• Chromatin Compaction
• Epigenetic Silencing
• Imaging
• PD-L1
• PHC2
• BMI1
• Polycomb
• Glioblastoma

ABSTRACT

Glioblastoma multiforme (GBM) is the most common and aggressive malignant form of primary brain tumor in adults, with a median age of onset of approximately 55 to 60 years. Despite the use of multiple therapies, including total surgical resection combined with adjuvant postoperative radiotherapy and adjuvant chemotherapy (TMZ - Temozolomide), glioblastoma has a high rate of recurrence and poor prognosis. The survival rate of the patients has not changed over the past decade, with median survival time of 12-15 months and with an overall 5-year survival rate of 5%. Therefore, one significant challenge of the modern medicine is a better understanding of the molecular basis and to improve the treat¬ment outcome of this disease.
In recent times, epigenetic aberrations have been implicated in the development of GBM. Particularly, the dysregulation of Polycomb group (PcG) proteins can influence the malignant evolution of various cancers, including GBM. PcG family members typically assemble into one of two large multiprotein complexes (Polycomb Repressive Complex: PRC1 and PRC2) that post-translationally modify histones and influence chromatin modification and remodeling, locally repressing transcription. PRC1 compacts chromatin and catalyses the monoubiquitination of histone H2Aat lysine 119 while PRC2 catalyses the methylation of histone H3 at lysine 27 to repress gene expression. Recent studies have shown that PRC1 component PCGF4, better known as BMI1, is a remarkable molecular crossroad in several kind of cancers, possibly including GBM. Accordingly, BMI1 recently became increasingly investigated as target for epigenetic therapeutic strategies. This activity has recently led to the development of several drugs able to modulate BMI1 expression, including siRNA. Targeting BMI1 could represent an effective strategy to modify the activity of other PRC1 components, including those that were found to be largely overexpressed in GBM cells compared to normal glia. Among those, the PRC1 protein PHC2 is intriguing, since it participates in the remodeling of extended chromatin regions by forming 3D protein networks embedding the nucleic acid chains. The formation of these membraneless organelles, often referred to as (liquid) condensates might represent a crucial factor in the onset of neoplasia such as GBM.
Additionally, epigenetic drugs were proposed as coadjuvants of established strategies in oncology, including immunotherapy. Actually, PD-L1 and PD-1, the two most important molecular players in cancer-hijacked immune checkpoint, were demonstrated to be under tight epigenetic control. Yet poor knowledge on the effect of polycomb transcription factors on these molecules is available to date.
In this work, we set two specific aims. The first one targeted the putative role of BMI1 in modulating the biological and biophysical features of PHC2. The second one targeted the putative role of BMI1 in modulating the biological and biophysical features of membrane PD-L1. Both aims were pursued by a combined strategy targeting both intracellular mRNA and protein expression. In the latter case, detection was mostly carried out by immunofluorescence, to obtain some insight on the spatial organization of PHC2 and PD-L1.
rtPCR analysis revealed a moderate decrease in PD-L1 and PHC2 transcripts when effectively suppressing BMI1 in U87 cells. However, only the reduction in PD-L1 showed statistical significance. Intriguingly, BMI1 suppression led to a significant decrease in PD-L1 and PHC2 expression. Employing direct and indirect immunofluorescence (IIF) staining in conjunction with confocal laser scanning fluorescence microscopy (CLSM) proved to be a valuable method for investigating the interaction and spatial characteristics of PHC2. This is especially pertinent due to PHC2's capacity to form small submicron condensates ("foci") linked to chromatin. Detailed confocal 3D imaging demonstrated that BMI1 silencing nearly halved the concentration of PHC2 in the condensate phases, countering the upregulation seen in GBM. However, there were no observed alterations in condensate descriptors such as volume or relative distance from the nuclear border. Additionally, BMI1 silencing did not disturb the monoexponential increase of PHC2 concentration with the volume of the formed foci, nor the nucleation-coalescence formation mechanism of condensate phases, as evidenced by the monoexponential shape of their cell-normalized volume distribution. Overall, these findings underscored the remarkable resilience of PHC2 condensates in the nucleus when PHC2 concentration was altered by BMI1. Further experiments are warranted to validate whether this pattern is indeed linked to a change in the tumoral phenotype. Confocal imaging exhibited a notable concentration of PD-L1 at the cell membrane level in non-silenced cells. Notably, enhanced resolution techniques like Airyscan microscopy and Single Molecule Localization Microscopy in dSTORM mode strongly indicated the organization of PD-L1 into small nanoclusters, reminiscent of observations in NSCLC cell lines from the research group where this thesis was conducted. The subsequent step involves a comprehensive characterization of these nanoclusters, likely associated with lipid-raft regions, and an assessment of whether BMI1 silencing induces a morphological alteration in PD-L1 assembly. This is critical, as recent suggestions propose the pivotal role of PD-L1 nanocluster organization in the immune checkpoint system. Moreover, employing ChIP and immunoFISH techniques may yield additional insights into the disparity between PD-L1 gene regulation at the transcriptional level and at the translational or post-translational level.
This research is meant as a first step towards new therapeutic strategies for healing the otherwise poorly drug-addressable GBM.