Conference Agenda

Glioblastoma progression remains elusive, especially in its first stages. By simultaneous transfer of PDGFB and genetic barcode in mouse brain, we triggered gliomagenesis in univocally labelled cells in vivo, hence enabling direct tracing of glioblastoma evolution from the earliest possible stage. We observed an unexpectedly high incidence of clonal extinction events and progressive clonal size divergence, even after the acquisition of a malignant phenotype. Computational modelling suggests these dynamics as the consequence of a clonal-based cell-cell competition. Bulk and single-cell transcriptome analyses, coupled with lineage tracing, revealed that the strongest correlation with clonal size unbalancing is shown by Myc transcriptional targets. Moreover, the downregulation of Myc expression is sufficient to drive competitive dynamics in intracranially transplanted gliomas. Our findings shed light on aspects of glioblastoma evolution inaccessible by conventional retrospective approaches and highlight the potential of the combined use of clonal tracing and transcriptomic analyses in this field.

Current therapies remain unsatisfactory in preventing the recurrence of glioblastoma multiforme (GBM), which leads to poor patient survival. It has been proposed that, even if the surgery is radical as possible, some cancer cells with tumor-initiating potential endure in preserved tissue, leading to the re-appearance of the disease. In particular, cancer stem cells (CSCs), defined as cells able to self-renew and to initiate or re-growth the tumor, remain quiescent or at very low proliferative activity, therefore capable to resist the adjuvant treatments.

By rational engineering of oncogenic transcription factors, key promoters of GBM malignancy, together with functional domains from endogenous epigenetic enzymes, we generated a family of synthetic repressors (Epigenetic Silencer Factors, ESFs). ESFs induce the transcriptional silencing of the targets of the original transcription factors which likely sustain the malignancy of tumor cells including CSCs.

The new factors kill both glioma cell lines and patient-derived CSCs in vitro. Mechanistically, by doing so ESFs follow precise molecular steps dictated by the epigenetic domains included in the platform. ESF expression, through local viral delivery in mouse xenografts, induces strong regression of human tumors and survival rescue. Conversely, ESFs are not harmful to neurons and glia, also thanks to a minimal promoter that restricts its expression in mitotically active cells, rarely present in the brain parenchyma. Collectively, ESFs produce a significant silencing of a large fraction of the oncogenic transcriptional network, achieving high levels of efficacy in repressing experimentally modeled aggressive brain tumors.

Gliomas are a vast group of tumours with heterogenous characteristics. Even if clinical course varies considerably according to tumour grade and patient clinical status, gliomas tend to recur over time and acquire resistance to treatments. In the last two decades, knowledge of molecular mechanisms fostering tumourigenesis and tumour progression has strikingly increased, and the identification of actionable molecular alterations has become a major goal.

In glioblastoma (GBM), hyperactivation of VEGF pathway and EGFR hyperexpression (or presence of EGFR vIII) are common. Therefore, antiangiogenic and EGFR-targeted therapies have been widely explored: we will present lights and shadows of those strategies from initial experiences to current applications.

Then, we will discuss the possibility of targeting rare molecular alterations, such as alterations of BRAF/MEK, MET, FGFR, and NTRK. Even if uncommon, some of these prevail in specific glioma subtypes, or in younger patients, and should be considered as compassionate options at tumour progression and/or for inclusion in clinical trials. In particular, the phase 2 trial “ROAR”, which explored the use of BRAF/MEK inhibitors (dabrafenib/trametinib) in patients with BRAF^V600E-mutant gliomas, provided encouraging results, with 33% and 69% MRI objective response rate among high-grade and low-grade gliomas cohorts, respectively; similarly, a potential efficacy of a NTRK inhibitor (larotrectinib) emerged from basket trials that included small cohorts of adult patients with NTRK-fused gliomas; also, two clinical trials investigating the efficacy and safety of inhibitors of MET (NCT03175224) and FGFR-fusions (NCT05267106) in glioma patients are currently ongoing.

Finally, we will discuss the role of the IDH1/2 inhibitor vorasidenib, which proved to significantly prolong time-to-progression (27.7 vs 11.1 months) and time-to-next-intervention as compared to placebo in IDH-mutant low-grade gliomas in the recent phase 3 INDIGO trial. Based on those dramatic preliminary results, a new paradigm of treatment for patients with IDH-mutant gliomas is probably on the horizon.

Introduction

Medulloblastoma (MB) and gliomas are the most frequent high-grade brain tumors (HGBT) in children and adulthood, respectively. The general treatment for these tumors consists in surgery, followed by radiotherapy and chemotherapy. Despite the improvement in patient survival, these therapies are only partially effective, and many patients still die, making these diseases an unmet medical challenge. Citron kinase (CITK), product of the primary microcephaly gene MCPH17, is required in neural progenitor cells for cytokinesis, mitotic spindle positioning and chromosomal stability. In vivo studies in xenograft models and in SHH MB arising in transgenic mice have shown that CITK deletion inhibits tumor progression. On this basis, we are working on the development of CITK inhibitors as a possible strategy for HGBT treatment

Methods

Stemming from published binding data between kinase inhibitors and the kinome, we discovered Lestaurtinib as CITK inhibitor. We therefore tested the biological effects of this inhibitor on different MB and GBM patient derived cell lines and in vivo injecting the drug in MBs arising in SmoA1 transgenic mice. In parallel, we developed a screening aimed at obtaining CITK specific inhibitors.

Results

Similar to CITK knockdown, treatment of MB cells with 100 nM Lestaurtinib reduces phospho-INCENP levels at the midbody and leads to cytokinesis failure. Moreover, Lestaurtinib impairs cell proliferation, leads to accumulation of DNA double strand breaks and cell death in MB and GBM cells. Finally Lestaurtinib treatment reduces tumor growth and increases mice survival. Moreover, our screening campaign produced several interesting hits that we are functionally validating.

Conclusions

Our data indicate that Lestaurtinib produces in MB cells and in GBM cells phenotypes that recapitulate CITK knockdown effects. Reduced cell proliferation and increased mice survival indicate that Lestaurtinib and more specific CITK inhibitors are promising candidates for HGBT treatment, deserving deeper investigation.