Centrosome Amplification Research Articles

Preliminary Results from a Phase 2 Open-Label, Multicenter, Dose Optimization Clinical Study of the Safety, Tolerability, and Pharmacokinetic (PK) and Pharmacodynamic (PD) Profiles of Cfi-400945 As a Single Agent or in Combination with Azacitidine or Decitabine in Patients with Acute Myeloid Leukemia, Myelodysplastic Syndrome or Chronic Myelomonocytic Leukemia (TWT-202)

Background: CFI-400945 is a potent, selective, orally administered, first-in-class inhibitor of the serine/threonine kinase, Polo-like kinase 4 (PLK4). PLK4 is a highly conserved master upstream regulator of centriole duplication and is critical for maintenance of genomic integrity. Aberrant expression of PLK4 results in centrosome amplification, often seen in aneuploid cancers, pointing to a potentially causative role for PLK4 in genome instability and cancer progression. An investigator-initiated study using a 28-day continuous CFI-400945 dosing schedule at the Princess Margaret Cancer Center (PMCC) demonstrated clinical activity in AML [Murphy et al., 2020] and responses were seen in high-risk pts with adverse cytogenetics, including those with TP53 mutations. CFI-400945 was tolerated with clinically manageable toxicities and the dose limiting toxicity (DLT) was colitis. The recommended phase 2 dose was 96mg. A newer crystal form of CFI-400945 was introduced after the PMCC study. Consequently, the optimal dosing of CFI-400945, including as a combination agent, are not yet clinically defined and are the rationale for this study. Study Design and Methods: This study is a multi-part phase 1/2 study of CFI-400945 as a single agent and in combination with hypomethylating agents (HMA) (NCT04730258). For Part 1, pts with relapsed and/or refractory AML, MDS, or CMML after >1 prior therapy will be included. Pts with MDS or CMML must have progressed or had a lack of response after at least 4 cycles of hypomethylating agents. For Part 2, pts should have relapsed and/or refractory AML or untreated MDS or CMML. Untreated pts who decline or are ineligible for intensive therapy may be included. The study uses a standard 3 + 3 design, and the dosing schedule is 21 days on and 7 days off (28-day cycle) - this is to potentially avoid the colitis seen in the study at PMCC. The maximum tolerated dose (MTD) will be defined as the dose level where the number of dose-limiting toxicities (DLTs) is <1 out of 6 at highest dose level at or below the maximally administered dose. Pharmacokinetics (PK) and pharmacodynamic (PD) markers will be assessed. Results: As of the 30Jun22 data cut off, 9 pts had been enrolled into the study. Four (44%) pts had AML, 2 (22%) had MDS, 3 (33%) had CMML. The median number of prior therapies was 2 (range 1-4), and 2 (22%) pts had received a stem cell transplant. Six (66%) were male and the median age was 69 (range 22-75). Three of 4 AML pts had unfavorable risk cytogenetics by ELN 2017 criteria, and 2 pts (1 each with MDS, CMML) with TP53 mutations. Five pts received 32mg and 4 pts 48mg. There have been no DLTs. There have been 8 (89%) SAEs with the following occurring in >2 pts (febrile neutropenia (6 pts), pneumonia (2 pts)). Common treatment emergent adverse events (TEAE) in > 2 pts were febrile neutropenia 6 pts (67%), hypokalemia 5pts (56%), anemia and hypomagnesemia 4 pts each (44%), hypophosphatemia, diarrhea, abdominal pain, nausea, vomiting, dyspnea, peripheral edema 3 pts each (33%). Thirty-three percent (20 events, 3 pts) of the grade 3 or greater TEAE's were considered related to CFI-400945. There have been no CR or PR per ELN response criteria observed to date, however, there were 2 (50%) SD per IWG criteria at the 48 mg dose level (1-MDS, 1-CMML). Pharmacokinetic (PK) evaluations indicated that the mean terminal-life ranged from 7 - 10 hours. Low accumulation was observed after 21 days of daily dosing (AR <2-fold). In general, exposure in this study is overlapped with that observed in the PMCC study. PD studies evaluating the effect of CFI-400945 on markers of mitosis, ploidy, and centriole function are ongoing. Conclusion: CFI-400945 has been generally well tolerated and TWT-202 continues to enroll in Part 1. There have been no responses per ELN to date but there was SD in 2 pts (48 mg dose). Lack of DLTs suggest dose level is still suboptimal at time of submission. PK characteristics support daily dosing of CFI-400945 and PD studies are ongoing. Dose escalation continues in this study and updated data will be presented.

RAD6 inhibition enhances paclitaxel sensitivity of triple negative breast cancer cells by aggravating mitotic spindle damage

BackgroundPaclitaxel (PTX), a first-line therapy for triple negative breast cancers (TNBC) induces anti-tumor activity by microtubule stabilization and inhibition of cell division. Its dose-limiting toxicity and short half-life, however, pose clinical challenges underscoring the need for strategies that increase its efficiency. RAD6, a E2 ubiquitin conjugating enzyme, is associated with centrosomes at all phases of cell cycle. Constitutive overexpression of the RAD6B homolog in normal breast cells induces centrosome amplification and multipolar spindle formation, indicating its importance in centrosome regulation.MethodsTNBC centrosome numbers were scored by pericentrin immunostaining. PTX sensitivities and interactions with SMI#9, a RAD6-selective small molecule inhibitor, on TNBC cell survival were analyzed by MTT and colony forming assays and an isogenic MDA-MB-468 TNBC model of PTX resistance. The molecular mechanisms underlying PTX and SMI#9 induced cytotoxicity were determined by flow cytometry, immunoblot analysis of cyclin B1 and microtubule associated protein TAU, and dual immunofluorescence staining of TAU and α-tubulin.ResultsOur data show aberrant centrosome numbers and that PTX sensitivities are not correlated with TNBC BRCA1 status. Combining PTX with SMI#9 synergistically enhances PTX sensitivities of BRCA1 wild-type and mutant TNBC cells. Whereas SMI#9/PTX combination treatment increased cyclin B1 levels in MDA-MB-468 cells, it induced cyclin B1 loss in HCC1937 cells with accumulation of reproductively dead giant cells, a characteristic of mitotic catastrophe. Cell cycle analysis revealed drug-induced accumulation of tetraploid cells in S and G2/M phases, and robust increases in cells with 4 N DNA content in HCC1937 cells. TAU overexpression is associated with reduced PTX efficacy. Among the six TAU isoforms, both SMI#9 and PTX downregulated 1N3R TAU in MDA-MB-468 and HCC1937 cells, suggesting a common mechanism of 1N3R regulation. Dual TAU and α-tubulin immunostaining showed that SMI#9 induces monopolar mitotic spindles. Using the isogenic model of PTX resistance, we show that SMI#9 treatment restores PTX sensitivity.ConclusionsThese data support a common mechanism of microtubule regulation by SMI#9 and PTX and suggest that combining PTX with RAD6 inhibitor may be beneficial for increasing TNBC sensitivities to PTX and alleviating toxicity. This study demonstrates a new role for RAD6 in regulating microtubule dynamics.

P029: Are Reed-Sternberg cells stuck in mitosis by short nucleoplasmic bridges as a consequence of centromeric instability?

Background: Multinucleated giant tumor cells are frequently observed in lymph nodes of untreated lymphoma patients. Malignant Hodgkin lymphoma (HL) cells i. e. small Hodgkin and large bi- or multinucleated Reed-Sternberg (HRS) cells are characterized by genomic instability and downregulation of numerous key regulators of e.g. cell cycle, spindle apparatus, cytokinesis and cell lineage differentiation. So far, the formation of HRS cells remains obscure. We have demonstrated previously that telomere instability is involved in the proliferation of small cells into large binucleated cells. Here, we have used a collection of HL cell lines to analyze the successive steps involved in the transformation of the mononucleated into the multinucleated RS-like cells. Materials and Methods: Seven HL cell lines and 30 HL lymph nodes were used. Cytokinesis-block micronucleus assay and telomere and centromere staining followed by the M-FISH technique were employed to analyze chromosomal instability. DNA repair pathways were investigated. Results: We demonstrate that telomere dysfunction in HL cell lines is associated with the formation of dicentric chromosomes with both centromeres in close proximity, but also with a high rate of formation of micronuclei. Two morphologically different types of nucleoplasmic bridges (NPBs) were observed: (1) Long NPBs related to telomere dysfunction and chromosome fusions, and (2) short NPBs related to centromere breakpoints with configurations of cells looking like “stuck” together as binucleated cells. Short NPBs and binucleated cells were seen in the cultures of all HL cell lines. However, each HL cell line was characterized by an individual signature of the proportion of long and short NPBs, which correlated with centrosome amplification and formation of stable binucleated cells. Similar mechanisms were identified in HL patient lymph nodes prior to treatment, thus underscoring the biological significance of our findings in cell cultures. Transcriptome analysis confirmed the variations in the DNA repair pathways regarding the rate of large cells among the different HL cell lines. Conclusion: The formation of dicentric chromosomes related to centromere instability, and short NPBs were associated with the emergence of binucleated cells through different mechanisms. Our findings open a novel route to understanding the transition from mononucleated cells to multinucleated cells in HL and in other B-cell lymphomas.

CEP192 is a novel prognostic marker and correlates with the immune microenvironment in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) responds poorly to standard chemotherapy or targeted therapy; hence, exploration for novel therapeutic targets is urgently needed. CEP192 protein is indispensable for centrosome amplification, which has been extensively characterized in both hematological malignancies and solid tumors. Here, we combined bioinformatics and experimental approaches to assess the potential of CEP192 as a prognostic and therapeutic target in HCC. CEP192 expression increased with tumor stage and was associated with poor clinicopathologic features, frequent recurrence, and higher mortality. Upon single-cell RNA sequencing, CEP192 was found to be involved in the proliferation and self-renewal of hepatic progenitor-like cells. This observation was further evidenced using CEP192 silencing, which prevented tumor cell proliferation and self-renewal by arresting cells in the G0/G1 phase of the cell cycle. Notably, CEP192 was highly correlated with multiple tumor-associated cytokine ligand–receptor axes, including IL11–IL11RA, IL6–IL6R, and IL13–IL13RA1, which could promote interactions between hepatic progenitor-like cells, PLVAP+ endothelial cells, tumor-associated macrophages, and CD4+ T cells. Consequently, CEP192 expression was closely associated with an immunosuppressive tumor microenvironment and low immunophenoscores, making it a potential predictor of response to immune checkpoint inhibitors. Taken together, our results unravel a novel onco-immunological role of CEP192 in establishing the immunosuppressive tumor microenvironment and provide a novel biomarker, as well as a potential target for therapeutic intervention of HCC.

BRCA1 transports the DNA damage signal for CDDP-induced centrosome amplification through the centrosomal Aurora A.

Breast cancer gene 1 (BRCA1) plays roles in DNA repair and centrosome regulation and is involved in DNA damage-induced centrosome amplification (DDICA). Here, the centrosomal localization of BRCA1 and the kinases involved in centrosome duplication were analyzed in each cell cycle phase after treatment with DNA crosslinker cisplatin (CDDP). CDDP treatment increased the centrosomal localization of BRCA1 in early S-G2 phase. BRCA1 contributed to the increased centrosomal localization of Aurora A in S phase and that of phosphorylated Polo-like kinase 1 (PLK1) in late S phase after CDDP treatment, resulting in centriole disengagement and overduplication. The increased centrosomal localization of BRCA1 and Aurora A induced by CDDP treatment involved the nuclear export of BRCA1 and BRCA1 phosphorylation by ataxia telangiectasia mutated (ATM). Patient-derived variants and mutations at phosphorylated residues of BRCA1 suppressed the interaction between BRCA1 and Aurora A, as well as the CDDP-induced increase in the centrosomal localization of BRCA1 and Aurora A. These results suggest that CDDP induces the phosphorylation of BRCA1 by ATM in the nucleus and its transport to the cytoplasm, thereby promoting the centrosomal localization Aurora A, which phosphorylates PLK1. The function of BRCA1 in the translocation of the DNA damage signal from the nucleus to the centrosome to induce centrosome amplification after CDDP treatment might support its role as a tumor suppressor.

Computational benchmarking of putative KIFC1 inhibitors.

The centrosome in animal cells is instrumental in spindle pole formation, nucleation, proper alignment of microtubules during cell division, and distribution of chromosomes in each daughter cell. Centrosome amplification involving structural and numerical abnormalities in the centrosome can cause chromosomal instability and dysregulation of the cell cycle, leading to cancer development and metastasis. However, disturbances caused by centrosome amplification can also limit cancer cell survival by activating mitotic checkpoints and promoting mitotic catastrophe. As a smart escape, cancer cells cluster their surplus of centrosomes into pseudo-bipolar spindles and progress through the cell cycle. This phenomenon, known as centrosome clustering (CC), involves many proteins and has garnered considerable attention as a specific cancer cell-targeting weapon. The kinesin-14 motor protein KIFC1 is a minus end-directed motor protein that is involved in CC. Because KIFC1 is upregulated in various cancers and modulates oncogenic signaling cascades, it has emerged as a potential chemotherapeutic target. Many molecules have been identified as KIFC1 inhibitors because of their centrosome declustering activity in cancer cells. Despite the ever-increasing literature in this field, there have been few efforts to review the progress. The current review aims to collate and present an in-depth analysis of known KIFC1 inhibitors and their biological activities. Additionally, we present computational docking data of putative KIFC1 inhibitors with their binding sites and binding affinities. This first-of-kind comparative analysis involving experimental biology, chemistry, and computational docking of different KIFC1 inhibitors may help guide decision-making in the selection and design of potent inhibitors.

EP16.03-015 Centrosome Amplification Is a Prognostic Indicator and Potential Therapeutic Vulnerability in Non-small Cell Lung Cancer

Centrosomes are organelles that facilitate chromosome segregation. The number of centrosomes in a cell is tightly regulated to ensure the genome is divided equally during cell division. In normal cells, two centrosomes form a bipolar mitotic spindle for faithful chromosome segregation. Cancers often exhibit centrosome amplification (CA), an increase in centrosome number that can be caused by cellular insults such as cell cycle defects and carcinogen exposure. Cells with CA are prone to forming abnormal, multi-polar mitotic spindles which can induce aneuploidies that promote tumour progression.

Postmitotic centriole disengagement and maturation leads to centrosome amplification in polyploid trophoblast giant cells.

DNA replication is normally coupled with centriole duplication in the cell cycle. Trophoblast giant cells (TGCs) of the placenta undergo endocycles resulting in polyploidy but their centriole state is not known. We used a cell culture model for TGC differentiation to examine centriole and centrosome number and properties. Before differentiation, trophoblast stem cells (TSCs) have either two centrioles before duplication or four centrioles after. We find that the average nuclear area increases approximately eight-fold over differentiation, but most TGCs do not have more than four centrioles. However, these centrioles become disengaged, acquire centrosome proteins, and can nucleate microtubules. In addition, some TGCs undergo further duplication and disengagement of centrioles, resulting in substantially higher numbers. Live imaging revealed that disengagement and separation are centriole autonomous and can occur asynchronously. Centriole amplification, when present, occurs by the standard mechanism of one centriole generating one procentriole. PLK4 inhibition blocks centriole formation in differentiating TGCs but does not affect endocycle progression. In summary, centrioles in TGC endocycles undergo disengagement and conversion to centrosomes. This increases centrosome number but to a limited extent compared with DNA reduplication.

Centromere protein J is overexpressed in human glioblastoma and promotes cell proliferation and migration.

Glioblastoma is the most common and malignant type of primary brain tumor. Previous studies have shown that alterations in centrosome amplification and its components are frequently found in treatment-resistant tumors and may be associated with tumor progression. A centrosome protein essential for centrosome biogenesis is the centromere protein J (CENPJ), known to control the proliferation of neural progenitors and hepatocarcinoma cells, and also neuronal migration. However, it remains unknown the role of CENPJ in glioblastoma. Here we show that CENPJ is overexpressed in human glioblastoma cell lines in comparison to human astrocytes. Using bioinformatics analysis, we find that high Cenpj expression is associated with poor prognosis in glioma patients. Examining Cenpj loss of function in glioblastoma by siRNA transfection, we find impairments in cell proliferation and migration. Using a Cenpj mutant version with the deleted PN2-3 or TCP domain, we found that a conserved PN2-3 region is required for glioblastoma migration. Moreover, Cenpj downregulation modulates glioblastoma morphology resulting in microtubules stabilization and actin filaments depolymerization. Altogether, our findings indicate that CENPJ controls relevant aspects of glioblastoma progression and might be a target for therapeutic intervention and a biomarker for glioma malignancy.

Abstract 1164: Somatic NF1 loss in breast cancer leads to centrosome amplification, aneuploidy and increased sensitivity to T-DM1

Abstract Background: Centrosome amplification (CA, the presence of &amp;gt;2 centrosomes) is a hallmark of several tumors. CA perturbs mitosis by generating nonphysiological pulling forces, leading to chromosome missegregation and aneuploidy. This condition may be advantageous for tumor outgrowth providing it gets corrected by clustering centrosomes into a pseudo-bipolar conformation for successful cell division. Molecular determinants of CA and therapeutic opportunities in breast cancer (BC) are still poorly understood. Preliminary data from our group show that somatic NF1 loss of function (LOF), common upon metastatic progression, is associated in vitro and in patients with selective sensitivity to maytansinoids such as T-DM1. Here, we explored the molecular basis of this increased sensitivity. Methods: Multiple CRISPR/Cas9-generated NF1KO or WT clones of HER2+ BC cell lines (BT474, SKBR3 and HCC1954) were assessed for sensitivity to T-DM1 or DM1 in BrdU-based and clonogenic assays, with RNAseq being performed. BT474 cells were furthered engineered with the FUCCI(Ca) reporter for live cell cycle imaging. Ploidy was assessed through flow cytometry. Aneuploidy in the GENIE cohort (restricted to BC patients analyzed with NF1-covering panels) was assessed by generating a segmentation score (sum of the absolute ploidy scores in segments covered by NGS panels). Centrosomes, spindle conformation and chromosome abnormalities were studied by confocal microscopy in cells synchronized both with RO3306 or thymidine block. Results: All lines showed increased sensitivity to T-DM1 in KO vs WT in BrdU-based and clonogenic assays; similar results were obtained with DM1 only, suggesting independence from HER2 targeting. RNAseq differential analysis showed significant enrichment for gene sets involved in mitotic spindle in KO but not WT cells upon T-DM1 treatment. FUCCI analysis showed significantly longer permanence in G2/M in NF1KO compared to WT cells (24.8 vs 17.9% in G2/M, p=1.81E-07). In vehicle-treated synchronized cells, significantly more KO cells showed &amp;gt;2 centrosomes (21.6 vs 4.7%, p&amp;lt;0.00001) and multiple pseudo-bipolar mitotic figures with narrow intercentriolar distances, indicative of efficient clustering. This was associated with more frequent chromosome misalignment (26.7 vs 6.7%, p=0.038). In the GENIE cohort, segmentation score was higher for patients with NF1 LOF mutations vs NF1 WT (median 46.7 vs 41.1, p=0.0023), indicative of more common aneuploidy. Upon T-DM1, KO cells exhibited significantly more non-bipolar spindles with massively wider intercentriolar distances. Conclusions: Somatic NF1 loss causes aneuploidy due to CA. This likely favors metastatization and can be exploited therapeutically. CA associated with other common oncogenic events (RAS and BRCA1/2 mutations, PTEN loss) may represent a general biomarker for drugs inhibiting centrosome clustering in BC. Citation Format: Bruno Achutti Duso, Elena Gavilan Dorronzoro, Giulia Tini, Maria de Filippo, Marica Ippolito, Chiara Soriani, Simona Rodighiero, Stefano Santaguida, Pier Giuseppe Pelicci, Luca Mazzarella. Somatic NF1 loss in breast cancer leads to centrosome amplification, aneuploidy and increased sensitivity to T-DM1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1164.

Abstract 779: A NRF1-driven JRK-mediated β-catenin transcriptional activity contributes to the aggressive growth of ovarian cancer

Abstract Ovarian cancer is the most lethal gynecological cancer, accounting for thousands of cancer-related deaths in the United States every year. Though there has been progress towards understanding ovarian cancer pathogenesis, much is not yet understood about the molecular mechanisms that facilitate chemoresistance and metastasis. Our work has shown that nuclear respiratory factor 1 (NRF1) is highly active in several human cancers, though the role of NRF1 remains largely unexplored in ovarian carcinomas. Therefore, our aim was to examine the role of this transcription factor in ovarian cancer, since understanding its transcriptional landscape may reveal NRF1 or its target genes as possible therapeutic targets for this highly aggressive cancer. The Cancer Genome Atlas (TCGA) was utilized to collect RNA-Seq data from 379 serous cystadenocarcinoma samples, where we found that about 20% of ovarian cancer patients had altered NRF1 expression, showing either high expression or amplification of this transcription factor. Patients with high NRF1 expression were found to have decreased survival compared to patients with low NRF1 expression. NRF1 amplification was also correlated with the overamplification of one of its target genes - Jrk helix-turn-helix protein (JRK) in 26% of the samples. JRK regulates centrosome amplification, and through the production of mitotic spindle abnormalities, centrosome amplification contributes to genomic instability. Further investigation of the relationship between NRF1 and JRK uncovered that protein levels of JRK and β-catenin were significantly higher in NRF1-induced stem-like cells compared to the control group, and JRK is known to bind to β-catenin. Our NRF1 ChIP-Seq analyses showed that promoters of these two genes - JRK and CTNNB1 - were bound to NRF1. The Flow Assorted Cell Sorting (FACS), ChIP-Seq, RNA Seq and confocal microscopic studies suggest that through JRK, NRF1 regulates β-catenin transcriptional activity. Amplified gene expression is the one of the first steps towards oncogene activation, and our findings illustrate that altered NRF1 expression (i.e., overexpression or amplification) coupled with that of JRK influences the prognosis of patients diagnosed with ovarian cancer. The Wnt/β-catenin pathway is also highly active in the aggressive ovarian cancer. In addition to JRK, through the activation of β-catenin, NRF1may also affect the development of ovarian tumors. Taken together, our findings provide a novel insight into the molecular basis of the contribution of NRF1-driven JRK-mediated β-catenin transcriptional activity to the susceptibility of highly aggressive ovarian cancer. A better understanding of how ovarian neoplasm formation depends on NRF1-JRK-β-catenin pathway may open new avenues for therapeutic strategy against ovarian cancer. Citation Format: Ana Ruas, Quentin Felty, Jayanta K. Das, Alok Deoraj, Changwon Yoo, Deodutta Roy. A NRF1-driven JRK-mediated β-catenin transcriptional activity contributes to the aggressive growth of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 779.

Abstract 2569: Development of potent dual PDK1/AurA kinase inhibitors for Ewing sarcoma therapy

Abstract Background. It has been estimated that more than 50% of cancers are related to the dysregulation of the PI3K-PDK1-Akt signaling axis. Within this pathway, phosphoinositide-dependent kinase-1 (PDK1) and Aurora kinase A (AurA) have key roles. PDK1 is a phosphorylation-mediated activator of Akt, the main apoptotic regulator, thereby acting at the crossroads of several oncogenic pathways. Overexpression and/or aberrant activation of AurA have been functionally linked to neoplastic transformation principally through the development of centrosome amplification and chromosomal instability. In this study, the molecular mechanisms involved in the dual inhibitory capacity of PDK1- and AurA-targeting OXID-pyridonyl compounds were explored in a panel of tumor cell lines carrying PI3KCA, K-RAS, PTEN, RB-1, EGFR and/or TP53 mutations Materials and Methods. To evaluate the enzymatic inhibitory activity, the oxindole-based derivatives were subjected to FRET-based Z′-Lyte assay (Invitrogen™) against PDK1 and AurA. All in vitro assays for ADME-Tox profiling were performed as published previously[1]. Anti-proliferative effects of OXID-pyridonyl compounds (SA16, IB35, VI8, VI18) were assessed by soft agar clonogenic formation and MTT assays after 72-h exposures with increasing concentrations (from 0.014 to 30 µM) in a panel of 15 cell lines. Protein levels were analyzed by Western Blot using commercial antibodies. OSU-03012 (PDK-1 inhibitor) and MK-5108 (AurA inhibitor) were used as reference compounds. Results. Among the four evaluated compounds, SA16 displayed nanomolar inhibitory potency against both PDK1 and AurA. In vitro, drugs SA16 and VI8 showed to exert anti-tumor activity in the 6647, TC71, SKNMC Ewing sarcoma (ES) cells lines and in the HCT116 colon carcinoma-derived cells (IC50s values between 2.6 and 25 µM after 72-h exposures), when tested in a panel of 15 cancer cell lines. These results were confirmed by the evaluation of 3D-clonogenic capacity in soft agar. The effect of 1 µM SA16 at the protein level was evaluated after 5 to 120 min exposure in the ES cell line SKNMC. No modulation of Aur-A protein or PDK-1 phosphorylation (Ser241) were observed in SKNMC cells, whereas a decrease of downstream PDK1 targets such as p-AKT, p-RSK2, and p-p44/42 MAPK was observed after 30 min of exposure. In addition, SA16 displayed acceptable in vitro ADME-Tox properties. Our results on structural insights into the mode of binding[2], in vitro drug-intake kinetics and in vivo PK appear to indicate that SA16 presents adequate features as a scaffold for the design of PDK1/AurA dual-target molecules. Conclusion. We report the synthesis of novel first-in-class 2-oxindole-based derivatives as dual PDK1-AurA kinase inhibitors as a novel scaffold for the design and synthesis of a new multitarget strategy for Ewing sarcoma.[1] Runfola M. et al. Data Brief 29 (2020) 105206.[2] Sistito S. et al. Eu J Med Chem 226 (2021) 11389. Citation Format: Maria Eugenia Riveiro, Samuel Huguet, Ramiro Vazquez, Roberta Frapolli, Guido Puricelli, Simona Sestito, Olivier Madar, Keyvan Rezai, Gianpiero Garau, Simona Rapposelli. Development of potent dual PDK1/AurA kinase inhibitors for Ewing sarcoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2569.

The binding between NPM and H2B proteins signals for the diabetes-associated centrosome amplification.

Diabetes not only increases the risk for cancer but also promotes cancer metastasis. Centrosome amplification (CA) is sufficient to initiate tumorigenesis and can enhance the invasion potential of cancer cells. We have reported that diabetes can induce CA, with diabetic pathophysiological factors as the triggers, which involves the signaling of nucleophosmin(NPM). Thus, CA can serve as a candidate biological link between diabetes and cancer. In the present study, we attempted to identify the NPMbinding partners and investigated whether the binding between NPMand its partner mediated the CA. We confirmed that high glucose, insulin, and palmitic acid cancer could elicit CA in the HCT16 colon cancer cells and found that the experimental treatment increased the binding between NPMand H2B, but not between p-NPMand H2B. The molecular docking analysis supported the fact that NPMand H2B could bind to each other through various amino acid residues. The treatment also increased the colocalization of NPMand H2B in the cytosol. Importantly, disruption of the NPM1-H2B complex by individualknockdown of the protein level of NPMor H2B led to the inhibition of the treatment-evoked CA. In conclusion, our results suggest that the binding between NPMand H2B proteins signals for the CA by high glucose, insulin, and palmitic acid.

Proteomic Mapping and Targeting of Mitotic Pericentriolar Material in Tumors Bearing Centrosome Amplification.

This study identifies the proteome of pericentriolar material and reveals therapeutic vulnerabilities in tumors bearing centrosome amplification.

PCID2 assists in the nuclear export of nucleophosmin (NPM) in HeLa cells

PCI‐domain containing protein 2 (PCID2) plays a key role in facilitating the nuclear export of various proteins. PCID2 further functions by localizing to the centrosome, where it likely helps to regulate the process of centrosome duplication. Nucleophosmin (NPM) is a nucleolar protein that undergoes Crm1‐dependent nuclear export and also associates with centrosomes to prevent premature cell division. Other proteins reliant on Crm1 for nuclear export, including BRCA1, further require the presence of PCID2 for efficient nuclear export and subsequent centrosomal localization. Here, we show that NPM is also reliant on PCID2 for its nuclear export through the use of siRNA knockdown on HeLa cells. Immunofluorescence revealed a 34% increase in nuclear fluorescence of NPM without PCID2, while centrosomal localization of NPM exhibited no direct impact due to removal of PCID2. These results identify a supporting role for PCID2 in the regulation of centrosome duplication through the facilitation of NPM nuclear export to the cytoplasm. Ongoing studies seek to identify NPM and PCID2 complexes via immunoprecipitation as well as exploring the possible effects of PCID2 knockdown on the structure or composition of the nucleolus and on NPM subnuclear localization. Our research has identified a role for PCID2 in the regulation of NPM nuclear export, providing potential insight into the development of tumorigenesis due to centrosome amplification and possible changes to nucleolar structure and composition.

PCID2 Facilitates the Nuclear Export, but not the Centrosomal Localization, of BRCA2 in Hs578T Breast Cancer Cells

PCI‐domain containing protein 2 (PCID2) assists in the export of proteins from the nucleus to the cytoplasm. PCID2 also localizes to the centrosome, where it is present more frequently in cells exhibiting an abnormally high number of centrosomes, an aberration associated with the development of cancer. Several proteins that negatively regulate the centrosome cycle are typically found in the nucleus, and we have previously discovered that PCID2 is involved in the nuclear export and centrosomal localization of one key centrosome cycle regulator, BRCA1. Therefore, PCID2 may play a role in the regulation of the centrosome cycle by delivering key regulators of duplication. BRCA2 is a nuclear tumor suppressor that travels to the centrosome, where it helps to arrest the centrosome cycle and prevent overduplication. BRCA2 mutations are associated with the development of breast and ovarian cancers, both of which are also associated with centrosomal overduplication. We proposed that PCID2 aids in the transport of BRCA2 from the nucleus to the centrosome. Using siRNA knockdown of PCID2 and subsequent immunofluorescence studies of BRCA2 and γ‐Tubulin, we found that the loss of PCID2 significantly decreased the amount of BRCA2 able to be exported from the nucleus to the cytoplasm, with Hs578T breast cancer cells depleted of PCID2 demonstrating a 37% increase in nuclear BRCA2 retention. However, the loss of PCID2 demonstrated no significant impact on the ability of BRCA2 to localize to centrosomes. Our results indicate that while PCID2 affects the cellular localization of BRCA2 by assisting its nuclear transport, it likely does not directly influence the recruitment of cytoplasmic BRCA2 to the centrosome. Ongoing studies are exploring the relationship of PCID2 to BRCA2 export and centrosomal localization in ovarian cancer cells and are attempting to confirm the presence of a PCID2‐BRCA2 nuclear export complexes in breast and ovarian cancer cells. These findings will shed light on the process by which PCID2 role in nuclear transport and the centrosome cycle, leading to a better understanding of centrosomal amplification and tumorigenesis in a variety of cancer types.

Aggressive uveal melanoma displays a high degree of centrosome amplification, opening the door to therapeutic intervention.

Uveal melanoma (UM) is the most common intraocular cancer in adults. Whilst treatment of primary UM (PUM) is often successful, around 50% of patients develop metastatic disease with poor outcomes, linked to chromosome 3 loss (monosomy 3, M3). Advances in understanding UM cell biology may indicate new therapeutic options. We report that UM exhibits centrosome abnormalities, which in other cancers are associated with increased invasiveness and worse prognosis, but also represent a potential Achilles' heel for cancer‐specific therapeutics. Analysis of 75 PUM patient samples revealed both higher centrosome numbers and an increase in centrosomes with enlarged pericentriolar matrix (PCM) compared to surrounding normal tissue, both indicative of centrosome amplification. The PCM phenotype was significantly associated with M3 (t‐test, p < 0.01). Centrosomes naturally enlarge as cells approach mitosis; however, whilst UM with higher mitotic scores had enlarged PCM regardless of genetic status, the PCM phenotype remained significantly associated with M3 in UM with low mitotic scores (ANOVA, p = 0.021) suggesting that this is independent of proliferation. Phenotypic analysis of patient‐derived cultures and established UM lines revealed comparable levels of centrosome amplification in PUM cells to archetypal triple‐negative breast cancer cell lines, whilst metastatic UM (MUM) cell lines had even higher levels. Importantly, many UM cells also exhibit centrosome clustering, a common strategy employed by other cancer cells with centrosome amplification to survive cell division. As UM samples with M3 display centrosome abnormalities indicative of amplification, this phenotype may contribute to the development of MUM, suggesting that centrosome de‐clustering drugs may provide a novel therapeutic approach.

NEDD9 links anaplastic thyroid cancer stemness to chromosomal instability through integrated centrosome asymmetry and DNA sensing regulation.

Stemness and chromosomal instability (CIN) are two common contributors to intratumor heterogeneity and therapy relapse in advanced cancer, but their interplays are poorly defined. Here, in anaplastic thyroid cancer (ATC), we show that ALDH+ stem-like cancer cells possess increased CIN-tolerance owing to transcriptional upregulation of the scaffolding protein NEDD9. Thyroid patient tissues and transcriptomic data reveals NEDD9/ALDH1A3 to be co-expressed and co-upregulated in ATC. Compared to bulk ALDH- cells, ALDH+ cells were highly efficient at propagating CIN due to their intrinsic tolerance of both centrosome amplification and micronuclei. ALDH+ cells mitigated the fitness-impairing effects of centrosome amplification by partially inactivating supernumerary centrosomes. Meanwhile, ALDH+ cells also mitigated cell death caused by micronuclei-mediated type 1 interferon secretion by suppressing the expression of the DNA-sensor protein STING. Both mechanisms of CIN-tolerance were lost upon RNAi-mediated NEDD9 silencing. Both in vitro and in vivo, NEDD9-depletion attenuated stemness, CIN, cell/tumor growth, while enhancing paclitaxel effectiveness. Collectively, these findings reveal that ATC progression can involve an ALDH1A3/NEDD9-regulated program linking their stemness to CIN-tolerance that could be leveraged for ATC treatment.

Synthesis and Evaluation of Novel Carboxamides Capable of Causing Centrosome Declustering and Apoptosis in Breast Cancer Cells

AbstractThe fragility of cancer cells at the time of mitosis has served as an important target for the development of many successful chemotherapeutic agents. Many cancers cells have supernumerary centrosomes that they cluster during mitosis to form bipolar spindles. Inhibition of centrosome clustering in these cells results in multipolar spindle formation and apoptotic cell death, providing an opportunity to selectively target a subset of cancers with centrosome amplification. In the current work, we report synthesis of 29 novel tethered biaryls and biological evaluation of their ability to inhibit centrosome clustering in breast cancer cells (BT‐549). We have identified N‐benzhydryl‐5‐nitrofuran‐2‐carboxamide (5 h) as a centrosome declustering compound. 5 h has potent antiproliferative activity in centrosome amplified BT‐549 cells with GI50 value of 1.81±0.19 μM (n=2). Treatment of BT‐549 cells with 5 h causes centrosome declustering resulting in mitotic arrest due to multipolar spindle formation and misaligned chromosomes which ultimately leads to apoptotic cell death.

PLK4 is upregulated in prostate cancer and its inhibition reduces centrosome amplification and causes senescence.

BackgroundIdentification of novel molecular target(s) is important for designing newer mechanistically driven approaches for the treatment of prostate cancer (PCa), which is one of the main causes of morbidity and mortality in men. In this study, we determined the role of polo‐like kinase 4 (PLK4), which regulates centriole duplication and centrosome amplification (CA), in PCa.Materials and MethodsEmploying human PCa tissue microarrays, we assessed the prevalence of CA, correlated with Gleason score, and estimated major causes of CA in PCa (cell doubling vs. centriole overduplication) by staining for mother/mature centrioles. We also assessed PLK4 expression and correlated it with CA in human PCa tissues and cell lines. Further, we determined the effects of PLK4 inhibition in human PCa cells.ResultsCompared to benign prostate, human PCa demonstrated significantly higher CA, which was also positively correlated with the Gleason score. Further, most cases of CA were found to arise by centriole overduplication rather than cell doubling events (e.g., cytokinesis failure) in PCa. In addition, PLK4 was overexpressed in human PCa cell lines and tumors. Moreover, PLK4 inhibitors CFI‐400945 and centrinone‐B inhibited cell growth, viability, and colony formation of both androgen‐responsive and androgen‐independent PCa cell lines. PLK4 inhibition also induced cell cycle arrest and senescence in human PCa cells.ConclusionsCA is prevalent in PCa and arises predominantly by centriole overduplication as opposed to cell doubling events. Loss of centrioles is cellular stress that can promote senescence and suggests that PLK4 inhibition may be a viable therapeutic strategy in PCa.