistically significant, EFS when compared to that of patients allocated to the 1st+2nd+3rd quartiles (n=100), butwe observed that the 4th quartile was enriched for patients who did not reach complete remission (CR) after induction therapy (28% vs 12%, p=0.03). Supervised clustering analyses showed that cases belonging to the 4th quartile (n=18) clustered separately from the remaining quartiles (n=40), irrespectively of the genetics. In particular, we found that patients with high BALR-2 (4th quartile) had 57 coding and 12 non-coding RNAs significantly differentially expressed (Fold Change|2|, p<0.01), and an upregulation of processes regulating mitochondrial mass and activity was found (p<0.05). We silenced BALR-2 in SHI-1 AML cell line (high BALR-2 expression) and revealed a decrease of mitochondrial mass by TOM20 staining (p<0.001), an enhanced mitochondrial depolarizion by JC1 staining, and an higher sensibility to FCCP after 6 and 24h of BALR-2 knockdown. We further investigated CDK6, being chromosomally adjacent to BALR-2, and demonstrated a positive correlation of these genes expression levels (Pearson correlation>0.7, p<0.05). Silencing of BALR-2 in vitro reduced CDK6 mRNA and protein levels, as well as phospho-RB, its direct target. Noteworthy, we observed BALR-2 depletion increasing myelomonocytic differentiation, with CD11 and PU.1, both Runt-related transcription factor (RUNX1) targets, being upregulated. In agreement with these findings we showed that additional 81 RUNX1 target genes were down-regulated in patients with high BALR-2 (4th quartile) and-CDK6 expression.
Conclusion
Taken together, our data suggest that pediatric AML may have a broad heterogeneity in metabolic requirements and capacities as well as mitochondrial energetic through BALR-2 expression. We also highlight a cis-regulatory transcriptional relationship between BALR-2 and CDK6 linked to myeloid differentiation to be further dissected.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): AML, Differentiation, Gene expression, Mitochondria