Genesis, Progression and Recurrence of Glioblastoma: Role of the Diaphanous 3 gene.

Glioblastoma multiform (GBM) is one of the most aggressive brain tumors with a median survival of about one year in humans. The bad prognosis of GBM is due to its very high proliferation rate and to its invasiveness capability. Surgery remains the mainstay of treatment; yet the extremely infiltrative nature of the tumor makes the surgical removal challenging. Radiotherapy and chemotherapy also have a limited efficacy. Hence, it is essential to better understand the genesis and the progression of GBM, uncover driving mutations, identify markers implicated in invasiveness, enable early diagnosis, and improve treatment. We have identified Diaphanous (DIAPH) 3 as a key regulator of neural stem cells biology. DIAPH3 belongs to a molecular network that comprises components of the spindle assembly checkpoint (SAC) and Chromosomal Passenger Complex (CPC) machineries. This network regulates kinetochores-mitotic spindle interactions, controls the transition from metaphase to anaphase, and secures the accurate segregation of chromosomes between daughter cells. Loss of Diaph3 compromises SAC activation. Hence, mitotic errors are not properly “amended”, causing aneuploidy, chromosomal instability, and cell death. We then asked whether aneuploidy could promote neoplastic transformation. To test this, we produced double Diaph3/p53 double conditional knockout mice and found that these animals indeed develop GBM (unpublished, please see preliminary results). We assessed DIAPH3 expression in human GBM samples and uncovered that it is varies between samples, and most importantly it correlates with disease aggressiveness and survival of patients (unpublished, please see preliminary results). The main objectives of this proposal are to determine the transcriptomic profile of GBM as they appear and progress; role of adult neural stem cells in gliomagenesis; role of DIAPH3 in human glioblastoma, and understand the regulation of DIAPH3 expression as well as Rolethe relationship between DIAPH3 level and resistance to chemotherapy. The first set of experiments will be performed in mice and aims at generating new markers to diagnosis and assessing the role of neural stem cells as cell of origin of GBM and therefore as a target for radiotherapy. So far, most of the transcriptomic analyses were performed on advanced stages of GBM, when the tumors induce detectable behavioral changes. We combined our mouse models with ultra-high field MRI (capable of detecting tumors of 1 microliter) to collect very early stages of GBM, but also to classify tumors thus reducing grade-related variability. We will conduct RNA sequencing experiments, determine the molecular signature of glioma genesis, and identify markers for early diagnosis. We will also utilize MRI in a follow-up study in mice to assess if all tumors appear in neurogenic niches of the adult brain where neural stem cells reside. Our study will address the long-lasting question of contribution of neural stem cells to GBM. The second set of experiments will be conducted on human samples. It aims at screening GBM samples for mutations of DIAPH3 and its interactors, evaluating the role of DIAPH3 expression level in aggressiveness of GBM, and investigating the regulation of DIAPH3 expression. We DNA and RNA materials from 135 GBM samples. We will use MiSeq to screen GBM for mutations of DIAPH3 and test its candidacy as a tumor suppressor gene. DIAPH3 is localized at 13q21.2, a region deleted in many cancers. We will use real time RT-PCR to scrutinize the expression of DIAPH3 in GBM samples. Preliminary results (done on 74 samples) are extremely encouraging, as they emphasize a longer survival of patients with high levels of DIAPH3 compared to those with low levels. We will extend our analysis to the rest of samples and perform multivariate Cox proportional-hazards analysis to test DIAPH3 expression as an independent prognostic factor. Finally, we will determine how the expression of DIAPH3 is regulated in GBM cells and whether methylation is involved in its differential expression. The third set of experiments will be conducted in GBM 251 cell lines. The objective of these experiments is double. First, we want to gain knowledge on how DIAPH3 prevents genomic instability and safeguards cell division; and how its lack disrupts the assembly of spindle, attachment of chromosomes to microtubules during metaphase, and their segregation during anaphase/telophase. This will be achieved by comparative live imaging analyses of control and Diaph3 KO cells upon transfections with tagged proteins to visualize DIAPH3, its partners and its effectors. Second, given the correlation between the expression levels of DIAPH3 and survival of patients, we will determine whether this feature can be harnessed to adjust/personalize therapy and target those patients who can benefit from chemotherapy.

Hamad Bin Khalifa University (HBKU)