Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT06334835 |
| Other study ID # |
1/23 OSS SDN |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
April 30, 2023 |
| Est. completion date |
May 31, 2026 |
Study information
| Verified date |
March 2024 |
| Source |
IRCCS SYNLAB SDN |
| Contact |
Laura Pierri, Master |
| Phone |
0812408260 |
| Email |
laura.pierri[@]synlab.it |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
Long non-coding RNAs (lncRNAs) are a class of biomarkers of crescent interest in the
hematologic and oncologic field.
They do not encode proteins and can alter gene expression by acting on different steps of
regulation, including DNA methylation and chromatin structure. Recent data identified
recurrent somatic alterations in genes involved in DNA methylation and post-translational
histone modifications in T-ALL, suggesting that epigenetic homeostasis is critically required
in restraining tumor development in the T-cell lineage. Further, recent studies showed that
the expression levels of specific lncRNAs correlate with the prognosis of patients with Acute
Lymphoblastic Leukemia of T-cells (T-ALL). The objectives of this research project are to
identify T-ALL-specific lncRNAs to be used as new diagnostic and prognostic biomarkers of
disease and to explore their role on chromatin reorganization and transcriptional regulation
that may lead to the onset and progression of T-ALL.
Description:
Specific Aim 1 Evaluation of lncRNAs in a cohort of T-ALL patients in comparison with T-Naive
lymphocytes and PBMC from healthy subjects. Identification of the best suitable in vitro
models. To achieve the goal of Aim 1 we planned to enroll a cohort of pediatric patients
affected by T-ALL at the Pausilipon hospital, the UO2, which is a reference center for the
treatment of oncological diseases in Campania. For patient recruitment, specific and detailed
informed consent will be used according to the current General Data Protection Regulation.
The collected and processed biological samples will be stored in the SDN Biobank, which is
the institutional biobank of IRCCS Synlab SDN and is a member of the BBMRI-ERIC
infrastructure. Considering that childhood T-ALL is rare cancer, with an incidence in the
pediatric population of 1 case every 100000 children, we plan to enroll 16-20 T-ALL patients.
Starting from RNA-seq data previously obtained by our laboratory, comparing 6 pediatric T-ALL
with naïve T-cells derived from cord blood, we identified a set of differentially regulated
lncRNAs. From these, we selected 6 among the most up-regulated lncRNAs that have never been
associated with leukemia, in general, and pediatric acute T-cell leukemia in particular. The
6 up-regulated selected lncRNAs are: AC002454.1 (ENSG00000237819), PCAT18 (ENSG00000265369),
HHIP-AS1 (ENSG00000248890), LINC01222 (ENSG00000233410), AC116351.1 (ENSG00000215246),
AC247036.1 (ENSG00000271201). Using ours and other public datasets of RNA-seq experiments, we
will confirm our suggestions in silico, and then proceed to subsequent analyses on biological
samples. The expression levels of these lncRNAs will be analyzed ex vivo, using RT-PCR
experiments, in the selected cohort in comparison with PBMCs derived from healthy subjects
and pediatric Acute Lymphoblastic Leukemia of B-cells (B-ALL) patients (already present in
our Biobank) to ascertain over-expression in the T-ALL population and specificity with
respect to another pediatric hematological disease such as B-ALL. Moreover, the transcript
levels of the selected lncRNAs will be analyzed in a minimum of 5 cell lines model disease,
for both T-ALL and B-ALL. These data will allow us to select the best cellular models for
subsequent functional studies. The lncRNA expression data will be cross-referenced with
clinical data from T-ALL pediatric subjects in order to identify a correlation between the
expression of the lncRNAs and patient outcomes and to obtain a molecular signature of lncRNAs
specific for T-ALL that will be useful for the diagnosis and for the improvement of pediatric
patient management.
Specific Aim 2 Identification of cis-regulatory elements differentially accessible between
pediatric T-ALL patients and healthy subjects, evaluation of epigenetic modifications of the
loci identified above, and gene regulatory networks modeling to unravel the functional role
of candidate lncRNAs in childhood T-ALL. Current advances in high-throughput sequencing and
bioinformatic approaches highlighted that lncRNAs can regulate gene expression at the
epigenetical, transcriptional and post-transcriptional levels. As for the epigenetic side,
they can drive significant changes in chromatin architecture, DNA methylation and histone
modifications. For example, lncRNAs may guide transcriptional regulators to specific genomic
loci or facilitate the enhancer-promoter communication by operating a direct or indirect
remodeling of the chromatin folding. Today it is well known that these kinds of epigenetic
regulation mechanisms have a critical impact on different steps of malignancy onset and
progression, from uncontrolled proliferation to apoptosis resistance, altering the chromatin
accessibility and methylation state of cis-regulatory elements and therefore leading to
aberrant gene expression in tumors with respect to the healthy counterpart. Despite the fact
that epigenetic alterations are known to be a key factor in leukemia as well, scarce advances
have been made to elucidate the pivotal role of lncRNAs on the epigenomic landscape of ALL
patients, let alone pediatric ones. Only few specific lncRNAs have been identified and most
of the questions about their regulatory function as potential oncogenes or tumor-suppressors
remain unanswered and even unasked. For the Aim 2, we will unveil the regulatory dynamics
interplaying between lncRNAs, epigenetic factors and oncogene expression. We plan to couple
RNA-seq with ATAC-seq and MethylC-seq experiments in T-ALL pediatric patient samples in
comparison with naïve T-cells from cord blood. ATAC-seq captures open chromatin regions,
usually trimethylated at H3K4, H3K36, and H3K79, which typically correlate with
cis-regulatory elements. ATACseq of T-ALL samples will provide genomic regions of interests
to predict putative lncRNA-genome DNA binding sites via computational methods and, in
combination with transcriptomics and functional analyses, this technology will allow us to
bioinformatically model the dynamic interactions between genomic cis-regulatory elements and
trans-effectors such as transcription factors, chromatin remodeling complexes and lncRNAs.
Further, as emerging evidences have uncovered a crosstalk also between lncRNAs and DNA
methylation, we will perform a genome-wide identification of cytosine DNA methylation states
at single-base resolution through MethylC-seq technique in order to complement and enhance
our gene regulatory network model. Once the epigenetic landscapes of T-ALL patients have been
defined, we intend to characterize also the chromatin and transcriptomic landscape in T-ALL
cell line models, both in control conditions and upon the knockdown of the target lncRNAs
(see Aim3 for further details). This experiment would help us to corroborate and sharpen our
model of the operating mechanism for lncRNAs under investigation. This cross-firing strategy
of functional experiments and computational models will provide mechanistic understanding of
epigenetic and transcriptomic relations in pediatric TALL and, above all, will pave the way
to novel strategies for diagnostic and therapeutic intervention.
Specific Aim 3 Evaluation, using appropriate T-ALL cell line model systems, of the role of
identified lncRNAs on epigenome modifications.
Once evaluated the epigenome of T-ALL patients in comparison with healthy subjects, we will
proceed to assess the involvement of identified lncRNAs in leukemogenesis. The activities of
Aim3 will be conducted in collaboration with the UO3, which has all the tools and skills to
support this experimental phase. We will proceed to the transient silencing of the lncRNAs
identified, in the T-ALL model cell lines that express them at the highest levels. For the
silencing experiments, we will use traditional (lipofectamine or lentiviral vectors) or
innovative methods. In particular, a method recently developed by an American company
(FANA-ASO, oligonucleotide, AUMBiotech, USA) based on the use of modified oligonucleotides
able to enter into hematopoietic cells without the use of transfection agents, known to be
toxic to cells. The FANA-ASO is a technology abundantly used for the silencing of target
transcripts in hematopoietic models that are difficult to transfect using classical
transfection methods. They also provide high silencing efficiency for molecules such as
lncRNAs.
In the selected T-ALL model systems, each lncRNA will be silenced individually. After the
transient transfection, the efficiency of the silencing will be verified by q-RT-PCR at
different time points. Then, we propose to analyze the effects of lncRNA knockdown on tumor
progression by evaluating changes in biological behaviors of the transfected cells (i.e.
proliferation, cell cycle progression, and motility abilities), changes in cell phenotype (by
performing three-dimensional culture assay), epithelial-mesenchymal transition (EMT), stem
cell markers, oxidative stress, and drug sensitivity (by performing viability assay or IC50
calculation). Depending on the results obtained, coupled silencing experiments with two
lncRNAs simultaneously will also be considered. For the lncRNAs that will have had the
greatest effect on the selected models, we will evaluate what are the effects that lncRNAs
have on chromatin reorganization, through the application of the NGS methods reported in Aim
2, comparing silenced lncRNA models respect to the untreated control. These data will allow
us to evaluate the relationships between the expression levels of identified lncRNAs and
peculiar chromatin alterations in leukemic T cells. These experiments prompted us to identify
a specific gene regulatory network associated with the selected lncRNAs. Passing from the
laboratory bench to the patient beds, we will silence the selected lncRNAs directly in the
childhood T-Blasts and we will evaluate the variations in the levels of the gene identified
as part of the gene regulatory network of the selected lncRNA.
The identified alterations could be then correlated with the clinical features of the disease
in order to evaluate their impact on therapeutic response.
