Mutations and misregulation of long non-coding RNAs have been shown to play pivotal roles in oncogenesis. Alterations in LncRNA expression or mutations promote tumorigenesis and metastasis. LncRNAs carry considerable promise as cancer novel biomarkers and therapeutic targets [20].
Histones epigenetic modifications, such as acetylation and methylation, have been proved to be frequently deregulated in AML [21].
The effect of methylation is site-specific, for example, histone 3 lysine 4 (H3K4), activates gene expression. On the contrary, methylation of histone 3 lysine 9 (H3K9) or lysine 27 (H3K27) suppresses the transcription [22].
The PRC2 complex contains the three core protein subunits: enhancer of zeste homolog 2 (EZH2), embryonic ectoderm development, and SUZ12. EZH2 contains a SET domain that catalyzes trimethylation of histone H3 at lysine 27. HOTAIR binding to PRC2 is required for H3K27 trimethylation [23].
Through whole-genome sequencing, NPM1 mutations and FLT3-ITD were found to be associated with distinct LncRNA signatures [24].
HOTAIR was reported to act as a bridge between protein complexes, supporting chromatin silencing, including the PRC2 [5].
HOTAIR binds to and sequesters miR-193a competitively, modulates the expression of c-KIT in AML cells, promotes cell growth, and inhibits apoptosis [25].
Patients with NPM1 mutations proved to have a better outcome with increased complete remission and improved overall survival [26]. However, AML with FLT3 ITD carries a poor prognosis owing to its higher risk of relapse [27].
In this study, the expression of LncRNA and HOTAIR genes in 47 AML cases was estimated using the RT qPCR technique compared to controls. HOTAIR gene expression was higher in AML cases than in controls. These findings match those of a previous study, reporting that HOTAIR was overexpressed in AML patients [28].
According to HOTAIR median expression, the patients were classified into high and low expression groups. The high expression group showed a significant decrease after CR achievement (p = 0.024). These results mirror those of previous studies, which found that the HOTAIR expression was reduced after the achievement of CR [18]. However, a non-significant increase in the HOTAIR expression levels was noticed in the low expression group after CR achievement.
HOTAIR expression was affected by NPM1 and FLT3 mutations. All NPM1 mutant/FLT3-ITD wild cases were from the low HOTAIR expression group (p = 0.017), and 40% of the double NPM1/FLT3-ITD mutant cases were from the low expression group.
Since all NPM1 mutations increased cytoplasmic concentrations of NPM1 protein, NPM1 modulates stress response and growth suppression by binding to and stabilizing p53 and inhibiting MDM2 [29].
Zhai et al. had demonstrated that the p53 level was negatively correlated with HOTAIR in non-small cell lung carcinoma, suggesting a regulatory role for p53 in HOTAIR transcription. They discovered two p53 binding sites in the HOTAIR promoter region [30]. Similarly, the strong negative correlation between HOTAIR expression level and NPM1 mutation can be explained.
The HOTAIR expression was found to be positively correlated with hemoglobin level, which contradicts a previous study [18].
However, Shih and Kung explained that HOTAIR expression was upregulated by hypoxia, which is a common feature of all malignancies, including AML. This upregulation occurs through the hypoxia-responsive element in the HOTAIR promoter region, which is activated directly by hypoxia-induced factor-1 α (HIF1α) [31]. HIF was found to upregulate δ-aminolevulinate synthase, which catalyzes the first and rate-limiting reaction in the heme biosynthesis [32]. TLC was found to be inversely associated with HOTAIR expression (p = 0.022). Previous studies on TLC and HOTAIR expression levels were controversial. Some researchers rated the TLC as high and low according to the highest normal count (10 × 109/L) and found a positive correlation between HOTAIR expression and TLC [18].
However, Zhang et al. concluded that there is no correlation between the HOTAIR expression and TLC of AML patients. They classified the cases into high and low TLC groups by the count 100 × 109/L [33].
In this study, no significant correlation was found between patients’ age, gender, platelet count, CR achievement, and HOTAIR expression.
CR achievement correlation with combined HOTAIR expression and mutation status revealed that among the low expression group, CR was maximally achieved in the NPM1 mutant/FLT3-ITD mutant genotype (100%) and minimally in the NPM1 wild/FLT3-ITD mutant group. In contrast, among the high expression group, CR was maximally achieved in the NPM1-wild /FLT3-ITD mutant.
Just 20% of the NPM1-mutant/FLT-wild party achieved CR, which is probably explained by other risk factors. Two patients had died before ending their induction, one patient was not fit to start therapy, and one patient missed his follow-up.
Patients in the NPM1 wild/FLT3-ITD mutant group experienced an unexplained rise in CR rates, despite having two weak prognostic factors: the FLT3-ITD mutation and elevated levels of HOTAIR oncogenic LncRNA expression.
Göllner et al. found that when FLT3-ITD mutation-positive leukemic cell lines after they have been cultured in media containing the kinase inhibitor for an extended time, they developed resistance to the kinase inhibitor and standard chemotherapeutics. Western blot identified complete loss of H3K27me3 in resistant cells. They also established that the mechanism of action is not restricted to the FLT3-ITD mutation [34].
The increased CR achievement rates with high HOTAIR expression in the FLT3-ITD-Mutant group were due to the interaction between HOTAIR LncRNA and its PRC2 binding function, which allows trimethylation of H3K27 and thus promotes chemo-sensitivity even in the presence of FLT3 ITD.
In this study, the OS of patients showed a high positive association with CR achievement (p = 0.004) and a low TLC (p = 0.05). These findings corroborate a previous study [18].
The HOTAIR high expression group had a greater RFS (p = 0.024), surprisingly against previous literature [2]. This finding was corroborated with the post-chemotherapy HOTAIR expression analysis, in which we found that the low expression group after achieving CR had higher HOTAIR levels than at diagnosis, and 3/4 of patients relapsed during follow-up, implying that post-treatment HOTAIR expression levels may serve as an early predictor of the relapse susceptibility.
Poor outcome was found in all combinations of NPM1and FLT3-ITD results regardless of the HOTAIR expression level except patients with FLT3-ITD mutant/NPM1-wild populations, where the poor outcome ameliorated from 84% in patients with low HOTAIR expression levels to 62% in patients with high HOTAIR expression levels. However, this improvement was not statistically important. It can be attributed to our finding of increased chemo-sensitivity in the group with high HOTAIR expression in the presence of FLT3-ITD mutation.