Inhibiting centrosome clustering reduces cystogenesis and improves kidney function in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic disorder accounting for approximately 5% of patients with renal failure, yet therapeutics for the treatment of ADPKD remain limited. ADPKD tissues display abnormalities in the biogenesis of the centrosome, a defect that can cause genome instability, aberrant ciliary signaling, and secretion of pro-inflammatory factors. Cystic cells form excess centrosomes via a process termed centrosome amplification (CA), which causes abnormal multipolar spindle configurations, mitotic catastrophe, and reduced cell viability. However, cells with CA can suppress multipolarity via "centrosome clustering," a key mechanism by which cells circumvent apoptosis. Here, we demonstrate that inhibiting centrosome clustering can counteract the proliferation of renal cystic cells with high incidences of CA. Using ADPKD human cells and mouse models, we show that preventing centrosome clustering with 2 inhibitors, CCB02 and PJ34, blocks cyst initiation and growth in vitro and in vivo. Inhibiting centrosome clustering activates a p53-mediated surveillance mechanism leading to apoptosis, reduced cyst expansion, decreased interstitial fibrosis, and improved kidney function. Transcriptional analysis of kidneys from treated mice identified pro-inflammatory signaling pathways implicated in CA-mediated cystogenesis and fibrosis. Our results demonstrate that centrosome clustering is a cyst-selective target for the improvement of renal morphology and function in ADPKD.

Superenhancer activation of KLHDC8A drives glioma ciliation and hedgehog signaling.

Glioblastoma ranks among the most aggressive and lethal of all human cancers. Self-renewing, highly tumorigenic glioblastoma stem cells (GSCs) contribute to therapeutic resistance and maintain cellular heterogeneity. Here, we interrogated superenhancer landscapes of primary glioblastoma specimens and patient-derived GSCs, revealing a kelch domain-containing gene, specifically Kelch domain containing 8A (KLHDC8A) with a previously unknown function as an epigenetically driven oncogene. Targeting KLHDC8A decreased GSC proliferation and self-renewal, induced apoptosis, and impaired in vivo tumor growth. Transcription factor control circuitry analyses revealed that the master transcriptional regulator SOX2 stimulated KLHDC8A expression. Mechanistically, KLHDC8A bound chaperonin-containing TCP1 (CCT) to promote the assembly of primary cilia to activate hedgehog signaling. KLHDC8A expression correlated with Aurora B/C Kinase inhibitor activity, which induced primary cilia and hedgehog signaling. Combinatorial targeting of Aurora B/C kinase and hedgehog displayed augmented benefit against GSC proliferation. Collectively, superenhancer-based discovery revealed KLHDC8A as what we believe to be a novel molecular target of cancer stem cells that promotes ciliogenesis to activate the hedgehog pathway, offering insights into therapeutic vulnerabilities for glioblastoma treatment.

Cilium induction triggers differentiation of glioma stem cells.

Glioblastoma multiforme (GBM) possesses glioma stem cells (GSCs) that promote self-renewal, tumor propagation, and relapse. Understanding the mechanisms of GSCs self-renewal can offer targeted therapeutic interventions. However, insufficient knowledge of GSCs' fundamental biology is a significant bottleneck hindering these efforts. Here, we show that patient-derived GSCs recruit elevated levels of proteins that ensure the temporal cilium disassembly, leading to suppressed ciliogenesis. Depleting the cilia disassembly complex components is sufficient to induce ciliogenesis in a subset of GSCs via relocating platelet-derived growth factor receptor-alpha (PDGFR-α) to a newly induced cilium. Importantly, restoring ciliogenesis enabled GSCs to switch from self-renewal to differentiation. Finally, using an organoid-based glioma invasion assay and brain xenografts in mice, we establish that ciliogenesis-induced differentiation can prevent the infiltration of GSCs into the brain. Our findings illustrate a role for cilium as a molecular switch in determining GSCs' fate and suggest cilium induction as an attractive strategy to intervene in GSCs proliferation.

Trends and challenges in modeling glioma using 3D human brain organoids.

The human brain organoids derived from pluripotent cells are a new class of three-dimensional tissue systems that recapitulates several neural epithelial aspects. Brain organoids have already helped efficient modeling of crucial elements of brain development and disorders. Brain organoids' suitability in modeling glioma has started to emerge, offering another usefulness of brain organoids in disease modeling. Although the current state-of-the organoids mostly reflect the immature state of the brain, with their vast cell diversity, human brain-like cytoarchitecture, feasibility in culturing, handling, imaging, and tractability can offer enormous potential in reflecting the glioma invasion, integration, and interaction with different neuronal cell types. Here, we summarize the current trend of employing brain organoids in glioma modeling and discuss the immediate challenges. Solving them might lay a foundation for using brain organoids as a pre-clinical 3D substrate to dissect the glioma invasion mechanisms in detail.

Rapid and Efficient Invasion Assay of Glioblastoma in Human Brain Organoids.

Glioblastoma (GBM) possesses glioma stem cells (GSCs) that exhibit aggressive invasion behavior in the brain. Current preclinical GBM invasion assays using mouse brain xenografts are time consuming and less efficient. Here, we demonstrate an array of methods that allow rapid and efficient assaying of GSCs invasion in human brain organoids. The assays are versatile to characterize various aspects of GSCs, such as invasion, integration, and interaction with mature neurons of brain organoids. Tissue clearing and quantitative 3D imaging of GSCs in host organoids reveal that invasiveness is inversely correlated with the organoids' age. Importantly, the described invasion assays can distinguish the invasive behaviors of primary and recurrent GSCs. The assays are also amenable to test pharmacological agents. As an example, we show that GI254023X, an inhibitor of ADAM10, could prevent the integration of GSCs into the organoids.

Inhibition of CPAP-tubulin interaction prevents proliferation of centrosome-amplified cancer cells.

Centrosome amplification is a hallmark of human cancers that can trigger cancer cell invasion. To survive, cancer cells cluster amplified extra centrosomes and achieve pseudobipolar division. Here, we set out to prevent clustering of extra centrosomes. Tubulin, by interacting with the centrosomal protein CPAP, negatively regulates CPAP-dependent peri-centriolar material recruitment, and concurrently microtubule nucleation. Screening for compounds that perturb CPAP-tubulin interaction led to the identification of CCB02, which selectively binds at the CPAP binding site of tubulin. Genetic and chemical perturbation of CPAP-tubulin interaction activates extra centrosomes to nucleate enhanced numbers of microtubules prior to mitosis. This causes cells to undergo centrosome de-clustering, prolonged multipolar mitosis, and cell death. 3D-organotypic invasion assays reveal that CCB02 has broad anti-invasive activity in various cancer models, including tyrosine kinase inhibitor (TKI)-resistant EGFR-mutant non-small-cell lung cancers. Thus, we have identified a vulnerability of cancer cells to activation of extra centrosomes, which may serve as a global approach to target various tumors, including drug-resistant cancers exhibiting high incidence of centrosome amplification.

Novel insights into SMALED2: BICD2 mutations increase microtubule stability and cause defects in axonal and NMJ development.

Bicaudal D2 (BICD2) encodes a highly conserved motor adaptor protein that regulates the dynein-dynactin complex in different cellular processes. Heterozygous mutations in BICD2 cause autosomal dominant lower extremity-predominant spinal muscular atrophy-2 (SMALED2). Although, various BICD2 mutations have been shown to alter interactions with different binding partners or the integrity of the Golgi apparatus, the specific pathological effects of BICD2 mutations underlying SMALED2 remain elusive. Here, we show that the fibroblasts derived from individuals with SMALED2 exhibit stable microtubules. Importantly, this effect was observed regardless of where the BICD2 mutation is located, which unifies the most likely cellular mechanism affecting microtubules. Significantly, overexpression of SMALED2-causing BICD2 mutations in the disease-relevant cell type, motor neurons, also results in an increased microtubule stability which is accompanied by axonal aberrations such as collateral branching and overgrowth. To study the pathological consequences of BICD2 mutations in vivo, and to address the controversial debate whether two of these mutations are neuron or muscle specific, we generated the first Drosophila model of SMALED2. Strikingly, neuron-specific expression of BICD2 mutants resulted in reduced neuromuscular junction size in larvae and impaired locomotion of adult flies. In contrast, expressing BICD2 mutations in muscles had no obvious effect on motor function, supporting a primarily neurological etiology of the disease. Thus, our findings contribute to the better understanding of SMALED2 pathology by providing evidence for a common pathomechanism of BICD2 mutations that increase microtubule stability in motor neurons leading to increased axonal branching and to impaired neuromuscular junction development.

Losers of Primary Cilia Gain the Benefit of Survival.

In this issue, Zhao and colleagues demonstrate that loss of primary cilia in medulloblastoma cells confers resistance to the Smoothened (SMO) inhibitor sonidegib. When treated with sonidegib, medulloblastoma cells lost their cilia and gained resistance. Surprisingly, loss of cilia is associated with recurrent mutations in ciliogenesis genes that are eventually able to drive drug resistance. These findings uncover a previously unknown mechanism of cancer cells in gaining a "persister-like" state against anticancer agents at the expense of losing primary cilia. Cancer Discov; 7(12); 1374-5. ©2017 AACRSee related article by Zhao et al., p. 1436.

New Colchicine-Derived Triazoles and Their Influence on Cytotoxicity and Microtubule Morphology.

A series of new colchicinoids with a variable triazole unit at C-7 was synthesized through Cu(I)-catalyzed 1,3-dipolar cycloaddition (click-chemistry) of a colchicine-derived azide with various alkynes and the cytotoxicity against THP-1 and Jurkat cancer cell lines was used for structural optimization. Three particularly active compounds (IC50 ≤ 5 nM) were additionally investigated with respect to their efficacy against relevant solid tumor cell lines (HeLa, A549, and SK MES 1). Besides distorting the microtubule morphology by tubulin depolymerization, one compound also exhibited a pronounced centrosome declustering effect in triple negative breast cancer cells (MDA-MB-231) and nonsmall cell lung cancer cells (H1975).

Telomerase activation by genomic rearrangements in high-risk neuroblastoma.

Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours.

Synthesis and biological studies of silver N-heterocyclic carbene complexes derived from 4,5-diarylimidazole.

A novel class of silver N-heterocyclic carbene complexes (5a-f) were synthesized in high yield by reacting silver(I) oxide with 4,5-diarylimidazolium halides (4a-f). The complexes were characterized using NMR and IR spectroscopy. The structure was confirmed on the example of bromo[1,3-diethyl-4,5-bis(4-fluorophenyl)imidazol-2-ylidene]silver(I) (5c) by crystal structure analysis. The X-ray structure indicated a three-dimensional coordination polymer with a repeating unit consisting of a C(carben)-Ag(2)-Br(2)-C(carben) cluster. Pharmacological investigations revealed that all silver complexes possessed growth inhibitory effects against breast cancer (MCF-7 and MDA-MB-231) as well as colon carcinoma (HT-29) cells. The most active compound 5c was slightly less active against MCF-7 cells, more active against MDA-MB-231 cells and comparable active as cisplatin against HT-29 cells. Further pharmacological investigations were performed with selected compounds on estrogen receptor (ER) binding, DNA intercalation, cyclooxygenase (COX) inhibition and antibacterial activity. The complexes were only marginally active at the DNA, ER and the COX enzymes, so these targets can be excluded to be involved in the mode of action. However, the growth of bacteria was significantly inhibited by 5c and 5f and opens a new application of this complex type.