We and others recently showed that coordinated expression of long non-coding (lnc)RNA is essential for myeloid differentiation and that, in turn, deregulation of lncRNA contributes to the pathogenesis of acute myeloid leukemia (AML).1 To better understand the relationship between lncRNA that control granulopoiesis and the block of differentiation in AML blasts, we sequenced stranded, non-poly-A-enriched cDNA libraries that were prepared from healthy human donor bone marrow (deposited under GEO accession number #GSE98946). Within this dataset, expression of the lncRNA Cancer Susceptibility 15 (CASC15) was significantly inversely correlated with myeloid differentiation and highly enriched in myeloblasts,1 which suggests a role in maintaining stem cell features such as an immature phenotype and/or self-renewal. Due to the reported proto-oncogenic role of CASC15 in childhood AML,2 we further explored CASC15 in the context of adult AML, especially in patients with mutated isocitrate dehydrogenase (IDH).
To assess the role of CASC15 in human AML cells, we genetically deleted the promoter and transcription start site of CASC15 in the human OCI-AML-5 cell line via a CRISPR/Cas9 approach (CASC15-KO). In brief, OCIAML- 5 cells were transduced with Cas9-ORF-carrying a DHC013 vector and selected with 5 mg/mL blasticidin. Cas9 expression was confirmed by western blotting. Single guide (sg)RNA were designed using CCTop (https://crispr.cos.uni-heidelberg.de/): T1: 5’-CACCGATGGATTACAATTTGATCGC- 3’fw, 5’-AAACGCGATCAAATTGTAATCCATC- 3’rev; T2: 5’-CACCgATTAATTAAAGTTAGCTTCC-3’fw, 5’- AAACGGAAGCTAACTTTAATTAATC-3’rev. Each sgRNA pair was cloned into either pL40CCRISPR. EFS.PAC or pL-CRISPR.EFS.tRFP, respectively (kindly provided by Dirk Heckl), each with the Cas9-ORF removed. Cells (1x106/mL) were spin-infected with an equimolar mix of virus-containing medium + 10 mg/mL polybrene. Single-cell clones were sorted, expanded in 1 mg/mL puromycin, and sent for Sanger sequencing. Lack of CASC15 expression was confirmed in heterozygous knock-out (KO) and control cell lines via polymerase chain reaction (PCR) (PlatinumTaq, ThermoFisher Scientific) and quantitative real-time PCR (qRT-PCR) (TBgreen, TaKaRa, Japan) using the following primer sets: CASC15 exon1: 5’-GGGTATCTCCCTCTCGCAAC- 3’fw, 5’-CGTTCGGACACTTTTTCCCG-3’rev; CASC15 exon2: 5’-TGACCTCCTTCATTCTGCGTT- 3’fw, 5’-GGGTATAAGCCCCAGACCAA-3’rev. Loss of CASC15 did not sustainably affect apoptosis or SOX4 RNA levels in these cells (Figure 1A, B), as was previously reported for CASC15-KO in a RUNX1-rearranged AML background,2 but increased the expression of myeloid surface markers (Figure 1C). To induce differentiation, we treated 0.75x106 cells with 1 nM 12-O-tetradecanoylphorbol- 13-acetate (TPA) or 100 nM all-trans retinoic acid (ATRA) (Sigma-Aldrich) and assessed the level of differentiation by flow cytometry using α-CD11b-APC (#17-0118-41, clone ICRF44, eBioscience, San Diego, CA, USA), α-CD11c-APC (#17-0116-42, clone 3.9, ThermoFisher Scientific, USA), α-CD13-FITC (#ab52461, clone WM15, abcam, Cambridge, UK), and α-CD14-APC (#17-0149-42, clone 61D3, eBioscience), α-CD15-FITC (#555401, lot 13878, Becton Dickinson, San Jose, CA, USA) antibodies.
In response to TPA and ATRA, CASC15-KO cells showed enhanced myeloid differentiation and reduced proliferation compared to the empty vector control cells (Figure 1D-F), suggesting that the presence of CASC15 contributes to the block of differentiation in human AML cells.
The highly complex genomic structure of the CASC15 locus pointed towards an equally complex transcriptional regulation and post-transcriptional processing of this (lnc)RNA (Figure 1G). Interrogating the entire transcription factor and co-factor footprinting data generated for the ENCODE project in K562 cells,4 we observed that the strongest binding intensities at the CASC15 site were for the erythroid differentiation factor TAL-1, the protooncogenic proteins JunD and c-MYC/MAZ, the co-activator p300, as well as for HEY-1, GATA-2, and members of the MAFF/BZIP family (Figure 2A). These transcription factors predominantly occupy the annotated CASC15 enhancer GH06J022044, which is located in intron 7 (GeneHancer v2, Jan 2019) (Figure 1G), share a strong enrichment for the basic helix-loop-helix motif, and are associated with transcriptional control in non-committed myeloid progenitors.5 This suggests that CASC15 is regulated by myeloid transcription factors, supporting our findings in human myeloblasts and AML cells.
In four independent transcriptome datasets (including the Beat AML and TCGA LAML cohorts as well as GEO datasets #GSE6891 and #GSE104099, for a total of 1,145 patients), we observed the highest CASC15 expression in AML patients with t(8;21) and in cytogenetically normal AML patients harboring IDH or TET2 mutations (shown for the 2 largest cohorts in Figure 2B, C). Mutations in IDH1/2 and TET2 represent early, almost mutually exclusive events in AML patients and are associated with a hypermethylation genotype as both mutations eventually reduce the cytosine demethylation activity of TET2.6 In contrast, most DNMT3A mutations are thought to result in genome-wide CpG hypomethylation by formation of non-functional mutant:wild-type DNMT3A heterodimeric complexes.7 IDH and DNMT3A mutations frequently co-occur in AML and genomes of IDH/DNMT3A double-mutant patients were found to exhibit an epigenetic antagonism, in which CpG that are affected by either mutation alone are no longer affected.8 Interestingly, we observed strong downregulation of CASC15 in patients carrying mutant DNMT3A or signaling- activating mutations (FLT3, NRAS, KRAS, PTPN11) (Figure 2B, C). We confirmed this by engineered overexpression of IDH2R172K mutant cDNA in human DNMT3Awild-type (HL-60) and DNMT3Amutant (OCI-AML2/3) AML cell lines. The IDH2R172K-mutation was introduced into human IDH2wild-type cDNA via PCR (pEX-C0462-M02-50, Genecopoeia), cloned into a third-generation “selfinactivating” vector (kindly provided by Tobias Mätzig), and confirmed by Sanger sequencing. Lentiviral production and transduction were performed as previously described.3 Whereas CASC15 levels did not change in DNMTAR635W OCI-AML2 and DNMTAR882C OCI-AML3 cells, CASC15 was significantly upregulated (P=0.01) in DNMT3Awild-type HL-60 cells in response to ectopic expression of mutant IDH2 (Figure 3A).
A more detailed analysis of CASC15 expression in subgroups of AML patients with different cytogenetic and molecular features revealed a generally balanced expression of two different CASC15 isoforms: a short 7-exon (ENST00000606336.5) and a long CASC15 transcript containing all 12 annotated exons (ENST00000606851.5) (Figure 1G). However, in IDH2mutant as well as in t(8;21) patients, in whom CASC15 expression is highest, we detected a significantly (P<0.003) enriched expression of its long isoform (Figure 3B). This isoform switch was not observed in groups of patients with low CASC15 expression, such as cytogenetically normal AML with FLT3-ITD (Figure 2B). This novel observation suggests differential or aberrant splicing in these subgroups of patients, with as of yet unknown cause or functional relevance.
In line with the elevated expression of CASC15 in lower-risk cytogenetic and molecular AML subtypes, we found that high CASC15 RNA expression was associated with improved overall survival in four independent adult AML datasets (n=584 patients) (Figure 3C).
Although we found CpG methylation to be generally increased in IDHmut and decreased in DNMT3Amut AML patients at the CASC15 transcription start site and gene body (Figure 1G), the finding that CASC15 is highly expressed in hypermethylated (IDHmut and TET2mut) and less expressed in patients with hypomethylated genotypes (DNMT3Amut and NPM1mut) suggests that these methylation changes do not directly affect CASC15 transcription. The molecular consequences of IDH and DNMT3A mutations are not limited to epigenetic changes but also affect cellular metabolism and disturb DNA damage responses,9,10 which is thought to promote genomic instability and to account for the high number of co-mutations observed in IDH- and DNMT3A-mutant leukemias. In comparison to the high CASC15 levels in IDH2R140 AML patients, we found that CASC15 expression was often lower in IDH1R132 and IDH2R172 patients (Figure 2B, C), which underlines the presence of different transcriptional landscapes in each mutational entity10 and suggests that the R140 and R172 mutations affect the cooperation of IDH2 with different interaction partners.
Finally, we also observed that the CASC15 genomic site possesses a potential topologically-associating domain boundary in THP-1 and K562 myeloid leukemia cells, which is located between two CASC15 enhancers and is not present in non-myeloid cell lines (Figure 3D). Aberrant methylation at this CTCF-insulator in response to methylation-affecting mutations could be a plausible mechanism responsible for the counterintuitive expression of CASC15 in hypo- versus hyper-methylated AML, as had already been shown in IDH-mutant gliomas,11 and could potentially affect regional splicing.
In summary, we report that the survival-associated lncRNA CASC15 acts as a proto-oncogene in AML cells, in which it affects proliferation and differentiation. We further found that its expression is indirectly linked to DNA methylation-affecting mutations and succumbs to splicing, potentially through a mechanism that involves an intragenic lineage-specific CTCF site. These novel insights warrant further investigation, especially in the context of treatment response to IDH inhibitors.
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