Long Non-Coding RNAs As Novel Therapeutic Targets in Juvenile Myelomonocytic Leukemia: Proof of Concept Study
Authors: Hofmans Mattias , Depreter Barbara , Lammens Tim , Cave Helene , Flotho Christian , Hasle Henrik , de Haas Valerie , Niemeyer Charlotte M. , Stary Jan , Van Vlierberghe Pieter , Philippé Jan , De Moerloose Barbara 
Published: November 2019

BACKGROUND
Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive myelodysplastic/myeloproliferative disorder of early childhood. Hematopoietic stem cell transplantation results in long-term overall survival of only 50-60% and is fraught with frequent relapse and toxicity. Consequently, there is need to develop novel treatments. Evidence is growing that non-coding RNAs can serve as alternative therapeutic targets. Long non-coding RNAs (lncRNAs) are a recently discovered class of RNAs, with a minimum length of 200 nucleotides, of which perturbation can impact the cancer phenotype in vivo. Recently, we documented for the first time the lncRNA landscape in 44 JMML patients using microarrays and have associated lncRNA expression with clinical and molecular characteristics. Also, we demonstrated that JMML patients exhibit a distinct lncRNA expression profile compared to healthy controls and we identified 15 lncRNAs overexpressed in JMML patients.
AIM
The aim of this study is to identify overexpressed lncRNAs in JMML and to provide proof-of-concept for lncRNA perturbation as novel therapeutic strategy in this disease.
METHODS
To further identify overexpressed lncRNAs in JMML, total RNA from isolated mononuclear cell preparations from 19 previously untreated JMML patients (median age: 2.02 years) and 3 normal pediatric bone marrow (NPBM) samples from healthy controls (siblings screened for transplantation) was sequenced (HiSeq, Illumina). Differential gene expression (adjusted P-value < 0.05 and FDR < 0.10) was performed with DESEQ2 and EdgeR (R Bioconductor). Functional analysis of differentially expressed lncRNAs was performed through a lncRNA-mRNA interaction network (lncPath, R Bioconductor). A subset of overexpressed lncRNAs, based on fold change ≥2 and low to absent expression in NPBM, were subsequently validated by quantitative reverse-transcriptase PCR (qPCR). Antisense LNA™ GapmeRs (Qiagen), potent antisense oligonucleotides used for highly efficient inhibition of mRNA and lncRNA function, were designed for a subset of significantly overexpressed lncRNAs. As no JMML cell lines are available, cell lines from other pediatric and adult haematopoietic malignancies were selected based on publically available RNA-seq data and lncRNA expression was verified on qPCR. The effect of GapmeR treatment on lncRNA expression and cell viability was tested with qPCR and flow cytometry viability assays (7-AAD and annexin V staining).
RESULTS
Total RNA sequencing additionally revealed presence of 122 upregulated and 47 downregulated lncRNAs, most of which not present on the microarray. JMML patients clustered together and divergent from healthy controls on principal component analysis using only lncRNAs. Different pathways associated with RAS-MAPK signaling, cancer development, DNA metabolism and cell cycle were identified as being synergistically regulated by these differentially expressed lncRNAs through a lncRNA-mRNA interaction network. Forty-two lncRNAs (55 different transcripts) showing overexpression in JMML were validated with qPCR and in 13 transcripts from 11 lncRNAs a significantly higher expression could be observed in JMML patients compared to NPBM (Figure 1A), thus providing potential therapeutic targets. A minimum of four different antisense LNA GapmeRs were subsequently designed for each of these validated lncRNAs, overexpressed in JMML. For each lncRNA, one or more hematopoietic cell lines, showing expression of that lncRNA, could be identified and validated with qPCR. In vitro incubation of these cell lines with GapmeRs against lncRNA with expression in these cell lines, revealed concentration-dependent molecular knockdown in ≥ 2 GapmeRs for 4/5 lncRNAs (Figure 1B). For two targets, lnc-THADA4-1 and lnc-ACOT9, additional cell viability assays were performed and an effect on cell viability could be observed in GapmeR treated wells (Figure 1C).
CONCLUSION
Using microarray and total RNA sequencing, we documented the lncRNA landscape in JMML, and identified deregulated lncRNAs associated with key processes in JMML pathogenesis. We further provided proof-of-concept that knockdown of these lncRNAs using LNA GapmeRs can be a feasible therapeutic strategy in vitro in hematopoietic cell lines. Subsequently, safety and efficacy of this novel therapeutic strategy needs to be validated in JMML xenograft models in vivo.
Figure 1

Disclosures
Niemeyer: Celgene: Consultancy.