Canonical Cell Cycle Research Articles

An alternative cell cycle coordinates multiciliated cell differentiation.

The canonical mitotic cell cycle coordinates DNA replication, centriole duplication and cytokinesis to generate two cells from one1. Some cells, such as mammalian trophoblast giant cells, use cell cycle variants like the endocycle to bypass mitosis2. Differentiating multiciliated cells, found in the mammalian airway, brain ventricles and reproductive tract, are post-mitotic but generate hundreds of centrioles, each of which matures into a basal body and nucleates a motile cilium3,4. Several cell cycle regulators have previously been implicated in specific steps of multiciliated cell differentiation5,6. Here we show that differentiating multiciliated cells integrate cell cycle regulators into a new alternative cell cycle, which we refer to as the multiciliation cycle. The multiciliation cycle redeploys many canonical cell cycle regulators, including cyclin-dependent kinases (CDKs) and their cognate cyclins. For example, cyclin D1, CDK4 and CDK6, which areregulators of mitotic G1-to-S progression, are required to initiate multiciliated cell differentiation. The multiciliation cycle amplifies some aspects of the canonical cell cycle, such as centriole synthesis, and blocks others, such as DNA replication. E2F7, a transcriptional regulator of canonical S-to-G2 progression, is expressed at high levels during the multiciliation cycle. In the multiciliation cycle, E2F7 directly dampens the expression of genes encoding DNA replication machinery and terminates the S phase-like gene expression program. Loss of E2F7 causes aberrantacquisition of DNA synthesis in multiciliated cells and dysregulation of multiciliation cycle progression, which disrupts centriole maturation and ciliogenesis. We conclude that multiciliated cells use an alternative cell cycle that orchestrates differentiation instead of controlling proliferation.

The impact of developmental stage, tissue type, and sex on DNA double-strand break repair in Drosophila melanogaster.

Accurate repair of DNA double-strand breaks (DSBs) is essential for the maintenance of genome integrity, as failure to repair DSBs can result in cell death. The cell has evolved two main mechanisms for DSB repair: non-homologous end-joining (NHEJ) and homology-directed repair (HDR), which includes single-strand annealing (SSA) and homologous recombination (HR). While certain factors like age and state of the chromatin are known to influence DSB repair pathway choice, the roles of developmental stage, tissue type, and sex have yet to be elucidated in multicellular organisms. To examine the influence of these factors, DSB repair in various embryonic developmental stages, larva, and adult tissues in Drosophila melanogaster was analyzed through molecular analysis of the DR-white assay using Tracking across Indels by DEcomposition (TIDE). The proportion of HR repair was highest in tissues that maintain the canonical (G1/S/G2/M) cell cycle and suppressed in both terminally differentiated and polyploid tissues. To determine the impact of sex on repair pathway choice, repair in different tissues in both males and females was analyzed. When molecularly examining tissues containing mostly somatic cells, males and females demonstrated similar proportions of HR and NHEJ. However, when DSB repair was analyzed in male and female premeiotic germline cells utilizing phenotypic analysis of the DR-white assay, there was a significant decrease in HR in females compared to males. This study describes the impact of development, tissue-specific cycling profile, and, in some cases, sex on DSB repair outcomes, underscoring the complexity of repair in multicellular organisms.

Abstract 5993: Temporal dynamics of ferroptosis vulnerability in a resistant cancer cell state

Abstract Once cancer has metastasized it remains incurable as a result of evolved resistance to nearly all systemic therapies. Our group has demonstrated that an endocycling cancer cell state is an underappreciated mechanism of therapeutic resistance in many cancer settings. Cells in this state uncouple DNA replication from cell division to obtain abnormally high genomic content and cell size (over 40x volume). Resulting polyploidy and cellular hypertrophy provide survival advantages such as elevated genomic content, higher oxidative buffer capacity, increased autophagy, and heightened resistance to chemotherapy. Endocycling cancer cells are induced by multiple different classes of chemotherapy and present in vivo at increased frequencies both in metastatic sites and following chemotherapy. We hypothesize elimination of this overlooked resistant cancer cell state is critical for sustained anticancer therapies. Cancer cells, including those in the endocycling cancer cell state, leverage canonical cell cycle and cell death programs to evade apoptosis from external stressors including all classes of chemotherapy. Engaging cell cycle-independent death pathways is an alternative therapeutic strategy that circumvents cancer’s trademark manipulation of the cell cycle1. Ferroptosis is a cell cycle agnostic form of cell death that occurs due to dysregulation of cellular iron and subsequent lipid peroxidation. While ferroptosis is emerging as a vulnerability in resistant cancers, efficacy of ferroptosis induction is highly heterogeneous depending on tissue type, cellular metabolism, and many other factors. We characterized oxidative damage, labile iron, lipid content, and signaling pathways in cells pre- and post-chemotherapy to assess vulnerability to ferroptosis. By inhibiting GPX4, the sole lipophilic antioxidant enzyme, we show induction of ferroptotic cell death across multiple cell lines and chemotherapies with cells in the endocycling state being more susceptible. Importantly, targeting cells in the endocycling cancer cell state (as opposed to untreated cell lines), also affords a more uniform elimination by ferroptosis. Ferroptosis also has a capacity to be immunogenic that may direct anti-cancer immune licensing in addition to direct cytotoxicity. These studies will directly contribute to development of ferroptosis engaging therapeutic strategies for resistant cancer cell states, particularly concerning the timing of intervention. 1Loftus, L.V.; Amend, S.R.; Pienta, K.J. Interplay between Cell Death and Cell Proliferation Reveals New Strategies for Cancer Therapy. Int. J. Mol. Sci. 2022, 23, 4723. https://doi.org/10.3390/ijms23094723 Citation Format: Luke Loftus, Ken Pienta, Sarah Amend. Temporal dynamics of ferroptosis vulnerability in a resistant cancer cell state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5993.

Rewiring of the phosphoproteome executes two meiotic divisions in budding yeast

The cell cycle is ordered by a controlled network of kinases and phosphatases. To generate gametes via meiosis, two distinct and sequential chromosome segregation events occur without an intervening S phase. How canonical cell cycle controls are modified for meiosis is not well understood. Here, using highly synchronous budding yeast populations, we reveal how the global proteome and phosphoproteome change during the meiotic divisions. While protein abundance changes are limited to key cell cycle regulators, dynamic phosphorylation changes are pervasive. Our data indicate that two waves of cyclin-dependent kinase (Cdc28Cdk1) and Polo (Cdc5Polo) kinase activity drive successive meiotic divisions. These two distinct phases of phosphorylation are ensured by the meiosis-specific Spo13 protein, which rewires the phosphoproteome. Spo13 binds to Cdc5Polo to promote phosphorylation in meiosis I, particularly of substrates containing a variant of the canonical Cdc5Polo motif. Overall, our findings reveal that a master regulator of meiosis directs the activity of a kinase to change the phosphorylation landscape and elicit a developmental cascade.

Comparative genomics incorporating translocation renal cell carcinoma mouse model reveals molecular mechanisms of tumorigenesis.

Translocation renal cell carcinoma (tRCC) most commonly involves an ASPSCR1-TFE3 fusion, but molecular mechanisms remain elusive and animal models are lacking. Here, we show that human ASPSCR1-TFE3 driven by Pax8-Cre (a credentialed clear cell RCC driver) disrupted nephrogenesis and glomerular development, causing neonatal death, while the clear cell RCC failed driver, Sglt2-Cre, induced aggressive tRCC (as well as alveolar soft part sarcoma) with complete penetrance and short latency. However, in both contexts, ASPSCR1-TFE3 led to characteristic morphological cellular changes, loss of epithelial markers, and an epithelial-mesenchymal transition. Electron microscopy of tRCC tumors showed lysosome expansion, and functional studies revealed simultaneous activation of autophagy and mTORC1 pathways. Comparative genomic analyses encompassing an institutional human tRCC cohort (including a hitherto unreported SFPQ-TFEB fusion) and a variety of tumorgraft models (ASPSCR1-TFE3, PRCC-TFE3, SFPQ-TFE3, RBM10-TFE3, and MALAT1-TFEB) disclosed significant convergence in canonical pathways (cell cycle, lysosome, and mTORC1) and less established pathways such as Myc, E2F, and inflammation (IL-6/JAK/STAT3, interferon-γ, TLR signaling, systemic lupus, etc.). Therapeutic trials (adjusted for human drug exposures) showed antitumor activity of cabozantinib. Overall, this study provides insight into MiT/TFE-driven tumorigenesis, including the cell of origin, and characterizes diverse mouse models available for research.

Abstract A021: Pre and post treatment transcriptomic analysis provide new insights on the mechanisms underlying the efficacy of PARPi and Androgen blockage in prostate cancer

Abstract Aim: Recent phase 3 trials (PROPEL, TALAPRO2) have demonstrated survival benefits when combining PARP inhibitors (PARPi) with androgen pathway inhibitors (API) in prostate cancer (PC) treatment. Importantly, this benefit is observed regardless of homologous repair deficiency (HRD) status, suggesting alternative underlying mechanisms beyond synthetic lethality. However, the complete identification of all the PC patients who can benefit from this combination therapy remains an unmet need. Therefore, further investigation into the distinct molecular pathways involved and identification of biomarkers of response is critical and is the aim of this study. Methods: We have performed an exploratory transcriptomic analysis including RNA-seq, differential expression analysis and gene set enrichment analyses (GSEA) of pre and post treatment biopsies of localized PC patients (pts) with higher risk of relapse and that were included in a window of opportunity (WoO) study. We have included pts recruited to the CANCAP-3 study that were exposed to either two weeks of olaparib alone or in combination with degarelix (1:1 randomization). A corresponding transcriptomic analysis of pre and post enzalutamide biopsies of patients recruited to the WoO study DARANA was also included. Results: GSEA results have shown p53 hallmark upregulation more pronounced in the combination cohort. In the combination cohort the androgen response genes were downregulated. Additionally, canonical cell cycle progression hallmarks (E2F and G2M checkpoint) were suppressed with a significant downregulation of the Prolaris signature. In the olaparib monotherapy cohort pts with the greatest PSA decline demonstrated large reductions in the Prolaris score. Furthermore, in the olaparib monotherapy cohort the two patients that relapsed within 4 years of follow up demonstrated greater Prolaris scores. Interestingly, a significant positive correlation between HR deficiency signatures and reduced prostate cancer proliferation index was observed across the study cohorts, although there was only minimal overlap in the genes constituting these two signatures. This correlation was maintained excluding HRD pts (n=2). Finally, this relationship was also observed following analysis of the DARANA trial. Conclusions: Tissue transcriptomic data analysis of PC pts can provide further insights on the possible biological mechanisms that underlie the effect of this drug combination. In this study the expression of HRD signature appeared to be in direct proportion to the reduction in cellular proliferation (Prolaris). This was seen in the combination treatment cohort, as well as in a separate API monotherapy study (DARANA). This suggests that androgen inhibition is the main driver for the reduction in HR signature. However, this phenomenon may represent a consequence of cell cycle arrest occurring more readily in cells with the greatest HR defect. Further studies are needed to further explain the unique upstream processes that contribute to an AR-supported DNA damage response, in the context of PARP inhibition. Citation Format: Ana Filipa Palma dos Reis, Toby Milne-Clark, Amit Dipak Amin, Liliya Nazlamova, Simon Pacey, Harveer Dev. Pre and post treatment transcriptomic analysis provide new insights on the mechanisms underlying the efficacy of PARPi and Androgen blockage in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A021.

Structure-Function Analysis of p57KIP2 in the Human Pancreatic Beta Cell Reveals a Bipartite Nuclear Localization Signal.

Mutations in CDKN1C, encoding p57KIP2, a canonical cell cycle inhibitor, underlie multiple pediatric endocrine syndromes. Despite this central role in disease, little is known about the structure and function of p57KIP2 in the human pancreatic beta cell. Since p57KIP2 is predominantly nuclear in human beta cells, we hypothesized that disease-causing mutations in its nuclear localization sequence (NLS) may correlate with abnormal phenotypes. We prepared RIP1 insulin promoter-driven adenoviruses encoding deletions of multiple disease-associated but unexplored regions of p57KIP2 and performed a comprehensive structure-function analysis of CDKN1C/p57KIP2. Real-time polymerase chain reaction and immunoblot analyses confirmed p57KIP2 overexpression, construct size, and beta cell specificity. By immunocytochemistry, wild-type (WT) p57KIP2 displayed nuclear localization. In contrast, deletion of a putative NLS at amino acids 278-281 failed to access the nucleus. Unexpectedly, we identified a second downstream NLS at amino acids 312-316. Further analysis showed that each individual NLS is required for nuclear localization, but neither alone is sufficient. In summary, p57KIP2 contains a classical bipartite NLS characterized by 2 clusters of positively charged amino acids separated by a proline-rich linker region. Variants in the sequences encoding these 2 NLS sequences account for functional p57KIP2 loss and beta cell expansion seen in human disease.

Targeting transcriptional kinase of CDK7 halts proliferation of multiple myeloma cells by modulating the function of canonical NF-kB pathway and cell cycle regulatory proteins

Multiple myeloma (MM) is an incurable plasma cell neoplasm. Despite several effective frontline therapeutic regimens, including Bortezomib (BTZ), relapse is almost inevitable; therefore, better therapeutic modalities to improve the outcomes are needed. Cyclin-dependent kinases (CDKs) are an essential constituent of the cellular transcriptional machinery and tumors including MM are critically dependent on transcription to maintain their oncogenic state. In the present study, we explored the efficacy of THZ1, a covalent CDK7 inhibitor in MM treatment using Bortezomib resistant (H929BTZR) cells and zebrafish xenografts. THZ1 showed anti-myeloma activity in the models of MM but had no effect on healthy CD34+ cells. THZ1 suppresses phosphorylation of carboxy-terminal domain of RNA polymerase II and downregulates the transcription of BCL2 family of proteins both in H929BTZS and H929BTZR cells leading to G1/S arrest and apoptosis. THZ1 mediates inhibition of bone marrow stromal cells-induced proliferation and activation of NF-kB signaling. The data derived from zebrafish xenografts of MM demonstrate that THZ1 combined with BTZ synergistically reduces tumor growth in zebrafish embryos. Collectively, our results reveal that THZ1 alone as well as in combination with BTZ has effective anti-myeloma activity.

Plasmodium falciparum CRK4 links early mitotic events to the onset of S-phase during schizogony.

Plasmodium falciparum proliferates through schizogony in the clinically relevant blood stage of infection. During schizogony, consecutive rounds of DNA replication and nuclear division give rise to multinucleated stages before cellularization occurs. Although these nuclei reside in a shared cytoplasm, DNA replication and nuclear division occur asynchronously. Here, by mapping the proteomic context of the S-phase-promoting kinase PfCRK4, we show that it has a dual role for nuclear-cycle progression: PfCRK4 orchestrates not only DNA replication, but in parallel also the rearrangement of intranuclear microtubules from hemispindles into early mitotic spindles. Live-cell imaging of a reporter parasite showed that these microtubule rearrangements coincide with the onset of DNA replication. Together, our data render PfCRK4 a key factor for nuclear-cycle progression, linking entry into S-phase with the initiation of mitotic events. In part, such links may compensate for the absence of canonical cell cycle checkpoints in P. falciparum. IMPORTANCE The human malaria parasite Plasmodium falciparum proliferates in erythrocytes through schizogony, forming multinucleated stages before cellularization occurs. In marked contrast to the pattern of proliferation seen in most model organisms, P. falciparum nuclei multiply asynchronously despite residing in a shared cytoplasm. This divergent mode of replication is, thus, a good target for therapeutic interventions. To exploit this potential, we investigated a key regulator of the parasite's unusual cell cycle, the kinase PfCRK4 and found that this kinase regulated not only DNA replication but also in parallel the rearrangement of nuclear microtubules into early mitotic spindles. Since canonical cell cycle checkpoints have not been described in P. falciparum parasites, linking entry into S-phase and the initiation of mitotic events via a kinase, may be an alternative means to exert control, which is typically achieved by checkpoints.

84-OR: Structure–Function Analysis of p57KIP2 in the Human Pancreatic Beta Cell Reveals a Canonical Bipartite Nuclear Localization Sequence (NLS)

p57KIP2 is a canonical cell cycle inhibitor encoded by the CDKN1C gene on chromosome 11. Mutations in CDKN1C underlie multiple pediatric endocrine syndromes including Beckwith-Wiedemann Syndrome (BWS), the Focal Variant of Congenital Hyperinsulinism (FoCHI), Insulinoma, Russel-Silver Syndrome and IMAGe Syndrome. Despite this central role in disease, little is known about the function of p57KIP2 in any cell type, including the human pancreatic beta cell. We hypothesized that p57KIP2 has functions that extend beyond cell cycle control and are unique to the human beta cell. We prepared RIP1 insulin promoter-driven adenoviruses encoding deletions of multiple putatively important and disease-associated but unexplored regions of p57KIP2 (WTp57, p57ΔPAPA, p57ΔCDKI, p57ΔPCNA and p57ΔNLS), and performed the first comprehensive structure-function analysis of CDKN1C/p57KIP2 in the human beta cell. RT-PCR and immunoblot analyses confirmed p57KIP2 overexpression, construct size and beta cell specificity in human islets. By immunocytochemistry, all constructs demonstrated nuclear localization, with the exception of a deletion mutant removing a putative NLS at amino acids 278-281. Unexpectedly, we identified a second NLS at amino acids 312-316. Further analysis showed that each individual NLS is required for nuclear localization of p57KIP2, but neither alone is sufficient to permit nuclear localization of p57KIP2. Finally, constructs containing both the 278-281 and 312-316 sequences were necessary and sufficient to target heterologous proteins to the nucleus of human beta cells. In summary, we discovered that p57KIP2 contains a classic bipartite NLS characterized by two clusters of positively charged amino acids 278KRKRS281 and 312RKRLR316 separated by a proline-rich linker region. Variants in the sequences encoding these two NLS sequences account for functional p57KIP2 loss and beta cell expansion seen in human disease. Disclosure L. Choleva: None. P. Wang: None. H. Liu: None. O. Wood: None. L. Lambertini: None. E. Karakose: None. A.F. Stewart: None.

A novel transcription factor, BmZFP67, regulates endomitosis switch by controlling the expression of cyclin B in silk glands

Endomitosis is involved in developmental processes associated with an increase in metabolic cell activity, which is characterized by repeated rounds of DNA replication without cytokinesis. Endomitosis cells are widespread in protozoa, plants, animals and humans. Endomitosis cell cycle is currently viewed as a variation of the canonical cell cycle and transformed from mitotic cell cycle. However, the meaningful question about how endomitosis transformed from mitosis is still unclear. Herein, we identified a novel transcription factor in silk glands, ZFP67, which is gradually reduced in silk glands during the transition of mitosis to endomitosis. In addition, over-expressed ZFP67 in silk glands led to the transition delayed. And, knock-out of ZFP67 led to abnormal chromatin division and unsuccessful cell division. These data reveled that ZFP67 played an important role in transition of mitosis to endomitosis. Furthermore, ZFP67 can regulate the transcription of cyclin B, a key cyclin related to cell division and G2/M phase, which is demonstrated by chromatin immunoprecipitation and dual luciferase reporter system in this article. In conclusion, it can be speculated that the decreasing expression of ZFP67 in silk glands during the transition stage of mitosis-to-endomitosis resulted in the lack of cyclin B, which further led to unsuccessful cytokinesis and then promoted the transition from mitosis to endomitosis of silk gland cells.

Abstract LB281: HIRA suppression is a mechanism of chemotherapy resistance in ovarian cancer

Abstract Ovarian cancer accounts for the fifth most common cause of cancer death among women, despite being relatively rare. This is in large because majority of cases are diagnosed at advanced stage and frequently recur after primary treatment. Despite numerous efforts to develop effective targeted therapies and immunotherapies for ovarian cancer the standard of care remains chemotherapies. We postulate that part of the problem is the paucity of preclinical research and clinical trials focused on dormant persister ovarian cancer cells that remain after chemotherapy treatments. Emerging evidence supports the idea that persister cancer cells undergo a period of dormancy following chemotherapy which enables their survival and drive late recurrence. Thus, there is dire need to understand the pathways that empower the establishment of dormancy following chemotherapies in ovarian cancer. Epigenetic reprogramming is notoriously known to enable cell fate transitions and the establishment of drug resistance. An important layer of epigenetic regulation frequently overlooked in cancer is the regulation of nucleosome composition via histone variants. Importantly, analysis of data from the human protein atlas revealed that histone H3.3 chaperone, Histone Regulator Protein A (HIRA) expression inversely correlated with poor prognosis, suggesting that HIRA suppression might play a role in the development of chemotherapy resistance. Here, we demonstrate that HIRA is a major regulator of cell fate transitions in ovarian cancer cells in response to chemotherapies. Our data indicates that HIRA levels are suppressed in response chemotherapies in cancer cells. Genetic suppression of HIRA in ovarian cancer cells triggered a non-poliferative state characterized by the induction of canonical dormancy markers (p27, an increase in p38 phosphorylation) and cell cycle arrest. Accordingly, HIRA suppression in these cancer cells renders them less sensitive to the standard of care chemotherapy agent paclitaxel. Together, our data demonstrates that HIRA levels play an important role in controlling cell fate decisions in ovarian cancer and regulate chemotherapy sensitivity. Citation Format: Maya Yasukawa, Stanislav Drapela, Didem Ilter, Mian M. Shahzad, Ana P. Gomes. HIRA suppression is a mechanism of chemotherapy resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB281.

Abstract 6144: Polyploidization enables initial survival of chemotherapy but a progressively increased susceptibility to ferroptosis

Abstract Once cancer has metastasized it remains incurable owing to evolved resistance to nearly all systemic therapies. Classical views of therapeutic resistance include tumor heterogeneity and genetic instability, but our group and others have identified an unappreciated cancer cell phenotype, the polyaneuploid cancer cell (PACC) state, that provides an alternative model. The PACC state is induced upon exposure to a variety of applied stressors, including multiple different classes of chemotherapy, and is characterized by polyploidization of the aneuploid cancer genome. We have shown that this polyploidization is accomplished through endocycling that leads to an enlarged cell size with increased transcriptomic and protein content, higher oxidative buffer capacity, and increased autophagy. We have demonstrated that PACCs are present in a variety of cell lines, arise in response to multiple stressors in vitro, and are resistant to subsequent treatment with several classes of chemotherapy. Thus, we hypothesize that the PACC state is driving therapeutic resistance to current treatment regimens. Elimination of this overlooked phenotype is critical for sustained anticancer therapy response. Cancer cells, including those in the PACC state, control canonical cell cycle and cell death programs to evade apoptosis from external stressors including chemotherapy, highlighting an opportunity to leverage cell cycle-independent death pathways as an alternative therapeutic strategy1. Ferroptosis is a cell cycle agnostic immunogenic form of cell death that is an ideal pathway for elimination of cells in the PACC state. Characterization of ROS levels, glutathione content, labile iron homeostasis, and lipid content indicate that cells in the PACC state have a mounting susceptibility to ferroptosis. Our data suggests that cells in the PACC state keep ferroptosis in check through elevated levels of reduced glutathione and proficient redox activity, including turn over of GPX4, the sole enzyme counteracting lipid peroxidation. Direct inhibition of GPX4 is highly toxic to cells in the PACC state, particularly at later timepoints post therapy removal when the immediate stress response is largely resolved. These studies will directly lead to development of a therapeutic strategy specifically targeting the PACC state with the intention of eliminating cancer resistance. 1Loftus, L.V.; Amend, S.R.; Pienta, K.J. Interplay between Cell Death and Cell Proliferation Reveals New Strategies for Cancer Therapy. Int. J. Mol. Sci. 2022, 23, 4723. https://doi.org/10.3390/ijms23094723 Citation Format: Luke Loftus. Polyploidization enables initial survival of chemotherapy but a progressively increased susceptibility to ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6144.

Targeting Transcriptional Kinase of CDK7 Halts Proliferation of Multiple Myeloma Cells By Modulating the Function of Canonical NF-Kβ Pathway and Cell Cycle Regulatory Proteins

Targeting Transcriptional Kinase of CDK7 Halts Proliferation of Multiple Myeloma Cells By Modulating the Function of Canonical NF-Kβ Pathway and Cell Cycle Regulatory Proteins

Uncoupling cell division and cytokinesis during germline development in metazoans.

The canonical eukaryotic cell cycle ends with cytokinesis, which physically divides the mother cell in two and allows the cycle to resume in the newly individualized daughter cells. However, during germline development in nearly all metazoans, dividing germ cells undergo incomplete cytokinesis and germ cells stay connected by intercellular bridges which allow the exchange of cytoplasm and organelles between cells. The near ubiquity of incomplete cytokinesis in animal germ lines suggests that this is an ancient feature that is fundamental for the development and function of this tissue. While cytokinesis has been studied for several decades, the mechanisms that enable regulated incomplete cytokinesis in germ cells are only beginning to emerge. Here we review the current knowledge on the regulation of germ cell intercellular bridge formation, focusing on findings made using mouse, Drosophila melanogaster and Caenorhabditis elegans as experimental systems.

Cohesin Mutations Induce Chromatin Conformation Perturbation of the H19/IGF2 Imprinted Region and Gene Expression Dysregulation in Cornelia de Lange Syndrome Cell Lines.

Traditionally, Cornelia de Lange Syndrome (CdLS) is considered a cohesinopathy caused by constitutive mutations in cohesin complex genes. Cohesin is a major regulator of chromatin architecture, including the formation of chromatin loops at the imprinted IGF2/H19 domain. We used 3C analysis on lymphoblastoid cells from CdLS patients carrying mutations in NIPBL and SMC1A genes to explore 3D chromatin structure of the IGF2/H19 locus and evaluate the influence of cohesin alterations in chromatin architecture. We also assessed quantitative expression of imprinted loci and WNT pathway genes, together with DMR methylation status of the imprinted genes. A general impairment of chromatin architecture and the emergence of new interactions were found. Moreover, imprinting alterations also involved the expression and methylation levels of imprinted genes, suggesting an association among cohesin genetic defects, chromatin architecture impairment, and imprinting network alteration. The WNT pathway resulted dysregulated: canonical WNT, cell cycle, and WNT signal negative regulation were the most significantly affected subpathways. Among the deregulated pathway nodes, the key node of the frizzled receptors was repressed. Our study provides new evidence that mutations in genes of the cohesin complex have effects on the chromatin architecture and epigenetic stability of genes commonly regulated by high order chromatin structure.

GCN-5/PGC-1α signaling is activated and associated with metabolism in cyclin E1-driven ovarian cancer.

Aim: We previously found Cyclin E1-driven high grade serous ovarian cancer (HGSOC) showed metabolic shift. In this study, we aimed to elucidate signaling pathway therein.Methods: In silico reproduction of TCGA ovarian cancer dataset and pathway analysis were performed. Candidate metabolic pathway was validated using in vitro and in vivo assays.Results: We found CCNE1-amplified HGSOC showed significant metabolic alteration besides canonical cell cycle control. Using CCNE1-amplified OVCAR-3 and A2780 OvCa cells, we found that knockdown of CDK2 and GCN5 resulted in decreased G6PC and increased PGC-1α level, and that both genetic and pharmaceutical (MB-3) inhibition of GCN5 resulted in significant decrease in acetylation of PGC-1α. Silencing of CDK2 also resulted in significant decrease in acetylation of both PGC-1α and Rb. GCN5-KD significantly decreased glucose uptake, increased lactate production and decreased SDH activity. Western blots showed hierarchy of the elements indicating Cyclin E1-CDK2/GCN5/PGC-1α regulatory axis from up- to down- stream. Inhibitory effect of Dinaciclib was similar to that of GCN5 silencing, whereas combination therapy further inhibited cell proliferation significantly. Similar findings were noted also in cell cycle arrest, apoptosis, invasion, migration and colony formation assays. Xenograft experiments showed that GCN5-KD alone did not alter tumor growth yet combination therapy of Dinaciclib and GCN5-KD conferred significant inhibition of tumor growth compared with either therapy alone with no toxicity observed.Conclusion: GCN-5/PGC-1α signaling is activated and associated with metabolism in Cyclin E1-driven HGSOC. Targeting GCN5 hold promise to augment current CDK2-targeting strategy and further studies are warranted for clinical translation.

Primary neurons can enter M-phase

Differentiated neurons can undergo cell cycle re-entry during pathological conditions, but it remains largely accepted that M-phase is prohibited in these cells. Here we show that primary neurons at post-synaptogenesis stages of development can enter M-phase. We induced cell cycle re-entry by overexpressing a truncated Cyclin E isoform fused to Cdk2. Cyclin E/Cdk2 expression elicits canonical cell cycle checkpoints, which arrest cell cycle progression and trigger apoptosis. As in mitotic cells, checkpoint abrogation enables cell cycle progression through S and G2-phases into M-phase. Although most neurons enter M-phase, only a small subset undergo cell division. Alternatively, neurons can exit M-phase without cell division and recover the axon initial segment, a structural determinant of neuronal viability. We conclude that neurons and mitotic cells share S, G2 and M-phase regulation.

Novel Methods to Quantitate and Identity Dividing Platelets in Culture Conditions

Background: Unlike dividing cells, platelets are usually regarded as unproductive in that they are anuclear fragments of megakaryocyte and incapable of the canonical cell cycle. However, recent evidence showed that platelets could form progeny or at least undergo fission. The observation indicated the existence of a non-hematopoietic mechanism to determine platelet count in peripheral blood. The assessing the kinetic aspects of platelet division is not easy because automated cell counters often give equivocal results after culture not supporting those previous findings. Manual chamber counting is impractical because of the difficulty in differentiating singlets from any small sized clumps.Objective: We examined platelet division under in-vitro culture conditions to assess how many platelets and how fast they divide.Methods: We devised a method to track platelet division based on flow cytometry and fluorescent microscopic image. Briefly, buffered human platelet suspension was prepared and stained with cell tracking dyes followed by sorting and resuspension in cell culture medium and incubation. Platelet division was assessed quantitatively by observing a decrease in fluorescent intensity by flow cytometry after culture in a liquid medium or counting the number of single colored platelet doublets after culture in hydrogel medium.Results: After 6-hour culture of CFSE labelled platelet in M199 medium, we observed the appearance of a separate cluster of platelets with reduced CFSE intensity adjacent to the original platelet cluster, as is the typical finding of CFSE dilution assay for lymphocyte proliferation. We continued the culture for 20 hours. The division fraction of strongly labeled platelets was 48.7 % (range: 27.5 - 70.4) after 6 hours and 85.6 % (79.1 - 92.0) after 20 hours. For weakly labelled platelets, the division fraction after 6 hours was 35.0 % (24.6 - 43.4) and 75.5 % (34.5 - 97.0) after 20 hours. The division fractions at the two time points were much higher than expected and the effect of external stimuli such as shearing force exposure during the sorting procedure was to be considered. In another assay, we divided platelet suspension into two and stained with labelling dyes of different colors (CFSE and FarRed). The two fractions were mixed and cultured in hydrogel medium. At the beginning of the culture and after 6 hours, we counted platelet singlets and doublets in ten randomly selected fields of con focal microscopy. Single colored doublets can form both by division and contact. Those with two colors can form only by contact. The count of single- and two-colored doublets can be estimated by binary probability prediction if the platelets don't divide. Initially, the doublet counts follow the binary probability prediction (P: 0.65 - 0.88), but after 6 hours, single colored doublets were observed more frequently than two colored doublets (P<0.01, in the order of 10-6). The division was suppressed by treatment with taxol (P =0.26), nocodazole (P=0.38), and cytochalasin D (P=0.61). From the counted numbers of doublets and singlets of the two colors, we could derive a formula for the platelet division fraction. The division fraction of FarRed stained platelets were 8.3 % (95% CI: 2.2 - 14.4), and that of the CFSE stained platelets were 11.9 % (5.4 - 18.4).Conclusion: We introduce novel methods to assess platelet division credibly in culture conditions which can be easily combined with functional studies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract B040: Differential impact of RB status on E2F1 reprogramming in human cancer

Abstract Recent examination of advanced prostate cancer (PCa) has suggested a major mechanism of progression to castration-resistant disease (CRPC) to be loss of the retinoblastoma (RB) protein. Along with its critical role in controlling cell cycle progression, RB is known to have important tumor-suppressor functions, and has been shown in PCa to be lost exclusively in late-stage disease. Additionally, loss of RB has been shown to correlate with increased E2F1 transcript and protein expression, via E2F-dependent mechanisms. Despite the vital role RB loss has been shown to play in this fatal stage of disease, the molecular underpinnings remain undefined. Thus, in order to elucidate these CRPC specific alterations, the current study utilizes isogenic models of RB loss in combination with genome-wide binding and transcriptional studies. Data presented herein demonstrate that loss of RB is frequent in CRPC, and represents the main mechanism of RB pathways disruption in PCa as detected through analyses of tumor samples and cell-free DNA. However, this phenomenon is not correlated with changes in proliferative indices, suggesting a role for RB loss outside of canonical cell cycle control. Further, RB loss induces significant genome-wide transcriptional alterations, including upregulation in Myc, E2F, and DNA-repair related pathways. Additionally, loss of RB significantly expands E2F1 binding capacity in castrate conditions, while largely maintaining the RB-intact E2F1 cistrome. Strikingly, while the current RB/E2F1 paradigm suggests that E2F1 exclusively occupies promoter regions of DNA in order to regulate transcriptional changes, RB loss induces marked reprogramming of E2F1 occupied regions, with a distinct increase in enhancer-bound E2F1. Further, motif analyses suggest divergence away from canonical E2F1 binding motifs after RB loss, specifically in regions of expanded E2F1 binding, and additionally suggest likely interaction of novel E2F1 cofactors under RB loss conditions. Interestingly, changes in E2F1 binding capacity after RB loss were seen to be distinct from those detected after androgen-induced RB inactivation, suggesting that the molecular alterations underlying RB loss are discrete from those resulting from functional inactivation. With respect to putative mechanism, it is of note that chromatin accessibility was not significantly altered to sufficiently explain the widespread changes in E2F1 cistrome, regardless of RB status, suggesting a mechanism outside simple opportunistic E2F1 binding after RB loss. Finally, interrogation of a CRPC patient tumor cohort showed predictive capacity for an “Expanded E2F1 Signature,” resulting from genes exhibiting gained E2F1 binding and differential expression after RB loss, in predicting loss of RB in patient samples, and indicating a novel E2F1-driven set of targets vital for CRPC transition in human disease. Together, these data present the first insight into E2F1 activity resulting from RB loss, and the role these changes play in progression to CRPC. Citation Format: Christopher McNair, Kexin Xu, Amy C. Mandigo, Matteo Benelli, Benjamin Leiby, Daniel Rodrigues, Johan Lindberg, Henrik Gronberg, Mateus Crespo, Bram De Laere, Luc Dirix, Tapio Visakorpi, Fugen Li, Felix Y. Feng, Johann de Bono, Francesca Demichelis, Mark A. Rubin, Myles Brown, Karen E. Knudsen. Differential impact of RB status on E2F1 reprogramming in human cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B040.