Cdk2 Activity Research Articles

Chromosome alignment and Kif18A action rely on spindle-localized control of Cdk1 activity

Chromosome alignment and Kif18A action rely on spindle-localized control of Cdk1 activity

Aurora B inhibitors promote RB hypophosphorylation and senescence independent of p53-dependent CDK2/4 inhibition

Aurora B kinase (AURKB) inhibitors have been trialled in a range of different tumour types but are not approved for any indication. Expression of the human papilloma virus (HPV) oncogenes and loss of retinoblastoma (RB) protein function has been reported to increase sensitivity to AURKB inhibitors but the mechanism of their contribution to sensitivity is poorly understood. Two commonly reported outcomes of AURKB inhibition are polyploidy and senescence, although their relationship is unclear. Here we have investigated the major cellular targets of the HPV E6 and E7, p53 and RB, to determine their contribution to AURKB inhibitor induced polyploidy and senescence. We demonstrate that polyploidy is a universal feature of AURKB inhibitor treatment in all cell types including normal primary cells, but the subsequent outcomes are controlled by RB and p53. We demonstrate that p53 by regulating p21 expression is required for an initial cell cycle arrest by inhibiting both CDK2 and CDK4 activity, but this arrest is only triggered after cells have undergone two failed mitosis and cytokinesis. However, cells can enter senescence in the absence of p53. RB is essential for AURKB inhibitor-induced senescence. AURKB inhibitor induces rapid hypophosphorylation of RB independent of inhibition of CDK2 or CDK4 kinases and p53. This work demonstrates that p53 activation determines the timing of senescence onset, but RB is indispensable for senescence.

Anti colorectal cancer activity and in silico studies of novel pyridine nortopsentin analog as cyclin dependent kinase 6 inhibitor

Nortopsentins are a vital class of deep-sea sponge metabolites which can be used as leads for antitumor agents. Although their action has been studied in several diseases’ contexts, their cytotoxic activity against colorectal carcinoma has not yet been fully investigated. Therefore, a series of 2,6-bis(1H-indol-3-yl)-4-(substituted-phenyl)pyridin-5-carbonitriles 4a–j (nortopsentin analogs) was investigated for their cytotoxic activity against colorectal carcinoma. The analog 4i showed the highest antitumor activity via inducing cell cycle arrest at G1 phase. Cell cycle arrest was induced due to expression downregulation of CDK2, CDK4, and CDK6. In addition, 4i suppressed the enzymatic activity of CDK6. The theoretical study of some basic quantum factors and the geometric shape of compound 4i proved that the compound is stable and a soft molecule, in which the EHOMO and ELUMO energies were negative and had a small ∆E gap. 4i also demonstrated a high potential for oral bioavailability due to its adherence to Lipinski’s rule of five. The molecular docking studies of 4i analog showed good binding mode with CDK6 active pocket through the formation of multiple interactions with its key amino acids.

Skrining fitokimia tumbuhan potensi obat kanker kolorektal Begonia medicinalis Ardi & D.C. Thomas

Cancer is a condition in which cells or tissues undergo abnormal progressive growth. Colorectal cancer is cancer that occurs in the colon to rectum organs of humans. Alternative therapy for colorectal cancer treatment is the use of herbs such as Begonia medicinalis Ardi & D.C. Thomas. This study aimed to screen the bioactive compounds in the Begonia medicinalis Ardi & D.C. Thomas. extract and its potential as a colorectal cancer drug. This research consists of two stages, namely the extraction stage using the maceration method and the screening stage. The results showed that the extract of Begonia medicinalis Ardi & D.C. Thomas. positively contains flavonoids, alkaloids, and saponins. These bioactive compounds have potential activity as preventive and curative agents for colorectal cancer through several mechanisms, including suppressing oxidative stress, inhibiting CDK activity, suppressing intestinal inflammation, and increasing floranormal diversity and probiotic barrier activity in the intestine.

Enhancing sensitivity to oxaliplatin in tongue squamous cell carcinoma: mechanistic insights and therapeutic potential of DHA combination therapy.

Squamous cell carcinoma of the tongue, a common and aggressive malignancy, poses a substantial threat to health and well-being. Despite the promising results of combination therapy with dihydroartemisinin (DHA) and oxaliplatin (Oxa) in various cancers, its effectiveness in treating tongue squamous cell carcinoma had not been explored prior to this study. Our research found that DHA significantly enhances the sensitivity of tongue squamous cell carcinoma cells to Oxa, even at very low concentrations. The combination treatment was observed to modulate the activity of CDK1 and Cyclin B1, arresting the cell cycle in the G2 phase. Additionally, it reduces mitochondrial membrane potential, prompting the release of cytochrome c and activating cleaved caspase-3, which promotes apoptosis. Notably, surface plasmon resonance and immunoprecipitation experiments revealed that DHA targets and attenuates CDK1 modification, weakening its interaction with STAT3 protein. This leads to reduced expression of anti-apoptotic genes and facilitates programmed cell death in CAL-27 cells. The findings underscore the potential of DHA and Oxa as a potent therapeutic strategy for tongue squamous cell carcinoma, opening avenues for clinical application and further exploration into its mechanistic pathways.

Non-fused Pyrimidine Derivatives as Potential Pharmacological Entities: A Review.

Non-fused pyrimidine scaffold is a significant component for designing new drugs. The review emphasizes the pharmacological importance of non-fused pyrimidine-containing moieties based on the broad spectrum of activities such as antiprotozoal, antibacterial, antimycobacterial, anticancer, anti-inflammatory activity, and CNS depressant. Pyrimidine derivatives are fascinating entities that display biological activities for the treatment of cancer. It also highlights the tendency of non-fused pyrimidine derivatives to suppress cell growth by obstructing the activity of VCP, CDK-2, EGFR, ATR, EphB4 & EphA2, PDGF as well as inhibitory action towards different cell lines such as MCF-7, HeLa, NCI/ADR-RES, NCIH23, HOP-92, HCT-116, OV-3, MOLT-4, PC-3, MDA-MB-231, MALME-3M, K562 and Bcr-Abl. The review details the importance of morpholine, piperidine, and pyrrolidine ring substitutions on pyrimidine moiety as well as the role of H-bonding and amino linkage along with antibacterial activity due to the presence of pleuromutilin and tetrazole molecules. Researchers were motivated to develop and enhance the non-fused pyrimidine scaffold to uncover novel medicines by reading this review article.

Hepatocyte-derived tissue extracellular vesicles safeguard liver regeneration and support regenerative therapy

Tissue-derived extracellular vesicles (EVs) are emerging as pivotal players to maintain organ homeostasis, which show promise as a next-generation candidate for medical use with extensive source. However, the detailed function and therapeutic potential of tissue EVs remain insufficiently studied. Here, through bulk and single-cell RNA sequencing analyses combined with ultrastructural tissue examinations, we first reveal that in situ liver tissue EVs (LT-EVs) contribute to the intricate liver regenerative process after partial hepatectomy (PHx), and that hepatocytes are the primary source of tissue EVs in the regenerating liver. Nanoscale and proteomic profiling further identify that the hepatocyte-specific tissue EVs (Hep-EVs) are strengthened to release with carrying proliferative messages after PHx. Moreover, targeted inhibition of Hep-EV release via AAV-shRab27a in vivo confirms that Hep-EVs are required to orchestrate liver regeneration. Mechanistically, Hep-EVs from the regenerating liver reciprocally stimulate hepatocyte proliferation by promoting cell cycle progression through Cyclin-dependent kinase 1 (Cdk1) activity. Notably, supplementing with Hep-EVs from the regenerating liver demonstrates translational potential and ameliorates insufficient liver regeneration. This study provides a functional and mechanistic framework showing that the release of regenerative Hep-EVs governs rapid liver regeneration, thereby enriching our understanding of physiological and endogenous tissue EVs in organ regeneration and therapy.

Design, synthesis and molecular modeling of pyrazoline based coumarin derivatives as tubulin polymerization inhibitors

A series of coumarin-pyrazoline hybrids was synthesized by cyclization of the coumarin chalcones with hydrazine hydrate and phenyl hydrazine through 1,4-addition on α,β-unsaturated carbonyl system. The synthesized derivatives were evaluated for their cytotoxicity against HCT116, MCF7 and BJ-1 cell lines. Among them, compounds 5d and 5f showed the best selectivity indices (IC50= 13.2 and 12.3 µM (HCT116), 5.0 and 5.5 µM (MCF7) and 26.2 and 26.5 µM (BJ-1), respectively). Derivative 5d revealed an inhibitory effect on tubulin assembly (IC50= 6.53 µM), and CDK2 activity (IC50= 0.47 µM). Induction of apoptosis was observed in MCF-7 cells after treatment with derivative 5d which was associated with a significant decrease in Bcl-2 level and 3-fold increase in Bax level. Moreover, derivative 5d revealed an anti-migration activity as detected by wound healing assay. The active compound 5d was docked within the β tubulin active site to investigate the protein-ligand interactions illustrating a unique binding mode with better score than colchicine (-8.5 kcal/mol). Those studies, along with pharmacokinetics predictions, provided a base for further optimization of compound 5d as a potential anti-tubulin agent.

Abstract PO-012: Gain-of-function small molecules to reprogram oncogenic transcription in lymphoma

Abstract Conventional pharmacotherapy in oncology centers on target-centric silencing of aberrant protein function through small molecule inhibitors, degraders, or CRISPR/RNAi. In contrast, here we leverage induced proximity to develop pharmacology that activates dominant signaling pathways for a therapeutic effect. To develop this concept, we describe a new class of bivalent small molecules that reprogram cancer-driving gene expression to activate cell death signaling in diffuse large B cell lymphomas (DLBCL). These molecules, called Transcriptional Chemical Inducers of Proximity (TCIPs), operate at fractional occupancy of their protein targets, without genomic modifications to the cell or organism. The first TCIPs transiently re-localized elongation factors such as BRD4 (TCIP1, PMID 37495688) to loci regulated by the transcription factor BCL6, inducing the expression of pro-apoptotic genes regulated by BCL6. In new, unpublished work, CDK-TCIPs have been made to re-direct the activity of transcriptional elongation kinases such as CDK9, CDK12, or CDK13 to induce the expression of pro-apoptotic BCL6-target genes. Profiling in ∼900 barcoded cancer cell lines show that the molecules exhibit potent cell killing potency in a highly DLBCL lineage-restricted manner. Cytotoxicity to healthy lymphocytes is 100- to 10,000-fold lower, although germinal center B cells are ablated in a dose-dependent manner consistent with the role of BCL6 in the germinal center. Biochemical, cell biological, proteomic, and genomic studies demonstrate that CDK-TCIPs operate via a gain-of-function mechanism reliant on the ternary complex formed by the bivalent molecule. Instead of global enzyme inhibition or degradation, CDK-TCIPs borrow and re-localize a fraction of total CDK activity to BCL6-bound chromatin, producing pharmacology from the induction of pro-apoptotic gene transcription. The findings suggest a path to rational design of new gain-of-function chemotherapeutic strategies to rewire cell signaling using induced proximity. Citation Format: Sai Gourisankar, Roman Sarott, Basel Karim, Sabin Nettles, Haopeng Yang, Brendan Dwyer, Juste Simanauskaite, Jason Tse, Hind Abuzaid, Stephen M Hinshaw, Michael R Green, Gerald R Crabtree, Nathanael S Gray. Gain-of-function small molecules to reprogram oncogenic transcription in lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-012.

Multiplexed Profiling of CDK Kinase Activities in Tumor Biopsies with Fluorescent Peptide Biosensors.

Detection of disease biomarkers constitutes a major challenge for the development of personalized and predictive diagnostics as well as companion assays. Protein kinases (PKs) involved in the coordination of cell cycle progression and proliferation that are hyperactivated in human cancers constitute attractive pharmacological targets and relevant biomarkers. Although it is relatively straightforward to assess the relative abundance of PKs in a biological sample, there is not always a direct correlation with enzymatic activity, which is regulated by several posttranslational mechanisms. Studies of relative abundance therefore convey limited information, and the lack of selective, sensitive, and standardized tools together with the inherent complexity of biological samples makes it difficult to quantify PK activities in physio-pathological tissues. To address this challenge, we have developed a toolbox of fluorescent biosensors that report on CDK activities in a sensitive, selective, dose-dependent, and quantitative fashion, which we have implemented to profile CDK activity signatures in cancer cell lines and biopsies from human tumors. In this study, we report on a standardized and calibrated biosensing approach to quantify CDK1,2,4, and 6 activities simultaneously through a combination of four different biosensors in a panel of 40 lung adenocarcinoma and 40 follicular lymphoma samples. CDK activity profiling highlighted two major patterns which were further correlated with age, sex of patients, tumor size, grade, and genetic and immunohistochemical features of the biopsies. Multiplex CDKACT biosensing technology provides new and complementary information relative to current genetic and immunohistochemical characterization of tumor biopsies, which will be useful for diagnostic purposes, potentially guiding therapeutic decision. These fluorescent peptide biosensors offer promise for personalized diagnostics based on kinase activity profiling.

Abstract PO3-18-06: First-in-human phase 1A study of RGT-419B, a next generation CDK4 inhibitor, in patients (pts) with hormone receptor positive (HR+) HER2- advanced/metastatic breast cancer (ABC) who progressed on prior CDK4/6 inhibitors (CDK4/6i)

Abstract Introduction: RGT-419B is a novel next generation CDK inhibitor. It has high potency against CDK4 with additional activity against CDK2 and selectivity against CDK6 to overcome resistance and reduce hematologic toxicity. In addition to anti-cancer activity in preclinical breast cancer models that are resistant to approved CDK4/6 inhibitors, we report the interim data from the first-in-human, multicenter trial of RGT-419B as a single agent in post-menopausal pts with HR+ HER2- ABC. Methods: Eligible pts received ≥2 lines of treatment and progressed on endocrine therapy (ET) and at least 1 line of an FDA approved CDK4/6i. Prior treatments of fulvestrant, other targeted agents and/or chemotherapy were allowed. Dose escalation of RGT-419B as oral (PO) monotherapy was administered in continuous 28-day cycles in 4 cohorts (25, 75,150 mg QD and 150 mg BID). A standard 3+3 design was employed to determine the maximum tolerated dose (MTD)/recommended dose for expansion (RDE). The primary objective was to assess safety and tolerability. The secondary objectives included pharmacokinetic (PK) assessment and exploratory efficacy. Results: At time of abstract submission, 12 eligible post-menopausal pts with HR+/HER2- ABC received RGT-419B. The median age was 64.8y (range 50-80y). All had prior palbociclib + ET (2 pts had abemaciclib or ribociclib after palbociclib); a majority received fulvestrant (67%) and prior chemotherapy (50%). Across the 4 cohorts (12 pts), the most observed treatment-emergent adverse events (TEAEs; all causality) with RGT-419B were nausea, reduced white blood cell counts (neutrophils and/or lymphocytes) and diarrhea. At data cutoff on June 30th 2023, there have been no CTCAE Grade 3 or higher treatment related AE (TRAEs). No ocular toxicity has been observed. No pts have discontinued treatment due to RGT-419B toxicity. Preliminary PK analyses for RGT-419B showed a dose-dependent increase of exposure at steady state with long half-life. Tumor assessments (RECIST v1.1) of pts in the first 3 cohorts treated with RGT-419B monotherapy (25-150 mg QD) showed partial responses in 2 pts (7 pts with measurable disease), 28.6% partial response (PR) (RECIST v. 1.1) and clinical benefit rate (CBR) 44%. Updated safety, PK and efficacy data will be presented. Conclusions: RGT-419B, with potent selective CDK4 activity, CDK2 activity and CDK6 selectivity, demonstrated a favorable safety and PK profile in an ongoing phase I study, with no grade 3 or higher TRAEs observed thus far. RGT-419B administered as once daily monotherapy also demonstrated preliminary evidence of efficacy as well. Dose expansions of RGT-419B as a single agent and in combination with ET in pts with HR+/HER2- ABC following prior CDK4/6i progression are planned. Citation Format: Seth Wander, Dave Valacer, Richard Zuniga, Heather Yeckes-Rodin, Christopher Perkins, Lisa Ge, Lili Yao, Jing Lin, Dawn Begley, Feifei Sun, Andie Zheng, Jing Han, Zhi (Julie) Xie, Aditya Bardia, Kevin Kalinsky. First-in-human phase 1A study of RGT-419B, a next generation CDK4 inhibitor, in patients (pts) with hormone receptor positive (HR+) HER2- advanced/metastatic breast cancer (ABC) who progressed on prior CDK4/6 inhibitors (CDK4/6i) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-18-06.

Abstract PO3-20-02: Olaparib front line therapy with endocrine therapy in locally advanced unresectable or in metastatic breast cancer: OFELIA TRIAL

Abstract INTRODUCTION: Currently, patients with recurrent or metastatic hormone-sensitive breast cancer are treated with endocrine therapy (ET) in combination with cyclin inhibitors, as it has been shown to improve both overall survival and progression-free survival. However, it has been observed that patients with gBRCA1 or gBRCA2 mutations, who receive treatment with endocrine therapy and CDK4/6 inhibitors, have lower OS and lower PFS compared to those without mutations. These findings could be due to the presence of higher cyclin E mRNA levels, which encode the protein that stimulates CDK2 activity provoking a resistance to CDK4/6 inhibitors. Having this said, the use of PARP inhibitors a potential first-line therapeutic option in combination with endocrine therapy could be an option to be re evaluated. JUSTIFICATION: PARP inhibitors (iPARP) are recommended in international guidelines as second-line palliative treatment and also as adjuvant management in the case of Olaparib. The use of iPARP is approved only as a second-line treatment in HER2 hormone-sensitive breast cancer, according to the results of the EMBRACA and OlympiAD clinical trials. However, these two clinical trials were not designed to evaluate its effectiveness as first-line palliative treatment. Given that patients with gBRCA mutations have lower overall survival when administered cyclin inhibitors (iCDK 4/6) + Endocrine therapy (ET) compared to those without mutations, it is necessary to reevaluate the use of iPARP as first-line palliative treatment in conjunction with ET, with the expectation of achieving a similar objective response rate and progression-free survival. OBJECTIVE: To Determine the ORR in patients with recurrent or metastatic hormone-sensitive HER2-negative breast cancer managed with PARP inhibitors + Endocrine therapy. HYPOTHESIS: The use of PARP inhibitors in combination with endocrine therapy will have an objective response rate of 60% or higher in both early stages and advanced disease. METHODOLOGY: Phase 2 open-label, multicenter clinical trial. Study Population: Cohort : All pre- or postmenopausal patients with a diagnosis of recurrent or metastatic hormone-sensitive HER2-negative breast cancer with gBRCA1 and/or gBRCA2 mutation. Sample: The inclusion of patients will be based on meeting the inclusion criteria. We plan to achieve an ORR of at least 60% with an acceptable minimum of 40%. Assuming a one-sided alpha level of 0.05 and statistical power of 80%, a two-stage Simon design was used to calculate the required sample size. In the first stage, a minimum of 23 patients is assumed, of which 14 patients need to achieve the desired response rate to proceed to the second stage. In the second stage, an additional 28 patients will be added to obtain a total sample size of 51 patients. The objective response rate will be evaluated using RECIST v1.1 criteria at 8 weeks after initiating treatment, with continued assessment during the first year, followed by assessments every 12 weeks during the second and third year, and then every 24 weeks until progression or toxicity. EXPECTED RESULTS: PARP inhibitors + Endocrine therapy will have a similar ORR as reported with cyclin inhibitors + Endocrine therapy, as well as a benefit in progression-free survival. Citation Format: Carlos Arturo González Núñez, Paula Cabrera-Galeana, Juan Enrique Bargalló Rocha, Rafael Vazquez-Romo, Sergio Aguilar-Villanueva, Alexandra Garcilazo, Areli Velazquez-Martinez, Andrea Maliachi, Ruben Rodriguez. Olaparib front line therapy with endocrine therapy in locally advanced unresectable or in metastatic breast cancer: OFELIA TRIAL [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-20-02.

Network pharmacology, single gene survival analysis and molecular docking to study the mechanism of Sotetsuflavone in the treatment of pancreatic cancer

Pancreatic cancer is a highly lethal cancer with limited treatment options. The number of pancreatic cancer patients is increasing rapidly worldwide. Many natural products have been shown to have anticancer activity in a range of studies. Sotetsuflavone is derived from Cycas revoluta Thunb. and exhibits anticancer activity. The present study incorporates network pharmacology, single gene survival analysis, gene expression analysis and molecular docking to reveal the mechanism of Sotetsuflavone in the treatment of pancreatic cancer. We have acquired 31 hub targets for the treatment of pancreatic cancer by Sotetsuflavone, namely ABCB1, AURKA, CDK1, and so on. Kaplan-Meier survival analyses demonstrated that ABCB1, AURKA, CDK1, HDAC6, MET, and MMP3 are promising hub targets that can be used as biomarkers for pancreatic cancer diagnosis and prognosis. These hub targets are highly expressed in pancreatic cancer tissues compared to normal tissues. The molecular docking results showed a strong binding capacity of Sotetsuflavone to these hub targets. In summary, it is proposed that Sotetsuflavone is a new anticancer drug, which can regulate pancreatic cancer-related signalling pathways by inhibiting the activities of ABCB1, AURKA, CDK1, HDAC6, MET, and MMP3, which are hub targets with up-regulated expression in pancreatic cancer tissues, in order to treat pancreatic cancer. However, it also requires a series of in vivo and in vitro studies to ensure its safety and efficacy.

Kinase activities in pancreatic ductal adenocarcinoma with prognostic and therapeutic avenues.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a limited number of known driver mutations but considerable cancer cell heterogeneity. Phosphoproteomics provides a direct read-out of aberrant signaling and the resultant clinically relevant phenotype. Mass spectrometry (MS)-based proteomics and phosphoproteomics were applied to 42 PDAC tumors. Data encompassed over 19 936 phosphoserine or phosphothreonine (pS/T; in 5412 phosphoproteins) and 1208 phosphotyrosine (pY; in 501 phosphoproteins) sites and a total of 3756 proteins. Proteome data identified three distinct subtypes with tumor intrinsic and stromal features. Subsequently, three phospho-subtypes were apparent: two tumor intrinsic (Phos1/2) and one stromal (Phos3), resembling known PDAC molecular subtypes. Kinase activity was analyzed by the Integrative iNferred Kinase Activity (INKA) scoring. Phospho-subtypes displayed differential phosphorylation signals and kinase activity, such as FGR and GSK3 activation in Phos1, SRC kinase family and EPHA2 in Phos2, and EGFR, INSR, MET, ABL1, HIPK1, JAK, and PRKCD in Phos3. Kinase activity analysis of an external PDAC cohort supported our findings and underscored the importance of PI3K/AKT and ERK pathways, among others. Interestingly, unfavorable patient prognosis correlated with higher RTK, PAK2, STK10, and CDK7 activity and high proliferation, whereas long survival was associated with MYLK and PTK6 activity, which was previously unknown. Subtype-associated activity profiles can guide therapeutic combination approaches in tumor and stroma-enriched tissues, and emphasize the critical role of parallel signaling pathways. In addition, kinase activity profiling identifies potential disease markers with prognostic significance.

Sustained ERK signaling promotes G2 cell cycle exit and primes cells for whole-genome duplication

Whole-genome duplication (WGD) is a frequent event in cancer evolution that fuels chromosomal instability. WGD can result from mitotic errors or endoreduplication, yet the molecular mechanisms that drive WGD remain unclear. Here, we use live single-cell analysis to characterize cell-cycle dynamics upon aberrant Ras-ERK signaling. We find that sustained ERK signaling in human cells leads to reactivation of the APC/C in G2, resulting in tetraploid G0-like cells that are primed for WGD. This process is independent of DNA damage or p53 but dependent on p21. Transcriptomics analysis and live-cell imaging showed that constitutive ERK activity promotes p21 expression, which is necessary and sufficient to inhibit CDK activity and which prematurely activates the anaphase-promoting complex (APC/C). Finally, either loss of p53 or reduced ERK signaling allowed for endoreduplication, completing a WGD event. Thus, sustained ERK signaling-induced G2 cell cycle exit represents an alternative path to WGD.

Abstract LB265: IpY.20: Innovative strategies for post-CDK estrogen receptor (ER)+ metastatic breast cancer (mBC)

Abstract Cyclin-dependent kinases (CDK) 4/6 are key cell cycle regulators that form complexes with cyclin D1 and the master regulator p27Kip1. This ternary complex acts downstream of the ER- and HER2-pathways, which drive cell proliferation. CDK4/6 phosphorylates the Retinoblastoma protein and other substrates, and keeps p27 from inhibiting G1-associated CDK2. Targeted inhibition of CDK4/6 (CDK4/6i) blocks the growth of ER+ and HER2+ BC by dissolving the CDK4/6-cyclin D1 complex and permitting the shuttling of p27 to inhibit CDK2 complex. Approval of CDK 4/6i such as palbociclib, though a game changer for ER+ mBC, resulted in tumor resistance, leaving patients without effective treatments and a 5-yr survival of only 30%. Although CDK2 inhibitors (CDK2i) address compensatory CDK2 activity bypassing CDK4/6i, a key resistance mechanism, they lack selectivity due to CDK homology, resulting in off-target toxicity. To inhibit CDK2/4/6 simultaneously, combinations of CDK4/6i and CDK2i monotherapies were attempted, but the lack of coordination between CDK4/6 and CDK2 inhibition resulted in suboptimal efficacy. Concarlo presents a unique therapeutic rationale for directly targeting the CDK master regulator p27 to simultaneously inhibit CDK2/4/6 in a concerted manner, one that is guided by the role of p27 to turn all of the G1 CDKs ON and OFF during periods of proliferation and quiescence, respectively. This transition is facilitated by BRK (Breast tumor kinase)-mediated phosphorylation of p27. When p27 is Y phosphorylated, it converts CDK4/6 and CDK2 into open, active complexes. An endogenous BRK-spliced variant ALT can competitively bind to p27, block Y phosphorylation and lock CDK2/4/6 in the OFF conformation. Concarlo engineered a shorter variant of ALT (CCL20) and packaged in cationic liposomes for intracellular delivery, creating a novel IpY.20 drug product. As a single agent, IpY.20 inhibits cell proliferation in treatment-naïve and CDK4i-resistant HR+ cells with reduced non-tumor cell activity. In combination, IpY.20 enhances CDK4/6i responses in treatment-naïve HR+ cells, making it a potential front-line mBC therapy. While other CDKi activity is limited to cytostasis, IpY.20 shows dual cytostasis-cytotoxicity, potentially a more durable option. IpY.20 does not cause neutropenia commonly seen with other CDKis, and rescues the CDK4/6i-induced neutropenia in mice, suggesting better clinical tolerability. These findings underscore the significance of coordinated targeting of CDK2/4/6, making IpY.20 a first-in-class product uniquely exploiting p27-mediated CDK2/4/6 regulation. Direct targeting of p27 presents a new treatment paradigm bypassing the challenges of ATP-competitive CDKis, with potential to expand into tumor types with aberrant p27, CDK2/4/6 activity, or RAS/MAPK pathways that increase CDK4/6 activity. Citation Format: Grace Chen, Natalie Dudas, Kate Coleman, Carolina Guido, Natalia Zisman, Krishna Allamneni, Stacy Wister Blain. IpY.20: Innovative strategies for post-CDK estrogen receptor (ER)+ metastatic breast cancer (mBC) [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 LB265.

Abstract 5903: MASTL small-molecule inhibition to target mitosis for cancer therapy

Abstract Cell cycle/tumor repressors can be pharmacologically activated to repress uncontrollable proliferation of cancer cells. However, targeting cell cycle regulators of G2/M and mitotic checkpoints to achieve maximum therapeutic benefit remains a challenge due to a limited therapeutic index. Here, we identified highly potent and selective oral bioavailable clinical candidate inhibitors to the first-in-class oncology target Microtubule Associated Serine/Threonine Kinase-Like (MASTL) aimed at expanding therapeutic control of the cell cycle for cancer therapy. Breath of efficacy screening in multiple tumor indications in vitro followed by computational analyses identified the regulatory subunit PPP2R2A as a potential patient selection biomarker that strongly correlates to MASTL inhibitor response irrespective of cancer genetic alterations. Functional genetics studies confirmed that sensitivity to MASTL inhibition is defined by PPP2R2A expression. To identify genetic mediators of MASTL inhibitor resistance, a whole genome CRISPR knockout screen with the MASTL inhibitor was performed and further established that the MASTL inhibitor response is dependent on PPP2R2A containing PP2A complexes. Mechanistic studies (proteomics, resistance, functional genetics, and time-lapse microscopy) demonstrated that MASTL inhibition disrupts mitosis by activating the tumor suppressor PP2A, leading to a switch from CDK1 activity to CDK1 inactivity, mitotic defects, and cancer cell death. MASTL inhibition treatment in tumor models in vivo and in patient-derived organoid models results in potent tumor growth inhibition. Our results demonstrate that the first identified highly selective MASTL clinical candidate inhibitors represent a potential new cancer therapeutic strategy to target mitosis and mitotic exit by activating the tumor repressor PP2A to counter CDK1 activity. All procedures performed on animals were in accordance with regulations and established guidelines and were reviewed and approved by an Institutional Animal Care and Use Committee or through an ethical review process. Citation Format: Chad M. Toledo, Koleen J. Eisele, Dong Hyun Kim, Xinmeng Mu, Sonja Brun, Carl Davis, Eric C. Greenwald, Fion Hui, Meilan He, Yanling Yang, Ana Flores-Bojorquez, Yuxin Li, Penney L. Khamphavong, Joseph H. Lee, Jian Li, Dana J. Ramms, Greg Jones, Marin Auth, Cory L. Painter, Andrew Nager, Jon Oyer, Sergei L. Timofeevski, William E. Pierceall, Anwar Murtaza, Jonathan R. Heyen, Rebecca Gallego, Casey Quinlan, Indrawan McAlpine, Todd L. VanArsdale. MASTL small-molecule inhibition to target mitosis for cancer therapy [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 5903.

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 1813: Discovery, biomarker validation, and pre-clinical profiling of a novel PKMYT1 inhibitor

Abstract Protein kinase membrane-associated tyrosine/threonine 1 (PKMYT1) is a negative regulator of CDK1 activity and has been identified as a putative synthetic lethal target in cancers which upregulate cyclin E (CCNE1). We used high-throughput virtual screening to identify inhibitors of PKMYT1 with high lipophilic ligand efficiency (LLE), which were rapidly optimized to potent leads with exceptional selectivity over a key off target, WEE1. Machine learning-driven optimization rapidly reduced mouse hepatocyte clearance to acceptable levels, delivering a compound with best-in-class potency, selectivity, and pharmacokinetic profiles in relevant animal models. Tool compounds from this series were used to validate a differentiated biomarker for sensitivity to PKMYT1 inhibition in cellular models, which could significantly expand the potential patient population. In vivo proof-of-concept studies are ongoing in a variety of cell line- and patient-derived xenograft models which represent the novel biomarker previously identified, to further validate our therapeutic strategy. Citation Format: Alfie Brennan, Jan Lanz, Daniel S. Miller, Oliver Vipond, Noah Harrison, James Aaronson, Anna Hercot. Discovery, biomarker validation, and pre-clinical profiling of a novel PKMYT1 inhibitor [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 1813.

Abstract 591: Discovery of ZM-2322, a highly selective, potent inhibitor of membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase (PKMYT1)

Abstract The genome DNA is consistently threatened by a variety of intrinsic and exogenous DNA damage factors. Cell cycle checkpoint is one important mechanism in response to DNA damage and maintaining genome stability. In tumor cells, gene alterations, such as CCNE1 amplification (encoding for Cyclin E1), result in G1/S checkpoint loss and replication stress, leading to high dependency on the G2/M checkpoint. PKMYT1 inhibits crucial checkpoint protein CDK1 activity by phosphorylating CDK1 at the residues of Thr-14 and Tyr-15 in the G2/M phase. In CCNE1 amplification cells, the inhibition of PKMYT1 leads to the reduction of pCDK1 and loss of G2/M checkpoint, consequently results in premature mitosis and the accumulation of DNA damage, then finally induces cell death. Here we identify a novel small molecular ZM-2322, which displays robust intracellular binding to PKMYT1 and inhibits the specific downstream phosphorylation of CDK1 Thr-14 with IC50 values of 14 nM and 29 nM, respectively. ZM-2322 strongly inhibits the proliferation of the HCC1569 cell line with CCNE1 high-level amplification and shows a >400 × selectivity folds over a CCNE1 wildtype cell line SKOV3. ZM-2322 also shows significant inhibition of tumor growth in a dose-dependent manner in the HCC1569 xenograft model, and the efficacy correlates well with PK and target inhibition. There is evidence showing that loss-of-function mutations of FBXW7, a cyclin E E3 ligase, lead to cyclin E accumulation and are synthetic-lethal with PKMYT1 inhibition. Consistently, a potent anti-proliferation of ZM-2322 is observed in DLD-1 FBXW7 -/- cells, but not DLD-1 parental cells. These data suggest a high potency of ZM-2322 on PKMYT1 inhibition and a potential good kinase selectivity which is further confirmed by using a kinase panel biochemical assay. As expected, when compared head-to-head with another clinical PKMYT1 inhibitor RP-6306 which shows inhibitory effect on 27 of 217 human kinases with inhibition rates >90% at 1 μM, ZM-2322 is found to be a much more PKMYT1-selective inhibitor and hits only several individual kinases under the same assay condition, suggesting a potentiality in better safety profile and higher dose tolerance. Furthermore, our data illustrates that in combination with gemcitabine, ZM-2322 elicits strong synergetic anti-proliferation activities on HCC1569 cells in vitro and results in tumor regression with a minor effect on body weight in vivo. Besides, a strong synergy effect is also observed in the combo of ZM-2322 and ATR inhibitor in HCC1569 in vitro. These data indicate that ZM-2322 has the potential of application expansion and good tolerance under continuous administration. Taken together, our data suggest that ZM-2322 is a potent and highly selective PKMYT1 inhibitor. Citation Format: Feng Zhou, Lu Liu, Lei Jiang, Baoying Cheng, Zhentao Li, Dongxing Zhu, Jianbin Xue, Liting Xue, Renhong Tang. Discovery of ZM-2322, a highly selective, potent inhibitor of membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase (PKMYT1) [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 591.