Overexpression of SNORD114-3 marks acute promyelocytic leukemia

A global downregulation of small nucleolar RNAs (snoRNA) was reported in acute lymphoblastic (ALL) and acute myeloid leukemias (AML) by Valleron et al.1 The authors also identified a specific snoRNA signature in acute promyelocytic leukemia (APL) with overexpression of snoRNA clusters SNORD112-114. Recently, Cohen et al.2 reported a marked overexpression of SNORD113-3, SNORD113-4, SNORD114-2 and SNORD114-3 in three additional cases of APL.

We have compiled a hematological gene expression data set of neoplastic and non-neoplastic samples hybridized to Affymetrix HG-U133 Plus 2.0 GeneChips, all downloaded from the GEO microarray repository. The data were collected as described earlier by Heinäniemi et al.3 Of all the genes on the microarray, 15 were identified as snoRNAs (Table 1). They represent various types of snoRNAs, including H/ACA and CD box types, small Cajal body-specific RNAs (scaRNA), intergenic and intronic snoRNAs, and even one snoRNA without any known target RNA (so-called ‘orphan’ snoRNA).

Table 1 Characteristics of 15 selected snoRNAs

To study the expression of the 15 snoRNAs, we focused on 883 cases of pediatric leukemias, which were subdivided into three classes: early B-ALL, T-ALL and AML. In addition, we considered 35 non-neoplastic samples consisting of hematopoietic stem cells (HSCs) along with naive B- and naive T-lymphocytes. In most cases, the snoRNAs were expressed in a relatively uniform manner across all leukemia subtypes and normal blood cells (Figure 1a). In our limited set of 15 snoRNAs, we did not observe a lower expression in acute leukemias as compared with HSCs and naive lymphocytes. Two snoRNAs, SNORA70 and SNORD104, were expressed consistently higher than other snoRNAs in leukemic as well as healthy samples. They were expressed at a level approximately fourfold higher relative to other snoRNAs (Figure 1a). Interestingly, SNORA25 and SNORA61 were expressed strongly in naive, or unstimulated, B cells compared to HSCs, naive T cells and leukemias, suggesting B-cell differentiation-dependent regulation of expression (Figure 1a).

Figure 1
figure1

Overexpression of SNORD114-3 in APL. (a) Expression of the 15 studied snoRNAs in three types of pediatric leukemias and four healthy cell types. (b) The expression of SNORD114-3 in pediatric leukemias. (c) When pediatric and adult AML patients are sorted according to SNORD114-3 expression, APL cases are clearly enriched among the patients with high expression. By using Mann–Whitney U-test, differential SNORD114-3 expression between APL and other AML was deemed statistically significant. (d) SNORD114-3 expression is strongly correlated with MEG3 expression, but weakly with DLK1 and DIO3. Pearson’s correlation coefficients were calculated using log2 expressions.

At single patient level, the expression of individual snoRNAs remained again rather constant except for SNORD114-3 (Figure 1a and Supplementary Figure S1). Among the 237 pediatric AML patients, 15 were found to have increased expression of SNORD114-3 when the cut-off level in log2-expression was set at 7 (Figure 1b). Interestingly, 13 out of these 15 patients harbored t(15;17) translocation, the hallmark of APL. In the two remaining t(15;17)-negative patients, the expression was only slightly above the threshold level of 7. These two samples did not fall into any major cytogenetic subtype of AML, but one of them had an internal tandem duplication of FLT3 and the other carried a mutation in either NRAS or KRAS. Among the cases with normal SNORD114-3 expression, only six patients out of 222, or 2.7%, were identified as t(15;17)-positive patients. Four of these six APL cases were carrying FLT3-ITD. A similar pattern emerged also in the cohort of 1117 adult AML patients in our database, 69 of which were of promyelocytic subtype (Figure 1c). One-hundred and nine adult AML samples were found with an elevated SNORD114-3 expression (keeping the threshold at 7). Out of them, 50 were from APL patients. From the adult samples with normal SNORD114-3 expression, only 19, or 1.9%, were classified as APL. The expression level of SNORD114-3 differentiated the APL-positive and -negative cases among both pediatric (P<10−8) and adult AML (P<10−28). Mann–Whitney U-test was used to assess statistical significance.

The snoRNA cluster SNORD114 lies in the genomically imprinted domain DLK1-DIO3 in the 14q32 region. Genomic imprinting gives rise to mono-allelic, parent-of-origin-specific gene expression. Imprinted genes are susceptible to errors, as a single genomic or epigenetic change may alter or even ablate their function. Imprinted small RNAs are involved in the development and metabolism, and are also frequently perturbed in malignancies.4 The paternal allele of DLK1-DIO3 domain expresses three protein-coding genes (DLK1, RTL1 and DIO3), whereas the maternal counterpart expresses two non-coding transcripts (MEG3 and RTL1AS) accompanied by 53 microRNAs and two snoRNA clusters (SNORD113 and -114) constituting 9 and 31 snoRNAs, respectively.4 To gain some insight on how deregulation of DLK1-DIO3 affects other genes it houses, we looked at the expressions of DLK1, DIO3 and MEG3. Besides SNORD114-3, these were the only genes in this domain to be represented on the microarray. The expression of MEG3 (Maternally Expressed Gene 3) was strongly correlated with that of SNORD114-3, whereas such correlation was not observed between SNORD114-3 and DLK1 or DIO3 (Figure 1d). This reflects the fact that MEG3 is expressed from the same (maternal) allele as SNORD114, whereas DLK1 and DIO3 are expressed from the other (paternal) one. Furthermore, it suggests that the deregulation may affect the maternal DLK1-DIO3 locus as a whole.

As it happens, SNORD114-3, like all the copies in SNORD114, is orphan. The function of such snoRNAs lacking RNA targets is unknown but they might have gene regulatory roles as microRNA precursors or by being involved in gene splicing events.5 Valleron et al.1 showed that SNORD114-1 is capable of modulating cell growth and proliferation in APL cells, thus suggesting leukemogenicity of SNORD114. Recently, Chu et al.6 found that ACA11, an orphan snoRNA encoded in an intron of the WHSC1 gene, is overexpressed in multiple myeloma patients with the translocation t(4;14). In these cells, overexpression of ACA11 increased proliferation, suppressed oxidative stress and conferred chemoresistance.

Our findings in regard to APL support and increase the robustness of those reported by Valleron et al.1 and Cohen et al.,2 as we used data obtained with a different platform and from a substantially larger cohort of patients. Collectively, they reveal an intricate regulation of snoRNA expression7 and add to the mounting evidence implicating DLK1-DIO3 locus in tumorigenesis. Seven microRNAs of this locus were found to be upregulated in APL by Dixon-McIver et al.,8 and other transcripts of DLK1-DIO3 domain have been associated with lung cancer,9 hepatocellular carcinoma10 and the pluripotency levels of stem cells.11

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Liuksiala, T., Teittinen, K., Granberg, K. et al. Overexpression of SNORD114-3 marks acute promyelocytic leukemia. Leukemia 28, 233–236 (2014). https://doi.org/10.1038/leu.2013.250

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