Background:
Acute myeloid leukemia (AML) is thought to be sustained by sub-populations of leukemia stem cells (LSCs), which possess the capacity for self-renewal and differentiation and are believed to be responsible for disease initiation, relapse and chemoresistance. There is therefore an urgent need to develop therapies that target the LSC population to achieve effective AML treatment. To date there has been limited success in this endeavor, highlighting the importance of gaining a more comprehensive understanding of the mechanistic elements that underpin LSC function.
Aberrant alternative splicing is recognized as a key driver of cancer. In the context of AML, genome-wide sequencing studies have shown that approximately one third of genes are differentially spliced in primitive CD34+ cells in AML patients compared to those obtained from normal controls. In particular, LSCs also have a unique splicing profile when compared to normal aging HSCs, underscoring the importance of understanding the mechanistic controls of aberrant splicing in LSC function.
Results:
Previous studies examining the link between aberrant splicing and AML focused on spliceosome genes with somatic mutations in AML patients. To examine mechanisms that mediate aberrant alternative splicing in LSC beyond splicing factor mutations, we performed a data-mining survey of 203 known mRNA splicing factors. Strikingly, RNA-binding motif protein 17 (RBM17) is the only splicing factor that is both strongly linked to poor AML prognosis and significantly elevated in LSC-enriched subsets of primary AML samples based on a recent study of 78 AML samples with normal karyotype. RBM17 has been implicated in regulating alternative splicing and cancer chemotherapy resistance. However, its function in AML or LSCs is not known.
From our studies, we found that the level of RBM17 protein is elevated in the phenotypically primitive subsets of primary AML samples (n=8, RBM17+%: 67.42% in CD34+ versus 34.66% in CD34-fractions). Depletion of RBM17 with shRNAs in 3 human primary AML samples resulted in reduced colony formation compared to shScramble controls. More importantly, knockdown of RBM17 in a primary AML sample greatly impeded AML engraftment in immune-deficient mice (mean of 0.89 control versus mean of 0.1671 and 0.3171 shRNAs), suggesting that RBM17 is required for the stem and progenitor potential of AML and maintenance of LSC populations.
Intriguingly, in contrast to the situation for the malignant hierarchy, the level of RBM17 in normal HSCs is lower than that in more committed cell populations in the normal hematopoietic system. To determine if RBM17 plays different roles in malignant LSC and normal hematopoietic stem and progenitor cells (HSPCs), we depleted RBM17 with shRNAs in human cord blood (CB) derived CD34+ HSPCs, and found RBM17 knockdown had negligible adverse impact on both CB total colony and primitive GEMM colony outputs and yielded no increase in apoptosis, indicating no defects were apparent to primitive cells of CB as read out in vitro.
To uncover the molecular mechanisms underlying the role of RBM17 in LSC functions, we preformed RBM17 eCLIP-seq in the K562 and HL60 human leukemic cell lines. We then cross-analyzed the CLIP-seq datasets with a published ENCODE RNA-seq dataset (RBM17 knockdown in K562 cells), where we found RBM17 directly binds to transcripts of stem cell program-related genes and regulates the splicing of these genes, including MADD (MAP kinase activating death domain) and MRPS18C (mitochondrial ribosomal protein S18C). We further demonstrated that the splicing patterns of MADD and MRPS18C are mediated by RBM17 in primary AML samples. In our ongoing functional validation experiments, isoform-specific knockdown of MADD or MRPS18C splice variants downstream of RBM17 impeded colony forming capacity and induced myeloid differentiation in the MOLM13 AML cell line. These results suggest that RBM17-mediated splicing events impact primitive cell function in AML.
Conclusion:
We have identified RBM17 as a novel LSC-regulating factor, plays an important role in maintaining AML LSC function through regulating the alternative splicing of stem cell program-related genes. The potential LSC-selective role for RBM17, along with its downstream splicing events, represent promising putative therapeutic targets whose modulation could offer attractive therapeutic windows in AML treatment.
Disclosures
No relevant conflicts of interest to declare.
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