Cell Cycle Regulatory Mechanisms Research Articles

DNA damage response and cell cycle regulation in bacteria: a twist around the paradigm.

The co-protease activity in the RecA-ssDNA complex cleaves the autorepressor LexA, resulting in the derepression of a large number of genes under LexA control. This process is called the SOS response, and genes that are expressed in response to DNA damage are called SOS genes. The proteins encoded by the SOS genes are involved in both DNA repair and maintaining the functions of crucial cell division proteins (e.g., FtsZ) under check until the damaged DNA is presumably repaired. This mechanism of SOS response is the only known mechanism of DNA damage response and cell cycle regulation in bacteria. However, there are bacteria that do not obey this rule of DNA damage response and cell cycle regulation, yet they respond to DNA damage, repair it, and survive. That means such bacteria would have some alternate mechanism(s) of DNA damage response and cell cycle regulation beyond the canonical pathway of the SOS response. In this study, we present the perspectives that bacteria may have other mechanisms of DNA damage response and cell cycle regulation mediated by bacterial eukaryotic type Ser/Thr protein kinases as an alternate to the canonical SOS response and herewith elaborate on them with a well-studied example in the radioresistant bacterium Deinococcus radiodurans.

Abstract 473: PVT1 exon 9 overexpression induces AR null phenotype and cellular reprogramming in prostate cancer

Abstract Plasmacytoma variant translocation 1 (PVT1) is an oncogenic long-noncoding RNA (lncRNA) gene located directly downstream of MYC on chromosome 8q24. PVT1 is an oncogene overexpressed in prostate cancer (PCa) and can promote oncogenesis by altering gene expression directly or acting as a noncoding RNA sponge. Beyond functioning as a lncRNA, PVT1 gene encodes six microRNAs that each have independent functions. Our group identified overexpression of PVT1 exon 9 in PCa samples derived from men of African Ancestry and overexpression may explain why this population group experiences more aggressive disease. We overexpressed PVT1 exon 9 in RWPE1 prostate epithelial cells (RWPE1_ex9) and found enhanced migration and invasion compared to empty vector (RWPE1_ev). Implantation of RWPE1_ex9 cells into mice led to the formation of tumors with the neuroendocrine PCa (NEPC) phenotype that was histopathologically confirmed. RNA-sequencing analysis of PVT1 exon 9 overexpression showed significant (p-value < 0.05) upregulation of 141 genes. The majority of pathways impacted by PVT1 exon 9 overexpression were interferon type 1 signaling (RSAD2, CMPK2), developmental NOTCH signaling (HES7), cargo-transport (STX16) and tumor microenvironment promoting factors (HS3ST3A1). We found loss of androgen receptor (AR) expression at mRNA and protein levels in RWPE1_ex9 cells, a hallmark of NEPC, which further supports our hypothesis that PVT1 exon 9 overexpression plays a role in NEPC development. Validation of RNA-sequencing confirmed upregulation of RSAD2, CMPK2, STX16, HES7 and HS3ST3A1 within RWPE1_ex9 cells compared to RWPE1_ev and RWPE1 wild-type, suggestive of cellular reprogramming of prostate epithelial cells. We further analyzed expression profile in a novel circulating tumor cell model of castration-resistant 22RV1 cells (C22OH) and found enhanced expression of PVT1 exon 9, suppression of AR, phosphorylation of AR serine residue 308 and enhanced CDK11 activity compared to the primary 22RV1 cell line T22OH suggestive of a cell-cycle mediated regulatory mechanism. We further found enhanced expression of chromogramin A in the C22OH cell line, a marker of NEPC. These results suggest PVT1 exon 9 overexpression may play a critical role in NEPC development from normal epithelial cells via cellular reprogramming as well as facilitating a transition to NEPC from a castration resistant phenotype. PVT1 exon 9 may be a useful predictive biomarker that can identify populations which are likely to have disease progression from castration resistance to NEPC and further highlights the need to identify therapeutic strategies to target PVT1 exon 9 overexpression. Citation Format: Rachel E. Sexton-Bonacci, Chinedum Udekwu, Baris Boylu, Olorunseun O. Ogunwobi. PVT1 exon 9 overexpression induces AR null phenotype and cellular reprogramming in prostate cancer [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 473.

Exploration of Potential Cyclin-dependent Kinases and their Inhibitors to Combat Abnormal Signalling and Cancer

Abstract: Regulation of cell proliferation depends on stimulatory and inhibitory factors that act in a coordinated manner in response to external signals. Various agents, including mitogens, growth factors, cytokines, and other external factors, can impact the mitotic cell cycle, resulting in either provoking growth, differentiation, or apoptosis. Many kinases, such as protein kinases, regulate mitotic cell proliferation through normal signalling. One of the major protein kinase family members is cyclin-dependent kinases (CDK), which are responsible for the regulation of cell cycle progression. If the cell cycle-regulatory mechanisms are permanently altered, it can cause abnormal proliferation that leads to neoplasia. This can result in tumour development, where the availability and expression of CDKs become altered, contributing significantly to impaired cell proliferation. Changes like these are often a characteristic of cancer. CDK inhibitors have shown significant clinical benefits in treating various types of tumours in recent years. The output has been achieved by the clinical approvals of particular CDK inhibitors. Researchers have also been studying the proteolysis-targeting chimera (PROTAC) molecule for the last two decades. This molecule uses ubiquitin-mediated proteasome mechanisms to break down specific targets, making it a promising method for targeted protein degradation (TPD). TPD has become a promising therapeutic option for tackling disease-causing proteins that are otherwise challenging to treat using traditional small molecules. This review provides an overview of the state of knowledge and a general understanding of selective or nonselective CDK inhibitors and PROTAC degraders currently under development or clinically approved congeners, focusing on improving cancer therapy.

Discovery of molecular and genetic mechanisms of cell cycle regulation: 2001 Nobel laureates leland Hartwell, Timothy Hunt and Paul Nurse

Leland Hartwell, Paul Nurse and Timothy Hunt in 2001 were awarded the Nobel Prize for their discovery of molecular and genetic mechanisms of the cell cycle. The review analyzes the features of the objects chosen by scientists, a wide range of methods from conventional light microscopy to recombinant DNA technology and complementation test, used for research, and covers a history of the discoveries made. Thanks to the work of these scientists, a modern­ understanding of the cell cycle checkpoints, the complexes formed by cyclin and cyclin-dependent­ kinases at different phases of the cell cycle, as well as the mechanism of periodic cyclin degradation and the universality of the cyclin mechanism of cellular division in all living organisms was gained. Keywords: cdc mutants, cdc2/cdc28, cdk protein kinase, cdk/cyclin complex, cell cycle, cyclin, Saccharomyces cere­visiae, Schizosaccharomyces pombe

Protein Phosphatase 6 Complex: Novel Regulator of Fetal Hemoglobin and Potential Therapeutic Target in Sickle Cell Disease

Although increasing levels of fetal hemoglobin (HbF) in sickle cell disease (SCD) significantly reduces mortality, effective pharmacologic induction of HbF has remained elusive. To identify potentially druggable molecules involved in HbF control, we carried out a domain-focused CRISPR-Cas9-based genetic screen in HUDEP2 cells targeting all 218 serine/threonine protein phosphatases with 6 independent sgRNAs each. A single phosphatase, PPP6C, emerged as a potential HbF repressor. PPP6C is the catalytic subunit of protein phosphatase 6 (PP6), a serine/threonine phosphatase complex that has been implicated in cell cycle regulation, autophagy, and innate immunity mechanisms in other cell types, but its role in erythropoiesis and HbF regulation has not previously been described. CRISPR-Cas9-based depletion of PPP6C in primary human erythroid cells elevated mRNA levels of HBG (the fetal form of β-globin) in a dose-dependent manner up to 3-4 fold, increased HbF levels 3-fold as measured by HPLC (up to 15-20% of total hemoglobin), and doubled the number of HbF-expressing cells. PPP6C depletion caused relatively few changes in the erythroid transcriptome by RNA-seq analysis, and did not measurably impair erythroid maturation. In addition, PPP6C loss in primary SCD patient-derived cells was well-tolerated, led to robust levels of HbF induction, and reduced cell sickling in vitro by up to 60%. Mechanistically, loss of PPP6C reduced the levels of the HbF repressor BCL11A by nearly 50% but left unchanged the levels of other HbF regulators, such as HRI, LRF, EKLF, NFIA/X, ZNF410, or HIC2, suggesting that PPP6C-mediated HbF regulation proceeds at least in part via loss of BCL11A. Importantly, xenotransplantation data (NBSGW) showed ~4-fold induction of HBG at 16 weeks post-transplant, suggesting that PPP6C deficiency leads to effective, sustained HbF induction in vivo. We next set out to determine whether any PP6 subunits have erythroid specificity that could be exploited therapeutically. In contrast to PPP6C, which is highly expressed in all hematopoietic lineages, the PP6 facultative subunit PPP6R1 appears to be enriched in erythroid precursors, suggesting that depletion of PPP6R1 may function as an erythroid-selective target to increase HbF levels. CRISPR-Cas9-based depletion of PPP6R1 in primary human erythroid cells showed a 2-3-fold increase in HBG and near-doubling of HbF-expressing cells, recapitulating the majority of PPP6C-mediated HbF induction. Functional studies are currently in progress to specifically determine whether additional PP6 subunits play a key role in HbF regulation. Furthermore, ongoing work utilizing dTAG-based acute depletion of PPP6C and PPP6R1 paired with phospho-proteomic studies will narrow down direct targets of the PP6 complex and provide insights into erythropoietic and HbF regulatory pathways impacted by PP6. Taken together, our data indicate that PP6, including its catalytic subunit PPP6C and erythroid-enriched regulatory subunit PPP6R1, inhibit HbF production in part via modulating BCL11A levels, and may serve as a red-cell specific therapeutic target in the treatment of SCD.

Arsenite Impairs BRCA1-Dependent DNA Double-Strand Break Repair, a Mechanism Potentially Contributing to Genomic Instability.

BRCA1 is a key player in maintaining genomic integrity with multiple functions in DNA damage response (DDR) mechanisms. Due to its thiol-rich zinc-complexing domain, the protein may also be a potential target for redox-active and/or thiol-reactive (semi)metal compounds. The latter includes trivalent inorganic arsenic, which is indirectly genotoxic via induction of oxidative stress and inhibition of DNA repair pathways. In the present study, we investigated the effect of NaAsO2 on the transcriptional and functional DDR. Particular attention was paid to the potential impairment of BRCA1-mediated DDR mechanisms by arsenite by comparing BRCA1-deficient and -proficient cells. At the transcriptional level, arsenite itself activated several DDR mechanisms, including a pronounced oxidative stress and DNA damage response, mostly independent of BRCA1 status. However, at the functional level, a clear BRCA1 dependency was observed in both cell cycle regulation and cell death mechanisms after arsenite exposure. Furthermore, in the absence of arsenite, the lack of functional BRCA1 impaired the largely error-free homologous recombination (HR), leading to a shift towards the error-prone non-homologous end-joining (NHEJ). Arsenic treatment also induced this shift in BRCA1-proficient cells, indicating BRCA1 inactivation. Although BRCA1 bound to DNA DSBs induced via ionizing radiation, its dissociation was impaired, similarly to the downstream proteins RAD51 and RAD54. A shift from HR to NHEJ by arsenite was further supported by corresponding reporter gene assays. Taken together, arsenite appears to negatively affect HR via functional inactivation of BRCA1, possibly by interacting with its RING finger structure, which may compromise genomic stability.

Various LncRNA Mechanisms in Gene Regulation Involving miRNAs or RNA-Binding Proteins in Non-Small-Cell Lung Cancer: Main Signaling Pathways and Networks.

Long non-coding RNAs (lncRNAs) are crucial players in the pathogenesis of non-small-cell lung cancer (NSCLC). A competing binding of lncRNAs and mRNAs with microRNAs (miRNAs) is one of the most common mechanisms of gene regulation by lncRNAs in NSCLC, which has been extensively researched in the last two decades. However, alternative mechanisms that do not depend on miRNAs have also been reported. Among them, the most intriguing mechanism is mediated by RNA-binding proteins (RBPs) such as IGF2BP1/2/3, YTHDF1, HuR, and FBL, which increase the stability of target mRNAs. IGF2BP2 and YTHDF1 may also be involved in m6A modification of lncRNAs or target mRNAs. Some lncRNAs, such as DLGAP1-AS2, MALAT1, MNX1-AS1, and SNHG12, are involved in several mechanisms depending on the target: lncRNA/miRNA/mRNA interactome and through RBP. The target protein sets selected here were then analyzed using the DAVID database to identify the pathways overrepresented by KEGG, Wikipathways, and the Reactome pathway. Using the STRING website, we assessed interactions between the target proteins and built networks. Our analysis revealed that the JAK-STAT and Hippo signaling pathways, cytokine pathways, the VEGFA-VEGFR2 pathway, mechanisms of cell cycle regulation, and neovascularization are the most relevant to the effect of lncRNA on NSCLC.

HypoxamiR-210-3p regulates mesenchymal stem cells proliferation via P53 & Akt.

Transplanted stem cells (˃95%) into ischemic myocardium die because of unfavourable conditions. Moreover, hypoxia role in the cell cycle regulation has been studied in transformed/immortalized cell lines which may have altered cell cycle regulators and/or mutated and, can't be transplanted in patients. We quest to find out the mechanism of cell cycle regulation in mesenchymal stem cells (MSC) to regulate its survival and proliferation in repair processes. Additionally, critically analysed role of hypoxamiR-210-3p, and cell cycle regulators that can regulate cell proliferation under hypoxic conditions. Bone marrow-derived MSC (BM-MSC) isolated from young male Fischer-344 rats by flushing the cavity of femur and propagated in vitro under 1% hypoxia for 72h showed an increased in cell proliferation ([Formula: see text] 30%, p < 0.05) compared to normoxia. miR-210-3p, role in cell proliferation under hypoxic condition was confirmed by knockdown. Loss of function studies with transfection of anti-mir-210-3p, we observed decrease in proliferation of BM-MSC under hypoxia. Furthermore, BM-MSC proliferation due to miR-210-3p was confirmed using CFSE assay and flow cytometry, in which more cells were observed in S-phase. Mechanistically, western blot analysis showed miR-210-3p inhibition upregulates p53 and p21 expression and subsequent decrease in pAkt under hypoxia. On contrary, CFSE and Western blot under normoxic conditions showed downregulation of p53 and p21 whilst upregulation of pAkt indicated the key role of miR-210-3p in BM-MSC proliferation. Our results demonstrate the role of miR-210-3p in BM-MSC proliferation under both hypoxic and normoxic conditions and illustrate the potential mechanism via the regulation of pAkt, p53 and p21.

HDAC-an important target for improving tumor radiotherapy resistance.

Radiotherapy is an important means of tumor treatment, but radiotherapy resistance has been a difficult problem in the comprehensive treatment of clinical tumors. The mechanisms of radiotherapy resistance include the repair of sublethal damage and potentially lethal damage of tumor cells, cell repopulation, cell cycle redistribution, and reoxygenation. These processes are closely related to the regulation of epigenetic modifications. Histone deacetylases (HDACs), as important regulators of the epigenetic structure of cancer, are widely involved in the formation of tumor radiotherapy resistance by participating in DNA damage repair, cell cycle regulation, cell apoptosis, and other mechanisms. Although the important role of HDACs and their related inhibitors in tumor therapy has been reviewed, the relationship between HDACs and radiotherapy has not been systematically studied. This article systematically expounds for the first time the specific mechanism by which HDACs promote tumor radiotherapy resistance in vivo and in vitro and the clinical application prospects of HDAC inhibitors, aiming to provide a reference for HDAC-related drug development and guide the future research direction of HDAC inhibitors that improve tumor radiotherapy resistance.

The secret life of lactate: A novel cell-cycle regulatory mechanism

Recently, Liu etal. uncovered an unexpected L-lactate-Zn2+ interaction in the active site of the deSUMOylating enzyme SENP1 that triggers a sequence of events that lead to mitotic exit. This study opens thedoor to new research avenues of metabolite-metal interactions controlling cellular decisions and functions.

SNHG18 inhibits bladder cancer cell proliferation by increasing p21 transcription through destabilizing c-Myc protein

BackgroundLong non-coding RNAs (lncRNAs) have been confirmed to play important roles in various cancers including bladder cancer (BC). The precise expression pattern of lncRNA small nucleolar RNA host gene 18 (SNHG18) in BC and its mechanisms of action have not been fully explored.Materials and methodsThe expression of SNHG18 was evaluated by RT-qPCR in bladder cancer clinical samples and human bladder cancer cell lines, and stable cell lines overexpressing SNHG18 were constructed. The effect of SNHG18 on the proliferation of bladder cancer cells was detected by soft agar colony formation test, ATP activity test and subcutaneous tumorigenesis model in nude mice. The specific mechanism of SNHG18 inhibition of bladder cancer proliferation was studied by flow cytometry, western blotting, dual luciferase reporter gene assay and protein degradation assay.ResultsWe found that SNHG18 is significantly downregulated in BC tissues and cell lines. Kaplan–Meier analysis showed that SNHG18 expression is positively correlated with survival in BC patients. Ectopic overexpression of SNHG18 significantly inhibited the proliferation of BC cells in vitro and in vivo. Further mechanistic investigations demonstrated that SNHG18 inhibited c-Myc expression by modulating the ubiquitination-proteasome pathway and that c-Myc is the critical transcription factor that mediates SNHG18 inhibition of BC growth by directly binding to the p21 promoter, which was attributed with significant p21 accumulation.ConclusionsSNHG18 promotes the transcription and expression of p21 by inhibiting c-Myc expression, leading to G0-G1 arrest and inhibiting the proliferation of bladder cancer cells. These findings highlight a novel cell cycle regulatory mechanism involving the SNHG18/c-Myc/p21 pathway in BC pathogenesis and could potentially lead to new lncRNA-based diagnostics and/or therapeutics for BC.

Lactate regulates cell cycle by remodelling the anaphase promoting complex.

Lactate is abundant in rapidly dividing cells owing to the requirement for elevated glucose catabolism to support proliferation1-6. However, it is not known whether accumulated lactate affects the proliferative state. Here we use a systematic approach to determine lactate-dependent regulation of proteins across the human proteome. From these data, we identify a mechanism of cell cycle regulation whereby accumulated lactate remodels the anaphase promoting complex (APC/C). Remodelling of APC/C in this way is caused by direct inhibition of the SUMO protease SENP1 by lactate. We find that accumulated lactate binds and inhibits SENP1 by forming a complex with zinc in the SENP1 active site. SENP1 inhibition by lactate stabilizes SUMOylation of two residues on APC4, which drives UBE2C binding to APC/C. This direct regulation of APC/C by lactate stimulates timed degradation of cell cycle proteins, and efficient mitotic exit in proliferative human cells. This mechanism is initiated upon mitotic entry when lactate abundance reaches its apex. In this way, accumulation of lactate communicates the consequences of a nutrient-replete growth phase to stimulate timed opening of APC/C, cell division and proliferation. Conversely, persistent accumulation of lactate drives aberrant APC/C remodelling and can overcome anti-mitotic pharmacology via mitotic slippage. In sum, we define a biochemical mechanism through which lactate directly regulates protein function to control the cell cycle and proliferation.

DivIVA Phosphorylation Affects Its Dynamics and Cell Cycle in Radioresistant Deinococcus radiodurans.

DivIVA is a member of the Min family of proteins that spatially regulates septum formation at the midcell position and cell pole determination in Bacillus subtilis. Deinococcus radiodurans, a Gram-positive coccus-shaped bacterium, is characterized by its extreme resistance to DNA-damaging agents, including radiation. D. radiodurans cells exposed to gamma radiation undergo cell division arrest by as-yet-uncharacterized mechanisms. divIVA is shown to be an essential cell division gene in this bacterium, and DivIVA of D. radiodurans (drDivIVA) interacts with genome segregation proteins through its N-terminal region. Earlier, RqkA, a gamma radiation-responsive Ser/Thr quinoprotein kinase, was characterized for its role in radioresistance in D. radiodurans. Here, we showed that RqkA phosphorylates drDivIVA at the threonine 19 (T19) residue. The phospho-mimetic mutant with a mutation of T19 to E19 in DivIVA (DivIVAT19E) is found to be functionally different from the phospho-ablative mutant (DivIVAT19A) or the wild-type drDivIVA. A DivIVAT19E-red fluorescent protein (RFP) fusion expressed in the wild-type background showed the arrest in the typical dynamics of drDivIVA and the loss of its interaction with the genome segregation protein ParA2. The allelic replacement of divIVA with divIVAT19E-rfp was not tolerated unless drDivIVA was expressed episomally, while there was no phenotypic change when the wild-type allele was replaced with either divIVAT19A-rfp or divIVA-rfp. These results suggested that the phosphorylation of T19 in drDivIVA by RqkA affected its in vivo functions, which may contribute to the cell cycle arrest in this bacterium. IMPORTANCE Deinococcus radiodurans, a radioresistant bacterium, lacks LexA/RecA-mediated DNA damage response and cell cycle regulation as known in other bacteria. However, it adjusts its transcriptome and proteome upon DNA damage. In eukaryotes, the DNA damage response and cell cycle are regulated by Ser/Thr protein kinases. In D. radiodurans, we characterized a gamma radiation-responsive Ser/Thr quinoprotein kinase (RqkA) that phosphorylated DNA repair and cell division proteins in this bacterium. In previous work, the effect of S/T phosphorylation by RqkA on activity improvement of the DNA repair proteins has been demonstrated. This study reports that Ser phosphorylation by RqkA attenuates the function of a cell polarity and plane of cell division-determining protein, DivIVA, and its cellular dynamics in response to DNA damage, which might help to understand the mechanism of cell cycle regulation in this bacterium.

Impact of local anesthetics on epigenetics in cancer.

Defective silencing of tumor suppressor genes through epigenetic alterations contributes to oncogenesis by perturbing cell cycle regulation, DNA repair or cell death mechanisms. Reversal of such epigenetic changes including DNA hypermethylation provides a promising anticancer strategy. Until now, the nucleoside derivatives 5-azacytidine and decitabine are the sole DNA methyltransferase (DNMT) inhibitors approved by the FDA for the treatment of specific hematological cancers. Nevertheless, due to their nucleoside structure, these inhibitors directly incorporate into DNA, which leads to severe side effects and compromises genomic stability. Much emphasis has been placed on the development of less toxic epigenetic modifiers. Recently, several preclinical studies demonstrated the potent epigenetic effects of local anesthetics, which are routinely used during primary tumor resection to relief surgical pain. These non-nucleoside molecules inhibit DNMT activity, affect the expression of micro-RNAs and repress histone acetylation, thus exerting cytotoxic effects on malignant cells. The in-depth mechanistic comprehension of these epigenetic effects might promote the use of local anesthetics as anticancer drugs.

Autophagy in Cancer Cell Transformation: A Potential Novel Therapeutic Strategy.

Basal autophagy plays a crucial role in maintaining intracellular homeostasis and prevents the cell from escaping the cell cycle regulation mechanisms and being cancerous. Mitophagy and nucleophagy are essential for cell health. Autophagy plays a pivotal role in cancer cell transformation, where upregulated precancerous autophagy induces apoptosis. Impaired autophagy has been shown to upregulate cancer cell transformation. However, tumor cells upregulate autophagy to escape elimination and survive the unfavorable conditions and resistance to chemotherapy. Cancer cells promote autophagy through modulation of autophagy regulation mechanisms and increase expression of the autophagyrelated genes. Whereas, autophagy regulation mechanisms involved microRNAs, transcription factors, and the internalized signaling pathways such as AMPK, mTOR, III PI3K, and ULK-1. Disrupted regulatory mechanisms are various as the cancer cell polymorphism. Targeting a higher level of autophagy regulation is more effective, such as gene expression, transcription factors, or epigenetic modification that are responsible for the up-regulation of autophagy in cancer cells. Currently, the CRISPR-CAS9 technique is available and can be applied to demonstrate the potential effects of autophagy in cancerous cells.

P324: PHARMACOLOGIC INHIBITION OF DYRK1A RENDERS HIGH-RISK KMT2A-R ALL SENSITIVE TO VENETOCLAX

Background:KMT2A-rearranged (R) ALL is a high-risk disease with a frequency of 70% in infants and 10% in children and adults with ALL and is associated with chemoresistance, relapse, and poor survival. Current intensive multiagent chemotherapy regimens induce significant side effects, yet fail to cure many patients, demonstrating continued need for novel therapeutic approaches. Aims: Determine if pharmacologic DYRK1A inhibition may be a novel treatment strategy for patients with KMT2A-R ALL. Methods: To identify novel targets in KMT2A-R leukemia, we performed a domain-specific kinome-wide CRISPR screen and identified multiple kinases required for cell growth. We focused on DYRK1A as it met the following three criteria: 1) Growth inhibition upon kinase targeting was greater in KMT2A-R leukemic cells than in non-KMT2A-R cells, 2) DYRK1A was not found to be common essential gene assessed through the Cancer Dependency Map, 3) Small molecule inhibitors are available. Results: We analyzed multiple ChIP-Seq experiments and identified that KMT2A-fusions directly bind to the DYRK1A promoter. Our RT-PCR and Western blot analyses demonstrate that KMT2A-R ALL cells treated with a menin inhibitor to disrupt the transcriptional activity of the KMT2A-R complex, downregulate DYRK1A, indicating direct regulation of DYRK1A by the KMT2A-fusion. We further observed that pharmacologic inhibition of DYRK1A with EHT1610 induced leukemic cell growth inhibition in vitro and in vivo, demonstrating that DYRK1A could be a new therapeutic target in KMT2A-R ALL cells. To further elucidate the mechanism of DYRK1A function, we treated several KMT2A-R ALL cell lines in vitro with EHT1610, which surprisingly resulted in the upregulation of MYC and hyperphosphorylation of the RAS/MAPK target ERK. Given that ERK hyperactivation stops B cell proliferation during early B cell development to allow them to rearrange their B cell receptor, we hypothesized that cell cycle inhibition upon ERK hyperactivation remains as a conserved mechanism of cell cycle regulation in KMT2A-R ALL. Strikingly, combining DYRK1A inhibition with the MEK inhibitor trametinib antagonistically rescued KMT2A-R ALL cell proliferation, indicating that ERK hyperactivation is the main driver of DYRK1A inhibitor mediated cell cycle arrest. Given that DYRK1A inhibitor does not induce apoptosis and cells restart cell proliferation after EHT1610 withdrawal we concluded that a DYRK1A monotherapy may not be an ideal new treatment option. However, it has been reported that increased MYC activity induces the accumulation of BIM in Burkitt’s Lymphoma. Given the increased expression of MYC following DYRK1A inhibition we performed a new Western blot analysis and validated increased expression of BIM in our KMT2A-R ALL cell lines after EHT1610 treatment. To test if targeting the interaction of BIM with BCL2 will induce an apoptotic effect when combined with EHT1610, we treated four KMT2A-R ALL cell lines with increasing concentrations of EHT1610 and the BCL2 inhibitor venetoclax. Strikingly, the combination of DYRK1A inhibition with BCL2 inhibition synergistically killed KMT2A-R ALL cells and significantly reduced the leukemia burden in vivo. Image:Summary/Conclusion: Our results validate DYRK1A as an important molecule to regulate cell proliferation via inhibition of MYC and ERK. Targeting DYRK1A results in the accumulation of BIM, which renders the cells sensitive to BCL2 inhibition via venetoclax. While further in vivo validation is needed, we predict that combining DYRK1A inhibition with venetoclax may be a novel precision medicine strategy for the treatment of KMT2A-R ALL.

A hierarchical transcriptional network activates specific CDK inhibitors that regulate G2 to control cell size and number in Arabidopsis

How cell size and number are determined during organ development remains a fundamental question in cell biology. Here, we identified a GRAS family transcription factor, called SCARECROW-LIKE28 (SCL28), with a critical role in determining cell size in Arabidopsis. SCL28 is part of a transcriptional regulatory network downstream of the central MYB3Rs that regulate G2 to M phase cell cycle transition. We show that SCL28 forms a dimer with the AP2-type transcription factor, AtSMOS1, which defines the specificity for promoter binding and directly activates transcription of a specific set of SIAMESE-RELATED (SMR) family genes, encoding plant-specific inhibitors of cyclin-dependent kinases and thus inhibiting cell cycle progression at G2 and promoting the onset of endoreplication. Through this dose-dependent regulation of SMR transcription, SCL28 quantitatively sets the balance between cell size and number without dramatically changing final organ size. We propose that this hierarchical transcriptional network constitutes a cell cycle regulatory mechanism that allows to adjust cell size and number to attain robust organ growth.

Modeling the temporal dynamics of master regulators and CtrA proteolysis in Caulobacter crescentus cell cycle.

The cell cycle of Caulobacter crescentus involves the polar morphogenesis and an asymmetric cell division driven by precise interactions and regulations of proteins, which makes Caulobacter an ideal model organism for investigating bacterial cell development and differentiation. The abundance of molecular data accumulated on Caulobacter motivates system biologists to analyze the complex regulatory network of cell cycle via quantitative modeling. In this paper, We propose a comprehensive model to accurately characterize the underlying mechanisms of cell cycle regulation based on the study of: a) chromosome replication and methylation; b) interactive pathways of five master regulatory proteins including DnaA, GcrA, CcrM, CtrA, and SciP, as well as novel consideration of their corresponding mRNAs; c) cell cycle-dependent proteolysis of CtrA through hierarchical protease complexes. The temporal dynamics of our simulation results are able to closely replicate an extensive set of experimental observations and capture the main phenotype of seven mutant strains of Caulobacter crescentus. Collectively, the proposed model can be used to predict phenotypes of other mutant cases, especially for nonviable strains which are hard to cultivate and observe. Moreover, the module of cyclic proteolysis is an efficient tool to study the metabolism of proteins with similar mechanisms.

Pharmacologic Inhibition of DYRK1A Results in Hyperactivation and Hyperphosphorylation of MYC and ERK Rendering KMT2A-R ALL Cells Sensitive to BCL2 Inhibition

Background: KMT2A-rearranged (R) ALL is a high-risk disease with a frequency of 75% in infants and 10% in children and adults with ALL and is associated with chemoresistance, relapse, and poor survival. Current intensive multiagent chemotherapy regimens induce significant side effects, yet fail to cure many patients, demonstrating continued need for novel therapeutic approaches. We performed a kinome-wide CRISPR screen and identified that DYRK1A is specifically required for the survival of KMT2A-R ALL cell. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as negatively regulator of cell proliferation.Results: We performed a kinome-wide CRISPR screen in human ALL cell lines and PDX models and identified DYRK1A as a novel target in KMT2A-R ALL. DYRK1A is a serine-threonine kinase with a proposed, but poorly defined role in cell cycle regulation. We performed a meta-analysis of multiple ChIP-Seq experiments and identified that oncogenic KMT2A fusions directly bind to the DYRK1A promoter. Our RT-PCR and Western blot analyses of KMT2A-R ALL cells treated with a menin inhibitor (MI-503) to disrupt the transcriptional activity of the KMT2A-R complex resulted in the downregulation of DYRK1A, indicating that DYRK1A is directly regulated by the KMT2A fusion complex. We further observed that pharmacologic inhibition of DYRK1A with EHT1610 induced potent leukemic cell growth inhibition in vitro and in vivo, demonstrating that DYRK1A could be a new therapeutic target in KMT2A-R ALL cells. To further elucidate the mechanism of DYRK1A function, we treated several KMT2A-R ALL cell lines in vitro with EHT1610, which surprisingly resulted in the upregulation of MYC and hyperphosphorylation of the RAS/MAPK target ERK. Given that ERK hyperactivation stops B cell proliferation during early B cell development to allow them to rearrange their B cell receptor, we hypothesized that cell cycle inhibition upon ERK hyperactivation remains as a conserved mechanism of cell cycle regulation in KMT2A-R ALL. Strikingly, combining DYRK1A inhibition with the MEK inhibitor trametinib antagonistically rescued KMT2A-R ALL cell proliferation, indicating that ERK hyperactivation is the main driver of DYRK1A inhibitor mediated cell cycle arrest. Given that DYRK1A inhibitor does not induce apoptosis and cells restart cell proliferation after EHT1610 withdrawal we concluded that a DYRK1A monotherapy may not be an ideal new treatment option. However, it has been reported that increased MYC activity induces the accumulation of BIM in Burkitt's Lymphoma. Given the increased expression of MYC following DYRK1A inhibition we performed a new Western blot analysis and validated increased expression of BIM in our KMT2A-R ALL cell lines after EHT1610 treatment. To test if targeting the interaction of BIM with BCL2 will induce an apoptotic effect when combined with EHT1610, we treated four KMT2A-R ALL cell lines with increasing concentrations of EHT1610 and the BCL2 inhibitor venetoclax. Strikingly, the combination of DYRK1A inhibition with BCL2 inhibition synergistically killed KMT2A-R ALL cells.Conclusion: Our results validate DYRK1A as an important molecule to regulate cell proliferation via inhibition of MYC and ERK. Targeting DYRK1A results in the accumulation of BIM, which renders the cells sensitive to BCL2 inhibition via venetoclax. While further in vivo studies are needed, we predict that combining DYRK1A inhibition with venetoclax may be a novel precision medicine strategy for the treatment of KMT2A-R ALL. [Display omitted] DisclosuresCrispino: Forma Therapeutics: Research Funding; Scholar Rock: Research Funding; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy. Tasian: Aleta Biotherapeutics: Consultancy; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Incyte Corporation: Research Funding. Carroll: Incyte Pharmaceuticals: Research Funding; Janssen Pharmaceutical: Consultancy.

TT-10-loaded nanoparticles promote cardiomyocyte proliferation and cardiac repair in a mouse model of myocardial infarction.

The meager regenerative capacity of adult mammalian hearts appears to be driven by the proliferation of endogenous cardiomyocytes; thus, strategies targeting mechanisms of cardiomyocyte cell cycle regulation, such as the Hippo/Yes-associated protein (Hippo/Yap) pathway, could lead to the development of promising therapies for heart disease. The pharmacological product TT-10 increases cardiomyocyte proliferation by upregulating nuclear Yap levels. When intraperitoneal injections of TT-10 were administered to infarcted mouse hearts, the treatment promoted cardiomyocyte proliferation and was associated with declines in infarct size 1 week after administration, but cardiac function worsened at later time points. Here, we investigated whether encapsulating TT-10 into poly-lactic-co-glycolic acid nanoparticles (NPs) before administration could extend the duration of TT-10 delivery and improve the potency of TT-10 for myocardial repair. TT-10 was released from the TT-10–loaded NPs for up to 4 weeks in vitro, and intramyocardial injections of TT-10–delivered NPs stably improved cardiac function from week 1 to week 4 after administration to infarcted mouse hearts. TT-10–delivered NP treatment was also associated with significantly smaller infarcts at week 4, with increases in cardiomyocyte proliferation and nuclear Yap abundance and with declines in cardiomyocyte apoptosis. Thus, NP-mediated delivery appears to enhance both the potency and durability of TT-10 treatment for myocardial repair.