Contributions
Abstract: EP409
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
High long-term relapse after induction chemotherapy is frequently observed among acute myeloid leukemia (AML) patients largely due to the presence of a heterogenous population of leukemic stem cells (LSC) with uncontrolled self-renewal ability and capable of maintaining an undifferentiated state. Based on a 17-gene stemness signature for high-risk AML (Ng SW, Nature. 2016), we identified FAM30 as a novel long non-coding RNA (lncRNA) highly expressed in the most primitive hematopoietic lineage. Nonetheless, the role of FAM30A in hematopoiesis as well as its clinical significance in AML remain unknown.
Aims
We aimed to characterize FAM30A at a molecular level and unravel the functional roles of this lncRNA in differentiation and chemoresistance processes in AML.
Methods
Colony-forming unit (CFU) methylcellulose assays were performed to assess the role of FAM30A in differentiation. FAM30A gain- or loss-of function studies were used to monitor RNA expression levels of LSC- and myeloid markers. Protein and RNA immunoprecipitation and RNA pulldown (biotinylated-tagged RNA) assays were performed in order to demonstrate RNA-protein specific interactions. To determine whether FAM30A has any protein-coding potential, we used sucrose gradient-based fractionation and performed targeted polysome profiling. Localization of FAM30A was revealed by cell fractionation.
Results
FAM30A expression levels were compared between AML patients and healthy donors based on a RNAseq dataset of a TCGA cohort (PanCancerAtlas) and revealed a significantly higher expression of FAM30A in AML samples compared to healthy donors (p<0.001, n=186). Importantly, high levels of FAM30 significantly correlated with unfavourable karyotypes and refractory disease. AML cell lines expressing high levels of FAM30A were used as a mechanistic model to study the molecular mechanisms in AML LSC residing in the CD34+ CD38- fraction as these cells have been shown to be most therapy-resistant and least immunogenic. Polysome profiling showed that FAM30A is indeed a long multi-exonic non-coding protein transcript, predominantly not found in polysomal fractions. However, we observed that FAM30A is mainly cytoplasmic at steady state. In silico prediction of potential binding sites of RNA-binding proteins (RBP) binding sites showed several putative conserved binding motifs for Musashi-2 (MSI2), a well-characterised oncoRBP known to play a critical role in regulating chemoresistance and LSC fate. Accordingly, detailed analysis of FAM30A sequence revealed MSI2-binding sites organized in tandem repeats. CFU-assays showed that exogenous expression of the MSI2-interacting region of FAM30A massively enhanced the potential of colony-formation (p<0.001) and promoted a monocyte-like differentiation status in AML LSC-like cell lines. Reversely, FAM30A knockdown in FAM30Ahigh cell lines was able to impair differentiation. Nonetheless, rescue experiments of the MSI2-binding region of FAM30A could not restore the original stem cell-like phenotype, suggesting a MSI2-dependent regulation of LSC stemness by FAM30A.
Conclusion
We identified the novel lncRNA FAM30A and its binding partner MSI2 as a potential novel regulatory axis of leukemic stem cell phenotypes which mediates differentiation and chemoresistance in AML. Moreover, we suggest that modulation of FAM30A expression could be exploited as a new therapeutic avenue in refractory and relapse AML.
Keyword(s): Acute myeloid leukemia, CD34+ cells, Chemoresistance, Leukemic stem cell
Abstract: EP409
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
High long-term relapse after induction chemotherapy is frequently observed among acute myeloid leukemia (AML) patients largely due to the presence of a heterogenous population of leukemic stem cells (LSC) with uncontrolled self-renewal ability and capable of maintaining an undifferentiated state. Based on a 17-gene stemness signature for high-risk AML (Ng SW, Nature. 2016), we identified FAM30 as a novel long non-coding RNA (lncRNA) highly expressed in the most primitive hematopoietic lineage. Nonetheless, the role of FAM30A in hematopoiesis as well as its clinical significance in AML remain unknown.
Aims
We aimed to characterize FAM30A at a molecular level and unravel the functional roles of this lncRNA in differentiation and chemoresistance processes in AML.
Methods
Colony-forming unit (CFU) methylcellulose assays were performed to assess the role of FAM30A in differentiation. FAM30A gain- or loss-of function studies were used to monitor RNA expression levels of LSC- and myeloid markers. Protein and RNA immunoprecipitation and RNA pulldown (biotinylated-tagged RNA) assays were performed in order to demonstrate RNA-protein specific interactions. To determine whether FAM30A has any protein-coding potential, we used sucrose gradient-based fractionation and performed targeted polysome profiling. Localization of FAM30A was revealed by cell fractionation.
Results
FAM30A expression levels were compared between AML patients and healthy donors based on a RNAseq dataset of a TCGA cohort (PanCancerAtlas) and revealed a significantly higher expression of FAM30A in AML samples compared to healthy donors (p<0.001, n=186). Importantly, high levels of FAM30 significantly correlated with unfavourable karyotypes and refractory disease. AML cell lines expressing high levels of FAM30A were used as a mechanistic model to study the molecular mechanisms in AML LSC residing in the CD34+ CD38- fraction as these cells have been shown to be most therapy-resistant and least immunogenic. Polysome profiling showed that FAM30A is indeed a long multi-exonic non-coding protein transcript, predominantly not found in polysomal fractions. However, we observed that FAM30A is mainly cytoplasmic at steady state. In silico prediction of potential binding sites of RNA-binding proteins (RBP) binding sites showed several putative conserved binding motifs for Musashi-2 (MSI2), a well-characterised oncoRBP known to play a critical role in regulating chemoresistance and LSC fate. Accordingly, detailed analysis of FAM30A sequence revealed MSI2-binding sites organized in tandem repeats. CFU-assays showed that exogenous expression of the MSI2-interacting region of FAM30A massively enhanced the potential of colony-formation (p<0.001) and promoted a monocyte-like differentiation status in AML LSC-like cell lines. Reversely, FAM30A knockdown in FAM30Ahigh cell lines was able to impair differentiation. Nonetheless, rescue experiments of the MSI2-binding region of FAM30A could not restore the original stem cell-like phenotype, suggesting a MSI2-dependent regulation of LSC stemness by FAM30A.
Conclusion
We identified the novel lncRNA FAM30A and its binding partner MSI2 as a potential novel regulatory axis of leukemic stem cell phenotypes which mediates differentiation and chemoresistance in AML. Moreover, we suggest that modulation of FAM30A expression could be exploited as a new therapeutic avenue in refractory and relapse AML.
Keyword(s): Acute myeloid leukemia, CD34+ cells, Chemoresistance, Leukemic stem cell