In humans, low expression of DIAPH3 is associated with poor prognosis in glioblastoma patients. In mice, DIAPH3 loss induces aberrant mitosis, chromosomal instability, and cell death; whereas the combined loss of DIAPH3 and p53 causes high-grade diffuse glioma and accelerates tumor onset. In this study, we used Diaph3/p53 double conditional knockout mice (dcKO) and ultra-high-resolution MRI to detect and characterize the development and progression of glioma. We transcriptionally profiled early and advanced gliomas and identified stage-dependent alterations in molecular signatures as the tumor progresses. Notably, genetic pathways underlying self-renewal and extracellular matrix (ECM) interactions were upregulated at the expense of neural activity signature, which was severely shrunk. CKAP2L was identified as a differentially expressed gene at both early and advanced tumor stages in the dcKO mice. A subsequent siRNA screen to identify genes involved in proliferation and migration of glioblastoma cells identified CKAP2L as a top hit. Functional studies showed that CKAP2L depletion reduced proliferation and migration and induced cell accumulation in G₂/M phase of the cell cycle. We also used data mining and thoroughly characterized the relationship between CKAP2L expression and glioma development. We found that CKAP2L expression correlates with proliferative and progenitor-associated markers, pointing to a potential role in maintaining highly proliferative and progenitor-like tumor cell state. We also identified a differentially methylated CpG site in the CKAP2L promoter that was associated with changes in gene expression across glioma datasets. Overall, our findings show that CKAP2L may contribute to glioma progression, help distinguish tumor grade, and represent a potential therapeutic target in glioblastoma treatment.

Diaphanous (DIAPH) 3 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 polarity of the mitotic spindle. DIAPH3-deficient cells display disorganized cytoskeleton, multipolar spindles, and supernumerary centrosomes. DIAPH3-deficiency disrupts the expression and/or stability of microtubule-associated proteins SPAG5 and KNSTRN. SPAG5 and DIAPH3 have similar expression patterns in the developing brain and overlapping subcellular localization during mitosis. Knockdown of SPAG5 phenocopies the DIAPH3 deficiency, whereas its overexpression rescues the DIAH3 phenotype. Conditional inactivation of Diaph3 in the cerebral cortex profoundly disrupts neurogenesis depleting cortical progenitors and neurons; and leading to cortical malformation and autistic-like behavior. Our data uncover 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.