Page 34 - Haematologica May 2022

D. Papaioannou et al.
Discussion
lncRNA are gaining recognition as important molecular mediators and regulators of key cellular functions in health and disease.22-29 In AML, lncRNA have been shown to asso- ciate with the clinical outcome of both younger and older patients.30-33 However, these previous studies have focused on the expression levels of the lncRNA transcripts; the effect of genetic variants within lncRNA has not been extensively studied. Accumulating evidence suggests that such genetic variants could impact on the function of the lncRNA and be relevant for disease pathogenesis.47 In sup- port of this view, disease-associated SNP are more frequent- ly found in regions of the genome that encode for non-cod- ing RNA transcripts in several types of solid tumors34,35 and in AML.34,36 These findings suggest that the presence of a variant in the non-coding transcriptome could be the func- tional link that explains how genetic variants that do not alter the structure of protein molecules associate with malignant phenotypes. Recently, Klco et al.37 have reported the acquisition of somatic mutations in lncRNA transcripts of AML patients, as demonstrated by analyses of the leukemic blasts in parallel with germline material. However, the prognostic value of these mutations could not be tested due to sample size limitations. Herein, we ana- lyzed total RNA Seq data of younger adult patients with CN-AML with the goal to detect recurrent lncRNA variants and evaluate their prognostic and biologic significance.
We used a stringent approach for detecting and filtering sequence variations of lncRNA. We generated a list of 981 recurrent variants, which are located within lncRNA in younger adult patients constituting the CALGB/Alliance cohort, and which had adequate sample sizes for meaning- ful survival analyses. In order to study potential associations of the detected variants with clinical outcome, we individ- ualized analysis for each variant and limited comparisons to the group of patients that were expressers of the correspon- ding lncRNA transcript (i.e., we compared the expressers of a variant to the expressers of the wild-type lncRNA). Of the 981 candidate variants, a subset of 41 significantly associat- ed with at least two clinical outcome endpoints of younger adults with CN-AML. LncRNA genetic variants RP5- 1074L1.4varT and SNHG15varT were significantly associ- ated with prognosis. RP5-1074L1.4 has not been previously associated with cancer pathogenesis or clinical outcome of cancer patients. In contrast, SNHG15, a MYC-regulated lncRNA, which harbors a small nucleolar RNA in one of its introns, has been implicated in the pathogenesis of multiple solid malignancies. SNHG15 has been shown to interact with the protein AIF and to associate with a clinically aggressive and prognostically unfavorable subset of colorec- tal carcinomas.48 SNHG15 has also been associated with aggressive phenotypes of hepatocellular and breast carcino- mas via sponging and inhibiting the function of microRNAs 141-3p and 211-3p.49,50
Patients who expressed the RP5-1074L1.4varT had more favorable outcome (i.e., longer DFS, EFS and a trend for longer OS) than RP5-1074L1.4wt expressers. SNHG15varT also associated with better prognosis and SNHG15varT expressers had longer DFS and EFS compared with patients who expressed the SNHG15wt lncRNA.
Further, we examined whether recurrent genetic variants could be detected in lncRNA previously reported to be prognostic in younger adults with CN-AML.30 A G-to-C variant in the prognostic lncRNA AL122127.25
(AL122127.25varC) significantly associated with clinical outcome of CN-AML patients. Specifically, expression of the AL122127.25varC associated with shorter DFS, OS and EFS. Notably, the expression levels of the AL122127.25 lncRNA were not significantly affected by the presence of the AL122127.25varC. Thus, both the abundance and the nucleotide sequence of the AL122127.25 lncRNA associate with the clinical outcome of CN-AML patients via poten- tially independent mechanisms.
Currently, there is limited availability of AML datasets analyzed with RNA Seq techniques (i.e., with total RNA Seq) that are suitable for in-depth analyses of the non-cod- ing transcriptome. In order to examine whether our obser- vations could be reproduced in other datasets, we exam- ined the presence of our curated variant list in an independ- ent cohort of CN-AML patients who were treated on AMLCG protocols. As expected, the use of a different RNA Seq technique significantly limited the number of lncRNA transcripts that we could interrogate. Nevertheless, SNHG15varT was detectable and associated with longer DFS and EFS of the AMLCG patients, similarly to its prog- nostic effect in the CALGB/Alliance dataset.
Given the constantly increasing number of prognostic molecular alterations in AML, it is important to examine the prognostic value of novel markers in the context of other established clinical and molecular prognosticators. It is thus noteworthy that the lncRNA variants that we exam- ined (RP5-1074L1.4varT, SNHG15varT, and AL122127.25varC) did not show associations with gene mutations that are currently used for the risk stratification of the treatment of AML patients.11 Mutations in the CEBPA, RUNX1, ASXL1 and NPM1 genes as well as the presence of the FLT3-ITD were similarly distributed between expressers of the lncRNA variants and expressers of the wild-type transcripts. In addition, in formal multi- variable analyses that included prognostic clinical and molecular parameters, RP5-1074L1.4varT, SNHG15varT, and AL122127.25varC retained their prognostic significance after adjusting for other covariates. Consequently, detection of these variants could provide additional prognostic infor- mation and further refine risk-stratification of CN-AML patients.
In addition to testing their prognostic significance, we sought to evaluate whether the presence of recurrent vari- ants in lncRNA sequences has functional implications. We focused on SNHG15 lncRNA and performed overex- pression experiments with SNHG15wt- and SNHG15varT-containing vectors in the K-562 and THP-1 cell lines. Overexpression of the SNHG15varT had no sig- nificant effect on cell viability and no evident impact on cell proliferation, compared with controls. In contrast, overexpression of the SNHG15wt led to a significant increase in the proliferative capacity of the leukemic blasts in both cell lines that were tested. Despite the lim- itations of in vitro assays, these results indicate that expression of SNHG15wt associates with a more aggres- sive disease phenotype, when compared with SNHG15varT, and are in line with the inferior clinical outcome of SNHG15wt-expressers.
In summary, we have performed comprehensive charac- terization of genetic variants within lncRNA in younger adults with CN-AML. We present analyses that support the prognostic and potential biologic significance of lncRNA variants in CN-AML. We believe that our work will serve as a useful starting point for further studies on the role of
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