Long non-coding RNA HOXB-AS3 promotes myeloid cell proliferation and its higher expression is an adverse prognostic marker in patients with acute myeloid leukemia and myelodysplastic syndrome.

BACKGROUND: Long non-coding RNAs (lncRNAs) represent the majority of cellular transcripts and play pivotal roles in hematopoiesis. However, their clinical relevance in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) remains largely unknown. Here, we investigated the functions of HOXB-AS3, a lncRNA located at human HOXB cluster, in the myeloid cells, and analyzed the prognostic significances in patients with AML and MDS.
METHODS: shRNAs were used to downregulate HOXB-AS3 in the cell lines and the effect was evaluated by quantitative polymerase chain reaction. The proliferation of the cell lines was illustrated by proliferation and BrdU flow assays. Further, we retrospectively analyzed the HOXB-AS3 expression in 193 patients with AML and 157 with MDS by microarray analysis, and evaluated its clinical importance.
RESULTS: Downregulation of HOXB-AS3 suppressed cell proliferation. Mechanistically, HOXB-AS3 potentiated the expressions of several key factors in cell cycle progression and DNA replication without affecting the expressions of HOX genes. In AML, patients with higher HOXB-AS3 expression had shorter survival than those with lower HOXB-AS3 expression (median overall survival (OS), 17.7 months versus not reached, P <  0.0001; median relapse-free survival, 12.9 months versus not reached, P = 0.0070). In MDS, patients with higher HOXB-AS3 expression also had adverse prognosis compared with those with lower HOXB-AS3 expression (median OS, 14.6 months versus 42.4 months, P = 0.0018). The prognostic significance of HOXB-AS3 expression was validated in the TCGA AML cohort and another MDS cohort from our institute. The subgroup analyses in MDS patients showed that higher HOXB-AS3 expressions could predict poor prognosis only in lower-risk (median OS, 29.2 months versus 77.3 months, P = 0.0194), but not higher-risk group.
CONCLUSIONS: This study uncovers a promoting role of HOXB-AS3 in myeloid malignancies and identifies the prognostic value of HOXB-AS3 expression in AML and MDS patients, particularly in the lower-risk group.

Background

Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are two myeloid malignancies [1] which share similar somatic gene mutations [2]. A portion of MDS patients eventually progresses to AML as the disease progresses. Several prognostic models have been developed to better risk stratify AML and MDS patients, such as European Leukemia Net (ELN) risk classification for AML [3] and international prognostic scoring system (IPSS) or revised IPSS (IPSS-R) for MDS [4-8]. However, patients may have different prognosis even in the same risk group [9-12]. Exploration of more markers that have prognostic significance are warranted to better risk stratify patients with the diseases.

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides without protein coding ability. The functions of most lncRNAs remain poorly characterized, but some of them have been demonstrated to be involved in the hematopoiesis [13-15]. For example, the expression levels of HOTAIRM1 and NEAT1 are elevated during myeloid differentiation, and downregulation of HOTAIRM1 or NEAT1 delays myeloid maturation [13, 14]. Expression of XIST, a lncRNA at X chromosome, inactivates X chromosome in the female cells. The female mice with deletion of Xist develop a highly aggressive disease mimicking MDS/MPN [15]. However, the roles of lncRNAs in MDS remain largely unknown [16], and only a few research investigated the role of lncRNAs in de novo AML [17-19]. In the research aimed to find prognostic biomarkers in acute myeloid leukemia (AML), we found expression of HOXB-AS3, a lncRNA located at human HOXB cluster, is a potential risk factor. However, its clinical relevance and pathogenesis in AML and MDS remain to be determined.

Here, we demonstrate that high expression of HOXB-AS3 is an adverse prognostic factor for both de novo AML and primary MDS patients. Furthermore, the expression of HOXB-AS3 promotes cell proliferation in myeloid cells.

Methods

Patients

We retrospectively included the adult patients with newly diagnosed primary MDS and de novo AML at the National Taiwan University Hospital (NTUH) from 1992 to 2010. Among them, 157 MDS and 193 AML patients, who had available cryopreserved bone marrow (BM) cells for RNA array analysis and comprehensive clinical information, were recruited for this study. The Cancer Genome Atlas (TCGA) AML cohort available on the TCGA website (https://cancergenome.nih.gov/) and an independent cohort of 30 MDS patients subsequently diagnosed between January 2011 and May 2012 at the NTUH was served as the validation cohorts.

All patients with AML other than acute promyelocytic leukemia (non-APL AML, n = 174) underwent standard induction chemotherapy (Idarubicin 12 mg/m2 per day for two to 3 days and Cytarabine 100 mg/m2 per day for five to 7 days), and consolidation chemotherapy with two-to-four courses of high-dose Cytarabine (2000 mg/m2 every twelve hours for 4 days, total eight doses), with or without an anthracycline (idarubicin or mitoxantrone), after they achieved complete remission (CR) as described in our previous studies [20]. Nineteen APL patients received concurrent all-trans retinoic acid (ATRA) and Idarubicin or Mitoxantrone as induction chemotherapy and ATRA with Idarubicin, Mitoxantrone or high dose Cytarabine as consolidation chemotherapy when they achieved CR. If the patients had relapsed or refractory AML, or adverse prognostic factors at diagnosis, such as adverse-risk cytogenetic abnormalities or somatic gene mutations, they underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) when they had feasible hematopoietic stem cell donors.

In the NTUH MDS training cohort, most MDS patients (70.7%) only received supportive care. Eight patients (5.1%) received AML-directed intensive chemotherapies, 18 (11.5%) received hypomethylating agent (azacitadine or decitabbine), and 20 patients (12.7%) underwent allo-HSCT.

The BM samples from AML and MDS patients were collected at diagnosis, and the mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation and cryopreserved as previously described [21, 22]. The BM cells from 20 healthy transplantation donors were used as the normal controls to compare the gene expressions with those of AML and MDS patients. This study was approved by the Institutional Review Board of NTUH (IRB number: 201507084RINA and 201503072RINC). All the patients have signed informed consents for the collection of samples and clinical information.

Analysis of cytogenetic abnormalities and gene mutations

The BM cells were harvested directly or after one to 3 days of un-stimulated cultures. The metaphase chromosomes were banded by the G-banding method as previously described. [23] The determination of mutations in NPM1 [21, 24], AML1 (RUNX1) [25], ASXL1 [26], DNMT3A [20], EZH2 [27], IDH2 [28], NRAS [29], KRAS [29], TP53 [30], SETBP1 [31], SRSF2 [32], TET2 [33], MLL/PTD [34], SF3B1 [35], U2AF35 [36], and ZRSR2 [35] was performed as described previously.

Microarray experiments and analysis

The raw data of TCGA AML cohort was downloaded from TCGA website (https://cancergenome.nih.gov/). The detail methods of microarrays for NTUH AML and NTUH MDS cohorts were described in Additional file 1.

The expression levels of two transcript clusters, TC17002254.hg.1 and TC17002858.hg.1, on Affymetrix GeneChip® HTA 2.0 represent HOXB-AS3 (NCBI Reference Sequence: NC_000017.11) expression. TC17002254.hg.1 detects variants 1 to 5 of HOXB-AS3, and TC17002858.hg.1 detects all variants of HOXB-AS3. Because TC17002858.hg.1 detects all HOXB-AS3 variants and the expression pattern was similar between the two transcript clusters, expression of TC17002858.hg.1 was used to stratify patients.

Cell lines, cell cultures, and associated experiments

OCI/AML3 and TF-1 were human myeloid leukemia cell lines. TF-1 cell line (BCRC number 60323) was purchased from Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan on Sep 29, 2014. BCRC (http://www.bcrc.firdi.org.tw/) is a nation-wide cell bank in Taiwan, and it provides the service of preservation, identification, and selling of cell lines. OCI/AML3 was a gift from Dr. Minden (Ontario Cancer Institute/Princess Margaret Hospital, Canada) in 2008. The detailed methods of cell cultures, constructions of lentiviral vectors with shRNA and lncRNA, lentiviral production, lentiviral infections, quantitative real time PCR, proliferation assay and nuclear-cytoplasm fractionation were described in Additional file 1.

BrdU flow assay

BD Pharmingen™ APC BrdU Flow Kits (Cat. NO. 552598) was used for BrdU flow assay. Cells were incubated with 10 μM BrdU at 37 °C for three hours, and then harvested for BrdU flow assay. The detailed method was described in the user manual of the manufacture. The flow cytometry was performed on LSR II (BD Bioscience, San Jose, CA) through the service provided by the Flow Cytometric Analyzing and Sorting Core Facility at the NTUH, and on FACS Canto II (BD Bioscience, San Jose, CA) through the service provided by the Molecular and Immune Function Laboratory at Tai Cheng Stem Cell Therapy Center at the National Taiwan University.

Statistical analysis

Mann-Whitney test was used to calculate the significance if the continuous data were not normally distributed, and Kruskal-Wallis test was used for comparing the difference between more than two groups. Chi-square test was used to calculate the significance of association between HOXB-AS3 expression and other categorical parameters, including sex, IPSS risk groups, 2016 WHO subtypes, cytogenetic abnormalities, and gene mutations. Fisher exact test was used if any expected value of the contingency table was less than five.

For patients with de novo AML in the NTUH AML cohort, overall survival (OS) was measured from the date of diagnosis to the date of last follow-up or death. The patients were censored on the date of last follow-up if they were alive. Relapse free survival (RFS) was defined from the date of complete remission to the date of relapse, last follow-up, or death. Relapse and death were defined as events in the RFS analysis. If the patients were alive and in complete remission, they would be censored. For patients with primary MDS, OS was measured from the date of diagnosis to the date of last follow-up or death. The patients were censored on the date of last follow-up if they were alive.

Kaplan-Meier (KM) estimation was used to plot survival curves, and log-rank tests were used to calculate the difference of OS and RFS between different groups in AML patients and OS in MDS patients. Median follow-up duration was calculated by reverse KM estimation. Multivariate Cox proportional hazard regression analysis was used to investigate independent prognostic factors for OS. A P value less than 0.05 was considered statistically significant. All statistical analyses were performed with MedCalc® 15.6.1 software (https://www.medcalc.org/).

Results

HOXB-AS3 is an anti-sense lncRNA in the HOXB cluster

In order to find lncRNAs with significant prognostic implications in myeloid malignancies, we analyzed the microarrays data from two cohorts: one AML cohort from the NTUH (NTUH AML cohort), and another one from The Cancer Genome Atlas (TCGA AML cohort) [37]. The expression of 2824 probes in TCGA AML cohort and 4454 transcript clusters in NTUH AML cohort had prognostic significance (Fig. 1a). The intersect between the two cohorts was 907 probes/transcript clusters, among which eleven lncRNAs were validated as non-coding RNAs according to National Center for Biotechnology Information (NCBI) database (Fig. 1a). Among these lncRNAs, HOXB-AS3 is particularly interesting, because of the crucial roles of HOX genes in cell proliferation, hematopoiesis and leukemogenesis [36, 38, 39] and the clinical significance of an anti-sense lncRNA in the HOX clusters, i.e., HOTAIRM1, in leukemia [40]. Since the role of lncRNAs in myeloid malignancies remains unclear, the following studies were focused on the role of HOXB-AS3 in myeloid malignancies.

Fig. 1

Identification of HOXB-AS3 as a prognostic biomarker and overview of its locus in human HOXB. a The numbers of genes influenced overall survival in TCGA AML cohort and NTUH AML cohort. b The neighborhood of HOXB-AS3 locus in human HOXB cluster. The genomic data from ENCODE project was taken from UCSC genome browser. The exon localizations of eight HOXB-AS3 isoforms and HOXB5, HOXB6, and HOXB7 genes are shown. Arrows indicate the transcription direction of these genes. The NCBI Reference Sequence (RefSeq) numbers were labeled in the end of each variant. c PhyloCSF analysis for predicting the noncoding nature of HOXB-AS3. Exon 1, exon 4, exon 5 and exon 6 are zoom-in to see the details of PhyloCSF scores of the specific exons. All PhyloCSF scores are negative in the exons of HOXB-AS3

HOXB-AS3 is a lncRNA located at human HOXB cluster on the chromosome 17q21.32 and has eight transcriptional variants generated by alternative splicing (Fig. 1b). The entire gene is overlapped with HOXB5 and HOXB6 genes, and the transcriptional direction is antisense to the overlapping HOXB genes (Fig. 1b). The exons of HOXB-AS3, except for exon 2, are not overlapped with the exons of HOXB5 or HOXB6 (Fig. 1b). By using PhyloCSF pipeline, HOXB-AS3 is predicted to be a noncoding RNA (Fig. 1c), and the coding probability is low (0.126) by Coding Potential Assessment Tool (CPAT) [41]. Therefore, HOXB-AS3 is an anti-sense lncRNA in the HOXB cluster, similar to HOTAIR in the HOXC cluster, and HOTTIP and HOTAIRM1 in the HOXA cluster [13, 42, 43].

A similar anti-sense transcript, Hoxb5os (NCBI Reference Sequence: NR_131758.1), exists in the mouse HOXB cluster on chromosome 11 (NCBI Reference Sequence: NC_000077.6 Chromosome 11 Reference GRCm38.p4 C57BL/6 J), and is also predicted to be a lncRNA (Additional file 1: Figure S1). We aligned the sequences of human HOXB-AS3 with that of mouse Hoxb5os by plalign (http://fasta.bioch.virginia.edu/) and observed two conserved regions (Additional file 1: Figure S2), one in exon 1 and the other in exon 6 of HOXB-AS3. These analyses indicated that HOXB-AS3 might be an evolutionarily conserved anti-sense transcript in the HOXB cluster in mammals.

HOXB-AS3 is a cytoplasmic lncRNA and promotes cell proliferation by increasing the expressions of genes involved in cell cycle progression and DNA replication

To investigate the function of HOXB-AS3, we infected the myeloid cell line OCI/AML3 using lentivirus carrying control or each of the two HOXB-AS3 shRNAs together with GFP. The infected cells were sorted for proliferation assay. We found that the cells carrying HOXB-AS3 shRNA grew more slowly than those carrying control shRNA (Fig. 2a). The result suggested the anti-proliferative effect resulted from HOXB-AS3 depletion. To corroborate this effect, we stably knocked down HOXB-AS3 in two myeloid cell lines, OCI/AML3 and TF-1 (Fig. 2b and d). Downregulation of HOXB-AS3 suppressed the proliferation of OCI/AML3 and TF-1 cells by decreasing the cells entering the S phase (Fig. 2c and e; Additional file 1: Figure S3a and S3b). Compared with OCI/AML3 cells, the milder effect of HOXB-AS3 depletion in TF-1 cells was possibly resulted from the very low expression of HOXB-AS3 in TF-1 cells (Additional file 1: Figure S3d). In the reciprocal experiments, overexpression of HOXB-AS3 in TF-1 cell line enhanced cell proliferation by promoting cells entering the S phase (Fig. 2f and g; Additional file 1: Figure S3c). These findings indicated that HOXB-AS3 promoted cell proliferation in the myeloid cell lines.

Fig. 2

HOXB-AS3 promotes cell proliferation in myeloid cells. a Proliferation assay of OCI/AML3 cells infected with lentivirus carrying pLKO-vector (control), shHOXB-AS3#1, or shHOXB-AS3#2. The infected cells were sorted by the expression of GFP. The sorted GFP-positive cells were seeded with a number of 1 × 106 in 5 mL culture medium on Day 0. Cell number was counted for 6 days. P values were calculated by Kruskal-Wallis test. b Quantitative PCR analysis of HOXB-AS3 relative to β2 microglobulin expression in OCI/AML3 cells stably carrying pLKO-vector (control), shHOXB-AS3#1, or shHOXB-AS3#2 by lentivirus infection. c The percentage of OCI/AML3 cells in the S phase of cell cycle. The result was derived from triple repeats. P values were calculated by Kruskal-Wallis test. d Quantitative PCR analysis of HOXB-AS3 relative to β2 microglobulin expression in TF-1 cells stably carrying pLKO-vector (control) or shHOXB-AS3#1 by lentivirus infection. e The percentage of TF-1 cells in the S phase of cell cycle. The result was derived from triple repeats. P values were calculated by Student t test. f Quantitative PCR analysis of HOXB-AS3 relative to β2 microglobulin expression in TF-1 cells infected with lentivirus carrying pLAS5w.Pbsd vector (control) or pLAS5w.Pbsd-HOXB-AS3 (HOXB-AS3 overexpression). g The percentage of cells in the S phase of cell cycle. The result was derived from the triple repeats. P values were calculated by Student t test. (* meant that P value was less than 0.05)

To explore the mechanisms of HOXB-AS3 in proliferation, we investigated the expression of genes influenced by HOXB-AS3 depletion through the microarray analysis. We compared the gene expression profiles of OCI/AML3 cells infected by virus carrying shHOXB-AS3 (two stable cell lines infected with different shHOXB-AS3, shHOXB-AS3#1 or shHOXB-AS3#2) with those infected by control virus (two stable cell lines infected with shLacZ or pLKO vector).

We compared the gene expressions of HOX clusters between OCI/AML3 cells with and without HOXB-AS3 depletion because previous studies showed that most lncRNAs in HOX clusters are located in the nucleus and regulate the expressions of HOX genes in cis- or trans-manner [42, 43]. However, we found that the expressions of HOX clusters were not influenced by HOXB-AS3 depletion (Fig. 3a). Instead, we identified 147 genes and 790 transcript clusters differentially expressed when HOXB-AS3 was knocked down in OCI/AML3 cells (Fig. 3b; the criteria were that the fold change of average log2-transformed gene expressions was more than 1.5, and that ANOVA P value was less than 0.05. The analysis was computed by BRB array tool.). GSEA analysis revealed that these differentially expressed genes were involved in several pathways associated with cell cycle progression and DNA replication (Fig. 3c and Additional file 1: Figure S4). Wikipathway analysis computed by Affymetrix® TAC also showed that many differentially expressed genes were involved in cell cycle pathway, DNA replication, G1-S transition, and RB pathway (Additional file 1: Figure S5-S8). Moreover, the expressions of these genes were mostly downregulated when HOXB-AS3 was knocked down, consistent with a promoting function of this lncRNA in cell proliferation.

Fig. 3

HOXB-AS3 regulates genes involving in cell cycle progression and DNA replication instead of HOX clusters. a The expressions of HOX genes were not influenced by HOXB-AS3 expression. RNA was purified from OCI/AML3 infected by lentivirus carrying pLKO-shLacZ (control#1), pLKO-vector (control#2), pLKO-shHOXB-AS3#1 or pLKO-shHOXB-AS3#2. b Differentially expressed genes between the control cells and HOXB-AS3 knockdown cells were revealed by microarray analysis. c GSEA analysis of the differentially expressed genes in HOXB-AS3 knockdown cells compared to the control cells. d Quantitative PCR analysis of the expressions of indicated genes in OCI/AML3 cells infected with lentivirus carrying pLKO-vector (control), shHOXB-AS3#1, or shHOXB-AS3#2. Data were derived from the triple repeats of experiments. P values were calculated by Kruskal-Wallis test. e Quantitative real time PCR analysis of RNA18S5 RNA (18 s rRNA), HOXB-AS3, MALAT1, NEAT1 and GAPDH in the nuclear and cytoplasmic fractions of OCI/AML3 cells. (* meant that P value was less than 0.05)

We validated a set of HOXB-AS3 regulated genes by RT-qPCR and confirmed that several genes involved in the cell cycle progression (CDK1, CCNB2, and CDC25A), DNA replication (PCNA), and assembly of pre-replicative complex (CDC6, MCM4, MCM6) were indeed downregulated when HOXB-AS3 was knocked down (Fig. 3d). Conversely, these genes were upregulated when HOXB-AS3 was overexpressed (Additional file 1: Figure S9). These findings suggested that HOXB-AS3 induced the expression of a number of genes involving in cell cycle progression and DNA replication to contribute to myeloid cell proliferation.

Furthermore, to identify the subcellular location of lncRNA HOXB-AS3, we used nuclear-cytoplasm fractionation to purify RNA from the nucleus and cytoplasm of OCI/AML3 cells, followed by reverse transcription and quantitative real time PCR. Our analysis demonstrated that the majority of HOXB-AS3 was located at the cytoplasm, different from the nuclear-residing MALAT1 and NEAT1 (Fig. 3e) [44, 45]. Therefore, our results suggest that HOXB-AS3 regulate the expressions of downstream genes through an indirect mechanism, which was different from other anti-sense lncRNAs in the HOX clusters.

Higher HOXB-AS3 expression is associated with shorter overall survival and progression free survival in AML patients

To determine the clinical significance of HOXB-AS3 in de novo AML patients, we analyzed the microarrays and clinical data of the NTUH AML cohort and validated the results with TCGA AML cohort. In the NTUH AML cohort, HOXB-AS3 expression was determined based on the expression levels of TC17002858.hg.1, the transcript cluster representing all variants of HOXB-AS3 on Affymetrix GeneChip® HTA 2.0. The AML patients were stratified into higher- and lower-expression groups with the median expression of HOXB-AS3 as the cutoff level. The HOXB-AS3 levels in the higher-expression group were much higher than those of the healthy donors (Additional file 1: Figure S10; P <  0.000001), while the expression levels were similar between lower group and the healthy donors (Additional file 1: Figure S10). The clinical characteristics of the NTUH AML patients were listed in Additional file 1: Table S1 Patients with higher HOXB-AS3 expression were older and had higher frequencies of mutated NPM1/wild FLT3-ITD, MLL-PTD, and RUNX1 mutations, but lower frequency of CEBPAɑ double mutations than patients with lower expression.

Patients with higher HOXB-AS3 expression had similar complete remission rate to those with lower expression, but had higher relapse rate (Additional file 1: Table S1). With a median follow-up time of 88.1 months, the AML patients with higher HOXB-AS3 expressions had shorter OS and RFS than those with lower HOXB-AS3 expressions (Fig. 4a and b; median OS, 17.7 months versus not reached, P value < 0.0001; median RFS, 12.9 months versus not reached, P value 0.0070, respectively). When we stratified patients according to the expression levels of the other transcript cluster representing HOXB-AS3, TC17002254.hg.1, the results were similar (Additional file 1: Figure S11). Subgroup analysis showed that higher HOXB-AS3 expression was also a poor-risk factor for OS in the intermediate-risk cytogenetic group (Fig. 4c). We validated our findings in the TCGA AML cohort in whom higher HOXB-AS3 expression was also an adverse prognostic biomarker (Fig. 4d) [37]. Multivariate analysis showed that higher HOXB-AS3 expression tended to be an independent adverse prognostic factor for OS in AML patients (P = 0.0634, Table 1).

Fig. 4

Survival analysis of de novo AML patients stratified by the expressions of HOXB-AS3. a Overall survival (OS) in the NTUH AML cohort. b Relapse free survival in the NTUH AML cohort. c OS in the AML patients with intermediate-risk cytogenetic changes. d OS in the TCGA AML cohort. The patients in the NTUH AML cohort were stratified by the expressions of transcript cluster: TC17002858.hg.1 on Affymetrix GeneChip® HTA 2.0 arrays

Table 1

Univariate and multivariate analyses (Cox regression) for overall survival in AML patients

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	P value	HR	95% CI	P value
Age > 60 years	2.7201	1.5917 to 4.6484	< 0.0001*	2.0425	1.2585 to 3.3148	0.0038*
WBC > 100 k μ/L	1.0529	0.6026 to 1.8399	0.8534	–	–	–
Favorable cytogenetics	0.4552	0.2905 to 0.7133	0.0051*	0.4579	0.2315 to 0.9058	0.0248*
NPM1+/ FLT3-ITD-	0.5674	0.3346 to 0.9625	0.0839	0.2972	0.1421 to 0.6216	0.0013*
FLT3-TKD	1.1062	0.5419 to 2.2580	0.7722	–	–	–
CEBPα double mutation	0.3093	0.1839 to 0.5201	0.0030*	0.3893	0.1585 to 0.9561	0.0396*
PTPN11 mutation	1.2426	0.5008 to 3.0836	0.6042	–	–	–
KRAS mutation	1.6638	0.6351 to 4.3587	0.1885	–	–	–
MLL/PTD	3.5868	1.0660 to 12.0688	0.0001*	2.3662	1.0555 to 5.3047	0.0365*
KIT mutation	1.0609	0.4827 to 2.3319	0.8797	–	–	–
RUNX1 mutation	2.2459	1.1285 to 4.4697	0.0013*	1.2736	0.6998 to 2.3179	0.4285
WT1 mutation	1.4062	0.7398 to 2.6728	0.2326	–	–	–
ASXL1 mutation	0.8468	0.3932 to 1.8236	0.6921	–	–	–
IDH1 mutation	0.5760	0.2364 to 1.4033	0.3395	–	–	–
IDH2 mutation	0.9412	0.5109 to 1.7341	0.8492	–	–	–
TET2 mutation	1.1970	0.6549 to 2.1879	0.5309	–	–	–
TP53 mutation	4.3752	0.5761 to 33.2257	0.0015*	3.1834	1.0992 to 9.2198	0.0328*
DNMT3A mutation	1.3679	0.8133 to 2.3004	0.1908	–	–	–
HOXB-AS3 expression	2.3003	1.5530 to 3.4072	<  0.0001*	1.6401	0.9727 to 2.7654	0.0634

Age, elder than 60 years versus younger; WBC, higher than 100 k μ/L versus lower; HOXB-AS3 expression, higher versus lower; NPM1+/ FLT3-ITD-, mutated NPM1 without FLT3-ITD versus others; other mutations, mutation versus wild type; favorable cytogenetics, favorable versus others

Abbreviations: HR hazard ratio, CI confidence interval, MLL/PTD partial tandem duplication of MLL gene

* P value < 0.05

We further analyzed the patients with AML other than acute promyelocytic leukemia (non-APL), and stratified them according to the median expression of HOXB-AS3 (Additional file 1: Figure S12). Patients with higher HOXB-AS3 expression still had shorter OS than those with lower HOXB-AS3 expression (Additional file 1: Figure S13a). The subgroup analysis in the intermediate-risk patients based on the 2017 European LeukemiaNet (ELN) risk classification also illustrated adverse prognosis for the patients with higher HOXB-AS3 expression (Additional file 1: Figure S13b).

Higher HOXB-AS3 expression also predicts adverse prognosis in MDS patients

To investigate the clinical relevance of HOXB-AS3 expressions in primary MDS, we analyzed the microarrays and clinical data of the NTUH MDS training cohort and validation cohort. HOXB-AS3 expression was determined based on the expression levels of TC17002858.hg.1. According to the expression levels of HOXB-AS3, the MDS patients was stratified into four groups, lowest, intermediate low, intermediate high, and highest HOXB-AS3 expression groups (Additional file 1: Figure S14). Only the patients in the highest group had distinct higher HOXB-AS3 expressions compared with the healthy donors (Additional file 1: Figure S14; P <  0.000001), while patients in other groups had similar HOXB-AS3 expressions to normal controls.

With a median follow-up time of 39.2 months, the MDS patients with highest HOXB-AS3 expressions had the shortest OS compared to others (Fig. 5a). The OS was similar among the patients with lowest, intermediate low, and intermediate high expressions of HOXB-AS3. Similar results were obtained when we stratified the patients according to the expressions of another transcript cluster, TC17002254.hg.1, which also represented HOXB-AS3 (Additional file 1: Figure S15a). If we used the cutoff point between the highest and intermediate high groups (Fig. 5a) to stratify the NTUH MDS training cohort into two groups, higher HOXB-AS3 expression predicted shorter OS in the MDS patients (Fig. 5b). The clinical characteristics of the MDS patients were listed in Additional file 1: Table S2, and the correlations of HOXB-AS3 expressions and somatic gene mutations were listed in Additional file 1: Table S3. Patients with higher HOXB-AS3 expression had a high rate of leukemic transformation, higher bone marrow blast percentage and more frequently WHO higher-risk subtypes, but similar distribution of IPSS subtypes, compared to those with lower HOXB-AS3 expression. Higher HOXB-AS3 expression was associated with RUNX1, ASXL1, and IDH2 mutations. When we applied the same cutoff point to the MDS validation cohort, the prognostic significance of HOXB-AS3 expression for OS in MDS patients was also confirmed (Fig. 5c and Additional file 1: Figure S15b). Multivariate analysis demonstrated that higher HOXB-AS3 expression was an independent poor prognostic factor for OS in primary MDS patients, irrespective of other poor prognostic factors, including higher IPSS scores and adverse risk mutations (Table 2).

Fig. 5

Overall survival of MDS patients stratified by the expressions of HOXB-AS3. a OS in the NTUH MDS training cohort stratified into 4 groups: HOXB-AS3 expression highest, intermediate high, intermediate low and lowest groups. b OS in the NTUH MDS training cohort stratified into 2 groups: HOXB-AS3 expression higher group (HOXB-AS3 expression highest group in Fig. 5a) and lower group (HOXB-AS3 expression lowest, intermediate low, and intermediate high groups in Fig. 5a). c OS in the NTUH MDS validation cohort stratified into 2 groups as Fig. 5b. The patients were stratified by the expressions of transcript cluster: TC17002858.hg.1 on Affymetrix GeneChip® HTA 2.0 arrays

Table 2

Univariate and multivariate analyses (Cox regression) for overall survival in MDS patients

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	P value	HR	95% CI	P value
Age > 60 years	1.7988	1.1298 to 2.8641	0.0223*	1.5433	0.8392 to 2.8383	0.1628
IPSS	3.4616	1.9730 to 6.0735	< 0.0001*	2.5755	1.4292 to 4.6412	0.0016*
EZH2 mutation	2.0164	0.7036 to 5.7785	0.0709	1.1877	0.4371 to 3.2271	0.7358
RUNX1 mutation	1.6949	0.7996 to 3.5925	0.0880	0.7265	0.3155 to 1.6731	0.4528
ASXL1 mutation	1.9909	1.0927 to 3.6274	0.0059*	1.3616	0.6273 to 2.9554	0.4350
IDH2 mutation	1.5777	0.2748 to 9.0568	0.5195	–	–	–
ZRSR2 mutation	2.5929	1.0588 to 6.3500	0.0016*	1.9474	0.8973 to 4.2263	0.0918
U2AF35 mutation	1.0584	0.4771 to 2.3480	0.8861	–	–	–
TET2 mutation	1.3481	0.6591 to 2.7572	0.3579	–	–	–
SRSF2 mutation	1.9654	0.9379 to 4.1184	0.0196*	1.5304	0.7107 to 3.2954	0.2769
SF3B1 mutation	0.7602	0.4237 to 1.3640	0.3970	–	–	–
TP53 mutation	7.1283	1.2057 to 42.1423	< 0.001*	6.1091	2.3732 to 15.7260	0.0002*
DNMT3A mutation	1.2781	0.6513 to 2.5080	0.4344	–	–	–
HOXB-AS3 expression	2.1624	1.1681 to 4.0033	0.0018*	1.8992	1.0606 to 3.4009	0.0309*

Age, elder than 60 years versus younger; HOXB-AS3 expression, highest versus others; mutations, mutation versus wild type; IPSS, intermediate-2 and high, versus low and intermediate-1

Abbreviations: HR hazard ratio, CI confidence interval

* P value < 0.05

Furthermore, we analyzed prognostic implications of HOXB-AS3 expressions in subgroups of patients with different IPSS risk. Among the patients with IPSS lower risk MDS (low and intermediate-1 risks), those with higher HOXB-AS3 expression had shorter OS than those with lower expression (median, 29.2 months vs 77.3 months, P value 0.0194; Fig. 6a). On the other hand, HOXB-AS3 expression did not influence OS in the patients with IPSS higher risk MDS (intermediate-2 and high risks; Fig. 6b). Therefore, high HOXB-AS3 expression could further identify a subgroup of patients with high risk in the IPSS lower risk group.

Fig. 6

OS of MDS patients stratified by the expressions of HOXB-AS3 in different IPSS risk groups. a OS in the patients with IPSS low or intermediate-1 risk, stratified by HOXB-AS3 expressions. b OS in the patients with IPSS intermediate-2 or high risk, stratified by HOXB-AS3 expressions. The patients were stratified by the expressions of transcript cluster: TC17002858.hg.1 on Affymetrix GeneChip® HTA 2.0 arrays into 2 groups as Fig. 5b

Discussion

In this study, we reported the clinical relevance of lncRNA HOXB-AS3 in de novo AML and primary MDS. We demonstrated that higher HOXB-AS3 expression was an adverse prognostic factor for AML and MDS patients. To our knowledge, HOXB-AS3 represents the first reported lncRNA whose expression is able to predict the prognosis of MDS patients. We also found that HOXB-AS3 expression could further stratify IPSS lower risk patients into two subgroups with distinct prognosis. This would help us identify the IPSS lower risk patients who need to be treated aggressively.

In addition to identifying the prognostic value of HOXB-AS3 expression in myeloid malignancies, we explored the biological function of HOXB-AS3. We revealed that HOXB-AS3 promoted the proliferation of myeloid cells. Previous studies showed that anti-sense lncRNAs in HOX clusters influence the expressions of HOX genes by trans- or cis-regulation [13, 42, 43]. For examples, HOTAIR trans-regulates the expression of HOXD genes through PRC2 complex [42], whereas HOTAIRM1 cis-regulates the expressions of HOXA1 and HOXA4 genes [13]. Given that expressions of HOX genes are important in cell proliferation, hematopoiesis and leukemogenesis [36, 38, 39], it is possible that HOXB-AS3 promotes cell proliferation by regulating the expressions of certain HOX genes. However, our microarray analysis indicated that downregulation of HOXB-AS3 in OCI/AML3 cells did not significantly alter the expression of any HOX gene (Fig. 3a). Instead, HOXB-AS3 potentiated the expressions of a set of genes critical for cell cycle progression and DNA replication (Fig. 3c and Additional file 1: Figure S4), and this finding was consistent with its ability to increase the S-phase cell population (Fig. 2c, e, and g). A recent study showed that knocking down HOXB-AS3 reduces the cells in S phase and the ability in colony formation, which is consistent with our findings [46]. Further, they demonstrated that HOXB-AS3 binds EBP1 to increase the EBP1-NPM1 complex [46]. Accordingly, overexpression of HOXB-AS3 increases the transcription of rRNA and de novo protein synthesis [46]. This mechanism might explain the effect of HOXB-AS3 on cell proliferation.

The clinical relevance of HOXB-AS3 in hematopoietic diseases remains poorly characterized. A previous study reported a positive correlation of HOXB-AS3 expression with NPM1 mutations in AML patients [47]. In our study, we not only illustrated high HOXB-AS3 expression as a poor prognostic biomarker in AML and MDS, but also disclosed its promotion effect on cell proliferation in two myeloid cell lines (Fig. 2). Further, we showed that higher HOXB-AS3 expression was an adverse prognostic marker in IPSS lower risk patients, but not higher risk ones. It may indicate that higher expressions of HOXB-AS3 influences the prognosis through enhancing proliferation of the abnormal hematopoietic cells in IPSS lower-risk patients, but has no implication when the patients already have many risk factors as in IPSS higher risk patients. These findings imply that HOXB-AS3 may have distinct clinical relevance in different myeloid malignancies.

Of note, a recent study reported that HOXB-AS3 can encode a small peptide to influence the alternative splicing of pyruvate kinase M, thereby inhibiting the proliferation of colon cancer cell lines [48]. In addition, HOXB-AS3 expression is downregulated in colorectal cancer (CRC) tissues and is correlated with favorable prognosis for CRC patients [48]. The seemingly discrepancies between this previous study and our findings are likely due to the different variants of HOXB-AS3 used. The previous study investigated exclusively HOXB-AS3 variant 1 (NR_033201.2), and the small peptide is encoded from the last two exons of this variant [48]. However, in leukemia cell lines, variant 1 is expressed at a very low level, and the majority of HOXB-AS3 transcripts are variants 2/3/5 (Additional file 1: Figure S16), which do not contain the last two exons (Fig. 1b) and therefore cannot encode the small peptide. Because of the abundant expression of variants 2/3/5, we used the longest variant, variant 2, for the overexpression studies and results are consistent with the conclusions derived from the knockdown studies, in which the two HOXB-AS3 shRNAs target variants 2, 3, 5, 8 and variants 2, 3, 5, 6, 7, 8, respectively. Therefore, the current and previous studies suggest the existence of variant-specific functions of HOXB-AS3. The relative abundance of HOXB-AS3 variants could determine its context-dependent roles in different cancer types.

Conclusions

Our study identifies that higher expression of HOXB-AS3 is an adverse prognostic marker for both de novo AML and primary MDS patients. Functionally, HOXB-AS3 promotes the proliferation of myeloid cells through upregulating the expressions of a set of genes critical for cell cycle progression and DNA replication. HOXB-AS3 can be a potential target for novel therapy in MDS and AML patients with higher HOXB-AS3 expression.

Table S1. Correlation of HOXB-AS3 expression with clinical characteristics and frequent somatic gene mutations in de novo AML patients. Table S2. Correlation of HOXB-AS3 expression with clinical characteristics in MDS patients. Table S3. Correlation of HOXB-AS3 expression with frequent gene mutations in MDS patients. Figure S1. Overview of Hoxb5os in mouse HOXB cluster. Figure S2. Alignment of mouse Hoxb5os and human HOXB-AS3. Figure S3. HOXB-AS3 promotes S phase entering in the cell cycle regulation. Figure S4. GSEA pathway analysis of the differentially expressed genes in HOXB-AS3 knockdown cells compared to the control cells. Figure S5. Cell cycle pathway from Wikipathway analysis of downstream pathways affected by downregulation of HOXB-AS3 in the myeloid cell lines. Figure S6. DNA replication pathway from Wikipathway analysis of downstream pathways affected by downregulation of HOXB-AS3 in the myeloid cell lines. Figure S7. RB pathway from Wikipathway analysis of downstream pathways affected by downregulation of HOXB-AS3 in the myeloid cell lines. Figure S8. G1-S pathway from Wikipathway analysis of downstream pathways affected by downregulation of HOXB-AS3 in the myeloid cell lines. Figure S9. Quantitative PCR analysis of the expressions of indicated genes in TF-1 cells infected with lentivirus carrying pAS5.1w-Pbsd (control), or pAS5.1w-Pbsd-HOXB-AS3 (HOXB-AS3 overexpression). Figure S10. HOXB-AS3 expressions of AML patients and health donors. Figure S11. Survival analysis of AML patients stratified by the expressions of HOXB-AS3. Figure S12. HOXB-AS3 expression of non-APL AML patients and health donors. Figure S13. Survival analysis of non-APL AML patients stratified by the expressions of HOXB-AS3 in the NTUH AML cohort. Figure S14. HOXB-AS3 expressions of MDS patients and health donors. Figure S15. Overall survival of MDS patients stratified by the expressions of HOXB-AS3. Figure S16. Quantitative PCR analysis of the expressions of different variants in TF-1 and OCI/AML3 cell lines. (PDF 2900 kb)