Molecular functions identified using subset-specific and relapse-specific lncRNAs

76

lncRNAs

The subtype-specific and relapse-specific lncRNAs showed significant correlation between genes enriched in key pathways associated with cell proliferation, growth, survival, metabolism, and autophagy based on the correlations with their neighboring and distinct protein-coding genes. These findings indicate that BCP-ALL subtype-specific and relapse-specific lnRNAs are associated with tumorigenesis of hematopoietic cells. LncRNAs are emerging as new players in cancer, due to their potential roles in both oncogenic and tumour suppressive pathways. They are frequently dysregulated in a variety of human cancers; however, the biological functions of a vast majority of them remain unknown. Recently, evidence of lncRNAs molecular mechanisms and function has begun to accumulate, providing insight into the functional roles they may play in tumorigenesis (Serviss, Johnsson, & Grandér, 2014).

The guilt-by-association method designates potential or putative functions to lncRNAs based on its co-expression of characterized PC genes. The certainty of the association is based on the condition of available expression data. For instance, time-dependent data can be notably crucial because aberrant regulation of expression can be informative of the certain pathways by which lncRNAs functions (Bartonicek, Maag, & Dinger, 2016). Our study consists of time-dependent dataset from two disease stages. The appropriateness and popularity of this approach have given rise to several subtypes of analyses including the cis and trans-correlation-based association.

We identified a remarkable fraction of subtype-specific lncRNAs (621 out 1534) with significant co-expression with their cis and trans located protein-coding genes. Notably, 32% of these lncRNAs are involved in pathways associated with proliferation, apoptosis and differentiation in leukemia, including, JAK-STAT, mTOR, PIK3-AKT, TGF-beta, MAPK, P53, hippo and NF-kappa B signaling pathways from both DUX4 and Ph-like subtypes. The co-expression between the protein-coding genes and subtype-specific lncRNAs provided a possible explanation of co-regulation or co-activation of lncRNAs, with their cis and trans PC genes. We also demonstrated that several lncRNAs were co-expressed with oncogenes associated with leukemias.

4.10.1 Potential functions of DUX4 specific DE lncRNAs associated with signaling pathways In the DUX4 subtype, we report lncRNAs signature (n = 185, both cis and trans based analysis) associated with pathways reported to play a key role in leukemogenesis, such as TGF-Beta signaling pathway, P53, Endocytosis, hippo, proteoglycans, and pathways in cancer. Considering the functional nexus between these lncRNAs and leukemia related pathways, targeting these lncRNAs provide novel insights for new therapeutic targets.

77

In ALL, TGFB has complex roles; it regulates the proliferation of the distinct myeloid stem cells (Dong

& Blobe, 2006). Recently there are some lncRNAs documented to be associated with TGFB gene. For instance, Lnc-ATB, a TGFB2 induced lncRNA that could mediate TGF-β-induced epithelial-mesenchymal transition and has been reported to promote metastasis in various solid cancer such as hepatocellular carcinoma, colorectal cancer, gastric cancer, and breast cancer. However, the lncRNAs associated with BCP-ALL subtypes are not reported. We identified the antisense lncRNA, RP11-224O19.2 and other novel lncRNAs significantly correlated with TGFB, and are enriched in TGF-beta pathway, indicating their functional relatedness or regulatory relationships. Interestingly, the subtype-specific lncRNAs and subtype-subtype-specific PC are globally predicted to activate or inhibit the same key signaling pathways in the DUX4 subtype.

4.10.2 Potential functions of Ph-like specific DE lncRNAs associated with signaling pathways The Ph-like subtype is both molecularly and functionally well characterized based on mRNAs/protein expression levels, whereas non-coding genes are not much studied. We have identified a list of 24 novel dysregulated Ph-like specific lncRNAs crucial in signaling pathways associated with Ph-like subtype.

The pathways controlling the cell proliferation, differentiation, and survival of hematopoietic cells were identified based on functional enrichment analysis, for example, the PI3K and mTOR signaling pathways. In addition to that, our functional predictions identified other prominent pathways which trigger chemotherapy resistance in BCP-ALL, including, JAK-STAT2, Cytokine-cytokine receptor and endocytosis pathways. The lncRNAs associated with these pathways are antisense or sense intronic to the mRNA genes with a significant co-expression pattern. Characterization of the lncRNAs involved in this pathway may be of interest in the search for new potential therapies.

Some of the functions predicted here have been validated by previous studies, suggesting that our guilt-by-association approach is valid. For example, lncRNA AC002454.1 was recently reported to regulate CDK6 to participate in cell cycle dysfunction in the endometriosis pathogenesis. LncRNA AC002454.1 is an antisense lncRNA of CDK6 gene. The results of our guilt-by-association study highlight an association of this lncRNA with the PIK3-Akt pathway. Both the CDK6 gene and antisense AC002454.1 are significantly co-expressed and up-regulated in the Ph-like subtype.

Interestingly, we observed a significant co-expression between oncogene IL2RA and its antisense lncRNA RP11-536K7.5. Recently, IL2RA gene was found to be specifically up-regulated by pre-B cell receptor (pre-BCR) signaling during early B cell development, and cells with oncogenically activated tyrosine kinases by a manifold to pre-BCR signalling in both in Ph+ALL and in Ph-like ALL (J.-W. Lee

78

et al., 2015). In Ph-like subtype, we observed IL2RA gene enriched in the cytokine-kinase signaling pathway. Both IL2RA and RP11-536K7.5 were up-regulated in Ph-like samples. The ability of IL2RA to stabilize oncogenic signaling strength in Ph-like ALL is important for leukemia initiation and development. Our analysis indicates the co-regulation or co-regulation of RP11-536K7.5 with IL2RA gene, which provided a new context for further characterization of RP11-536K7.5 lncRNA. We predicted the positive association between cytokine-kinase signaling pathway and RP11-536K7.5 lncRNA, but the mechanisms involved are still poorly understood, therefore, further studies are needed to better understand RP11-536K7.5/ cytokine-kinase signaling transduction.

It was noteworthy that subtype-specific lncRNAs and subtype-specific PC are globally predicted to activate or inhibit the same pathways. However, some exclusivity appeared. For example, lncRNAs specific to the Ph-like subtype particularly involved in the activation of mTOR and the PI3K-Akt signaling pathway. Considering the functional nexus between Ph-like specific lncRNAs and the activation of pathways such as mTOR and PI3K signaling pathways, targeting those lncRNAs may be a promising novel therapeutic option for BCP-ALL subtypes provided a new context for further characterization of RP11-536K7.5 lncRNA. We predicted the positive association between cytokine-kinase signaling pathway and RP11-536K7.5 lncRNA, but the mechanisms involved are still poorly understood, therefore, further studies are needed to better understand RP11-536K7.5/ cytokine-kinase signaling transduction.

4.10.3 Molecular and functional association of relapse-specific lncRNAs signature

We applied the “guilt-by-association” approach also on the relapse-specific lncRNAs markers within the subtypes for investigating their functions. However, the relapse-specific signature from the Ph-like and the NH-HeH subtypes did not show any significant enrichment of pathways. A potential reason can be that the DUX4 subtype is particularly perturbed in both relapse-specific and subtype-specific classification, and therefore the number of dysregulated lncRNAs are high compared to the other two subtypes.

In the DUX4 subtype, a notable observation was a strong correlation between relapse-specific lncRNAs with genes involved in the activation of metabolic pathways and signaling pathways. We identified 112 relapse-specific lncRNAs co-expressed with 29 PC genes activated in metabolic pathways. Out of this 112 lncRNA, eight lncRNAs were previously reported as biomarker lncRNAs in the context of various cancers. For example, we identified oncogenic lncRNA LUCAT1 reported to be associated with poor prognosis in lung cancer. In addition to that, eight relapse-specific lncRNAs associated with metabolic

79

pathways in the DUX4 subtype was previously reported due to their ability to dysregulate metabolic pathways in multiple tumor contexts. Taken together, the global co-expression analysis and gene-expression profiling suggest important and previously unappreciated roles of lncRNAs in the BCP-ALL subtypes.