Contributions
Abstract: EP328
Type: E-Poster Presentation
Session title: Acute lymphoblastic leukemia - Biology & Translational Research
Background
Treatment of pediatric acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) remains challenging, in particular after relapse. In both pediatric ALL and especially AML, death from intensity-maximized conventional chemotherapies is still unacceptably high, and outcome re-mains poor in both AML and high-risk ALL. Thus, there is still an urgent need to identify new targetable structures and/or pathways. Transcriptional regulation in leukemias has been extensively studied and targeted therapies are under evaluation, however, the post-transcriptional layer of gene-regulation remains underexplored. Mutations and aberrant expression patterns of RNA-binding proteins (RBPs) has been detected in leukemia and implicated in leukemic transformation, with notable examples like IGF2BP3 (IGF2 mRNA binding protein 3) and MSI2 (Musashi 2). However, the oncogenic potential of most of the more than 1500 RBPs has not been studied in pediatric acute leukemias.
Aims
We aim to identify oncogenic RBPs that play a significant and targetable role in hard-to-cure subtypes of leukemias.
Methods
We performed pooled CRISPR-Cas9 dropout screenings followed by next generation sequencing using a pool of sgRNAs directed against 1500 RBPs. The screens were performed in four AML and five ALL cell lines (CMK, NOMO1, M-07e, SKNO1, NALM6, HAL-01, SEM, REH, KOPN8), representing either common leukemic subtypes or those associated with poor prognosis. SgRNA frequencies were quantified at the outset of the experiment and after 16 doublings using deep sequencing (Illumina HiSeq 4000). MAGeCK analysis was conducted for each cell line, with negative scores indicating depleting sgRNAs.
Results
We identified 191 putative oncogenes whose knockout led to a significant loss of cell fitness in at least one of the 9 screens. We further observed overlaps of depleting RBPs between the ALL cell lines and the AML cell lines. To differentiate between a general essential role of a given RBP in any cell (e.g. essential in RNA/cellular metabolism) and a putative necessary function in initiating or promoting leukemic growth, we integrated our data with cell line data collected in the framework of the project Achilles. We compared essentiality scores in leukemic cell lines to those cell lines of all origins to derive those RBPs, which display oncogenic properties (as opposed to just representing core essential factors).
One of 191 identified putative oncogenes is CAD, which pro-proliferative potential was verified experimentally in both, AML and ALL. In terms of clinical relevance, we found a tendency towards reduced overall survival (OS) in AML patients with high CAD protein expression. Since CAD protein is a multi-domain enzyme and plays a pivotal role in pyrimidine synthesis, we will dissect its independent function as an RBP.
Conclusion
Our CRISPR-Cas9 screening in combination with functional validation states the pro-proliferative potential of many RBPs including CAD in AML and ALL cell lines and highlights its utility for identifying novel targetable mechanisms in leukemia.
Keyword(s): Acute leukemia, Screening
Abstract: EP328
Type: E-Poster Presentation
Session title: Acute lymphoblastic leukemia - Biology & Translational Research
Background
Treatment of pediatric acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) remains challenging, in particular after relapse. In both pediatric ALL and especially AML, death from intensity-maximized conventional chemotherapies is still unacceptably high, and outcome re-mains poor in both AML and high-risk ALL. Thus, there is still an urgent need to identify new targetable structures and/or pathways. Transcriptional regulation in leukemias has been extensively studied and targeted therapies are under evaluation, however, the post-transcriptional layer of gene-regulation remains underexplored. Mutations and aberrant expression patterns of RNA-binding proteins (RBPs) has been detected in leukemia and implicated in leukemic transformation, with notable examples like IGF2BP3 (IGF2 mRNA binding protein 3) and MSI2 (Musashi 2). However, the oncogenic potential of most of the more than 1500 RBPs has not been studied in pediatric acute leukemias.
Aims
We aim to identify oncogenic RBPs that play a significant and targetable role in hard-to-cure subtypes of leukemias.
Methods
We performed pooled CRISPR-Cas9 dropout screenings followed by next generation sequencing using a pool of sgRNAs directed against 1500 RBPs. The screens were performed in four AML and five ALL cell lines (CMK, NOMO1, M-07e, SKNO1, NALM6, HAL-01, SEM, REH, KOPN8), representing either common leukemic subtypes or those associated with poor prognosis. SgRNA frequencies were quantified at the outset of the experiment and after 16 doublings using deep sequencing (Illumina HiSeq 4000). MAGeCK analysis was conducted for each cell line, with negative scores indicating depleting sgRNAs.
Results
We identified 191 putative oncogenes whose knockout led to a significant loss of cell fitness in at least one of the 9 screens. We further observed overlaps of depleting RBPs between the ALL cell lines and the AML cell lines. To differentiate between a general essential role of a given RBP in any cell (e.g. essential in RNA/cellular metabolism) and a putative necessary function in initiating or promoting leukemic growth, we integrated our data with cell line data collected in the framework of the project Achilles. We compared essentiality scores in leukemic cell lines to those cell lines of all origins to derive those RBPs, which display oncogenic properties (as opposed to just representing core essential factors).
One of 191 identified putative oncogenes is CAD, which pro-proliferative potential was verified experimentally in both, AML and ALL. In terms of clinical relevance, we found a tendency towards reduced overall survival (OS) in AML patients with high CAD protein expression. Since CAD protein is a multi-domain enzyme and plays a pivotal role in pyrimidine synthesis, we will dissect its independent function as an RBP.
Conclusion
Our CRISPR-Cas9 screening in combination with functional validation states the pro-proliferative potential of many RBPs including CAD in AML and ALL cell lines and highlights its utility for identifying novel targetable mechanisms in leukemia.
Keyword(s): Acute leukemia, Screening