Patient's Derived Organoids (PDOs) from tumour surgical biopsies are an innovative tool
to test the response of individual patients to specific therapeutic strategies. Organoids
are three dimensional (3D) structures made of organ-specific and self-organizing cells
which can be maintained and propagated in culture. To date, PDOs have been established
from a great number of cancer types, including prostate, ovarian and breast cancers.
Glioblastoma PDOs have also been produced, and they were shown to maintain the
characteristics of their parent tumours, both at mutational level and in terms of gene
expression profiles and cellular heterogeneity. Since they recapitulate the
characteristics of the original tumour better than GBM cell lines, they represent an
important advancement for personalized medicine approaches. Thus, glioblastoma PDOs
represent useful pre-clinical models for drug screening, CAR-T cell testing and for the
generation of brain orthotopic xenografts in model models . In this perspective, the PDOs
offer an opportunity to better characterize the molecular heterogeneity of glioblastoma
patients and to test new therapeutic strategies in a context that mimic parent tumour
genetic properties.
Immunotherapy is emerging as a powerful anticancer approach in some cancer types.
Immunotherapy exploits the ability of the immune system to recognise non self-antigens to
target and destroy cancer cells. Immune checkpoints inhibitors (e.g. anti-PD-1 and
anti-CTLA-4 monoclonal antibodies) were shown effective in tumours exhibiting a high
mutational burden, such as melanoma. Unfortunately, glioblastoma has a low mutational
burden, resulting in a small amount of neoantigens. Moreover, glioblastoma is highly
heterogeneous, meaning that not all the patients produce the same antigens. Thus, higher
benefits could be achieved by developing immunotherapies that target multiple neoantigens
and by combining neoantigen recognition strategies with immune checkpoint blockade
inhibitors. In this perspective, epigenetic regulation to activate the transcription of
normally silent transposable elements (TEs) in glioblastoma by DNA demethylating agents
can enhance the production of neoantigens and trigger a specific immune response.
Transcription of TEs is low or absent in most adult cells, while it is more active during
embryonic development, in stem cells and, intriguingly, in tumors. TEs de-repression in
tumors occurs through multiple epigenetic changes to TE loci, including DNA demethylation
and histone deacetylation. Both epigenetic changes can be associated with oncogenesis,
resulting in different levels of epigenetic de-regulation. TEs overexpression in tumors
compared with healthy tissue has prompted the search for anti-TE T cell responses in
cancer. Proteogenomic approaches have identified tumor-specific, non-canonical open
reading frames (ORFs) that encode peptides presented by human leukocyte antigen (HLA)-I
molecules on tumour cells. Most of the identified peptides derived from non-coding
genomic regions. Interestingly some of these potential tumor-specific antigens are found
in multiple patients and can induce immune responses in vitro or in mouse models. The
investigators recently characterized a long non-coding RNA (lncRNA) in the antisense
direction of SOD1 gene locus (SOD1-DT), that includes several transposable elements. Some
of these (LTR and Alu) contain ORFs and could potentially encode different epitopes. By
in silico translation of these elements, the investigators identified peptides
corresponding to epitopes already tested as GBM-specific targets for cancer
immunotherapies. However, the DNA sequence of these transposable elements is highly
methylated in the nervous tissue and in the U87 GBM cell line (data from Genome Browser).
The investigators will focus on the study of the TEs belonging to the LTR12C family,
because they have been shown to act as enhancer-like and promoter-like elements, shaping
the transcriptomics landscape in a tissue-specific manner. It has been already
demonstrated that treatments with DNMTi and HDACi do not alter the expression of
canonical genes but induce de novo transcription of LTRs, which in turn drive the
expression of specific genes. In addition to producing the epitope, by activating
specific LTRs, it is therefore possible to activate the genes connected to them. Notably,
LTR12C was identified as regulator of proapoptotic genes, such as TP63 and TNFRSF10B.
Thus, the proposed strategy could represent a generally applicable means to produce
proapoptotic genes and immunogenic epitopes in a controlled manner, ensuring a very
specific outcome.
Another potential source of neoepitopes is defective splicing. Splicing is a fundamental
step in pre-messenger RNA (mRNA) maturation operated by a large macromolecular machinery
named the spliceosome. The spliceosome removes the introns and ligates the flanking exons
of the pre-mRNAs, yielding the mature mRNAs. Regulated alternative splicing (AS) of many
exons is exploited by cells to generate multiple protein isoforms from a single gene.
However, the altered splicing program is often deregulated in cancer cells, generating an
actionable vulnerability for tumours, including brain tumours. Profiling of primary and
recurrent GBM and non-malignant brain tissues datasets has identified AS events that are
differently regulated between in GBM and that could be translated into neoepitopes. These
results suggest that splicing modulation could represent a valid therapeutic strategy for
glioblastoma. Indeed, inhibition of the arginine methyl transferase PRMT5 in GBM cells
dysregulates splicing and leads to incremented intron retention and cell senescence both
in vitro and in vivo. Furthermore, PRMT5 has a role in the preservation of GSCs, which
are necessary for tumour self-renewal. Recently, it was shown that pharmacologic
inhibition of splicing generates splicing-derived immunogenic neoepitopes, which are
presented by MHC-I on tumour cells and induce a T cell immune response in vivo. Another
potential therapeutic target is the Splicing Factor 3b Subunit 1 (SF3B1), a core
component of the splicing machinery that is overexpressed in GBM. Taken together, these
results support the rationale of studying the effects of DNA demethylating agents and
splicing inhibitors in glioblastoma PDOs and GSCs to identify suitable candidates to
develop new therapeutic strategies for this disease.
The above-described approaches will be applied to prospectively enrolled patients
undergoing neurosurgery for glioblastoma. Neurosphere cultures and PDO will be
established from primary tumor tissue. Drug screening and cell manipulation to induce TE
expression and to modulate splicing will be applied. The results of in vitro tests will
be correlated with tumor molecular profile, response to treatments and overall patients
outcome.
