Overexpression Of Cyclin Research Articles

CCNE1 amplification as marker of poor prognosis and novel therapeutic target in advanced breast cancer.

1040 Background: Cyclin E1 (CCNE1) overexpression or gene amplification is linked to dismal outcomes in various tumors, including breast cancer (BC). Recent studies suggest that CCNE1 amplification is a potential target for new synthetic lethality-based treatments, including CDK2-selective and PKMYT1 inhibition. This study explores the clinical and genomic characteristics of CCNE1-amplified BCs. Methods: We analyzed genomic and clinical data from consecutive BCs that underwent clinical tumor-normal targeted panel sequencing (MSK-IMPACT) between April 2014 and December 2021. Allele-specific copy number, including CCNE1 amplification, and fraction genome altered (FGA) were calculated by FACETS. Mutual exclusivity and co-occurrence analyses were performed using CoMEt. Benjamini–Hochberg method for multiple testing correction (q) was applied. Real-world progression-free survival (rwPFS) was assessed by the Kaplan Meier method and Cox models, focusing on patients with available pre-treatment samples. Results: Out of 3,753 BCs, 125 (3.3%) had CCNE1 amplification. A higher occurrence of CCNE1 amplification was noted in post-treatment (n=2,368) compared to treatment-naïve tumors (n=1,385; 4% vs 2.4%, p=0.007). CCNE1 amplification was less common in hormone receptor (HR)+/HER2- BCs (2%) compared to HER2+ (7.6%) and triple-negative (7.2%) tumors (p<0.001). BCs with CCNE1 amplification showed higher median FGA, indicating increased genomic instability. In primary BC, TP53 alterations were more frequent in CCNE1-amplified tumors (q<0.001), while CDH1 alterations were mutually exclusive (q<0.001) suggesting that lobular BCs rarely harbor CCNE1 amplification. Similar trends were seen in post-treatment samples. CCNE1 amplification detected at baseline was linked to shorter median rwPFS in HR+/HER2- metastatic BCs treated with CDK4/6 inhibitors plus endocrine therapy (8.8 vs 15.2 months in CCNE1-amplified [n=9] vs CCNE1-non amplified [n=402]; hazard ratio [HR] 2.82, 95% CI 1.38-5.75), and in HER2+ metastatic BCs treated with first-line THP regimen (7.3 vs 20.8 months in CCNE1-amplified [n=5] vs CCNE1-non amplified [n=106]; HR 3.1, 95% CI 1.24-7.87). In a CCNE1-amplified triple-negative cell model (HCC1569), treatment with the PKMYT1 inhibitor RP-6306 significantly reduced tumor growth. Conclusions: CCNE1 amplification in BCs is associated with greater genomic instability and characterizes a group of metastatic BCs with poor response to standard of care therapies. Novel therapies targeting CCNE1-amplified tumors are under evaluation in preclinical and clinical studies.

Biomarkers for prognosis of meningioma patients: A systematic review and meta-analysis.

Meningioma is the most common primary brain tumor and many studies have evaluated numerous biomarkers for their prognostic value, often with inconsistent results. Currently, no reliable biomarkers are available to predict the survival, recurrence, and progression of meningioma patients in clinical practice. This study aims to evaluate the prognostic value of immunohistochemistry-based (IHC) biomarkers of meningioma patients. A systematic literature search was conducted up to November 2023 on PubMed, CENTRAL, CINAHL Plus, and Scopus databases. Two authors independently reviewed the identified relevant studies, extracted data, and assessed the risk of bias of the studies included. Meta-analyses were performed with the hazard ratio (HR) and 95% confidence interval (CI) of overall survival (OS), recurrence-free survival (RFS), and progression-free survival (PFS). The risk of bias in the included studies was evaluated using the Quality in Prognosis Studies (QUIPS) tool. A total of 100 studies with 16,745 patients were included in this review. As the promising markers to predict OS of meningioma patients, Ki-67/MIB-1 (HR = 1.03, 95%CI 1.02 to 1.05) was identified to associate with poor prognosis of the patients. Overexpression of cyclin A (HR = 4.91, 95%CI 1.38 to 17.44), topoisomerase II α (TOP2A) (HR = 4.90, 95%CI 2.96 to 8.12), p53 (HR = 2.40, 95%CI 1.73 to 3.34), vascular endothelial growth factor (VEGF) (HR = 1.61, 95%CI 1.36 to 1.90), and Ki-67 (HR = 1.33, 95%CI 1.21 to 1.46), were identified also as unfavorable prognostic biomarkers for poor RFS of meningioma patients. Conversely, positive progesterone receptor (PR) and p21 staining were associated with longer RFS and are considered biomarkers of favorable prognosis of meningioma patients (HR = 0.60, 95% CI 0.41 to 0.88 and HR = 1.89, 95%CI 1.11 to 3.20). Additionally, high expression of Ki-67 was identified as a prognosis biomarker for poor PFS of meningioma patients (HR = 1.02, 95%CI 1.00 to 1.04). Although only in single studies, KPNA2, CDK6, Cox-2, MCM7 and PCNA are proposed as additional markers with high expression that are related with poor prognosis of meningioma patients. In conclusion, the results of the meta-analysis demonstrated that PR, cyclin A, TOP2A, p21, p53, VEGF and Ki-67 are either positively or negatively associated with survival of meningioma patients and might be useful biomarkers to assess the prognosis.

Overexpression of cyclin F/CCNF as an independent prognostic factor for poor survival in clear cell renal cell carcinoma

Cyclin F (encoded by CCNF gene) has been reported to be implicated in the pathobiology of several human cancers. However, its potential clinical significance in clear cell renal cell carcinoma (ccRCC) remains unknown. The present study aimed to evaluate the potential significance of cyclin F, assessed by immunohistochemical (IHC) staining and molecular (bioinformatics) techniques, as a prognostic marker in ccRCC in relation to clinicopathological features and outcomes. IHC staining was performed using two independent ccRCC tissue array cohorts, herein called tissue macroarray (TMA)_1 and tissue microarray (TMA)_2, composed of 108 ccRCCs and 37 histologically normal tissues adjacent to the tumor (NAT) and 192 ccRCCs and 16 normal kidney samples, respectively. The mRNA expression data were obtained from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) public datasets, followed by bioinformatics analysis of biological mechanisms underlying prognosis. The relationship between immune cell infiltration level and CCNF expression in ccRCC was investigated using the Tumor Immune Estimation Resource 2.0 (TIMER2) and Gene Expression Profiling Interactive Analysis 2 (GEPIA2). Cyclin F expression was significantly elevated in ccRCC lesions compared to both NAT and normal renal tissues. Likewise, CCNF mRNA was markedly increased in ccRCCs relative to non-cancerous tissues. In all analyzed cohorts, tumors with features of more aggressive behavior were more likely to display cyclin F/CCNF-high expression than low. Furthermore, patients with high cyclin F/CCNF expression had shorter overall survival (OS) times than those with low expression. In addition, multivariable analysis revealed that cyclin F/CCNF-high expression was an independent prognostic factor for poor OS in ccRCC. Enrichment analysis for mechanistically relevant processes showed that CCNF and its highly correlated genes initiate the signaling pathways that eventually result in uncontrolled cell proliferation. CCNF expression was also correlated with immune cell infiltration and caused poor outcomes depending on the abundance of tumor-infiltrating immune cells in ccRCC. Our findings suggest that cyclin F/CCNF expression is likely to have an essential role in ccRCC pathobiology through regulating multiple oncogenic signaling pathways and affecting the tumor immune microenvironment and may serve as prognostic biomarker and promising therapeutic target in ccRCC.

Abstract 1649: Large scale multi-omics concordance study of cyclin E1 identifies high expressing tumors lacking CCNE1 gene alterations across multiple tumor indications

Abstract Cyclin E1 is a key regulator of the G1/S transition and is thought to cause excessive entry into the cell cycle. CCNE1 is amplified in a range of tumor indications, and its amplification is associated with poorer prognosis and resistance to chemotherapy. Cyclin E1 is also being investigated as a potential biomarker for inhibitors targeting the replication stress response. Understanding the best way to quantify cyclin E1 overexpression is fundamental to support patient selection approaches for these inhibitors. In many cases, gene amplification correlates with increased cyclin E1 protein expression; however, there are a proportion of high-grade serous ovarian cancer and basal-like breast cancer cases where cyclin E1 protein overexpression does not correlate with gene amplification. In this study, we investigate if this phenomenon is observed in other cancers and some of the potential mechanisms causing cyclin E1 overexpression. We used a multi-omics approach to assess cyclin E1 expression in 1417 formalin-fixed paraffin-embedded (FFPE) patient tumor resections across 6 indications (SCCHN, Bladder, NSCLC, TNBC, Ovarian & SCLC). We used copy number variation (CNV) determined by next generation sequencing (NGS), NanoString nCounter RNA analysis and protein expression by immunohistochemistry (IHC). 830 were evaluable for all 3 methods and demonstrated CCNE1 gene amplification by NGS (CNV normalized log2 ratio ≥1.5) in 3.3% cases. Using a threshold of mRNA z-score >2, 17% cases had high CCNE1 mRNA and 29% had high cyclin E1 protein (H-score ≥100). 15.7% (130/830) cases had high protein expression without gene amplification or gain, crucially this was found across all 6 indications (n=20/102 SCCHN, n=29/166 Bladder, n=34/232 NSCLC, n=15/76 SCLC, n=15/143 TNBC & n=17/118 Ovarian). Impaired protein degradation has been proposed as a mechanism that may result in high Cyclin E1 expression. From a panel of 1600 genes, we assessed the relationship between cyclin E1 and mRNA expression of 57 genes involved in the ubiquitin-proteasome pathway in 1239 samples across the 6 indications. We assessed mutations which could impair ubiquitination in 915 samples across the 6 indications. We also assessed the methylation status of 893 genes associated with ubiquitin ligases or deubiquitinases in a cohort of 170 TNBC samples. Further investigation into other mechanisms causing cyclin E1 overexpression is required. IHC provides an ideal platform to build our understanding of cyclin E1 expression spatially. Our results indicate that assessing Cyclin E1 protein overexpression instead of CCNE1 gene amplification could greatly broaden the population of patients who could benefit from replication stress response kinase inhibitors, as cyclin E1 overexpression correlates with genomic instability and replication stress. Citation Format: Sophie E. Willis, Gemma N. Jones, Shaan Gill, Emma V. Jones, Amelia Raymond, Barrett Nuttall, Sophie Kirschner, Sakshi Gulati, Paola Marco-Casanova, Benedetta Lombardi, David Jenkins, Felicia S. Ng, James Hadfield, Paul Waring, Helen K. Angell, Heike Laman. Large scale multi-omics concordance study of cyclin E1 identifies high expressing tumors lacking CCNE1 gene alterations across multiple tumor indications [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 1649.

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 7121: The novel WEE1i, APR-1051, is a potentially well tolerated and effective treatment for cyclin E-overexpressing cancers

Abstract Previous studies have demonstrated that WEE1 inhibitors (WEE1i) are a promising cancer therapeutic through synthetic lethality with Cyclin E overexpression. One challenge for two of the leading WEE1i, AZD1775 and ZN-c3, has been the observed off-targeting of PLK family members, namely, PLK1, PLK2 and PLK3. In addition, the therapeutic application of these inhibitors in clinical trials has been limited by adverse hematological effects, including anemia. Herein, we describe the testing of a novel WEE1i (APR-1051) that, based on preclinical studies: 1) demonstrates reduced off-targeting of PLK1, PLK2 and PLK3, and 2) has the ability to suppress the growth of CCNE1-overexpressing xenografted tumors while causing little impact on red blood cell and reticulocyte levels. APR-1051 has an IC50 of 2.2 nM for WEE1 in vitro and has shown the ability to limit the proliferation of various cancer cell lines in culture. Interestingly, the genotoxicity of APR-1051 is suppressed by increasing concentrations of PLK1 inhibitors, suggesting that off-targeting of PLK1 by other WEE1i may compromise efficacy of this targeted cancer treatment in addition to the risk of producing PLK1i-associated adverse effects. APR-1051 demonstrated effectiveness in suppressing the growth of Cyclin E-overexpressing breast and ovarian cancer cells lines, thus pinpointing Cyclin E as a potential biomarker for APR-1051 treatment. Importantly, dose and scheduling of APR-1051 that causes significant suppression of CCNE1-amplified high-grade serious ovarian cancer tumors in mice is well tolerated, with red blood cell and platelet counts remaining within non-pathogenic ranges after a 28-day treatment period. In addition, inhibition WEE1 by APR-1051 occurs at an IC50 that is 200-fold lower on average than the IC50 of hERG potassium channel inhibition, implying that APR-1051 will potentially exhibit low cardiotoxicity. APR-1051 is now progressing through IND-enabling studies. Together, these findings underscore the potential of APR-1051 as a novel WEE1i for the treatment of Cyclin E-overexpressing cancers. Citation Format: Molly M. Hansbarger, Stephen J. Rocca, Sonia Ritzmann, Joseph Vacca, Bridget Tokiwa, Jonathan Weinstein, Henry Zabierek, Justin Frye, Michael Carleton, Eric J. Brown, Oren Gilad. The novel WEE1i, APR-1051, is a potentially well tolerated and effective treatment for cyclin E-overexpressing 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 7121.

Abstract B169: Retrospective baseline biomarker analyses in a first-in-human Phase 1 trial of the PKMYT1 inhibitor lunresertib (RP-6306) in pts with advanced solid tumors harboring CCNE1 amplification and/or deleterious alterations in FBXW7 or PPP2R1A

Abstract Background: Lunresertib (RP-6306) is a first-in-class membrane-associated tyrosine- and threonine-specific Cdc2-inhibitory kinase (PKMYT1) inhibitor that disrupts the G2/M checkpoint leading to premature mitosis and catastrophic DNA damage in cells harboring synthetic lethal genomic alterations. The safety and tolerability of lunresertib alone and in combination with camonsertib (RP-3500) is being investigated in the Phase 1 MYTHIC study in patients with advanced solid tumors with either CCNE1 amplifications (amp) or deleterious alterations in FBXW7 or PPP2R1A (NCT04855656). Here, we present a comprehensive retrospective biomarker analysis aimed at understanding concordance between local vs. central and tissue- vs. plasma-based next generation sequencing (NGS). The correlation between CCNE1 copy number assessment by NGS and fluorescence in situ hybridization (FISH), as well as the relationship between CCNE1 amp and Cyclin E1 protein levels, was investigated. Methods: Patients were enrolled based on results from sponsor-approved local tumor NGS and plasma circulating tumor DNA (ctDNA) tests. To ensure molecular eligibility criteria were met, local NGS reports were centrally reviewed and functionally annotated prior to enrollment. Tumor tissue and plasma samples collected at screening were retrospectively analyzed by SNiPDx™ and Tempus xF(+), respectively. Tissue samples were further evaluated by FISH and immunohistochemistry. Results: As of June 1, 2023, 145 patients were screened with uterine (23%), colorectal (CRC, 15%), ovarian (13%), and upper GI (6%) as the most frequent cancers. Ninety-four percent (137/145) of patients screened had local NGS results available (98% tissue, 2% plasma). CCNE1 amp (40.1%) were most frequently observed in ovarian, uterine, and upper GI malignancies. FBXW7 (39.4%) and PPP2R1A (16.0%) alterations were enriched in CRC and uterine cancer, respectively. TP53 alterations were frequent across all biomarker groups (76% CCNE1, 54% FBXW7, and 88% PPP2R1A). Central tissue NGS confirmed 94% (47/50) of the enrollment alterations. Central plasma ctDNA confirmed 83.0% (39/47) of FBXW7 and PPP2R1A mutations and had high positive predictive value (100%, 17/17) but low sensitivity (42.5%; 17/40) for CCNE1 amp. CCNE1 amp call and copy number estimation by tissue NGS and FISH were highly correlated (Spearman ρ=0.65, p=0.006). Seventy-five percent of tumors with CCNE1 amp had Cyclin E1 overexpression (H-score ≥100; mean=168; range [0, 300]). Conclusion: In this study, local NGS tests were an efficient and robust strategy for directing patients into a molecularly defined Phase 1 trial. Plasma ctDNA reliably detected alterations in all 3 genes, but caution should be taken when interpreting CCNE1 amplification negative results due to a high false negative rate. FISH and genomics are appropriate methods to estimate CCNE1 amp copy number. These retrospective analyses enable the testing of the synthetic lethal hypothesis of lunresertib and CCNE1, PPP2R1A and FBXW7, assuring reliable interpretation of the clinical data. Citation Format: Elia Aguado-Fraile, Sunantha Sethuraman, Adam Petrone, Emeline Bacque, Alison Schram, Stephanie Lheureux, Elizabeth K Lee, Maria Koehler, Ian M Silverman, Victoria Rimkunas, Timothy A Yap. Retrospective baseline biomarker analyses in a first-in-human Phase 1 trial of the PKMYT1 inhibitor lunresertib (RP-6306) in pts with advanced solid tumors harboring CCNE1 amplification and/or deleterious alterations in FBXW7 or PPP2R1A [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B169.

Abstract B057: Preclinical development of PKMYT1 and ATR inhibitor combinations

Abstract Background: Inactivation of the PKMYT1 kinase is synthetically lethal in tumor cells with genomic alterations leading to high replication stress, such as CCNE1 amplification frequently found in high-grade serous ovarian, uterine, and gastro-esophageal cancers. On this basis, we developed lunresertib (RP-6306), a first-in-class, potent and selective oral PKMYT1 inhibitor currently in Phase I clinical trials (NCT04855656). We have shown previously that PKMYT1 inhibition abrogates an inhibitory phosphorylation of the cyclin-dependent kinase CDK1 at threonine 14 (CDK1 pT14), leading to premature mitotic entry and catastrophic chromosome shattering specifically in cyclin E-high tumor cells, but not in cells with normal cyclin E levels. This leads to robust tumor growth inhibition by lunresertib in cyclin E-high pre-clinical models. We hypothesized that inhibition of the DNA damage response kinase ATR may further potentiate the tumor cytotoxicity of lunresertib, as ATR is known to regulate CDK1 pT14 levels in a parallel fashion to PKMYT1 through inhibition of the CDC25 family of phosphatases. Here we present the pre-clinical validation of this hypothesis. Methods: We used in vitro cell viability studies to determine the level of synergy between lunresertib and the ATR inhibitor camonsertib (RP-3500) in various engineered as well as tumor-derived models. We investigated the mechanism of action of the lunresertib/camonsertib combination using high content imaging and immunoblotting for DNA damage and cell cycle markers in cultured cells. Finally, we have evaluated the pre-clinical efficacy and tolerability of lunresertib/camonsertib combinations in vivo using mouse xenograft models. Results: In CCNE1-overexpressing cells, combining lunresertib with camonsertib led to a synergistic increase in cytotoxicity and catastrophic DNA damage at doses that did not affect wild type counterparts. Increased DNA damage resulted primarily from enhanced CDK1 dephosphorylation when lunresertib was combined with camonsertib, leading to robust and rapid induction of premature mitosis. Altogether these data suggest that camonsertib synergistically potentiates lunresertib’s mechanism of action. Consistent with that, we observed synergy between camonsertib and lunresertib in models carrying genomic biomarkers predictive of lunresertib sensitivity (CCNE1 amplification, FBXW7 loss-of-function), and not unrelated DNA repair defects (ATM or BRCA1 loss-of-function). In mouse xenograft models of tumors carrying predictive molecular alterations, the combination of lunresertib/camonsertib was well tolerated, enhanced CDK1 dephosphorylation and DNA damage, and was highly efficacious, including generating tumor regressions at doses sub-therapeutic as monotherapy. Conclusions: Lunresertib and camonsertib elicit synergistic activity in pre-clinical models of CCNE1-amplified and cyclin E-overexpressing tumors due to a complementary mechanism of action. These data provide a rationale for clinical development of PKMYT1 and ATR inhibitor combinations. Citation Format: David Gallo, Marc L. Hyer, Giovanni Martino, Adam Pertrone, Elia Aguado-Fraile, Rosie Kryczka, Jimmy Fourtounis, Rino Stocco, Stephen J. Morris, C. Gary Marshall, Anne Roulston, Michael Zinda, Michal Zimmermann. Preclinical development of PKMYT1 and ATR inhibitor combinations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B057.

Tumor-suppressive microRNA-152 inhibits the proliferation of Ewing’s sarcoma cells by targeting CDK5R1

We elucidated the mechanism through which the reduced expression of miR-152 leads to the overexpression of its target cyclin-dependent kinase-5 activator 1 (CDK5R1) in Ewing’s sarcoma (ES) cells and the role of this mechanism in the proliferation of ES cells. To explore possible oncogenic factors in ES, we conducted microarray-based investigation and profiled the changes in miRNA expression and their effects on downstream mRNAs in five ES cell lines and human mesenchymal stem cells (hMSCs). miR-152 was significantly downregulated, while cyclin-dependent kinase-5 activator 1 (CDK5R1) expression was significantly upregulated in all tested ES cells as compared to hMSCs. The overexpression of CDK5R1 led to the activation of CDK5, enabling the phosphorylation of retinoblastoma protein and persistent overexpression of CCNE. Moreover, miR-152 suppressed cell proliferation via cell cycle retardation, and its upregulation reduced tumor size and CCNE expression in tumor tissues. The overexpression of cyclin E (CCNE) has been detected in ES cells, but the detailed mechanisms have not been previously elucidated. These findings identify the miR152-CDK5R1 signaling axis as a critical mechanism for tumorigenesis that may serve as a new therapeutic target in Ewing’s sarcoma. We believe that our results will aid in the development of effective treatment strategies for patients with ES.

Cyclin D1 extensively reprograms metabolism to support biosynthetic pathways in hepatocytes

Cell proliferation requires metabolic reprogramming to accommodate biosynthesis of new cell components, and similar alterations occur in cancer cells. However, the mechanisms linking the cell cycle machinery to metabolism are not well defined. Cyclin D1, along with its main partner cyclin-dependent kinase 4 (Cdk4), is a pivotal cell cycle regulator and driver oncogene that is overexpressed in many cancers. Here, we examine hepatocyte proliferation to define novel effects of cyclin D1 on biosynthetic metabolism. Metabolomic studies reveal that cyclin D1 broadly promotes biosynthetic pathways including glycolysis, the pentose phosphate pathway, and purine and pyrimidine nucleotide synthesis in hepatocytes. Proteomic analyses demonstrate that overexpressed cyclin D1 binds to numerous metabolic enzymes including those involved in glycolysis and pyrimidine synthesis. In the glycolysis pathway, cyclin D1 activates aldolase and GAPDH, and these proteins are phosphorylated by cyclin D1/Cdk4 in vitro. De novo pyrimidine synthesis is particularly dependent on cyclin D1. Cyclin D1/Cdk4 phosphorylates the initial enzyme of this pathway, CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase), and metabolomic analysis indicates that cyclin D1 depletion markedly reduces the activity of this enzyme. Pharmacologic inhibition of Cdk4 along with the downstream pyrimidine synthesis enzyme DHODH (dihydroorotate dehydrogenase) synergistically inhibits proliferation and survival of hepatocellular carcinoma cells. These studies demonstrate that cyclin D1 promotes a broad network of biosynthetic pathways in hepatocytes, and this model may provide insights into potential metabolic vulnerabilities in cancer cells.

PPM1D activity promotes the replication stress caused by cyclin E1 overexpression.

Oncogene-induced replication stress has been recognized as a major cause of genome instability in cancer cells. Increased expression of cyclin E1 caused by amplification of the CCNE1 gene is a common cause of replication stress in various cancers. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and has been implicated in termination of the cell cycle checkpoint. Amplification of the PPM1D gene or frameshift mutations in its final exon promote tumorigenesis. Here, we show that PPM1D activity further increases the replication stress caused by overexpression of cyclin E1. In particular, we demonstrate that cells expressing a truncated mutant of PPM1D progress faster from G1 to S phase and fail to complete licensing of the replication origins. In addition, we show that transcription-replication collisions and replication fork slowing caused by CCNE1 overexpression are exaggerated in cells expressing the truncated PPM1D. Finally, replication speed and accumulation of focal DNA copy number alterations caused by induction of CCNE1 expression was rescued by pharmacological inhibition of PPM1D. We propose that increased activity of PPM1D suppresses the checkpoint function of p53 and thus promotes genome instability in cells expressing the CCNE1 oncogene.

Prospects of new targeted nanotherapy combining liponiosomes with berberine to combat colorectal cancer development: An in vivo experimental model

Colorectal cancer (CRC) is one of the most identified and deadly malignancies worldwide. It presents a serious challenge due to its quick growth, which finally culminates in severe malignancy. It is critical to improve the efficacy of berberine (BR) as an anticancer agent to overcome its limited bioavailability. Implementation of a novel, effective nanocarrier system of liponiosomes for BR (LipoNio.BR) can support mechanistic actions associated with its anti-CRC role. Following CRC induction in rats using 1,2 Dimethylhydrazine (40 mg DMH/kg/week), the potency and mechanistic actions of LipoNio.BR were assessed by evaluating the lesion severity and molecular mechanisms controlling oxidative stress, apoptosis, autophagy, and inflammatory responses, and conducting histopathological and immunohistochemistry examinations of colonic tissues. The results indicated that the severity of clinical signs comprising weight gain loss, increased diarrhea and rectal bleeding, and reduced survivability were greatly restored in the LipoNio.BR-treated group. LipoNio.BR remarkably reduced CRC development compared to FBR (free berberine), as it induced apoptosis via upregulating apoptotic genes (Bax and caspase3, increased up to 7.89 and 6.25-fold, respectively) and downregulating the anti-apoptotic gene Bcl-2 by 2.25-fold. LipoNio.BR mitigated the oxidative stress associated with CRC and maintained redox homeostasis. Notably, the excessive inflammatory response associated with CRC was prominently reduced following administration of LipoNio.BR [which decreased iterleukin (IL-B, IL-6), tumor necrosis factor-alpha (TNF-α), cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS), proliferating cell nuclear antigen (PCNA), follistatin, and activin BA (beta-A) expression]. LipoNio.BR modulated the expression of nuclear factor kappa B (NF-κB) and mammalian target of rapamycin (mTOR), which impacted tumor vascularity (decreased Vascular endothelial growth factor (VEGF) expression by 2.36-fold). The severity of the histopathological alterations in the colonic tissues, including the development of neoplastic epithelium and the invasion of some neoplastic masses, was greatly reduced in the LipoNio.BR group compared to the FBR-(free berberine) administrated group. Following CRC induction, immunohistochemical staining revealed that the overexpression of cyclin and COX-2 in colonic tissues were suppressed in the LipoNio.BR group. Taken together, these findings suggest that LipoNio.BR has a potential role in reducing CRC progression to a greater extent compared to free BR and could be considered a promising and potent therapy against CRC.

FBXL12 degrades FANCD2 to regulate replication recovery and promote cancer cell survival under conditions of replication stress

Fanconi anemia (FA) signaling, a key genomic maintenance pathway, is activated in response to replication stress. Here, we report that phosphorylation of the pivotal pathway protein FANCD2 by CHK1 triggers its FBXL12-dependent proteasomal degradation, facilitating FANCD2 clearance at stalled replication forks. This promotes efficient DNA replication under conditions of CYCLIN E- and drug-induced replication stress. Reconstituting FANCD2-deficient fibroblasts with phosphodegron mutants failed to re-establish fork progression. In the absence of FBXL12, FANCD2 becomes trapped on chromatin, leading to replication stress and excessive DNA damage. In human cancers, FBXL12, CYCLIN E, and FA signaling are positively correlated, and FBXL12 upregulation is linked to reduced survival in patients with high CYCLIN E-expressing breast tumors. Finally, depletion of FBXL12 exacerbated oncogene-induced replication stress and sensitized cancer cells to drug-induced replication stress by WEE1 inhibition. Collectively, our results indicate that FBXL12 constitutes a vulnerability and a potential therapeutic target in CYCLIN E-overexpressing cancers.

Correlation of cyclin E1 expression and clinical outcomes in a phase 1b dose-escalation study of azenosertib (ZN-c3), a WEE1 inhibitor, in combination with chemotherapy (CT) in patients (pts) with platinum-resistant or refractory (R/R) epithelial ovarian, peritoneal, or fallopian tube cancer (EOC).

5513 Background: Azenosertib (ZN-c3) is a novel, selective, and orally bioavailable WEE1 inhibitor demonstrating single-agent antitumor activity. Azenosertib may inhibit CT-induced DNA damage repair and provide benefit in pts with platinum R/R EOC. Cyclin E1 amplification/overexpression is present in ≥35% of metastatic ovarian cancer. High expression/amplification of Cyclin E1 is a poor prognostic marker and predictive of lack of response to platinum-based CT. Ovarian models overexpressing Cyclin E1 have exquisite sensitivity to azenosertib in vitro and in vivo; forced Cyclin E1 overexpression sensitizes cell lines with low endogenous Cyclin E1 to azenosertib. Methods: This open-label, multicenter study (NCT04516447) assessed azenosertib + paclitaxel (PAC), carboplatin (Carbo), gemcitabine (GEM), or pegylated liposomal doxorubicin (PLD) in pts with metastatic high-grade serous EOC after ≤2 lines of CT including platinum CT. Primary endpoint is safety and identification of RP2D of each combination. Secondary endpoints include clinical activity. Azenosertib was given continuously or intermittently QD in 21 or 28D cycles until PD or unacceptable toxicity. Results: 103 pts were enrolled; at data cut-off, 94 were efficacy evaluable. 26.6% had a partial response (PR) and median progression free survival (PFS) = 9.03 mo (95%CI: 5.52-11.01). Azenosertib + PAC demonstrated the highest ORR [9/18 (50%)], followed by Carbo [9/27 (33.3%)]; ORR for azenosertib + PLD or + GEM was 14.3% (5/35, 2/14 respectively). 80 pts had available Cyclin E1 expression data by IHC; higher Cyclin E1 (H-score >50) correlated with higher ORR and longer PFS (ORR = 31.3% vs 7.7%; PFS = 10.35 vs 3.25 mo, HR=0.3; Table). Frequent Grade ≥3 TEAEs (%) were neutropenia (44.4), thrombocytopenia (30.3), anemia (12.1), leukopenia (11.1), fatigue (10.1), diarrhea (6.1), nausea (5.1), and vomiting (5.1). Conclusions: Azenosertib + CT is well tolerated and has encouraging clinical activity, with durable responses in pts with platinum R/R EOC. Pts with Cyclin E1 overexpressing tumors, a subgroup with suboptimal benefits from CT, demonstrated significant improvements in ORR and PFS vs pts with tumors having low expression. These data support a planned trial of azenosertib + CT vs CT alone in Cyclin E1 overexpressing platinum R/R EOC. Clinical trial information: NCT04516447 . [Table: see text]

Abstract 2153: Cyclin E1 protein overexpression sensitizes ovarian cancer cells to ZN-c3, a novel, selective and oral bioavailable inhibitor of Wee1

Abstract CCNE1 gene amplification is associated with overexpression of Cyclin E1 protein and is an important oncogenic driver. CCNE1 amplification is common in high grade serous ovarian carcinomas (HGSOC) and is associated with platinum resistance and poor patient outcomes. Importantly, Cyclin E1 overexpression can also occur in the absence of gene amplification in ovarian cancer. Overexpression of Cyclin E1 increases CDK2 activity and accelerates entry into S phase of cell cycle, resulting in replication stress and rendering cells more dependent on DNA repair. WEE1 participates in the DNA damage response by controlling essential checkpoints of the cell cycle, preventing cells from entering mitosis and allowing for DNA repair before cell cycle progression. Thus, we hypothesize that ovarian cancers overexpressing Cyclin E1, either via CCNE1 gene amplification or through other independent mechanisms, are more sensitive to WEE1 inhibition. Here, we tested this hypothesis in a range of preclinical cancer models using azenosertib, a novel, selective, and orally bioavailable WEE1 inhibitor currently in clinical development. Ovarian cancer cell lines overexpressing Cyclin E1 protein (Cyclin E1high) were more sensitive to azenosertib than ovarian cancer cell lines with lower levels of Cyclin E1 protein (Cyclin E1low) inducing significant cytotoxic effects (GRmax < -0.5). In Cyclin E1low ovarian cancer cell lines, overexpression of Cyclin E1 increased sensitivity to azenosertib. In contrast, silencing of CDK2 in Cyclin E1high cells reduced sensitivity to azenosertib. In vivo, higher levels of baseline expression of Cyclin E1 in tumor cells were associated with better response to azenosertib, with tumor growth inhibition (TGI) ranging from 51.5% in a Cyclin E1low SKOV3 model to 88% in a Cyclin E1high OVCAR3 model. Azenosertib synergized with chemotherapy agents in multiple ovarian cancer models. In vitro, we report that Cyclin E1high cancer cells were more sensitive to the combination of azenosertib and chemotherapeutics than Cyclin E1low cancer cells. In vivo, the synergy between azenosertib and paclitaxel was stronger in Cyclin E1high OVCAR3 model (46% reduction of initial tumor volume/104% TGI) than Cyclin E1low A2780 model (85% TGI). Taken together, these data suggest that Cyclin E1 overexpression via gene amplification or independent mechanisms sensitize ovarian cancer cells to azenosertib alone or in combination with chemotherapy. This observation supports the use of Cyclin E1 expression to enrich for azenosertib responders in ovarian cancer and possibly other tumor types. Two subsets of Cyclin E1-driven platinum resistant ovarian cancer patients, as defined by gene amplification or protein overexpression, are being enrolled in an azenosertib monotherapy trial. Citation Format: Jianhui Ma, Wen Liu, Heekyung Chung, Hooman Izadi, Petrus DeJong, Olivier Harismendy, Jia li, Fernando Doñate, Ahmed Samatar, Mark Lackner, Laure Escoubet, Kevin Bunker, Daehwan Kim, Nathan Jameson, Tugba Yildiran Ozmen, Kangjin Jeong, Dong Zhang, Wen-An Pan, Gordon Mills. Cyclin E1 protein overexpression sensitizes ovarian cancer cells to ZN-c3, a novel, selective and oral bioavailable inhibitor of Wee1 [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 2153.

Abstract 5994: INX-315, a potent and selective CDK2 inhibitor, demonstrates robust antitumor activity in CCNE1-amplified cancers

Abstract Cyclin-dependent kinases (CDK) are a family of serine/threonine kinases that heterodimerize with regulatory subunits called cyclins to drive cell cycle progression. Uncontrolled cellular proliferation is a hallmark of cancer commonly driven by dysregulated kinase activity of specific CDK family members, including cyclin-dependent kinase 2 (CDK2). Aberrant CDK2 activity most frequently occurs through amplification of CCNE1 and/or overexpression of its protein product cyclin E1, which is a canonical binding partner of CDK2. Overexpression of cyclin E1 is observed in many solid tumors including in patients with high grade serous ovarian cancer (HGSOC), gastric cancer, and ER-positive breast cancer patients whose tumors have progressed on a prior CDK4/6 inhibitor regimen. Selective inhibition of CDK2 is thus a compelling therapeutic approach to regain cell cycle control. Here, we report preclinical data supporting the development of INX-315 for patients with cancers in which proliferation is CDK2-dependent. INX-315 is a potent inhibitor of CDK2/cyclin E (0.6 nM biochemical IC50) with high selectivity over other CDK family members in both biochemical and intracellular NanoBRET assays. In CCNE1-amplified human ovarian and gastric cancer cell lines, INX-315 potently inhibited Rb phosphorylation, induced a G1 cell cycle arrest, and inhibited proliferation. INX-315 also showed potent anti-proliferative activity in luminal breast cancer cell lines that had been cultured in CDK4/6 inhibitor (+/- anti-estrogen therapy) for prolonged periods to the point of developing drug resistance. While these cell lines did not readily respond to either CDK4/6 or CDK2 inhibition alone, combination treatment again suppressed Rb phosphorylation, accompanied by G1 arrest and a senescent-like phenotype. Lastly, in CCNE1-amplified xenograft models of ovarian and gastric carcinomas, INX-315 inhibited Rb-phosphorylation and induced tumor regression. These data demonstrate INX-315 to be a potent and selective CDK2 inhibitor that may benefit patients with CDK2/cyclin E driven cancers. Citation Format: Alec G. Trub, John E. Bisi, Catherine Dietrich, Michael Taylor, Jay C. Strum, Shom Goel, Patrick J. Roberts. INX-315, a potent and selective CDK2 inhibitor, demonstrates robust antitumor activity in CCNE1-amplified cancers [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 5994.

Hgc1 Independence of Biofilm Hyphae in Candida albicans.

Biofilm and hypha formation are central to virulence of the fungal pathogen Candida albicans. The G1 cyclin gene HGC1 is required for hypha formation under diverse in vitro and in vivo growth conditions. Hgc1 is required for disseminated infection and is a linchpin in the argument that hyphal morphogenesis itself is required for pathogenicity. We report here that HGC1 is dispensable for hypha formation during biofilm formation both in vitro, under strong inducing conditions, and in vivo, in a mouse oropharyngeal candidiasis model. These findings are validated with two or more C. albicans isolates. Systematic screening of overexpressed cyclin genes indicates that CCN1 and CLN3 can compensate partially for Hgc1 function during biofilm growth. This conclusion is also supported by the severity of the hgc1Δ/Δ ccn1Δ/Δ double mutant biofilm defect. Our results suggest that hypha formation in biofilm is accomplished by combined action of multiple cyclins, not solely by Hgc1. IMPORTANCE The HGC1 gene encodes a cyclin that is required for virulence of the fungal pathogen Candida albicans. It is required to produce the elongated hyphal filaments of free-living planktonic cells that are associated with virulence. Here, we show that HGC1 is not required to produce hyphae in the alternative growth form of a biofilm community. We observe Hgc1-independent hyphae in two infection-relevant situations, biofilm growth in vitro and biofilm-like oropharyngeal infection. Our analysis suggests that hypha formation in the biofilm state reflects combined action of multiple cyclins.

NOTCH1 mutation in chronic lymphocytic leukaemia is associated with an enhanced cell cycle G1/S transition and specific cyclin overexpression: Preclinical ground for targeted inhibition.

Studies prior to next-generation sequencing (NGS) showed that the frequent indolent course of chronic lymphocytic leukaemia (CLL) is related to most cells remaining quiescent in the G0 -G1 cell cycle phase, due to the expression of dysregulated cyclin genes. Of note, the activating nature of the NOTCH1 mutation in T lymphoblastic leukaemia also drives the dysregulation of cell cycle genes. Our goal was to comprehensively revisit the cell cycle in NOTCH1-mutated CLL (NOTCH1MUT ) to test for potential therapeutic targets. Among 378 NGS-annotated CLL cases, NOTCH1MUT cells displayed a unique transcriptome profile of G0 -G1 cell cycle components, with an overexpression of early-phase effectors, reaching a 38-, 27- and ninefold change increase for the complex elements CCND3, CDK4 and CDK6, respectively. This NOTCH1MUT cells' profile was related to more cells traversing through the cell cycle. In-vitro targeted inhibition of NOTCH1 gamma-secretase and CDK4/6 reversed the distribution of cells through the cycle phases and enhanced the killing of NOTCH1MUT CLL cells, suggesting new therapeutic approaches.

CDK4/6 initiates Rb inactivation and CDK2 activity coordinates cell-cycle commitment and G1/S transition

External signaling controls cell-cycle entry until cells irreversibly commit to the cell cycle to ensure faithful DNA replication. This process is tightly regulated by cyclin-dependent kinases (CDKs) and the retinoblastoma protein (Rb). Here, using live-cell sensors for CDK4/6 and CDK2 activities, we propose that CDK4/6 initiates Rb inactivation and CDK2 activation, which coordinates the timing of cell-cycle commitment and sequential G1/S transition. Our data show that CDK4/6 activation induces Rb inactivation and thereby E2F activation, driving a gradual increase in CDK2 activity. We found that rapid CDK4/6 inhibition can reverse cell-cycle entry until CDK2 activity reaches to high levels. This suggests that high CDK2 activity is required to initiate CDK2-Rb positive feedback and CDK4/6-indpendent cell-cycle progression. Since CDK2 activation also facilitates initiation of DNA replication, the timing of CDK2-Rb positive feedback is coupled with the G1/S transition. Our experiments, which acutely increased CDK2 activity by cyclin E1 overexpression, indicate that cells commit to the cell cycle before triggering DNA replication. Together, our data suggest that CDK4/6 inactivates Rb to begin E2F and CDK2 activation, and high CDK2 activity is necessary and sufficient to generate a bistable switch for Rb phosphorylation before DNA replication. These findings highlight how cells initiate the cell cycle and subsequently commit to the cell cycle before the G1/S transition.

220 (PB100) - Discovery of INCB123667, a potent and selective cyclin-dependent kinase 2 (CDK2) inhibitor for the treatment of cyclin E dysregulated cancers

Background: Dysregulation of the CDK/cyclin complex causes aberrant cell cycle entry and progression that can lead to cancer. For instance, the CDK2/ cyclin E complex governs the G1-S transition and overexpression of cyclin E has been associated with ovarian and breast cancers. Our initial studies have demonstrated that genetic depletion of CDK2 inhibits cell growth and survival in cancer cells overexpressing cyclin E. Furthermore, inhibition of CDK2 may be effective in cancers resistant to clinically approved CDK4/6 therapies as increased CDK2/cyclin E activity has been reported as a compensatory mechanism.