G1 Cell Research Articles

Stable Dual miR-143 and miR-506 Upregulation Inhibits Proliferation and Cell Cycle Progression.

The mainstays of lung cancer pathogenesis are cell cycle progression dysregulation, impaired apoptosis, and unregulated cell proliferation. While individual microRNA (miR) targeting or delivering is a promising approach that has been extensively studied, combination of miR targeting can enhance therapeutic efficacy and overcome limitations present in individual miR regulations. We previously reported on the use of a miR-143 and miR-506 combination via transient transfections against lung cancer. In this study, we evaluated the effect of miR-143 and miR-506 under stable deregulations in A549 lung cancer cells. We used lentiviral transductions to either up- or downregulate the two miRs individually or in combination. The cells were sorted and analyzed for miR deregulation via qPCR. We determined the miR deregulations' effects on the cell cycle, cell proliferation, cancer cell morphology, and cell motility. Compared to the individual miR deregulations, the combined miR upregulation demonstrated a miR-expression-dependent G2 cell cycle arrest and a significant increase in the cell doubling time, whereas the miR-143/506 dual downregulation demonstrated increased cellular motility. Furthermore, the individual miR-143 and miR-506 up- and downregulations exhibited cellular responses lacking an apparent miR-expression-dependent response in the respective analyses. Our work here indicates that, unlike the individual miR upregulations, the combinatorial miR treatment remained advantageous, even under prolonged miR upregulation. Finally, our findings demonstrate potential advantages of miR combinations vs. individual miR treatments.

Toxicity of cannabidiol and its metabolites in TM3 mouse Leydig cells: a comparison with primary human Leydig cells

Cannabidiol (CBD), one of the major components extracted from the plant Cannabis sativa L., has been used as a prescription drug to treat seizures in many countries. CBD-induced male reproductive toxicity has been reported in animal models; however, the underlying mechanisms remain unclear. We previously reported that CBD induced apoptosis in primary human Leydig cells, which constitute the primary steroidogenic cell population in the testicular interstitium. In this study, we investigated the effects of CBD and its metabolites on TM3 mouse Leydig cells. CBD, at concentrations below 30 µM, reduced cell viability, induced G1 cell cycle arrest, and inhibited DNA synthesis. CBD induced apoptosis after exposure to high concentrations (≥ 50 µM) for 24 h or a low concentration (20 µM) for 6 days. 7-Hydroxy-CBD and 7-carboxy-CBD, the main CBD metabolites of CBD, exhibited the similar toxic effects as CBD. In addition, we conducted a time-course mRNA-sequencing analysis in both primary human Leydig cells and TM3 mouse Leydig cells to understand and compare the mechanisms underlying CBD-induced cytotoxicity. mRNA-sequencing analysis of CBD-treated human and mouse Leydig cells over a 5-day time-course indicated similar responses in both cell types. Mitochondria and lysosome dysfunction, oxidative stress, and autophagy were the major enriched pathways in both cell types. Taken together, these findings demonstrate comparable toxic effects and underlying mechanisms in CBD-treated mouse and primary human Leydig cells.

An arresten-derived anti-angiogenic peptide triggers apoptotic cell death in endothelial cells.

In recent years, anti-angiogenic peptides have received considerable attention as candidates for cancer treatment. Arresten is an angiogenesis inhibitor that cleaves from the α1 chain of type IV collagen and stimulates apoptosis in endothelial cells. We have recently indicated that a peptide corresponding to the amino acid 78 to 86 of arresten, so-called Ars, prevented the migration and tube formation of HUVECs and the colon carcinoma growth in mice significantly. The current study aimed to determine whether induction of apoptotic cell death in endothelial cells is one of the biochemical mechanisms of this anti-angiogenic peptide. This hypothesis was assessed using the MTT assay, cell cycle analysis, Annexin V-FITC/PI staining, BCL2, CASP8, CASP9, p53, and CDKN2A gene expression studies as well as evaluating apoptosis in tumor tissues by TUNEL assay. Results demonstrated that 40µM of Ars significantly stimulated 46.2% of early and late apoptosis in HUVECs compared to 13.6% in the untreated cells and did not significantly alter the cell cycle distribution. Moreover, BCL2 and CASP8 were down-regulated, while CASP9 and p53 were up-regulated in endothelial cells. CDKN2A gene expression, the regulator of G1 cell cycle arrest, was not significantly altered. It might be suggested that Ars induced apoptosis in endothelial cells through the mitochondrial pathway and had no effect on the cell cycle. Besides, Ars induced apoptosis significantly in vivo. However, further studies are required to confirm the detailed molecular mechanism of Ars, this peptide has the potential to be optimized for clinical translations.

Combination p53 activation and BCL-xL/BCL-2 inhibition as a therapeutic strategy in high-risk and relapsed acute lymphoblastic leukemia

Due to the rarity of TP53 mutations in acute lymphoblastic leukemia (ALL), p53 re-activation by antagonism of the p53-MDM2 interaction represents a potential therapeutic strategy for the majority of ALL. Here, we demonstrate the potent antileukemic activity of the MDM2 antagonist idasanutlin in high-risk and relapsed ex vivo coculture models of TP53 wildtype ALL (n = 40). Insufficient clinical responses to monotherapy MDM2 inhibitors in other cancers prompted us to explore optimal drugs for combination therapy. Utilizing high-throughput combination screening of 1971 FDA-approved and clinically advanced compounds, we identified BCL-xL/BCL-2 inhibitor navitoclax as the most promising idasanutlin combination partner. The idasanutlin-navitoclax combination was synergistically lethal to prognostically-poor, primary-derived and primary patient blasts in ex vivo coculture, and reduced leukemia burden in two very high-risk ALL xenograft models at drug concentrations safely attained in patients; in fact, the navitoclax plasma concentrations were equivalent to those attained in contemporary “low-dose” navitoclax clinical trials. We demonstrate a preferential engagement of cell death over G1 cell cycle arrest, mechanistically implicating MCL-1-binding pro-apoptotic sensitizer NOXA. The proposed combination of two clinical-stage compounds independently under clinical evaluation for ALL is of high clinical relevance and warrants consideration for the treatment of patients with high-risk and relapsed ALL.

Abstract LB172: A cytosolic complex between exportin-7(XPO7) and ste20-like kinase (SLK) regulates PI3K-AKT-mTOR signaling in cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) is a deadly malignancy with an evolving therapeutic landscape. Although nucleic acid-based evaluations have unveiled some druggable targets, limited options are available for the majority of patients with this disease. We therefore evaluated biliary exosomes by mass spectrometry from 16 patients with CCA, which revealed a 17-fold enhancement of the nuclear export protein XPO7 in CCA-derived biliary exosomes. IHC analysis of XPO7 expression in CCA tumors from 170 patients unexpectedly demonstrated intense cytoplasmic staining in 30% of the samples, which correlated with abbreviated survival. Within the cytosol, we demonstrate that XPO7 exists in a molecular complex with the serine/threonine kinase SLK. shRNA-mediated knockdown of either XPO7 or SLK abrogated tumor organoid formation in vitro, and reduced orthotopic tumor formation and substantially altered tumor morphology using athymic mice. A kinome screen of FDA-approved drugs revealed tivozanib inhibits SLK (IC50= 81.8 nM), which we confirmed using crystallography. Tivozanib treatment reduced tumor organoid formation in vitro and induced tumor regression in vivo using murine xenografts with resulting morphology similar to XPO7 and SLK knockdown tumors. Importantly, site-directed mutagenesis of SLK was used to confirm the observed phenotypic alterations secondary to SLK inhibition. Signal transduction studies with SLK knockdown in CCA cell lines demonstrated downregulation of PI3K-AKT-mTOR signaling with further effects on DNA damage resulting in G2 cell cycle arrest. Moreover, tivozanib demonstrated anti-tumor activity and increased apoptosis in established CCA patient-derived xenografts with cytoplasmic XPO7 expression. Using ex vivo tumor slice cultures, tivozanib significantly increased tumor cell apoptosis in XPO7/SLK-expressing tumors (N=3) but not in tumors without this expression pattern (N=6). Further evaluation of these tivozanib-treated tumor slice cultures demonstrated a reduction in the expression of phosphorylated mTOR (S2448), AKT (T308), S6 (S235/236) in the high XPO7/SLK-expressing tumors but not in tumors without this expression pattern. Lastly, tivozanib monotherapy demonstrated in vivo efficacy with arrest of tumor progression (RECIST stable disease) in an ongoing clinical trial (NCT04645160). Citation Format: Priyanka P. Desai, Tahsin M. Khan, Reed I. Ayabe, Emily Smith, Surajit Sinha, Ashley Rainey, Yuri Lin, Tracey Pu, Kenneth Lubrice, Leila Sarvestani, Sarfaraz Akmal, Shahyan Rehman, Kirsten Remmert, Michael Yaffe, Craig Thomas, John Brognard, Jonathan M. Hernandez. A cytosolic complex between exportin-7(XPO7) and ste20-like kinase (SLK) regulates PI3K-AKT-mTOR signaling in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB172.

Dbf4-dependent kinase promotes cell cycle controlled resection of DNA double-strand breaks and repair by homologous recombination

DNA double-strand breaks (DSBs) can be repaired by several pathways. In eukaryotes, DSB repair pathway choice occurs at the level of DNA end resection and is controlled by the cell cycle. Upon cell cycle-dependent activation, cyclin-dependent kinases (CDKs) phosphorylate resection proteins and thereby stimulate end resection and repair by homologous recombination (HR). However, inability of CDK phospho-mimetic mutants to bypass this cell cycle regulation, suggests that additional cell cycle regulators may be important. Here, we identify Dbf4-dependent kinase (DDK) as a second major cell cycle regulator of DNA end resection. Using inducible genetic and chemical inhibition of DDK in budding yeast and human cells, we show that end resection and HR require activation by DDK. Mechanistically, DDK phosphorylates at least two resection nucleases in budding yeast: the Mre11 activator Sae2, which promotes resection initiation, as well as the Dna2 nuclease, which promotes resection elongation. Notably, synthetic activation of DDK allows limited resection and HR in G1 cells, suggesting that DDK is a key component of DSB repair pathway selection.

Hepatoprotective effect of jojoba oil against lead-induced toxicity in rats

Background Previous investigations showed that jojoba oil exhibited a protective effect against hepatotoxicity caused by different toxicants, however, to the best of our knowledge, no prior research has been done to determine the effectiveness of jojoba oil in protecting against lead toxicity. Objective This study assessed the hepatoprotective properties of jojoba oil against lead toxicity in rats. Materials and methods The study included four groups, each consisting of six Sprague Dawley male rats, and orally administered jojoba oil (JO group), lead acetate (LA group), and lead acetate plus jojoba oil (protective group) Results and conclusion The results showed that lead acetate-induced hepatotoxic effects were revealed by increased serum alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activities with hepatic histomorphological changes. There was a significant increase in serum total cholesterol, triglycerides, and low-density lipoprotein-cholesterol, while levels of high-density lipoprotein-cholesterol significantly declined compared to normal rats. Additionally, lead acetate triggered oxidative damage of hepatocytes, evidenced by a significant increase of malondialdehyde levels and a decrease of reduced glutathione levels and activities of superoxide dismutase and glutathione S-transferase. Administration of lead was associated with a change in the distribution of cells over different cell cycle phases, characterized by a marked increase in the sub G1 cell population and a significant decrease in the G0/G1 cell population. Supplementation of jojoba oil with lead acetate relieved the toxic impacts of lead acetate with an enhancement of the liver enzyme activities, antioxidant status, lipid profile parameters, and histopathological alterations. In conclusion, jojoba oil might be an effective natural product that offers a promising preventive action towards lead-induced liver damage in rats.

Cynaroside Induces G1 Cell Cycle Arrest by Downregulating Cell Division Cycle 25A in Colorectal Cancer.

Natural chemicals derived from herbal plants have recently been recognized as potentially useful treatment alternatives owing to their ability to target a wide range of important biological molecules. Cynaroside is one of these natural compounds with promising anticancer activity for numerous tumor types. Nevertheless, the anticancer effects and molecular mechanisms of action of cynaroside on colorectal cancer (CRC) remain unclear. In this study, cynaroside was found to markedly inhibit CRC cell proliferation and colony formation in vitro. Cynaroside also inhibited cell proliferation in vivo and decreased the expression of KI67, a cell nuclear antigen. RNA sequencing revealed 144 differentially expressed genes (DEGs) in HCT116 cells and 493 DEGs in RKO cells that were enriched in the cell cycle signaling pathway. Cell division cycle 25A (CDC25A), a DEG widely enriched in the cell cycle signaling pathway, is considered a key target of cynaroside in CRC cells. Cynaroside also inhibited DNA replication and arrested cells in the G1/S phase in vitro. The expression levels of CDC25A and related G1-phase proteins were significantly elevated after CDC25A overexpression in CRC cells, which partially reversed the inhibitory effect of cynaroside on CRC cell proliferation and G1/S-phase arrest. In summary, cynaroside may be used to treat CRC as it inhibits CDC25A expression.

Design, synthesis, and activity evaluation of 2-iminobenzimidazoles as c-Myc inhibitors for treating multiple myeloma

Multiple myeloma (MM) is a plasma cell malignancy that remains incurable and poses a significant threat to global public health. The multifunctional transcription factor c-Myc plays a crucial role in various cellular processes and is closely associated with MM progression. As part of the basic-helix-loop-helix-leucine zipper (bHLHZip) family, c-Myc forms heterodimers with its obligate partner Max, binds to the Enhancer-box (E-box) of DNA, and ultimately co-regulates gene expression. Therefore, impeding the capacity for heterodimerization to bind to DNA represents a favored strategy in thwarting c-Myc transcription. In this study, we first synthesized a series of novel 2-iminobenzimidazole derivatives and further estimated their potential anti-MM activity. Notably, among all the derivatives, 5b and 5d demonstrated remarkable inhibitory activity against RPMI-8226 and U266 cells, with IC50 values of 0.85 μM and 0.97 μM for compound 5b, and 0.96 μM and 0.89 μM for compound 5d. Western blot and dual-luciferase reporter assays demonstrated that compounds 5b and 5d effectively suppressed both c-Myc protein expression and transcriptional activity of the c-Myc promoter in RPMI-8226 and U266 cells. Furthermore, these compounds induced apoptosis and G1 cell cycle arrest in the aforementioned MM cells. Molecular docking studies revealed that 5b and 5d exhibited strong binding affinity to the interface between c-Myc/Max and E-box of DNA. Taken together, our findings suggest that further investigations are warranted for potential therapeutic applications of 5b and 5d for c-Myc-related diseases.

Notch2 Regulates the Function of Bovine Follicular Granulosa Cells via the Wnt2/β-Catenin Signaling Pathway.

Ovarian follicular GCs are strongly implicated in the growth, development, and atresia of ovarian follicles. The Wnt/β-catenin and Notch signaling pathways participate in GC proliferation, differentiation, apoptosis, and steroid hormone production during follicular development. However, the crosstalk between Wnt and Notch signaling in GCs remains unclear. This study investigated this crosstalk and the roles of these pathways in apoptosis, cell cycle progression, cell proliferation, and steroid hormone secretion in bovine follicular GCs. The interaction between β-catenin and Notch2 in GCs was assessed by overexpressing CTNNB1, which encodes β-catenin. The results showed that inhibiting the Notch pathway by Notch2 silencing in GCs arrested the cell cycle, promoted apoptosis, reduced progesterone (P4) production, and inhibited the Wnt2-mediated Wnt/β-catenin pathway in GCs. IWR-1 inhibited Wnt2/β-catenin and Notch signaling, reduced GC proliferation, stimulated apoptosis, induced G1 cell cycle arrest, and reduced P4 production. CTNNB1 overexpression had the opposite effect and increased 17β-estradiol (E2) production and Notch2 protein expression. Co-immunoprecipitation assays revealed that Notch2 interacted with β-catenin. These results elucidate the crosstalk between the Wnt/β-catenin and Notch pathways and the role of these pathways in bovine follicular GC development.

Abstract 4722: Enhanced phenotypic screening: A kinetic, multiplexed approach to analyzing drug effects using Incucyte® Live-Cell Analysis System

Abstract Phenotypic screening is a valuable tool for analysis of the complex mix of effects that drug treatment has on cells. Traditionally image-based screening uses fixed cells stained with reagents at a pre-chosen end point, however our technique combines kinetic, label-free readouts with non-perturbing fluorescence reporters to enable information-rich quantification of compound effects. A549 lung cancer cells were treated with a library of over 800 FDA-approved drugs while images were acquired in the Incucyte® Live-Cell Analysis System every two hours for four days. Cell growth and viability were measured using label-free readouts of confluence and dead cell count while cell cycle, cell death and Akt pathways were investigated using multiplexed fluorescent readouts. Cytotoxicity was measured using label-free Incucyte® AI Cell Health Analysis Software Module which uses two neural networks to segment individual cells and classify them as live or dead. Assay robustness was quantified using the time course of positive and negative controls (camptothecin and vehicle, respectively). Z’ reached a maximal value >0.9 between 48h and 72h; at 72h 4.4% of the compounds within the library induced >50% cell death. Correlation between % Live cells and % Confluence indicated 3 types of effect: cytotoxic (low viability, low confluence), cytostatic (high viability, low confluence), and none (high viability, high confluence). Morphological changes were observed in a multitude of wells with 29 compounds inducing cell enlargement >1800 µm2, suggesting induction of senescence. Hits from fluorescent readouts were identified as the compounds which perturbed the quantified readout more than 3 standard deviations away from the mean value of the vehicle. Mechanisms of cell death were examined by correlative analysis of the total dead cell population (label-free cell death identification), caspase-active (caspase 3/7-reagent positive) and Annexin V positive cells. This enabled rapid identification of compounds which induced apoptosis by caspase-independent pathways, including the serotonin-selective reuptake inhibitor Sertraline. Kinetic plots of cell cycle stage (% S|G2|M or G1) revealed contrasting mechanisms. Mycophenolic acid induced cell cycle arrest, observed as a consistent increase in the percentage of cells in G1 over time. However flumazenil displayed repeated, transient peaks in the percentage of cells in S|G2|M over the same 72h period, indicating synchronisation behavior. Incucyte® Live-Cell Analysis System as a phenotypic screening platform provides valuable kinetic data from cells within the physiologically relevant environment of a standard incubator. Label-free readouts yield direct cellular information and can be combined with fluorescent data to provide vastly more information than a single endpoint readout. Citation Format: Kirsty McBain, Gillian Lovell, Jasmine Trigg, Nicola Bevan, Daniel Appledorn. Enhanced phenotypic screening: A kinetic, multiplexed approach to analyzing drug effects using Incucyte® Live-Cell Analysis System [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 4722.

Abstract 3229: IACS-16559, a CBP/P300 bromodomain inhibitor for the treatment of specific AML subsets

Abstract Block in differentiation and accumulation of undifferentiated blasts are hallmarks of Acute Myeloid Leukemia (AML) and epigenetic processes have been shown to play a critical role in hematological malignancies. Paralogs CREBBP and EP300, in association with co-factors, are hijacked during leukemogenesis by aberrant transcriptional factors, thus driving blast proliferation and maintaining the undifferentiated phenotype. Here, we introduce IACS-16559, a bromodomain inhibitor targeting CREBBP/EP300. IACS-16559 is highly selective for the bromodomains of CBP and EP300 versus BRD4, showing a dose-dependent inhibition of H3K27 acetylation residue in vivo and in vitro. In addition, IACS-16559 pharmacokinetic properties show low clearances, high oral exposures and predicted QD dosing in human. We screened AML cell lines in vitro for their response to IACS-16559 and identified specific subtypes of AML that exhibit heightened sensitivity to CBP/EP300 inhibition, specifically AML with AML1-ETO and MLL-AF9 rearrangements, and AML featuring mutations in the nucleophosmin (NPM1c) gene. IACS-16559 inhibited proliferation of AML responder cell lines in vitro, halted cell cycle progression, with an accumulation of cells in G1 and a decrease of cells in S phase. IACS-16559 did not induce apoptosis across a panel of AML cell lines, indicating that induction of apoptosis is not a major mechanism of action of CBP/EP300 inhibition. To augment the anti-tumor effects of IACS-16559, we prioritized the pharmacological combination of CBP/EP300 and MLL-Menin inhibitors. This combination synergistically impeded cell proliferation and potently triggered apoptosis in vitro and in vivo, whereas none of the individual agents alone had this effect. The changes in gene and surface marker expression produced by these treatments, as single agents and in combination with synergistic effects, were in line with an induced myeloid differentiation phenotype. Collectively, our findings provide a compelling basis for further exploring the use of a CBP/EP300 bromodomain inhibitor in conjunction with an inhibitor of the MLL-Menin interaction in a subset of AML patients with MLL rearrangements or NPM1 mutations. Citation Format: Ciro Zanca, Michael Soth, Guang Gao, Jason Gay, Ningping Feng, Christopher Bristow, Kang Le, Quanyun Xu, Phuong Nguyen, Yongying Jiang, Teresa Perry, Faika Mseeh, Paul Leonard, Jason Cross, Virginia Giuliani, Joe Marszalek, Philip Jones, Tim Heffernan. IACS-16559, a CBP/P300 bromodomain inhibitor for the treatment of specific AML subsets [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 3229.

Abstract 5701: AU2-94: A CDK4-specific inhibitor with the marked anti-tumor efficacy

Abstract Cyclin-dependent kinases (CDKs) are a vital family of proteins in the cell development. Several CDK4/6 inhibitor drugs significantly extended the lives of subtypes breast cancer patients. However, they have some shortcomings. Many patients do not respond to them well mainly due to off-target toxicity requiring dose reductions and treatment interruptions, and thus building resistance, which limit their effectiveness. Here, we describe AU2-94, a highly selective CDK4 inhibitor, for the treatment of CDK4-dependent tumours including breast cancer (ER+ and TNBC), colorectal cancer and glioblastoma (GBM). AU2-94 exhibits high selectivity for CDK4 (Ki = 2 nM) over CDK6 (Ki = 279 nM). Consistent with the CDK4-targeted mechanism, AU2-94 arrested the G1 cells and inhibited cancer cell proliferation. Interestingly, AU2-94 reduced cellular CDK2, Cyclin E2, Cyclin A2, Cyclin B1, and induced apoptosis via reduction of Bcl-2, FOXM1 etc. In contrast to cancer cells, AU2-94 has little impact on the cell cycle, senescence, or apoptosis of human bone marrow cells, whereas the known CDK4/6 inhibitors increased the G1 cells. AU2-94 showed little or no effect on lymphocyte and neutrophil counts whilst the clinical CDK4/6 inhibitors reduced them. This lower effect of AU2-94 on the bone marrow, neutrophils and lymphocytes might be due to its lesser effect on CDK6 compared to the clinical CDK4/6 inhibitors. AU2-94 was highly efficacious against multiple in vivo tumour models. In the ER+ breast cancer T47D xenografts, AU2-94 caused tumour regression. In the model of MDA-MB-453 AR+ luminal breast cancer (CDK4, KRASmut, PI3KCAmut), AU2-94 demonstrated remarkable efficacy, resulting in the 6 out of 7 (86%) mice tumour-free. In the colorectal cancer HCT116 (CDK4/6, KRASmut) xenografts, AU2-94 inhibited tumour growth markedly in a dose-dependent manner from 25 to 200 mg/kg without any overt toxicity. The pharmacokinetic and pharmacodynamic study revealed up to 500-fold higher concentration of AU2-94 in the tumours than in the plasma, suggesting its tumour targeting specificity. Several robust biomarkers/PD markers were identified. The Rb-CDK4-Cyclin D pathway is altered in the majority of GBM due to homozygous deletion of CDKN2A/B. AU2-94 has an excellent therapeutic potential as it crossed the blood-brain-barrier effectively. In the both U87 subcutaneous and orthotopic models, AU2-94 reduced the tumour burden and increased the survival of GBM bearing mice significantly compared to vehicle. Moreover, AU2-94 synergised the anti-cancer efficacy of PI3K/Akt/mTOR and HER2 inhibitors in combination. In summary, AU2-94 is a CDK4 inhibitor with high selectivity against a panel of 360 human kinases. This selectivity can be a reason for the better safety of AU2-94 compared to the clinical CDK4/6 inhibitors. The high oral bioavailability, robust antitumor efficacy and excellent safety profiles make AU2-94 a highly attractive candidate for clinical development. Citation Format: Laychiluh Bantie, Jimma Likisa, Muhammed Rahaman, Theodosia Teo, Ava Safaroghliazar, Sunita KC Basnet, Ramin Hassankhani, Benjamin Noll, Robert Milne, Shudong Wang. AU2-94: A CDK4-specific inhibitor with the marked anti-tumor efficacy [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 5701.

Abstract 518: The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation

Abstract Richter transformation (RT), characterized by the transformation of chronic lymphocytic leukemia (CLL) to an aggressive large-cell lymphoma, remains a challenge for the therapy. Genetic aberrations, such as Myc activation, TP53 abnormality, NOTCH1 gene mutation or loss of CDKN2A are associated with uncontrolled RT cell proliferation and escaping of apoptosis. Among those changes, gain of Myc activity, found in around 70% of RT, plays a nexus role in the pathogenesis of RT. As a transcription factor, Myc activation has a profound effect on cell survival, proliferation, adhesion and metabolism. Because of its essential role in lymphoma transformation and aggressiveness, Myc is a vital target for therapy toward RT. Fadraciclib (CYC065) is a second-generation CDK inhibitor with selective potency toward CDK2 and CDK9. We have shown previously that by inhibiting CDK9-mediated transcription, fadraciclib reduced expression of the short-lived anti-apoptotic protein Mcl-1 to initiate apoptosis in primary CLL cells. Like Mcl-1, Myc is a quintessential example of an oncogene with rapid turnover in both its mRNA and protein, therefore a promising target of CDK9 inhibition. Thus, we proposed that fadraciclib would target both the survival and proliferation pathways of RT pathogenesis through collaborative inhibition of Mcl-1, Myc and CDK2. First, our data showed that fadraciclib effectively induced apoptosis in the RT cell line HPRT1, as well as the lymphoma cell lines with Myc amplification, including Raji, Ramos and SU-DHL-2. The IC50 for apoptosis was 0.3 µM at 24h in the HPRT1 cells, which is less than the average IC50 for the primary CLL cells (0.8 µM). The IC50 for the lymphoma lines ranged from 0.8 µM to 1.3 µM. Immunoblots showed reduction of RNA Pol2 phosphorylation by fadraciclib, consistent with the inhibition of CDK9. As a result of loss of RNA Pol2 activity and pausing of transcription, there was a clear reduction of both Mcl-1 and Myc mRNA, measured by real-time RT-PCR, that was associated with the reduction of their protein levels. CDK2 was also inhibited by fadraciclib, shown by the reduction of the phosphorylation of both RB and NPM. Second, Fadraciclib inhibited cell proliferation of the HPRT1 cells at an IC50 of 0.25 µM measured by the CellTiter-Glo assay. The IC50 for growth inhibition in the lymphoma lines ranged from 0.5 to 0.7 µM. CFSE tracing demonstrated halting of cell division upon fadraciclib incubation. Cell cycle analysis by Click-iT EdU showed reduction of S-phase population and arresting of cells in both G1 and G2. We are currently working on dissecting the contributions of inhibiting Myc, Mcl-1 and CDK2 individually and together, in apoptosis induction and proliferation blockade. Taken together, these data suggest that fadraciclib is active in RT cells and Myc-dependent lymphoma cell lines and support expansion of its clinical testing in patients with RT. Citation Format: Rong Chen, Yuling Chen, Ping Xiong, William Plunkett. The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation [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 518.

Abstract 5965: First-in-class chemical induced proximity system achieves dose-dependent control of TP53 activation in gastric cancer

Abstract Background: Gastric cancer (GC) remains a common and deadly disease, and inactivating TP53 mutations occur in up to 68% of GC tumors. p53 is a master regulator of multiple cellular pathways, such as the cell cycle, apoptosis, and senescence. Previously, we demonstrated the ability to activate endogenous target gene expression using a novel chemical induced proximity system comprised of deactivated Cas9 (dCas9) and “chemical epigenetic modifiers” or “CEMs” (PMID: 31712774). In this study, we applied our dCas9-CEM system for the first time in oncology to activate TP53 gene expression and selectively kill targeted cells in a preclinical model of GC. Methods: AGS cells were derived from a gastric adenocarcinoma, and harbor wild-type TP53. They were transduced with our dCas9 system and either sgRNAs targeting TP53 or a non-targeting (NT) sgRNA. After 48 h CEM87 incubation (1-25 nM), expression of TP53 and downstream targets (CDKN1A and BBC3) were evaluated by RT-qPCR. After 48 h CEM87 incubation (0.1-25 nM), colony-forming potential was assessed by crystal violet staining after 10 days. After 24 h incubation (5-25 nM), cells were fixed and stained with propidium iodide (PI), and DNA content was assessed by flow cytometry to determine cell cycle fractions. After 48 h incubation (50-200 nM), cells were fixed and stained with Annexin V and PI, and apoptosis was assessed by flow cytometry. Last, cells were treated with nine ascending concentrations (10 pM-100 μM) for 48 h, and cell viability was measured using CellTiter-Glo™. IC50 values were calculated using four-parameter non-linear regression. Results: AGS-TP53 cells show significantly increased expression of TP53 and downstream target genes CDKN1A and BBC3 versus AGS-NT cells after 10 nM CEM87 (105%, 99%, and 43% increase, P=0.004, 0.002, and 0.03, respectively; n=3). Colony formation was significantly reduced in AGS-TP53 versus AGS-NT cells after 10 nM CEM87 (60.2% reduction, P=0.003; n=3). Treatment with 10 nM CEM87 also led to a G2/M stall after significantly reducing cells in both G1 (47.4% vs 38.9%, P<0.0001; n=3) and S-phase (25.1% vs 20.3%%, P<0.0001; n=3) in AGS-TP53 versus AGS-NT cells. Treatment with 200 nM CEM87 significantly increased early apoptosis in AGS-TP53 versus AGS-NT cells (28.7% vs 6.6%, respectively, P<0.0001; n=3). Last, CEM87 was 50 times more potent in AGS-TP53 cells than AGS-NT cells (IC50: 3 vs 146 nM; n=8). Conclusions: These data highlight how our innovative system upregulates TP53 expression and impacts downstream phenotypic effects of TP53 activation in a preclinical model of GC. Citation Format: Ryan M. Kemper, Travis J. Nelson, Nathaniel A. Hathaway, Daniel J. Crona. First-in-class chemical induced proximity system achieves dose-dependent control of TP53 activation in gastric 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 5965.

Abstract 5705: Discovery of a selective slow-off CDK2 inhibitor NKT3447 with distinct features of suppressing pCDK2, downregulating cyclin E, and achieving prolonged pathway inhibition

Abstract CDK2 is a cell cycle kinase that plays a critical role in controlling the G1 to S-phase transition and its abnormal activation through cyclin E1(CCNE1) amplification has been indicated as the primary oncogenic driver in advanced cancers including ovarian, endometrial, and gastric cancers. In addition, cyclin E1 overexpression has emerged as a key resistance mechanism to CDK4/6 inhibitors in hormone receptor-positive breast cancer patients. Therefore, CDK2 represents a promising novel oncology target. However, development of a potent and selective CDK2 inhibitor has been challenging due to the high homology of CDK2 with other CDKs, particularly CDK1, which is essential in normal cell cycle progression. NKT3447 is a novel, orally bioavailable small molecule CDK2 inhibitor with slow-off kinetics and is highly selective against CDK1 and other CDKs. In addition, NKT3447 exhibits a distinct mechanism of action, including suppression of the Thr160 activating phosphorylation on CDK2 and downregulation of cyclin E1. In biochemical assays, NKT3447 drives a concentration dependent disruption of the CDK2/cyclin E1 and CDK2/cyclin A2 complexes, but not the CDK1/cyclin B1 complex. Consequently, NKT3447 potently inhibits the phosphorylation of Rb (pRb) and induces G1 cell cycle arrest in CCNE1 amplified OVCAR3 cells, while showing minimal or no effect on G2/M phase distribution in all the cell lines tested, including OVCAR3 and KYSE520 (CDK1-dependent) cells. Importantly, NKT3447 has a slow-off kinetics, as demonstrated by NanoBRET target engagement assay, which leads to sustained pRb inhibition after treatment withdrawal. In addition, NKT3447 treatment reduces cyclin E1 protein levels and suppresses phospho-CDK2 (Thr160), which further represses CDK2 activity and prolongs the inhibition of CDK2 signaling. NKT3447 also demonstrates a selective anti-proliferation effect in a panel of human cancer cell lines with elevated cyclin E1 expression and/or dependency on CDK2 or cyclin E1 (DepMap). In line with cellular studies, pharmacodynamic study of NKT3447 in mouse tumor models recapitulates a significant repression of pRb, pCDK2 and cyclin E1. Furthermore, NKT3447 treatment results in a dose-dependent tumor growth inhibition or tumor regression in CCNE1 amplified OVCAR3 and MKN1 tumor models. In addition, the combination of NKT3447 with carboplatin exhibits enhanced anticancer effects in OVCAR3 and MFE-280 models. These results demonstrate that NKT3447 is a potent, selective CDK2 inhibitor with distinct properties and mechanism of action, which holds great potential to effectively treat patients with aberrant CDK2/Cyclin E activation in a variety of cancer types. NKT3447 is currently being investigated in a Phase 1 clinical trial in patients with advanced solid tumors. Citation Format: Jianlin Geng, Ke Liu, Zhiyong Yu, Wenfeng Sun, Hairong Wei, Wenjun Li, Jing Lu, Juan Deng, Liqing Geng, Zhihong Liu, Zhenhai Gao, Yan Lou. Discovery of a selective slow-off CDK2 inhibitor NKT3447 with distinct features of suppressing pCDK2, downregulating cyclin E, and achieving prolonged pathway inhibition [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 5705.

Abstract 5861: CDK4/6 therapy-induced senescence as a resistance mechanism-MiDAS touch

Abstract Background: Cyclin-dependent kinase inhibitors (CDK4/6i) have revolutionized the therapy for estrogen receptor positive (ER+) breast cancer. Despite the response to CDK4/6i, resistance and recurrence are still a clinical reality, resulting in death due to breast cancer. G1 cell cycle arrest has been considered as the main mechanism of action of CDK4/6i in luminal breast cancer. However, the response to therapy does not correlate with the expression of CDK4/6 or key cell cycle proteins. It is also unclear whether CDK4/6i-induced senescence enhances the therapeutic response or mediates development of resistance. Experimental Procedures: We have developed drug-tolerant persisters resistant to CDK4/6 inhibitors (CDK4/6i-DTPs) in a panel of estrogen receptor positive (ER+) cells to explore the molecular mechanisms associated with resistance. Using different cell phenotype assays (i.e., cell viability/cell cycle, senescence/senescence reversibility and wound healing) as well as senescence-associated secretory phenotype (SASP) human cytokine array of 105 markers, we characterized the phenotype and secretome of these CDK4/6i DTPs. Summary: CDK4/6i-DTPs induced by abemaciclib, palbociclib and ribociclib (all from Selleckchem) demonstrated resistance to these drugs and dramatically slowed growth. The percentage of senescent cells reached 30-89% of the total population for all cell lines in all three drugs, whereas control parental cells exhibited around 10%. CDK4/6i DTPs also exhibited significant cell migration, and increased lysosomal phenotype compared to their parental sensitive cells. Further analysis with human cytokine array revealed that protein levels of growth differentiation factor 15 (GDF-15) are elevated in both cell lysates and conditioned-media of CDK4/6i DTPs, whereas Serpin E1 (Plasminogen Activator Inhibitor, Type I, PAI-1) was unique to secreted CDK4/6i DTPs. Interestingly, we did not observe changes in pro-inflammatory senescence-associated secretory phenotype (SASP) markers such as interleukins. Apart from being a SASP protein, GDF-15 is a mitochondrial cytokine (mitokine), suggesting a role in mitochondrial dysfunction-associated senescence (MiDAS). HMGB1, and LMNB1 are also down-regulated in CDK4/6-DTPs supporting the MiDAS phenotype. CDK4/6i-induced MiDAS appears to be transient, as CDK4/6i-DTPs reverted to the parental phenotype in drug-free culture media 20 days after the removal of CDK4/6i's. This included growth characteristics and sensitivity to CDK4/6i. This reversion of phenotype was observed in all five cell line models tested with all three drugs. Conclusions: CDK4/6-induces a mitochondrial dysfunction-associated senescence (MiDAS). This appears to be transient in nature as withdrawal of the drug results reacquisition of parental cell phenotype and drug-sensitivity. Targeting MiDAS pathway might be critical to improve efficacy of CDK4/6 inhibitors. Citation Format: Yesim Gokmen-Polar, Yuan Gu, Ling Ruan, Sunil S. Badve. CDK4/6 therapy-induced senescence as a resistance mechanism-MiDAS touch [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 5861.

Abstract 4709: The RASMULTI(ON) inhibitor RMC-7977 blocks downstream MAPK and PI3K pathway activation in KRASG12X_mutant cancers

Abstract Oncogenic RAS binds to multiple effector proteins to activate downstream signaling pathways, including the MAPK and PI3K axes. While RAS-mediated MAPK activation has a well-validated role in driving cancer progression, the contribution of PI3K activation has been more difficult to assess. Understanding the degree to which different KRAS-mutant genotypes stimulate PI3K activity may inform biomarker and combination strategies for RAS inhibitors currently undergoing clinical evaluation.RMC-7977 is a RASMULTI(ON) inhibitor that targets GTP-bound RAS(ON) and blocks effector protein binding. RMC-7977 exhibits similar potency for wild-type and oncogenic mutant RAS and is a powerful preclinical tool, representative of the investigational RASMULTI(ON) inhibitor RMC-6236, for comparing how RAS-mutant genotypes differ with respect to signal activation. MAPK signaling proceeds through a protein phosphorylation cascade that is readily detectable using standard cell biology methods, but PI3K is a lipid kinase whose activity is not amenable to protein characterization techniques. An assay of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) was developed to directly measure the activity of PI3K in the context of RAS inhibition.In isogenic LIM1215 cell lines expressing different KRASG12X mutants (KRASG12D, KRASG12V, KRASG12C and KRASG12R), RAS inhibition by RMC-7977 completely suppressed PI3K activity in cells with wild-type KRAS and KRASG12D, had a moderate inhibitory effect in KRASG12C and KRASG12V, and had no effect in KRASG12R. To confirm that this inhibition is dependent on a direct interaction between RAS(ON) and PI3K, CRISPR engineering was used to introduce mutations in PIK3CA to perturb the RAS-binding domain. In cells with disrupted RAS-PI3K interactions, the basal levels of PIP3 are significantly reduced and no longer change in response to treatment with RMC-7977.Based on the isogenic cell line results, KRASG12D and KRASG12R-mutant pancreatic cancer cell lines were profiled for their response to RAS inhibition. RMC-7977 decreased the levels of MAPK signaling in both KRASG12D and KRASG12R genotypes, but PI3K activity was only inhibited in KRASG12D lines. MEK inhibitors only inhibited MAPK signaling and had no effect on PI3K activity in either genotype. The simultaneous inhibition of MAPK and PI3K signaling by RMC-7977 led to deeper suppression of cyclin D levels and a more pronounced G1 cell cycle arrest as compared with MAPK inhibitors.We demonstrate that the coupling between RAS activation and PI3K signaling varies between oncogenic mutants, with KRASG12D showing the greatest degree of PI3K stimulation. In KRASG12D-mutant pancreatic cancer cell lines, a RASMULTI(ON) inhibitor suppresses both PI3K and MAPK signaling. These preclinical results may be used to inform potential, rational, mechanism-based combination treatment strategies for RAS(ON) inhibitors. Citation Format: Priyanka S. Bapat, Kyle Seamon, Andrea Gould, David Wildes. The RASMULTI(ON) inhibitor RMC-7977 blocks downstream MAPK and PI3K pathway activation in KRASG12X_mutant cancers [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 4709.

Abstract 5617: Combined inhibition of ATR and ATM with tuvusertib and lartesertib (M4076) impacts the tumor microenvironment

Abstract Ataxia telangiectasia and Rad3-related protein (ATR) and ataxia telangiectasia mutated (ATM) are two of the main kinases of the DNA damage response (DDR). Recent work has demonstrated that the addition of ATM inhibitors enhances the cytotoxicity and in vivo anti-tumor efficacy of ATR inhibitors by abrogating the ATR inhibitor mediated G1 cell cycle arrest and enhancing chromosomal damage.1 Prior to causing cell death, damaged chromosome fragments can end up in the cytosol where they are detected by cytosolic nucleic acid sensors such as cGAS, resulting in the induction of inflammatory signaling pathways and an anti-tumor immune response. This suggests that the mode of action of DDR inhibitors may involve the tumor microenvironment (TME). We therefore aimed to assess the influence of combined pharmacological ATR and ATM inhibition on the TME to better understand how this could be exploited therapeutically. In line with published results in human tumor models, the treatment of murine colon cancer MC-38 cells in vitro with a combination of ATR inhibitor tuvusertib and ATM inhibitor lartesertib led to cell death. Further analysis revealed an activation of the cGAS-STING signaling pathway, an upregulation of PD-L1 and the release of inflammatory cytokines. These results confirmed the activation of immune signaling pathways upon combined inhibition of ATR and ATM. To analyze the stimulation of an anti-tumor immune response upon tuvusertib + lartesertib treatment in vivo, MC-38 cells were transplanted into immunocompetent C57BL/6J mice. Immunoprofiling by flow cytometry and immunohistochemistry staining confirmed that treatment with tuvusertib + lartesertib indeed altered the TME in this setting. In line with the in vitro findings, a significant upregulation of PD-L1 on immune and tumor cells could be observed after 24, 48, and 72 h of treatment even though MC-38 cells expressed high levels of PD-L1 already. Immunoprofiling did not reveal any changes in the TME immune cell content at these early time points. Interestingly, PD-L1 expression as well as tumor immune population changed after prolonged (23 days) treatment. PD-L1 was still expressed on tumor cells but to a significantly lesser extent and a depletion of CD8+ T-cells was observed. However, a significant increase in NK cells could be detected. In summary, we show that inhibition of the DDR through a combination of ATR and ATM inhibitors influences the TME in the MC-38 tumor model. These findings suggest an additional mode of action beyond direct cancer cell cytotoxicity. Further studies on how this may be leveraged therapeutically, for example through a combination with immune checkpoint inhibitors, are warranted. 1. Turchick A, Zimmermann A, et al. Mol Cancer Ther 2023;22(7):859-872. Citation Format: Karin Laaber, Audrey Turchick, Julia Jabs, Brian Elenbaas, Lyubomir T. Vassilev, Astrid Zimmermann. Combined inhibition of ATR and ATM with tuvusertib and lartesertib (M4076) impacts the tumor microenvironment [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 5617.

Abstract 5113: Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma

Abstract Radiation is one of the standard therapies for pediatric high-grade glioma (pHGG), of which the prognosis remains poor. To gain an in-depth understanding of the biological activities beyond the classic DNA damage, we treated 6 patient-derived orthotopic xenograft (PDOX) models, including one with DNA mismatch repair (MMR) deficiency, with fractionated radiations (2 Gy/day x 5 days). Extension of survival time was noted in 4/6 PDOX models (P<0.05) and widespread γH2AX positivity in >95% tumor cells in tumor core and >85% in the invasive foci, accompanied by ~30% apoptotic and mitotic catastrophic cell death. The model with DNA MMR (IC-1406HGG) was the most responsive to radiation with a reduction of Ki-67(+) cells. Altered metabolism, including mitochondria number elevation, COX IV activation and reactive oxygen species buildup, were detected together with the accumulation of CD133+ tumor cells. The latter was caused by the entry of quiescent G0 cells into cell cycle and the activation of self-renewal (SOX2 and BMI1) and epithelial mesenchymal transition (fibronectin) genes. These novel insights about the cellular and molecular mechanisms of fractionated radiation in vivo should support the development of new radio-sensitizing therapies. Citation Format: Zi-Lu Huang, Zhi-Gang Liu, Qi Lin, Ya-Lan Tao, Xinzhuoyun Li, Patricia Baxter, Jack MF Su, Adekunle M. Adesina, Chris Man, Murali Chintagumpala, Wan Yee Teo, Yu-Chen Du, Yun-Fei Xia, Xiao-Nan Li. Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma [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 5113.