Cdk1 Activation Research Articles

Notch signaling mediated repressive effects of resveratrol in inducing caspasedependent apoptosis in MCF-7 breast cancer cells.

Resveratrol, a potent anticancer bioactive compound, has been shown to trigger apoptosis in numerous cancer cells. Although Notch signaling promotes breastcancer apoptosis, it is unclear whether resveratrolinduces apoptosis in MCF-7cells via influencing the Notch pathway. This study aimed to evaluate the effect of resveratrolon modulating Notch signaling targetsand provide critical information for employing resveratrolin breastcancer therapy. Thus, in this study, we have deciphered the effect of resveratrol against three potent genes (Notch1, Jagged1, and DLL4) of the notch signaling pathway. For mechanistic studies, in silico, and in vitro analysis was executed to investigate the apoptotic-inducing potential of resveratrol against three selected oncogenes involved in the progression of breast cancer. Docking analysis revealed the inhibitory potential of resveratrol against all three selected targets of the Notch pathway (Notch1: -5.0; Jagged-1: -5.9; DLL4: -5.8). In vitro, findings further displayed a significant reduction in cell viability in resveratrol-treated MCF-7 cancer cells, which were concomitantly related to the downregulation of Notch-1, Jagged-1, and DLL4. Moreover, the antiproliferative efficacy of resveratrol was correlated with apoptosis and modulation in the expression of Bax, Bcl-2, cyclin D1, CDK4, p21, and caspase-3 activation. Taken together, these experimental findings suggested that apoptotic inducing potential of resveratrol was mediated through a novel mechanism involving suppression of the Notch signaling pathway.

GRK2-mediated AKT activation controls cell cycle progression and G2 checkpoint in a p53-dependent manner

Cell cycle checkpoints, activated by stressful events, halt the cell cycle progression, and prevent the transmission of damaged DNA. These checkpoints prompt cell repair but also trigger cell death if damage persists. Decision-making between these responses is multifactorial and context-dependent, with the tumor suppressor p53 playing a central role. In many tumor cells, p53 alterations lead to G1/S checkpoint loss and the weakening of the G2 checkpoint, rendering cell viability dependent on the strength of the latter through mechanisms not fully characterized. Cells with a strong pro-survival drive can evade cell death despite substantial DNA lesions. Deciphering the integration of survival pathways with p53-dependent and -independent mechanisms governing the G2/M transition is crucial for understanding G2 arrest functionality and predicting tumor cell response to chemotherapy. The serine/threonine kinase GRK2 emerges as a signaling node in cell cycle modulation. In cycling cells, but not in G2 checkpoint-arrested cells, GRK2 protein levels decline during G2/M transition through a process triggered by CDK2-dependent phosphorylation of GRK2 at the S670 residue and Mdm2 ubiquitination. We report now that this downmodulation in G2 prevents the unscheduled activation of the PI3K/AKT pathway, allowing cells to progress into mitosis. Conversely, higher GRK2 levels lead to tyrosine phosphorylation by the kinase c-Abl, promoting the direct association of GRK2 with the p85 regulatory subunit of PI3K and AKT activation in a GRK2 catalytic-independent manner. Hyperactivation of AKT is conditioned by p53’s scaffolding function, triggering FOXO3a phosphorylation, impaired Cyclin B1 accumulation, and CDK1 activation, causing a G2/M transition delay. Upon G2 checkpoint activation, GRK2 potentiates early arrest independently of p53 through AKT activation. However, its ability to overcome the G2 checkpoint in viable conditions depends on p53. Our results suggest that integrating the GRK2/PI3K/AKT axis with non-canonical functions of p53 might confer a survival advantage to tumor cells.

Structural basis of Cdk7 activation by dual T-loop phosphorylation

Cyclin-dependent kinase 7 (Cdk7) is required in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of transcription factor TFIIH. Cdk7 forms active complexes by associating with Cyclin H and Mat1, and is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates basic residues conserved in other CDKs, pS164 nucleates an arginine network unique to the ternary Cdk7 complex, involving all three subunits. We identify differential dependencies of kinase activity and substrate recognition on the individual phosphorylations. CAK function is unaffected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by T170 phosphorylation. Moreover, dual T-loop phosphorylation stimulates multisite phosphorylation of the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7 activation is a two-step process wherein S164 phosphorylation precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing processivity, while pT170 enhances activity towards key transcriptional substrates.

Caveolin-2 controls preadipocyte survival in the mitotic clonal expansion for adipogenesis

Here, we report that Caveolin-2 (Cav-2) is a cell cycle regulator in the mitotic clonal expansion (MCE) for adipogenesis. For the G2/M phase transition and re-entry into the G1 phase, dephosphorylated Cav-2 by protein tyrosine phosphatase 1B (PTP1B) controlled epigenetic activation of Ccnb1, Cdk1, and p21 in a lamin A/C-dependent manner, thereby ensuring the survival of preadipocytes. Cav-2, associated with lamin A/C, recruited the repressed promoters of Ccnb1 and Cdk1 for activation, and disengaged the active promoter of p21 from lamin A/C for inactivation through histone H3 modifications at the nuclear periphery. Cav-2 deficiency abrogated the histone H3 modifications and impeded the transactivation of Ccnb1, Cdk1, and p21, leading to a delay in mitotic entry, retardation of re-entry into G1 phase, and the apoptotic cell death of preadipocytes. Re-expression of Cav-2 restored the G2/M phase transition and G1 phase re-entry, preadipocyte survival, and adipogenesis in Cav-2-deficient preadipocytes. Our study uncovers a novel mechanism by which cell cycle transition and apoptotic cell death are controlled for adipocyte hyperplasia.

Highlighting the Major Role of Cyclin C in Cyclin-Dependent Kinase 8 Activity through Molecular Dynamics Simulations.

Dysregulation of cyclin-dependent kinase 8 (CDK8) activity has been associated with many diseases, including colorectal and breast cancer. As usual in the CDK family, the activity of CDK8 is controlled by a regulatory protein called cyclin C (CycC). But, while human CDK family members are generally activated in two steps, that is, the binding of the cyclin to CDK and the phosphorylation of a residue in the CDK activation loop, CDK8 does not require the phosphorylation step to be active. Another peculiarity of CDK8 is its ability to be associated with CycC while adopting an inactive form. These specificities raise the question of the role of CycC in the complex CDK8-CycC, which appears to be more complex than the other members of the CDK family. Through molecular dynamics (MD) simulations and binding free energy calculations, we investigated the effect of CycC on the structure and dynamics of CDK8. In a second step, we particularly focused our investigation on the structural and molecular basis of the protein-protein interaction between the two partners by finely analyzing the energetic contribution of residues and simulating the transition between the active and the inactive form. We found that CycC has a stabilizing effect on CDK8, and we identified specific interaction hotspots within its interaction surface compared to other human CDK/Cyc pairs. Targeting these specific interaction hotspots could be a promising approach in terms of specificity to effectively disrupt the interaction between CDK8. The simulation of the conformational transition from the inactive to the active form of CDK8 suggests that the residue Glu99 of CycC is involved in the orientation of three conserved arginines of CDK8. Thus, this residue may assume the role of the missing phosphorylation step in the activation mechanism of CDK8. In a more general view, these results point to the importance of keeping the CycC in computational studies when studying the human CDK8 protein in both the active and the inactive form.

The Role of Speedy/RINGO Protein in Breast Cancer as a Future Biomarker.

Cyclin-dependent kinases (CDKs) are proteins that require the binding of regulatory subunits called cyclins and play a key role in cell cycle progression and activation. CDKs play a key role in carcinogenesis of many solid malignancies, and inhibition of these proteins has produced anti-cancer effects demonstrated in preclinical studies. This narrative review was conducted to develop a hypothetical approach to determine whether Speedy/RINGO, a protein associated with CDK2, could be a possible predictive factor in breast cancer patients treated with a CDK4/6 inhibitor. A literature search was conducted in PubMed, Web of Science, Medline, and Google Scholars search engines to match the following words: "Speedy/RINGO" or "Spy1" and "CDKs" or "Cyclin-dependent kinases (CDKs)" and "CDK4/6 inhibitors" and "Regulation" and "Molecular" and "Breast cancer" and "Carcinogenesis". Only articles investigating the relationship between the Speedy/RINGO protein and CDKs at the molecular level were included. Literature information was compiled by trying to establish a relationship with our hypothesis question. Speedy/RINGO is a tightly regulated proto-oncogenic mammalian protein playing important roles in the somatic cell cycle. Studies have emphasized that although it does not have amino acid sequence homology with cyclins, it can activate CDK2. In addition, results showing molecular compensation of CDK4/6 inhibition through CDK2 activation, also showed that CDK2 can predict drug resistance. Another important finding was that overexpressed Speedy/RINGO, during CDK4/6 inhibitor treatment, could strongly activate CDK2, resulting in a negative response to treatment. Although many predictive factors have been investigated to indicate response to CDK4/6 inhibitors or determine drug resistance, a consensus biomarker has yet to be established. In light of the information obtained from our review, it can be concluded that the Speedy/RINGO protein may have an important role as a predictive biomarker in terms of response to treatment, continuity of treatment and drug resistance in patients treated with CDK4/6 inhibitors.

Slower CDK4 and faster CDK2 activation in the cell cycle

Dysregulation of cyclin-dependent kinases (CDKs) impacts cell proliferation, driving cancer. Here, we ask why the cyclin-D/CDK4 complex governs cell cycle progression through the longer G1 phase, whereas cyclin-E/CDK2 regulates the shorter G1/S phase transition. We consider available experimental cellular and structural data including cyclin-E's high-level burst, sustained duration of elevated cyclin-D expression, and explicit solvent molecular dynamics simulations of the inactive monomeric and complexed states, to establish the conformational tendencies along the landscape of the distinct activation scenarios of cyclin-D/CDK4 and cyclin-E/CDK2 in the G1 phase and G1/S transition of the cell cycle, respectively. These lead us to propose slower activation of cyclin-D/CDK4 and rapid activation of cyclin-E/CDK2. We provide the mechanisms through which this occurs, offering innovative CDK4 drug design considerations. Our insightful mechanistic work addresses a compelling cell cycle regulation question and illuminates the distinct activation speeds between the G1 and the G1/S phases, which are crucial for function.

Abstract GS01-08: CDK4/6 inhibition is a potential vulnerability in NF1-depleted ER+ breast cancer

Abstract CDK4/6 control a key checkpoint by phosphorylating Rb, leading to Rb degradation and subsequent S-phase entry. CDK4/6 inhibition, together with endocrine therapy, is standard of care for advanced ER+ breast cancer; however, CDK4/6 inhibition typically is cytostatic. This study aims to identify breast cancer patients who may better respond to treatments targeting CDK4/6. NF1/neurofibromin is a key tumor suppressor that we have shown can represses not only RAS as a GAP but also ER as a transcriptional co-repressor. Dual activation of RAS and ER promotes endocrine therapy resistance. We computed kinase activity scores from phosphor-proteomic data in the CPTAC breast cancer cohort and found that both CDK4/6 activity scores and Rb-pS780 levels negatively correlated with NF1 protein levels. Furthermore, NF1 mutations co-occur with mutations in CDK4/6 and CCND1-3 in breast tumors. These results suggest a functional dependency between NF1 loss and activation of the CDK4/6-Rb pathway. To delineate how NF1 and CDK4 interact molecularly, we depleted NF1 in ER+ breast cancer cells and found an increase in Rb-pS780 and in phosphorylation in CDK4’s activation loop (CDK4-pT172). Cyclin-D binding is required for CDK4 activation. CCND1 expression is under the direct control by ER, and upon NF1-depletion, Cyclin D1 expression was increased, partly due to enhanced ER recruitment to an ERE in CCND1. Cyclin-D-bound CDK4 must be phosphorylated at T172 to be fully active; however, what kinase is responsible for this is largely unknown. We have evidence that CDK4-T172 phosphorylation is RAF, but not ERK, dependent. When the NF1-depleted ER+ cells were seeded in the presence of fulvestrant together with either palbociclib or abemaciclib, apoptosis readily occurred. Remarkably, fulvestrant plus palbociclib efficiently caused durable tumor regression in two ER+ PDX models, in which NF1 is undetectable by IHC. In contrast, no tumor regression was seen in a PDX model with detectable NF1. Finally, greater growth inhibition and apoptosis were detected in NF1-low tumors in the neoadjuvant NeoPalAna trial when palbociclib was added after anastrozole. These data support a model whereby ER and RAS signaling converge upon CDK4/6, and CDK4/6 activation is a key survival mechanism when ER signaling is attenuated by treatment in NF1-depleted ER+ breast cancer cells. This apparent addiction for CDK4/6 activity makes NF1-depleted ER+ breast tumors vulnerable to CDK4/6 inhibition, thus creating a potential therapeutic opportunity to match CDK4/6 inhibition with patients who can benefit the most. Citation Format: Ze-Yi Zheng, Anran Chen, Eric Jaehnig, Meenakshi Anurag, Jonathan Lei, Chenwei Wang, Long Feng, Purba Singh, Hilda Kennedy, Jin Cao, Ghazal Yadav, Jill Tsai, Xi Chen, Charles Foulds, Bora Lim, Matthew Ellis, Bing Zhang, Eric Chang. CDK4/6 inhibition is a potential vulnerability in NF1-depleted ER+ breast cancer [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 GS01-08.

Abstract 5710: The brain penetrant CDK4/6 inhibitor, PRT3645, is highly effective in combination with other targeted therapies in preclinical models of breast cancer, CRC and NSCLC

Abstract Cell cycle deregulation is a hallmark of cancer and the hyperactivation and overexpression of cyclin-dependent kinase (CDK)s are often drivers of cancer pathogenesis. CDK4/6 are critical mediators of cellular transition into S phase and important for cancer initiation, growth, and survival. Previously we reported a novel brain penetrant CDK4/6 inhibitor, PRT3645, that exhibits single digit nanomolar biochemical potency against CDK4/6 and >2000-fold selectivity against other CDKs. PRT3645 inhibits cellular phosphorylation of Rb with nanomolar potency. Furthermore, PRT3645 exhibits favorable in-vivo safety pharmacology and ADME properties, including increased brain penetration, and demonstrates oral bioavailability across rodents, dog and non-human primates. Here, we explored the therapeutic potential of PRT3645 in tumor models harboring actionable genomic alterations but lacking effective targeted treatments. Specifically, we explored the combination of PRT3645 with a selective estrogen receptor degrader (SERD), an approved treatment for patients with HR+/HER2-, ESR1-mutated advanced or metastatic breast cancer. In an ESR1 mutated breast cancer PDX model, PRT3645 not only exhibited monotherapy activity but also induced significant tumor regression in combination with a SERD through inhibiting ER-mediated signaling. We also investigated PRT3645 single agent activity and combination with a clinically validated MEK inhibitor in a BRAF class III mutant colorectal CDX model. PRT3645 was well-tolerated and demonstrated significant anti-tumor efficacy, which was further enhanced by combination therapy. To further explore comprehensive inhibition of cell cycle progression and overcome potential resistance to CDK4/6 inhibition, we investigated dual inhibition of CDK4/6 and CDK2. CDK2 is a crucial player in regulating the cell cycle by controlling the late G1/S transition, promoting DNA replication, and contributing to the DNA repair processes. Additionally, CDK2 activation has been identified as a potential mechanism of resistance to CDK4/6 inhibition. We evaluated CDK2 and CDK4/6 dual inhibition in CDKN2A loss or CCNE amplified cancer cell lines and observed enhanced proliferation suppression, cell cycle arrest and senescence induction, underscoring the potential of this combination for improved therapeutic efficacy across various cancer types. In summary, PRT3645 displays an excellent balance of potency, selectivity, pharmacokinetic parameters across species as well as brain penetrance. In preclinical studies, PRT3645 was highly efficacious when combined with other targeted therapies, offering potential benefits across a wide spectrum of cancer types. In addition, the concept of dual inhibition of CDK4/6 and CDK2 presents a rational and promising approach to enhance the efficacy of cancer therapy. Citation Format: Yue Zou, Kirsten Gallagher, Srijita Dhar, Andrew Busking, Sarah Pawley, Ryan Holmes, Xiaowei Wu, Min Wang, Joseph Rager, Tom Emm, Stefan Ruepp, Miles Cowart, Andrew Combs, Kris Vaddi, Sandy Geeganage, Peggy Scherle, Sang Hyun Lee. The brain penetrant CDK4/6 inhibitor, PRT3645, is highly effective in combination with other targeted therapies in preclinical models of breast cancer, CRC and NSCLC [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 5710.

Abstract 1977: ACR-2316: A potentially first-in-class, potent, selective WEE1/PKMYT1 inhibitor rationally designed for superior single agent activity through synergistic disruption of cell cycle checkpoints

Abstract WEE1 and PKMYT1 kinases play essential roles in cell cycle checkpoints and DNA damage response. Genetic interaction has been documented between WEE1 and PKMYT1, and clinical trials have reported clinical activity with inhibitors of WEE1 (adavosertib, azenosertib) and PKMYT1 (lunresertib). Here, we report the discovery and characterization of ACR-2316, a dual inhibitor of WEE1 and PKMYT1 specifically designed for optimal selectivity through co-crystallography and superior single agent activity uniquely enabled by Acrivon Predictive Precision Proteomics (AP3). Mass spectrometry-based AP3 profiling was conducted across several novel WEE1 and PKMYT1 inhibitor leads generated through co-crystallography-based rational drug design. Selective leads originating from one series were further optimized using AP3 for biological structure-activity relationship analysis. AP3 profiling revealed WEE1 inhibitor-upregulated phosphorylation sites across a subset of phosphoproteins that were oppositely regulated (quenched) by PKMYT1 inhibitors. Consensus sites for these included CDK1 T14, a direct PKMYT1 phosphorylation site, as well as CHK1 S296. A particular lead compound, ACR-2316, demonstrated a desirable potent, balanced ratio of cellular WEE1 (IC50 = 2 nM, IC90 = 10 nM) and PKMYT1 (IC20 = 44 nM) inhibition resulting in superior activation of the mitotic kinases CDK1, CDK2, and PLK1 compared to adavosertib and lunresertib, based on annotated kinase substrate relationships. ACR-2316 is more selective than adavosertib, azenosertib, and lunresertib based on >200 kinases profiled by AP3 and 468 kinases assessed by KINOMEscan. Cell cycle analyses demonstrated a drastic ACR-2316-induced S-G2/M accumulation qualitatively distinct from adavosertib or lunresertib. In a 19-cancer cell line proliferation assay (CellTiter-Glo), ACR-2316 demonstrated greater potency in all cell lines tested compared to adavosertib and lunresertib (mean IC50 = 70, 252 and 364 nM, respectively). Superior anti-cancer activity of ACR-2316 was observed in 12 ovarian cancer patient-derived xenograft models tested ex vivo (CellTiter-Glo 3D) compared to azenosertib and lunresertib (mean IC50 = 9, 248 and 1620 nM, respectively). Across human tumor xenograft mouse models, oral administration of ACR-2316 demonstrated superior, durable, dose-dependent efficacy compared to azenosertib and lunresertib and was well tolerated at all doses. Complete responses observed with ACR-2316 were associated with strong WEE1 and intermediate PKMYT1 inhibition in tumors. In conclusion, ACR-2316 is a potent, selective dual WEE1/PKMYT1 inhibitor with superior single-agent activity compared to clinical WEE1 or PKMYT1 inhibitors. ACR-2316 is progressing through IND-enabling studies in preparation for clinical monotherapy development. Citation Format: Caroline Wigerup, Helén Nilsson, Lei Shi, Joon Jung, Joelle Baddour-Sousounis, Ruban Cornelius, Nina Lipjankic, Uthira Muralitharan, Valentina Siino, Ignacio Arribas Diez, Zachary Best, Martina Pasetto, William Dahlberg, Shahrzad Rafiei, Portia Lombardo, Magnus E. Jakobsson, Reina Improgo, Christina Scherer, John van Duzer, David A. Proia, Kristina Masson, Peter Blume-Jensen. ACR-2316: A potentially first-in-class, potent, selective WEE1/PKMYT1 inhibitor rationally designed for superior single agent activity through synergistic disruption of cell cycle checkpoints [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 1977.

Abstract 3309: Discovery of potent and selective bivalent CDK2 degraders that demonstrate activity in CCNE1amp driven tumors

Abstract Introduction: The Cyclin-Dependent Kinases (CDKs) with their cyclin binding partners, are associated with cell cycle progression and transcriptional regulation. Targeting CDKs is a key oncology therapeutic strategy with CDK4/6 inhibitors demonstrating significant clinical benefit in HR+/HER2− metastatic breast cancer, the most prevalent subtype. However, 30% of patients develop acquired resistance in the clinic with cyclin E1 (CCNE1) amplification/overexpression and CDK2 activation implicated as a major resistance mechanism to CDK4/6i breast cancer therapy. Additionally, CCNE1/CDK2 activation has also been associated with poor prognosis in ovarian and endometrial cancers. Selective targeting of CDK2 using small molecule inhibitor-based approaches have recently advanced into the clinic. Targeted protein degradation of CDK2 provides an alternative strategy that has the potential to eliminate the activity of the CCNE1/CDK2 complex, as well as CDK2 complexed with other cyclins, including cyclin A, and provide improved clinical benefit. Results: Discovery efforts at Plexium have identified CDK2 bivalent degraders that consist of a CDK2-binding moiety, a linker and a high affinity cereblon-binding ligand. Cereblon binding potency was found to correlate with potent and deep degradation of CDK2. Degradation was blocked in the presence of a proteasome inhibitor as well as in a cereblon knock-out cell line, confirming that CDK2 degradation is mediated by the ubiquitin proteasome system and through engaging cereblon. Proteome-wide analysis demonstrated that CDK2 was selectively depleted without significantly modulating other CDKs or known cereblon neo-substrates. Selective degradation was achieved despite lack of selectivity for binding/inhibition of other CDKs and was confirmed to be dependent on the formation of a CRBN-CDK2 ternary complex. Dose dependent CDK2 degradation resulted in dose dependent inhibition of Rb phosphorylation, cell cycle arrest, senescence-associated phenotypes and antiproliferative activity in CCNE1 amplified cancer cell lines. The in vitro data was used to evaluate the PK/PD drug exposure-response relationship in vivo and deep CDK2 degradation was demonstrated both in vitro and in vivo. Conclusions: These data provide validation for CDK2 degradation as a therapeutic approach. Potent and selective CDK2 bivalent degraders were exploited as tools for studying the sensitivity of CCNE1 amplified tumor models to CDK2 degradation and dependence on E3 ligase. This proof-of concept study supports Plexium’s current approach of discovering novel CDK2 molecular glue degraders for the treatment of CDK4/6 inhibitor-naïve and -resistant HR+/HER2− breast cancer, and CCNE1 amplified ovarian and endometrial cancers. Citation Format: Adina Gerson-Gurwitz, Pengyu Yang, Sarah Fish, Gabrielle Blanco, Hongfeng Gao, Kyohei Hayashi, Mike Hocker, Aleks Jamborcic, Andre Richters, Mary E. Spalding, Julia Toth, Duc Tran, Linette Yang, Shu You, Andrew Burritt, Alex Campos, Gregory Parker, Kevin Freeman-Cook, Peggy A. Thompson, Simon Bailey. Discovery of potent and selective bivalent CDK2 degraders that demonstrate activity in CCNE1amp driven tumors [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 3309.

Abstract 1959: BLU-222, a potent and highly selective CDK2 inhibitor, demonstrates antitumor activity as monotherapy and as combination treatment in CCNE1-aberrant endometrial cancer models

Abstract Background: Many cyclin-dependent kinases (CDK) bind regulatory cyclins to control cell cycle progression. Aberrant CDK2 activation occurs in selected cancers (e.g., ovarian cancer, endometrial cancer) through CCNE1 amplification, leading to Rb phosphorylation, inactivation, and cell cycle checkpoint dysregulation. CCNE1 amplification and subsequent cyclin E overexpression are associated with chemotherapy resistance and shorter progression-free and overall survival. Therefore, novel therapies are needed to inhibit aberrant CDK2 activation and elicit positive clinical benefit in these patients. BLU-222 is a potent, highly selective, orally bioavailable, investigational CDK2 inhibitor with demonstrated activity in preclinical CCNE1-amplified ovarian cancer models. Here, we explored additional, specific biomarkers to predict BLU-222 sensitivity in ovarian and endometrial cancer as monotherapy or in novel combination treatment strategies. Methods: In vitro antiproliferative effect of BLU-222 was assessed by CyQuant in a panel of CCNE1-aberrant or CCNE1-normal ovarian and endometrial cell lines. Combination of BLU-222 and paclitaxel was evaluated by dosing matrix; synergy was calculated using SynergyFinder. In vivo antitumor activity of BLU-222 as a single agent or combined with paclitaxel was measured in CCNE1-aberrant endometrial cancer patient-derived xenograft (PDX) models (XenoSTART). Results: In ovarian and endometrial cell lines, CCNE1 amplification (copy number ≥ 6) predicted strong antiproliferative response to BLU-222. Using multivariate biomarker analysis, high p16 protein expression and wild-type Rb protein were identified as additional predictive markers for response to BLU-222 in CCNE1-aberrant cell lines. BLU-222 with paclitaxel was broadly active in endometrial cancer cell lines, with the strongest combination effect in cells with high CCNE1 expression, wild-type Rb protein, and low p16 protein expression. In vivo, BLU-222 elicited strong monotherapy antitumor response in CCNE1 copy number-aberrant endometrial cancer PDX models, and this effect was strengthened with the combination of paclitaxel. Conclusions: In preclinical models of CCNE1-aberrant endometrial cancer, BLU-222 demonstrated enhanced activity in combination with paclitaxel. Furthermore, Rb and p16 protein expression can be used in conjunction with CCNE1 copy number to predict response to both BLU-222 monotherapy and combination treatment. These data provide a rationale for using BLU-222 in combination with paclitaxel and demonstrate the potential benefit of using specific biomarkers to predict response. Citation Format: Nealia House, Victoria Brown, Liang Yuan, Maxine Chen, Stephanie Lee, Rentian Wu, Lakshmi Muthuswamy, Scott Ribich, Philip Ramsden, Kerrie Faia. BLU-222, a potent and highly selective CDK2 inhibitor, demonstrates antitumor activity as monotherapy and as combination treatment in CCNE1-aberrant endometrial cancer models [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 1959.

A novel HDAC6 inhibitor interferes microtubule dynamics and spindle assembly checkpoint and sensitizes cisplatin-induced apoptosis in castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (CRPC), the most refractory prostate cancer, inevitably progresses and becomes unresponsive to hormone therapy, revealing a pressing unmet need for this disease. Novel agents targeting HDAC6 and microtubule dynamics can be a potential anti-CRPC strategy. Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay and anchorage-dependent colony formation assay. Flow cytometric analysis of propidium iodide staining was used to determine cell-cycle progression. Cell-based tubulin polymerization assay and confocal immunofluorescence microscopic examination determine microtubule assembly/disassembly status. Protein expressions were determined using Western blot analysis. A total of 82 novel derivatives targeting HDAC6 were designed and synthesized, and Compound 25202 stood out, showing the highest efficacy in blocking HDAC6 (IC50, 3.5 nM in enzyme assay; IC50, 1.0 μM in antiproliferative assay in CRPC cells), superior to tubastatin A (IC50, 5.4 μM in antiproliferative assay). The selectivity and superiority of 25202 were validated by examining the acetylation of both α-tubulin and histone H3, detecting cell apoptosis and HDACs enzyme activity assessment. Notably, 25202 but not tubastatin A significantly decreased HDAC6 protein expression. 25202 prolonged mitotic arrest through the detection of cyclin B1 upregulation, Cdk1 activation, mitotic phosphoprotein levels, and Bcl-2 phosphorylation. Compound 25202 did not mimic docetaxel in inducing tubulin polymerization but disrupted microtubule organization. Compound 25202 also increased the phosphorylation of CDC20, BUB1, and BUBR1, indicating the activation of the spindle assembly checkpoint (SAC). Moreover, 25202 profoundly sensitized cisplatin-induced cell death through impairment of cisplatin-evoked DNA damage response and DNA repair in both ATR-Chk1 and ATM-Chk2 pathways. The data suggest that 25202 is a novel selective and potent HDAC6 inhibitor. Compound 25202 blocks HDAC6 activity and interferes microtubule dynamics, leading to SAC activation and mitotic arrest prolongation that eventually cause apoptosis of CRPC cells. Furthermore, 25202 sensitizes cisplatin-induced cell apoptosis through impeding DNA damage repair pathways.

Epigenetic upregulation of MNAT1 by SMYD2 is linked to PI3K/AKT activation and tumorigenesis of pancreatic adenocarcinoma.

Dysregulation of histone methyltransferase SET and MYND domain-containing protein 2 (SMYD2) has been correlated with human developmental disorders and cancers. This research aims to investigate the roles of SMYD2 and its interacted molecules in pancreatic adenocarcinoma (PAAD). Two PAAD-related gene expression datasets were downloaded to screen key molecules involved in tumor progression. SMYD2 was expressed at high levels in PAAD tissues and cells. SMYD2 silencing suppressed while its overexpression promoted proliferation, invasiveness, migration, apoptosis resistance, and cell cycle progression of PAAD cells. Target molecules of SMYD2 were predicted by online tools and validated by chromatin immunoprecipitation and luciferase assays. SMYD2 catalyzed H3K36me2 modification at the promoter region of MNAT1 component of CDK activating kinase (MNAT1), to promote its transcription. MNAT1 was correlated with an unfavorable clinical outcome of PAAD patients. Alteration of MNAT1 alone also affected the malignant behavior of PAAD cells. Moreover, MNAT1 overexpression in cells rescued the malignant phenotype of cells suppressed by SMYD2 silencing. MNAT1 activated the phosphatidyl inositol 3-kinase/protein kinase B (PI3K/AKT) signaling. In vivo, SMYD2 silencing decreased the growth rate and weight of xenograft tumors in nude mice. Overall, this paper demonstrates that SMYD2-mediated MNAT1 upregulation is linked to PAAD tumorigenesis via PI3K/AKT pathway activation.

Ku70 senses cytosolic DNA and assembles a tumor-suppressive signalosome.

The innate immune response contributes to the development or attenuation of acute and chronic diseases, including cancer. Microbial DNA and mislocalized DNA from damaged host cells can activate different host responses that shape disease outcomes. Here, we show that mice and humans lacking a single allele of the DNA repair protein Ku70 had increased susceptibility to the development of intestinal cancer. Mechanistically, Ku70 translocates from the nucleus into the cytoplasm where it binds to cytosolic DNA and interacts with the GTPase Ras and the kinase Raf, forming a tripartite protein complex and docking at Rab5+Rab7+ early-late endosomes. This Ku70-Ras-Raf signalosome activates the MEK-ERK pathways, leading to impaired activation of cell cycle proteins Cdc25A and CDK1, reducing cell proliferation and tumorigenesis. We also identified the domains of Ku70, Ras, and Raf involved in activating the Ku70 signaling pathway. Therapeutics targeting components of the Ku70 signalosome could improve the treatment outcomes in cancer.

Abstract B173: The anti-tumor activity of CDK2 inhibition alone or in combination with other anti-cancer agents in human cancers

Abstract Cyclin-dependent kinase 2 (CDK2) plays a crucial role in the regulation of the cell cycle and is involved in a variety of biological processes. It is responsible for phosphorylating proteins involved in G1 and S phase cell cycle progression, DNA damage response, intracellular transport, protein degradation, signal transduction, and DNA and RNA metabolism. CDK2 activity is particularly significant in cells exhibiting CCNE1 amplification or overexpression. Additionally, CDK2 activity has been linked to potential resistance against chemotherapy or targeted therapy. Several studies have indicated that abnormal activation of CDK2 may contribute to resistance against targeted therapies, such as CDK4/6 inhibitors and KRAS inhibitors. To investigate this hypothesis, we conducted experiments testing the combined effect of ARTS-021, a highly selective and potent CDK2 inhibitor, with Palbociclib, a CDK4/6 inhibitor, or AMG510, a KRAS-G12C inhibitor, in various xenograft tumor models. The combination of CDK2 inhibition with CDK4/6 inhibition demonstrated superior efficacy compared to either agent alone. This combination induced tumor regression in both Palbociclib-sensitive T47D parental cell line models and Palbociclib-resistant T47D cells overexpressing CCNE1. Similar benefits were also observed when CDK2 inhibition was combined with KRAS G12C inhibition in multiple KRAS-G12C lung cancer cell line xenografts. Importantly, these combination treatments were well tolerated. These findings suggest that CDK2 may play a crucial role in cancer cell resistance to chemotherapy or targeted therapy, and a CDK2 inhibitor may have broader applications beyond CCNE1-amplified cancers, as it can be combined with different treatment modalities. Citation Format: Yaoyu Chen, Jinhong Chen, Yali Guo, Wei Pang, Tingru Zhou, Xipan Liu, Nathan Schomer, Fang Li, Jiaqi Liang, Qing Sheng. The anti-tumor activity of CDK2 inhibition alone or in combination with other anti-cancer agents in human cancers [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 B173.

Abstract A023: Preclinical development of PKMYT1 and WEE1 inhibitor combinations

Abstract Background: CCNE1-amplification or overexpression are prevalent alterations in multiple tumor types, including high-grade serous ovarian, endometrial, and gastro-esophageal cancers. CCNE1-amplification is associated with high levels of replication stress, genomic instability, and resistance to chemotherapy. Previously, CRISPR-enabled synthetic lethal screening identified that loss of the CDK1 regulator Membrane-associated tyrosine- and threonine-specific Cdc2-inhibitory kinase (PKMYT1) is synthetic lethal with CCNE1-overexpression, leading to the development of lunresertib - a first-in-class, potent, selective, orally bioavailable PKMYT1 inhibitor currently being investigated in phase I clinical trials (NCT04855656, NCT05147272, NCT05147350). In CCNE1 overexpressing cells, lunresertib treatment leads to reduction of inhibitory CDK1-T14 phosphorylation and activated CDK1 in S-phase, triggering premature mitosis, chromosome pulverization, and ultimately cell death. In CCNE1-amplified tumor models, lunresertib exhibits antitumor activity as monotherapy. We hypothesized that further activation of CDK1 by reducing the inhibitory phosphorylation site at CDK1-pY15 by WEE1 inhibition would increase the antitumor activity of lunresertib. Methods: In vitro cell viability studies were used to determine the level of synergy between lunresertib and the WEE1 inhibitor Debio-0123 in engineered as well as tumor-derived CCNE1-high models. The lunresertib/Debio-0123 combination mechanism of action was investigated using high content imaging and immunoblotting for DNA damage and cell cycle markers in cultured cells. Finally, the pre-clinical anti-tumor activity and tolerability of combination lunresertib/Debio-0123 was evaluated in vivo using CCNE1-amplified tumor xenografts in mice. Results: Combination of low doses of lunresertib with Debio-0123 selectively induced synergistic cytotoxicity and catastrophic DNA damage in CCNE1-overexpressing cells, without affecting wild type counterparts. Increased DNA damage resulted from robust activation of CDK1 in S-phase, causing rapid induction of premature mitosis. Combination of lunresertib and Debio-0123 abrogated phosphorylation of both Thr14 and Tyr15 inhibitory CDK1 sites at doses that led to only partial dephosphorylation in both monotherapy settings. Mechanistically, synergistic CDK1 dephosphorylation was partially mediated by activation of CDC25B phosphatase. In a mouse model, the combination of RP-6306 and Debio-0123 was well-tolerated and demonstrated tumor regressions in a CCNE1-overexpressing tumor xenograft model at sub-therapeutic monotherapy doses. Conclusions: Lunresertib and the WEE1 inhibitor Debio-0123 elicit strong synergistic activity in preclinical models of CCNE1-overexpressoin due to rapid and complete CDK1 activation. In a xenograft tumor model, intermittent dosing of lunresertib/WEE1i lead to tumor regressions at sub-therapeutic monotherapy doses. Together these findings provide mechanistic rationale for clinical development of PKMYT1 and WEE1 inhibitor combinations. Citation Format: David Gallo, Fenil Shah, Rino Stocco, Prasamit Baruah, Jimmy Fourtounis, Rosie Kryczka, Marc L Hyer, Anne Roulston, C Gary Marshall, Michal Zimmermann, Stephen Morris, Michael Zinda. Preclinical development of PKMYT1 and WEE1 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 A023.

Cyclin B Export to the Cytoplasm via the Nup62 Subcomplex and Subsequent Rapid Nuclear Import Are Required for the Initiation of Drosophila Male Meiosis.

The cyclin-dependent kinase 1 (Cdk1)-cyclin B (CycB) complex plays critical roles in cell-cycle regulation. Before Drosophila male meiosis, CycB is exported from the nucleus to the cytoplasm via the nuclear porin 62kD (Nup62) subcomplex of the nuclear pore complex. When this export is inhibited, Cdk1 is not activated, and meiosis does not initiate. We investigated the mechanism that controls the cellular localization and activation of Cdk1. Cdk1-CycB continuously shuttled into and out of the nucleus before meiosis. Overexpression of CycB, but not that of CycB with nuclear localization signal sequences, rescued reduced cytoplasmic CycB and inhibition of meiosis in Nup62-silenced cells. Full-scale Cdk1 activation occurred in the nucleus shortly after its rapid nuclear entry. Cdk1-dependent centrosome separation did not occur in Nup62-silenced cells, whereas Cdk1 interacted with Cdk-activating kinase and Twine/Cdc25C in the nuclei of Nup62-silenced cells, suggesting the involvement of another suppression mechanism. Silencing of roughex rescued Cdk1 inhibition and initiated meiosis. Nuclear export of Cdk1 ensured its escape from inhibition by a cyclin-dependent kinase inhibitor. The complex re-entered the nucleus via importin β at the onset of meiosis. We propose a model regarding the dynamics and activation mechanism of Cdk1-CycB to initiate male meiosis.

Myt1 overexpression mediates resistance to cell cycle and DNA damage checkpoint kinase inhibitors.

Cell cycle checkpoint kinases serve as important therapeutic targets for various cancers. When they are inhibited by small molecules, checkpoint abrogation can induce cell death or further sensitize cancer cells to other genotoxic therapies. Particularly aberrant Cdk1 activation at the G2/M checkpoint by kinase inhibitors causing unscheduled mitotic entry and mitotic arrest was found to lead to DNA damage and cell death selectively in cancer cells. Promising drugs inhibiting kinases like Wee1 (Adavosertib), Wee1+Myt1 (PD166285), ATR (AZD6738) and Chk1 (UCN-01) have been developed, but clinical data has shown variable efficacy for them with poorly understood mechanisms of resistance. Our lab recently identified Myt1 as a predictive biomarker of acquired resistance to the Wee1 kinase inhibitor, Adavosertib. Here, we investigate the role of Myt1 overexpression in promoting resistance to inhibitors (PD166285, UCN-01 and AZD6738) of other kinases regulating cell cycle progression. We demonstrate that Myt1 confers resistance by compensating Cdk1 inhibition in the presence of these different kinase inhibitors. Myt1 overexpression leads to reduced premature mitotic entry and decreased length of mitosis eventually leading to increased survival rates in Adavosertib treated cells. Elevated Myt1 levels also conferred resistance to inhibitors of ATR or Chk1 inhibitor. Our data supports that Myt1 overexpression is a common mechanism by which cancer cells can acquire resistance to a variety of drugs entering the clinic that aim to induce mitotic catastrophe by abrogating the G2/M checkpoint.

CDC6 as a Key Inhibitory Regulator of CDK1 Activation Dynamics and the Timing of Mitotic Entry and Progression.

Timely mitosis is critically important for early embryo development. It is regulated by the activity of the conserved protein kinase CDK1. The dynamics of CDK1 activation must be precisely controlled to assure physiologic and timely entry into mitosis. Recently, a known S-phase regulator CDC6 emerged as a key player in mitotic CDK1 activation cascade in early embryonic divisions, operating together with Xic1 as a CDK1 inhibitor upstream of the Aurora A and PLK1, both CDK1 activators. Herein, we review the molecular mechanisms that underlie the control of mitotic timing, with special emphasis on how CDC6/Xic1 function impacts CDK1 regulatory network in the Xenopus system. We focus on the presence of two independent mechanisms inhibiting the dynamics of CDK1 activation, namely Wee1/Myt1- and CDC6/Xic1-dependent, and how they cooperate with CDK1-activating mechanisms. As a result, we propose a comprehensive model integrating CDC6/Xic1-dependent inhibition into the CDK1-activation cascade. The physiological dynamics of CDK1 activation appear to be controlled by the system of multiple inhibitors and activators, and their integrated modulation ensures concomitantly both the robustness and certain flexibility of the control of this process. Identification of multiple activators and inhibitors of CDK1 upon M-phase entry allows for a better understanding of why cells divide at a specific time and how the pathways involved in the timely regulation of cell division are all integrated to precisely tune the control of mitotic events.