DIAPH3 predicts survival of patients with MGMT-methylated glioblastoma.

Background: Glioblastoma is one of the most aggressive primary brain tumors, with a poor outcome despite multimodal treatment. Methylation of the MGMT promoter, which predicts the response to temozolomide, is a well-established prognostic marker for glioblastoma. However, a difference in survival can still be detected within the MGMT methylated group, with some patients exhibiting a shorter survival than others, emphasizing the need for additional predictive factors. Methods: We analyzed DIAPH3 expression in glioblastoma samples from the cancer genome atlas (TCGA). We also retrospectively analyzed one hundred seventeen histological glioblastomas from patients operated on at Saint-Luc University Hospital between May 2013 and August 2019. We analyzed the DIAPH3 expression, explored the relationship between mRNA levels and Patient's survival after the surgical resection. Finally, we assessed the methylation pattern of the DIAPH3 promoter using a targeted deep bisulfite sequencing approach. Results: We found that 36% and 1% of the TCGA glioblastoma samples exhibit copy number alterations and mutations in DIAPH3, respectively. We scrutinized the expression of DIAPH3 at single cell level and detected an overlap with MKI67 expression in glioblastoma proliferating cells, including neural progenitor-like, oligodendrocyte progenitor-like and astrocyte-like states. We quantitatively analyzed DIAPH3 expression in our cohort and uncovered a positive correlation between DIAPH3 mRNA level and patient's survival. The effect of DIAPH3 was prominent in MGMT-methylated glioblastoma. Finally, we report that the expression of DIAPH3 is at least partially regulated by the methylation of three CpG sites in the promoter region. Conclusion: We propose that combining the DIAPH3 expression with MGMT methylation could offer a better prediction of survival and more adapted postsurgical treatment for patients with MGMT-methylated glioblastoma.

Long-term survival in patients with IDH-wildtype glioblastoma: clinical and molecular characteristics.

BACKG ROUND: Glioblastoma is an aggressive tumor that has a dismal prognosis even with multimodal treatment. However, some patients survive longer than expected. The objective of this study was to revisit patients diagnosed with glioblastoma according to the 2021 WHO classification and analyze clinical and molecular characteristics associated with long-term survival (LTS). METHODS: We retrospectively analyzed 120 IDH-wildtype glioblastomas operated on at our institution between 2013 and 2018. We divided them into LTS patients, surviving more than 3 years, and non-LTS patients, and then compared their features. Additionally, we performed DNA methylation-based brain tumor classification in LTS patients. RESULTS: Sixteen patients were long-term survivors. Age < 70 years, MGMT promoter methylation, extent of resection ≥ 95%, and administration of radiochemotherapy were associated with LTS (P = 0.005, P < 0.001, P = 0.048, and P = 0.008, respectively). In addition, when these factors were combined, the probability of LTS was 74% (95% CI: 62--84). The methylome analysis confirmed the diagnosis of glioblastoma in the majority of the tested LTS patients. Regarding subtypes, 29% of cases were mesenchymal (MES), 43% were RTK1, and 29% were RTK2. Interestingly, RTK1 and RTK2 cases tended to have longer overall survival than MES cases (P = 0.057). Moreover, the only tested LTS patient with an unmethylated MGMT promoter had an "adult-type diffuse high-grade glioma, IDH-wildtype, subtype E" rather than a glioblastoma. This tumor was characterized by multinucleated giant cells and a somatic mutation in POLE. CONCLUSIONS: We suggest that glioblastoma patients with a combination of favorable prognostic factors can achieve LTS in 74% of cases. In addition, methylome analysis is important to ascertain the type of glioma in LTS patients, especially when the MGMT promoter is unmethylated.

KIF2A deficiency causes early-onset neurodegeneration.

KIF2A is an atypical kinesin that has the capacity to depolymerize microtubules. Patients carrying mutations in KIF2A suffer from progressive microcephaly and mental disabilities. While the role of this protein is well documented in neuronal migration, the relationship between its dysfunction and the pathobiology of brain disorders is unclear. Here, we report that KIF2A is dispensable for embryogenic neurogenesis but critical in postnatal stages for maturation, connectivity, and maintenance of neurons. We used a conditional approach to inactivate KIF2A in cortical progenitors, nascent postmitotic neurons, and mature neurons in mice. We show that the lack of KIF2A alters microtubule dynamics and disrupts several microtubule-dependent processes, including neuronal polarity, neuritogenesis, synaptogenesis, and axonal transport. KIF2A-deficient neurons exhibit aberrant electrophysiological characteristics, neuronal connectivity, and function, leading to their loss. The role of KIF2A is not limited to development, as fully mature neurons require KIF2A for survival. Our results emphasize an additional function of KIF2A and help explain how its mutations lead to brain disorders.

DIAPH3 deficiency links microtubules to mitotic errors, defective neurogenesis, and brain dysfunction.

Diaphanous (DIAPH) three (DIAPH3) is a member of the formin proteins that have the capacity to nucleate and elongate actin filaments and, therefore, to remodel the cytoskeleton. DIAPH3 is essential for cytokinesis as its dysfunction impairs the contractile ring and produces multinucleated cells. Here, we report that DIAPH3 localizes at the centrosome during mitosis and regulates the assembly and bipolarity of the mitotic spindle. DIAPH3-deficient cells display disorganized cytoskeleton and multipolar spindles. DIAPH3 deficiency disrupts the expression and/or stability of several proteins including the kinetochore-associated protein SPAG5. DIAPH3 and SPAG5 have similar expression patterns in the developing brain and overlapping subcellular localization during mitosis. Knockdown of SPAG5 phenocopies DIAPH3 deficiency, whereas its overexpression rescues the DIAHP3 knockdown phenotype. Conditional inactivation of Diaph3 in mouse cerebral cortex profoundly disrupts neurogenesis, depleting cortical progenitors and neurons, leading to cortical malformation and autistic-like behavior. Our data uncover the uncharacterized functions of DIAPH3 and provide evidence that this protein belongs to a molecular toolbox that links microtubule dynamics during mitosis to aneuploidy, cell death, fate determination defects, and cortical malformation.

Pyridoxamine-phosphate oxidases and pyridoxamine-phosphate oxidase-related proteins catalyze the oxidation of 6-NAD(P)H to NAD(P).

6-NADH and 6-NADPH are strong inhibitors of several dehydrogenases that may form spontaneously from NAD(P)H. They are known to be oxidized to NAD(P)+ by mammalian renalase, an FAD-linked enzyme mainly present in heart and kidney, and by related bacterial enzymes. We partially purified an enzyme oxidizing 6-NADPH from rat liver, and, surprisingly, identified it as pyridoxamine-phosphate oxidase (PNPO). This was confirmed by the finding that recombinant mouse PNPO oxidized 6-NADH and 6-NADPH with catalytic efficiencies comparable to those observed with pyridoxine- and pyridoxamine-5'-phosphate. PNPOs from Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana also displayed 6-NAD(P)H oxidase activity, indicating that this 'side-activity' is conserved. Remarkably, 'pyridoxamine-phosphate oxidase-related proteins' (PNPO-RP) from Nostoc punctiforme, A. thaliana and the yeast S. cerevisiae (Ygr017w) were not detectably active on pyridox(am)ine-5'-P, but oxidized 6-NADH, 6-NADPH and 2-NADH suggesting that this may be their main catalytic function. Their specificity profiles were therefore similar to that of renalase. Inactivation of renalase and of PNPO in mammalian cells and of Ygr017w in yeasts led to the accumulation of a reduced form of 6-NADH, tentatively identified as 4,5,6-NADH3, which can also be produced in vitro by reduction of 6-NADH by glyceraldehyde-3-phosphate dehydrogenase or glucose-6-phosphate dehydrogenase. As 4,5,6-NADH3 is not a substrate for renalase, PNPO or PNPO-RP, its accumulation presumably reflects the block in the oxidation of 6-NADH. These findings indicate that two different classes of enzymes using either FAD (renalase) or FMN (PNPOs and PNPO-RPs) as a cofactor play an as yet unsuspected role in removing damaged forms of NAD(P).

[A five-year experience in conformational radiotherapy in the treatment of prostate cancer. Evaluation of acute toxicity in 131 patients]. / Radiothérapie conformationnelle dans le traitement du cancer de la prostate. Evaluation de la toxicité aiguë chez 131 patients.

PURPOSE: To evaluate the urinary and rectal toxicity secondary to 3D conformal radiotherapy for prostate cancer. MATERIAL AND METHODS: Between 1998 and 2003, 131 men with prostate cancer underwent 3D conformal radiotherapy with or without androgen deprivation. The different stages were: 2 T1b ; 40 T1c; 19 T2a; 16 T2b; 18 T2c; 33 T3a; 1 T3b and 2 T3c with Gleason score: 4-6 = 47%, 7 = 36% and 8-9 in 17% of the cases. The median patient age was 66 (48-87). Pretreatment PSA level was respectively < 10 ng/ml (41%). 10-20 ng/ml (30%) and > 20 ng/ml (29%). Of the 131 patients, 98 received androgen ablation therapy before radiation. The total radiation dose varied between 66 and 74 Gy, delivered with 18MV photons of the linear accelerator, the median follow up was 33 months (5-67). RESULTS: According to the RTOG grading (gr) for acute toxicity, we noticed 3gr 3 genitourinary (GU) toxicity and no gr3 gastro intestinal (GI) toxicity. There were 36 gr 1 and 12 gr 2 GI toxicity, 41 gr 1 and 22 gr 2 GU toxicity. The mean prostate volume was 41 cc for patients who received androgen ablation and 56 cc for the others (p < 0.002). The percentage of volume receiving more than 50 Gy (V50) was calculated, the median V50 was 32% (5-67) for the rectum and 35% (5-79) for the bladder CONCLUSION: The toxicity profile in this study is in the same range than those of the literature and of our previous study concerning our first 50 patients with prostate cancer treated with 3D conformal radiotherapy.