Cdk2 Activity Research Articles

Invivo visualization of fluorescence reflecting CDK4 activity in a breast cancer mouse model.

The CDK4/6‐Rb axis is a crucial target of cancer therapy and several selective inhibitors of it have been approved for clinical application. However, current therapeutic efficacy evaluation mostly relies on anatomical imaging, which cannot directly reflect changes in drug targets, leading to a delay in the selection of optimal treatment. In this study, we constructed a novel fluorescent probe, CPP30‐Lipo/CDKACT4, for real‐time monitoring of CDK4 activity and the therapeutic efficacy of its inhibitor in HR+/HER2– breast cancer. CPP30‐Lipo/CDKACT4 exhibited good optical stability and targetability. The signal of the probe in living cells decreased after CDK4 knockdown or palbociclib treatment. Moreover, the fluorescence intensity of the tumors after 7 days of palbociclib treatment was significantly lower than that before treatment, while no significant change in tumor diameter was observed under magnetic resonance imaging. Overall, we developed an innovative fluorescent probe that can monitor CDK4 activity and the early therapeutic response to CDK4 inhibitors in living cells and in vivo. It may provide a new strategy for evaluating antitumor therapeutic efficacy in a clinical context and for drug development.

RB loss determines selective resistance and novel vulnerabilities in ER-positive breast cancer models.

The management of metastatic estrogen receptor (ER) positive HER2 negative breast cancer (ER+) has improved; however, therapeutic resistance and disease progression emerges in majority of cases. Using unbiased approaches, as expected PI3K and MTOR inhibitors emerge as potent inhibitors to delay proliferation of ER+ models harboring PIK3CA mutations. However, the cytostatic efficacy of these drugs is hindered due to marginal impact on the expression of cyclin D1. Different combination approaches involving the inhibition of ER pathway or cell cycle result in durable growth arrest via RB activation and subsequent inhibition of CDK2 activity. However, cell cycle alterations due to RB loss or ectopic CDK4/cyclin D1 activation yields resistance to these cytostatic combination treatments. To define means to counter resistance to targeted therapies imparted with RB loss; complementary drug screens were performed with RB-deleted isogenic cell lines. In this setting, RB loss renders ER+ breast cancer models more vulnerable to drugs that target DNA replication and mitosis. Pairwise combinations using these classes of drugs defines greater selectivity for RB deficiency. The combination of AURK and WEE1 inhibitors, yields synergistic cell death selectively in RB-deleted ER+ breast cancer cells via apoptosis and yields profound disease control in vivo. Through unbiased efforts the XIAP/CIAP inhibitor birinapant was identified as a novel RB-selective agent. Birinapant further enhances the cytotoxic effect of chemotherapies and targeted therapies used in the treatment of ER+ breast cancer models selectively in the RB-deficient setting. Using organoid culture and xenograft models, we demonstrate the highly selective use of birinapant based combinations for the treatment of RB-deficient tumors. Together, these data illustrate the critical role of RB-pathway in response to many agents used to treat ER+ breast cancer, whilst informing new therapeutic approaches that could be deployed against resistant disease.

Fbxo7 promotes Cdk6 activity to inhibit PFKP and glycolysis in T cells.

Fbxo7 is associated with cancer and Parkinson's disease. Although Fbxo7 recruits substrates for SCF-type ubiquitin ligases, it also promotes Cdk6 activation in a ligase-independent fashion. We discovered PFKP, the gatekeeper of glycolysis, in a screen for Fbxo7 substrates. PFKP is an essential Cdk6 substrate in some T-ALL cells. We investigated the molecular relationship between Fbxo7, Cdk6, and PFKP, and the effect of Fbxo7 on T cell metabolism, viability, and activation. Fbxo7 promotes Cdk6-independent ubiquitination and Cdk6-dependent phosphorylation of PFKP. Importantly, Fbxo7-deficient cells have reduced Cdk6 activity, and hematopoietic and lymphocytic cells show high expression and significant dependency on Fbxo7. CD4+ T cells with reduced Fbxo7 show increased glycolysis, despite lower cell viability and activation levels. Metabolomic studies of activated CD4+ T cells confirm increased glycolytic flux in Fbxo7-deficient cells, alongside altered nucleotide biosynthesis and arginine metabolism. We show Fbxo7 expression is glucose-responsive at the mRNA and protein level and propose Fbxo7 inhibits PFKP and glycolysis via its activation of Cdk6.

Phase 3 ENABLAR-2 study to evaluate enobosarm and abemaciclib combination compared to estrogen-blocking agent for the second-line treatment of AR+, ER+, HER2- metastatic breast cancer in patients who previously received palbociclib and estrogen-blocking agent combination therapy.

TPS1121 Background: Targeting the androgen receptor (AR) may be the next important endocrine therapy for advanced breast cancer. The AR has been demonstrated to be a tumor suppressor when activated. Enobosarm is an oral selective AR targeting agonist that activates the AR in breast cancer. Preclinical studies in CDK4/6 inhibitor resistant PDX models demonstrated combinatorial synergistic activity of enobosarm plus CDK 4/6 inhibitors. An open-label, Phase 2 study, was conducted in 136 women with heavily pretreated ER+ HER2- metastatic breast cancer that were randomized to oral daily enobosarm at a dose of 9 or 18 mg. The efficacy evaluable (EE) group were patients that were AR positive (> 10% AR nuclear staining). In the EE population with measurable disease at baseline, 10 patients had received prior endocrine therapy + a CDK 4/6 inhibitor. Subsequent treatment with enobosarm resulted in a clinical benefit rate of 50% and the best overall response rate (ORR) was 30% (2CRs and 1 PR). Of the 10 patients, 7 had AR nuclear staining ≥40%. None of the patients with AR nuclear staining < 40% responded to enobosarm. Although a small subset of the study, it appears that enobosarm has activity in patients who had ≥40% AR staining and who had progressed on standard endocrine therapy with a CDK 4/6 inhibitor. Overall, treatment with enobosarm was well tolerated with significant positive effects on quality-of-life measurements. Methods: The ENABLAR-2 trial is an ongoing Phase 3, randomized, open-label, efficacy and safety study in patients with AR+ ER+ HER2- MBC with AR nuclear staining of ≥40%, who have progressed after one line of systemic therapy comprising estrogen blocking agent and palbociclib. The planned sample size is 186 patients randomized 1:1 to enobosarm + abemaciclib OR fulvestrant if the first line of therapy for MBC was a non-steroidal AI plus palbociclib, until disease progression, toxicity, or loss of clinical benefit. If first line therapy for metastatic breast cancer was fulvestrant plus palbociclib, then the patient will be randomized 1:1 to either enobosarm + abemaciclib OR an AI. Randomization will be stratified by AR% nuclear staining, ≥60% versus < 60%, as well as by estrogen blocking agent such that each cohort will have the same number of subjects previously receiving fulvestrant + palbociclib in first line therapy. The key objectives are to determine the safety and efficacy of enobosarm and abemaciclib combination versus an alternative estrogen blocking agent with the primary endpoint of PFS. Secondary endpoints include ORR, duration of response, overall survival, change from baseline in Short Physical Performance Battery (SPPB), change in EORTC Quality of Life Questionnaire (EORTC-QLQ) and change in body composition as measured by DEXA. Clinical trial information: NCT05065411.

A phase 1 dose-finding and dose-expansion study evaluating the safety, tolerability, pharmacokinetics, and efficacy of a highly selective WEE1 inhibitor (Debio 0123) in adult patients with advanced solid tumors.

TPS2702 Background: Debio 0123 is an oral, highly selective inhibitor of the tyrosine kinase WEE1. WEE1 is a key regulator of cell cycle progression that modulates the activity of CDK1 (CDC2), influencing entry into mitosis. WEE1 inhibition results in G2 checkpoint abrogation, triggering mitosis with unrepaired DNA leading to cell death. In vitro and in vivo tumor models have shown Debio 0123 antitumor activity. Continuous exposure seems to be needed to maximize monotherapy efficacy in preclinical models. Preliminary data from an ongoing phase 1 study of intermittent Debio 0123 combined with carboplatin, showed a manageable safety profile, and signals of antitumor activity in patients (pts) with advanced solid tumors. We present the design of a phase 1 study (NCT05109975) of continuous Debio 0123 administered as a single agent in pts with advanced solid tumors. Methods: The study comprises a dose escalation part and a dose expansion part. The primary objective of the dose escalation part of the study is to determine the maximum tolerated dose (MTD) and/or the recommended phase 2 dose (RP2D) of continuous Debio 0123 monotherapy in adults with previously treated advanced solid tumors. Key secondary objectives include characterization of safety and tolerability, pharmacokinetics (PK), and preliminary antitumor activity (overall response rate [ORR]). Key inclusion criteria are histologically or cytologically confirmed locally advanced or metastatic solid tumors, and ECOG-PS 0 or 1. Key exclusion criteria are asymptomatic or unstable brain metastases, history of cardiac disorders, inability to swallow oral medication or abnormalities affecting drug absorption. Approximately 30 dose-limiting toxicity (DLT)-evaluable pts are anticipated to be enrolled based on an escalation with overdose control (EWOC) approach for 21-day treatment cycles. When the pts of each cohort become evaluable, a safety monitoring committee will review safety and tolerability and based on EWOC recommendations will decide the next dose level or will declare the MTD and/or RP2D. Pharmacodynamic biomarkers will be correlated with tumor response and/or PK. The dose-expansion part may start after MTD and/or RP2D determination. The primary objective of this part will be to characterize the safety and tolerability (e.g. percentage of pts with DLTs, serious adverse events or discontinuations) of Debio 0123 monotherapy at the MTD/RP2D, and to evaluate the anti-tumor activity (ORR) in pts with selected recurrent/progressive solid tumors. Key inclusion criteria are measurable disease per RECIST 1.1 and specific tumor types. Based on a Simon 2-stage design, up to 34 pts per tumor type cohort may be enrolled. Currently accrual to the dose escalation is ongoing in the United States and Switzerland. Clinical trial information: NCT05109975.

Conserved Cdk inhibitors show unique structural responses to tyrosine phosphorylation

Balanced proliferation-quiescence decisions are vital during normal development and in tissue homeostasis, and their dysregulation underlies tumorigenesis. Entry into proliferative cycles is driven by Cyclin/Cyclin-dependent kinases (Cdks). Conserved Cdk inhibitors (CKIs) p21Cip1/Waf1, p27Kip1, and p57Kip2 bind to Cyclin/Cdks and inhibit Cdk activity. p27 tyrosine phosphorylation, in response to mitogenic signaling, promotes activation of CyclinD/Cdk4 and CyclinA/Cdk2. Tyrosine phosphorylation is conserved in p21 and p57, although the number of sites differs. We use molecular-dynamics simulations to compare the structural changes in Cyclin/Cdk/CKI trimers induced by single and multiple tyrosine phosphorylation in CKIs and their impact on CyclinD/Cdk4 and CyclinA/Cdk2 activity. Despite shared structural features, CKI binding induces distinct structural responses in Cyclin/Cdks and the predicted effects of CKI tyrosine phosphorylation on Cdk activity are not conserved across CKIs. Our analyses suggest how CKIs may have evolved to be sensitive to different inputs to give context-dependent control of Cdk activity.

Molecular Rearrangement of Pyrazino[2,3-c]quinolin-5(6H)-ones during Their Reaction with Isocyanic Acid.

New tetrahydropyrazino[2,3-c]quinolin-5(6H)-ones were prepared from 3-chloroquinoline-2,4(1H,3H)-diones and ethylene diamine. In their reaction with HNCO, an unprecedented molecular rearrangement produced new types of hydantoin derivatives. All prepared compounds were characterized on the basis of their 1H, 13C, and 15N NMR and ESI mass spectra and some were authenticated by X-ray analysis of single crystalline material. A proposed mechanism for rearrangement is discussed in this essay. The CDK and ABL inhibition activity as well as in vitro cytotoxicity of the prepared compounds was also tested.

Quantitative Mass Spectrometry Reveals a Proteome‐wide Role for Cyclin A and Cks1 in Multisite, Non‐Proline Directed Phosphorylation by CDK1

Cdk1 is a master kinase of mitotic cell division, phosphorylating thousands of substrates in a highly temporally regulated manner. How Cdk1 phosphorylates the right substrate at the right time remains an open question.Cdk1 activity requires complex formation with a cyclin subunit: cyclin A or cyclin B. Additionally, active Cdk1 is found in complex with a small adaptor protein called Cks1. Cyclin subunits activate Cdk1 and tune Cdk1 substrate choice through interactions with short linear motifs on substrates. While Cks1 is essential in all organisms tested to date, a general function for Cks1 in Cdk1 substrate choice has not been demonstrated.In this study, we investigated the roles of the non‐catalytic subunits of Cdk1 in substrate phosphorylation in human cells. We designed an in vitro assay in which we induced protein phosphorylation in fixed and permeabilized human lymphoblasts using active recombinant Cdk1 either in complex with cyclin A, cyclin B, or cyclin B+Cks1. Global phosphorylation levels were then measured using quantitative mass spectrometry.Our data demonstrate that, contrary to the textbook model of Cdk1 as a proline‐directed kinase, both cyclin A and Cks1 facilitate widespread Cdk1 phosphorylation of sites lacking a +1 proline motif. Our results also suggest a role for Cks1 in multisite phosphorylation. We propose a model whereby cyclin A and Cks1 have indispensable functions in promoting non‐proline directed, multisite phosphorylation of Cdk1 substrates to mediate dynamic, switch‐like changes in protein function required for mitotic entry, proper chromosome segregation and cell division.

Disrupted control of origin activation compromises genome integrity upon destabilization of Polε and dysfunction of the TRP53-CDKN1A/P21 axis

The maintenance of genome stability relies on coordinated control of origin activation and replication fork progression. How the interplay between these processes influences human genetic disease and cancer remains incompletely characterized. Here we show that mouse cells featuring Polε instability exhibit impaired genome-wide activation of DNA replication origins, in an origin-location-independent manner. Strikingly, Trp53 ablation in primary Polε hypomorphic cells increased Polε levels and origin activation and reduced DNA damage in a transcription-dependent manner. Transcriptome analysis of primary Trp53 knockout cells revealed that the TRP53-CDKN1A/P21 axis maintains appropriate levels of replication factors and CDK activity during unchallenged S phase. Loss of this control mechanism deregulates origin activation and perturbs genome-wide replication fork progression. Thus, while our data support an impaired origin activation model for genetic diseases affecting CMG formation, we propose that loss of the TRP53-CDKN1A/P21 tumor suppressor axis induces inappropriate origin activation and deregulates genome-wide fork progression.

Pharmacological reactivation of RBL2/p130 could be an effective antitumoral strategy for malignant pleural mesothelioma

Malignant mesothelioma (MM) is an aggressive tumor developing from the mesothelium covering the body cavities. The most common MM type affects the pleura surrounding the lungs. MM is mainly associated with asbestos exposure. At present, no curative modalities are effective against MM exist. The molecular mechanisms underlying MM development seem to depend mostly on the inactivation of tumor suppressors, among which also the dysfunction of the retinoblastoma (RB) protein pathway that is a hallmark of cancers. We previously demonstrated that RBL2/p130 is a direct AKT target and its reactivation following AKT inhibition mediates apoptosis in MM.So, we hypothesize that concomitant inhibition of CDK and AKT activity, both converging on the reactivation of RBL2/p130 tumor suppressor role, synergize in counteracting MM.We first assessed, through an MTS assay, the effect of three new generation CDK inhibitors (CDKi), palbociclib, ribociclib, abemaciclib, on the cell viability of MM (NCI‐H28, IST‐MES TWO, NCI‐H2452, MMB, MSTO‐211H, NCI‐H2052) and immortalized human normal mesothelial (MET‐5A) cells at 72h. Then, to verify whether CDKi exerted long‐term cell growth inhibition, we performed clonogenic assays upon treatment of all the MM All the MM and mesothelial cell lines. Finally, we also examined, by MTS, the possible synergistic effects of abemaciclib in combination with an AKT inhibitor (AKT1/2 VIII).Our preliminary data show that ribociclib and palbociclib only moderately reduce viability of the MPM cells, whereas abemaciclib shows cytotoxic effects at very low doses in all MM cell lines. Abemaciclib treatment induces a dose‐dependent cytotoxic effect in all the MM cell lines without meaningfully affecting the normal mesothelial cells. Interestingly, abemaciclib also induced a cytotoxic effect in the most aggressive histotype, the sarcomatoid NCI‐H2052 cells. To verify whether abemaciclib exerted long‐term cell growth inhibition, we performed clonogenic assays upon treatment with IC50 of abemaciclib and found that the CDKi dramatically reduced colony formation in all the MM cell lines. We also examined, by MTS, the possible synergistic effects of abemaciclib in combination with a kinase AKT inhibitor (iAKT1/2 VIII), so we treated the MM cell lines for 72 h with the two agents, both alone and in combination, at five different concentrations, in a constant ratio. The cell viability data were evaluated through the Chou–Talalay method and showed a strong synergism revealing a combination index (CI) values < 1 for all cell.Although reactivating oncosoppressor genes is difficult to achieve because they are hardly ‘druggable’, our preliminary findings will provide the rationale for pharmacological strategies able to unleash RBL2/p130 tumor suppressor potential. The pharmacological reactivation of RBL2/p130 could be an effective strategy against MM, which can be rapidly implemented in the clinical practice.

The oocyte spindle midzone pauses Cdk1 inactivation during fertilization to enable male pronuclear formation and embryo development.

Inactivation of cyclin-dependent kinase 1 (Cdk1), controlled by cyclin B1 proteolysis, orders events during mitotic exit. Here, we used a FRET biosensor to study Cdk1 activity while simultaneously monitoring anaphase II and pronuclear (PN) formation in live mouse eggs throughout fertilization. We find that Cdk1 inactivation occurs over two phases separated by a 3-h pause, the first induces anaphase II and the second induces PN formation. Although both phases require the inhibitory Cdk1 kinase Wee1B, only the first involves cyclin B1 proteolysis. Enforcing the 3-h pause is critical for providing the delay required for male PN formation and is mediated by spindle midzone-dependent sequestration of Wee1B between the first and second phases. Thus, unlike continuous Cdk1 inactivation driven by cyclin B1 proteolysis during mitotic exit, MII oocytes engineer a physiologically important pause during fertilization involving two different pathways to inactivate Cdk1, only the first of which requires proteolysis.

PKM2 Interacts With the Cdk1-CyclinB Complex to Facilitate Cell Cycle Progression in Gliomas.

PKM2 is a phosphotyrosine-binding glycolytic enzyme upregulated in many cancers, including glioma, and contributes to tumor growth by regulating cell cycle progression. We noted, however, that in multiple glioma cell lines, PKM2 knock-down resulted in an accumulation of cells in G2-M phase. Moreover, PKM2 knock-down decreased Cdk1 activity while introducing a constitutively active Cdk1 reversed the effects of PKM2 knock-down on cell cycle progression. The means by which PKM2 increases Cdk1 activity have not been described. Transient interaction of T14/Y15-phosphorylated Cdk1 with cyclin B allows Cdk7-mediated pT161 Cdk1 phosphorylation followed by cdc25C-mediated removal of pT14/Y15 and activation of Cdk1 in cycling cells. In the present course of investigation, PKM2 modulation did not influence Cdk7 activity, but phosphotyrosine binding forms of PKM2 co-immunoprecipitated with pY15-containing Cdk1-cyclinB and enhanced formation of active pT161 Cdk1-cyclin B complexes. Moreover, exogenous expression of phosphotyrosine binding forms of PKM2 reversed the effects of PKM2 knock-down on G2-M arrest. We here show that PKM2 binds and stabilize otherwise transient pY15-containing Cdk1-cyclinB complexes that in turn facilitate Cdk1-cyclin B activation and entry of cells into mitosis. These results, therefore, establish metabolic enzyme PKM2 as a direct interactor and activator of Cdk1-cyclin B complex and thereby directly controls mitotic progression and the growth of brain tumor cells.

Regulation of Myt1 kinase activity via its N-terminal region in Xenopus meiosis and mitosis.

Immature animal oocytes are naturally arrested at the first meiotic prophase (Pro-I), which corresponds to the G2 phase of the cell cycle. In Xenopus oocytes, Myt1 kinase phosphorylates and inactivates cyclin-dependent kinase 1 (Cdk1) at Pro-I, thereby preventing oocytes from entering meiosis I (MI) prematurely. Previous studies have shown that, upon resuming MI, Cdk1 and p90rsk, which is a downstream kinase of the Mos-MAPK pathway, in turn phosphorylate the C-terminal region of Myt1, to suppress its activity, thereby ensuring high Cdk1 activity during M phase. However, the roles of the N-terminal region of Myt1 during meiosis and mitosis remain to be elucidated. In the present study, we show that the N-terminal region of Myt1 participates in the regulation of Myt1 activity in the Xenopus cell cycle. In particular, we found that a short, conserved sequence in the N-terminal region, termed here as the PAYF motif, is required for the normal activity of Myt1 in oocytes. Furthermore, multiple phosphorylations by Cdk1 at the Myt1 N-terminal region were found to be involved in the negative regulation of Myt1. In particular, phosphorylations at Thr11 and Thr16 of Myt1, which are adjacent to the PAYF motif, were found to be important for the inactivation of Myt1 in the M phase of the cell cycle. These results suggest that in addition to the regulation of Myt1 activity via the C-terminal region, the N-terminal region of Myt1 also plays an important role in the regulation of Myt1 activity.

Circular RNA circLAMA3 inhibits the proliferation of bladder cancer by directly binding an mRNA

The circular RNA (circRNA) circLAMA3 is significantly downregulated in bladder cancer tissues and cell lines. However, its function in bladder cancer has not yet been explored, and further research is needed. In this study, functional experiments demonstrated that circLAMA3 significantly inhibited the proliferation, migration, and invasion of bladder cancer cells and inhibited bladder cancer growth in vivo. Mechanistically, circLAMA3 directly binds to and promotes the degradation of MYCN mRNA, thereby reducing the MYCN protein expression in bladder cancer cells. Decreased expression of the MYCN protein inhibits the promoter activity and expression of CDK6. Ultimately, circLAMA3 affects DNA replication by downregulating CDK6, resulting in G0/G1 phase arrest and inhibition of bladder cancer proliferation. In summary, we report a potential novel regulatory mechanism via which a circRNA directly binds an mRNA and thereby regulates its fate. Moreover, circLAMA3 significantly affects the progression of bladder cancer and has potential as a diagnostic biomarker and therapeutic target for bladder cancer.

Quantitative profiling of adaptation to cyclin E overproduction.

Cyclin E/CDK2 drives cell cycle progression from G1 to S phase. Despite the toxicity of cyclin E overproduction in mammalian cells, the cyclin E gene is overexpressed in some cancers. To further understand how cells can tolerate high cyclin E, we characterized non-transformed epithelial cells subjected to chronic cyclin E overproduction. Cells overproducing cyclin E, but not cyclins D or A, briefly experienced truncated G1 phases followed by a transient period of DNA replication origin underlicensing, replication stress, and impaired proliferation. Individual cells displayed substantial intercellular heterogeneity in cell cycle dynamics and CDK activity. Each phenotype improved rapidly despite high cyclin E-associated activity. Transcriptome analysis revealed adapted cells down-regulated a cohort of G1-regulated genes. Withdrawing cyclin E from adapted cells only partially reversed underlicensing indicating that adaptation is at least partly non-genetic. This study provides evidence that mammalian cyclin E/CDK inhibits origin licensing indirectly through premature S phase onset and provides mechanistic insight into the relationship between CDKs and licensing. It serves as an example of oncogene adaptation that may recapitulate molecular changes during tumorigenesis.

Abstract OT2-19-08: Phase IB/II trial of palbociclib and binimetinib in advanced triple-negative breast cancer with hyperactivation of ERK and/or CDK4/6

Abstract Background: Novel effective and safe therapies are required for advanced TNBC after progression to standard-of-care first line treatment with anti-PD-1/L1 + chemotherapy. We have found that the most aggressive TNBC variants are driven by a heterogeneous set of genetic aberrations that converge in the increased activity of 6 kinases: KIT, PNKP, PRKCE, P70S6K, ERK and CDK6 (Nat Commun; 9:3501-18). Although the inhibition of each kinase in monotherapy yielded little efficacy in preclinical models, the combinations targeting pairs of the former kinases led to therapeutic synergy in most cases. The combined inhibition of CDK6 and ERK led to 5-fold increase in overall survival in preclinical TNBC models (PDXs and spontaneous murine cancer models). The greatest activity was observed in models with increased activity of either CDK6 or ERK. Thus, we aimed to test the safety and preliminary efficacy of combined ERK and CDK6 inhibition with binimetinib and palbociclib in women with advanced TNBC with hyperactivation of ERK and/or CDK6. The combination has been preliminary tested in lung cancer, where a phase I dose-escalation trial established the RP2D in binimetinib 45 mg/BID plus Palbociclib 125 mg daily 21/7. Trial design: Single-arm, prospective, multicentric, open-label, phase IB/II trial with intra-patient dose-escalation. Patients candidate for pre-screening will have determined the activity of ERK and CDK6 with an in-house developed assay and acquisition algorithm at the central lab. Those testing positive for either marker will undergo full screening. Patients will start treatment with continuous oral binimetinib at 45 mg/BID and palbociclib 100 mg/day from days 1 to 21, in 28-day cycles. Patients experiencing ≤ grade 1 tolerable side effects as the greatest toxicity will be allowed to escalate to palbociclib to 125 in cycle 2. Fresh biopsies will be harvested at baseline and disease progression, in order to establish patient-derived organoids (PDOs) and perform WES. PBMCs will be harvested at baseline, +24 hours, and at the beginning of cycle 2, to study changes in the immunophenotype. RECIST 1.1 and NCI CTC AE V 5.0 criteria will be used for assessing disease control (q8 weeks) and toxicity. Eligibility criteria: Pre-screening: Women >18 year old diagnosed of advanced, non-curable TNBC; 2) who have received a minimum of 1 treatment line including immunotherapy; 3) no more than 2 treatment lines for advanced disease; 4) Adequate organ function and recovery from previous toxicity to < tolerable G2. Full screening: 5) Demonstration of hyper-activation of CDK6 and/or ERK in the tumor sample; 6) PD to the previous treatment regimen within the last 28 days. Specific aims: Primary: 1) To determine the progression-free survival time of the combination of binimetinib and palbociclib in TNBC patients with hyperactivation of ERK and/or CDK6 in second/third line 2) To assess the safety and tolerability of the former combination Secondary: 1) 6-month PFS rate, response rate, and overall survival of the combination 2) To study biomarkers of sensitivity and primary and acquired resistance to the combination taking advantage of PDOs 3) To determine the immunodynamics of the combination. Statistical methods: The null hypothesis is that the median PFS time for second/third line TNBC with best physician’s choice is 1.7 months (NEJM; 384:1529-41, 2021). With alpha and beta errors of 0.05 and 0.2, the minimum number of patients to demonstrate a 30% improvement in PFS to 2.5 months is 25. Preliminary data suggest that approximately 40% of the patients show hyper-activation of CDK6 and/or ERK; thus, and assuming a methodological failure of 10%, the minimum number of patients to screen is 69. Present accrual/target accrual: 27 screened of 69 planed; 11 accrued of 25 planned Citation Format: Luis Manso, Alfonso Cortes, Juan M Cejalvo, Serafin Morales, Jose A García Saenz, Ramon Colomer, Rodrigo Sanchez-Bayona, Jorge Silva, Juan A Guerra, Diego Malon, Silvana Mouron, Eduardo Caleiras, Miguel Quintela-Fandino. Phase IB/II trial of palbociclib and binimetinib in advanced triple-negative breast cancer with hyperactivation of ERK and/or CDK4/6 [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-19-08.

Abstract P5-16-18: Developing IpY: A novel inhibitor for the treatment of ER+ CDK4i-resistant breast cancer

Abstract Almost 270,000 US women will be diagnosed with breast cancer (BC) this year, anddespite advances in treatment, ~40,000 women will die. ER/PR+ (endocrineresponsive), Her2- tumors occur in approximately 40% of breast cancer patients andare candidates for drugs targeting estrogen responsiveness, such as letrozole orfulvestrant. CDK4 targeting drugs (CDK4i) like palbociclib, abemaciclib, or ribociclib arenow approved in combination with letrozole or fulvestrant as a front- and second-linetherapy for metastatic ER/PR+, Her2- patients. However, while combined ER andCDK4i treatment significantly extends Progression Free Survival (PFS), those treatedinvariably develop resistance. Thus, drug resistance remains an urgent unmet need, as current treatments only marginally improve Overall Survival (OS) for these patients.Resistance to palbociclib develops because of compensation by another kinase, CDK2, suggesting that to be effective, therapies must be developed to inhibit both CDK4 andCDK2. Concarlo has taken a different approach to inhibit CDK4 and CDK2 by targetingp27Kip1 (p27). p27 interacts specifically with CDK4/6 and CDK2, and is responsible forturning these kinases ON and OFF. This transition from ON to OFF is mediated by aspecific modification to p27 itself, by the tyrosine kinase BRK (breast tumor relatedkinase). Published work has shown that a naturally occurring ALTernatively splicedform of BRK, ALT, can bind to p27, blocking BRK's association, and preventing BRK'sphosphorylation of p27. This locks CDK4 and CDK2 into the OFF conformation andsimultaneously inhibits activities of both kinases. As the naked 144 aa ALT peptidedoes not enter cells, it was formulated within a lipid nano-particle (NP), and called IpY.1.IpY.1 inhibits proliferation of HR+ and triple negative (TN) BC cells, but not non-cancerous breast cells MCF10A. IpY.1 arrests CDK4i-resistant and endocrine-resistantBC cells and it is both cytostatic and cytotoxic, demonstrating that p27 is a viabletherapeutic target to combat drug resistance. In vivo, IpY reduces tumor volumes and increase OS in cell line-derived xenografts (CDX). In order to convert manufacturing of the therapeutic peptide portion of IpY from recombinant to synthetic production, Concarlo truncated the large ALT peptide and bioengineered it to a more stable form. IpY.20 has a comparable IC50 as IpY.1 in HR+and TNBC cells and does not cause growth arrest in non-cancerous MCF10A cells. Using the fluorescent-labeled variant of IpY.20 (fluo-IpY.20) injected into tumor-bearingsyngeneic CDX model, followed by IVIS imaging, we could detect fluo-IpY.20 in tumorsas early as 15 min and up to 24h post injection. 6-10% of the total fluorescent signal isallocated to tumor at all timepoints. In addition, fluo-IpY.20 inhibits the activation of itstarget in tumors within 24h, suggesting fluo-IpY.20 not only reaches tumors but alsoengages with its target. IpY.20 induces tumor regression in CDX models and animmunocompetent genetically engineered model that overexpresses the potent Erbb2oncogene. Overall, repeated dosing of IpY.20 does not exhibit significant adverseeffects and is well tolerated in immunocompetent mice. However, in these treated mice, there was an increase in platelets and production of some cytokines compared to vehicle-treated mice, suggesting that an immune response may have been initiated. By targeting p27 instead of the conserved CDK4 or CDK2 active sites, IpY.20 will bemore selective and have fewer off-target effects than the ATP-competitive smallmolecule CDK4is currently in use. As the majority of the drug resistance seen withCDK4 inhibitory drugs is a result of compensatory CDK2 activity, IpY.20 preventsacquired drug resistance by hitting both CDK4 and CDK2 simultaneously, resulting in amore durable response to the drug. Concarlo is currently in manufacturing with IpY.20. Citation Format: Grace Chen, Stacy W Blain, Irina Jilishitz, Allison Vanlnwegen, Lingyue Yan, Yun Wu. Developing IpY: A novel inhibitor for the treatment of ER+ CDK4i-resistant breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-16-18.

Abstract P4-01-10: A kinase inhibitor library screen reveals novel candidates that reverse CDK4/6 inhibitor resistance in CDK6 amplified HR(+) breast cancer

Abstract Background and rationale: Metastatic HR (+) breast cancer currently still remains an incurable disease. CDK4/6 inhibitors in combination with endocrine therapy have emerged as standard 1st and 2nd lines in metastatic HR (+) breast cancer. Resistance to CDK4/6 inhibitors eventually occurs, and currently the optimal subsequent treatment for these patients remains unsettled. Based on the excellent tolerability and efficacy of CDK4/6 inhibitors, it remains a clinical interest to identify mechanisms of resistance towards CDK4/6 inhibitors in breast cancer. This is currently an area of intense research, with many resistance models being proposed, including loss of Rb, upregulation of cell cycle components (CDK2, cyclin E, AURKA), activation of growth factors (HER2, FGFGR), and the Hippo pathway. The activation of the Hippo pathway downstream gene YAP resulted in CDK6 overexpression that further led to CDK4/6 inhibitor resistance. This was confirmed in preclinical models and in patients developing resistance towards CDK4/6 inhibitors. The mechanism for how increased CDK6 activity restricts efficacy towards CDK4/6 inhibitors is unknown. We sought to discover kinase inhibitors that could downregulate CDK6 expression and CDK6 overexpression mediated resistance. Our goal is to develop therapeutics that could be added on CDK4/6 inhibitors upon progression to reverse the resistance, and to uncover pathways that modulate the relevant interactions. Results: We individually overexpressed YAP and CDK6 in the HR(+) breast cancer cell line MCF-7 and T47D and confirmed that each could increase the IC50 of CDK4/6 inhibitors. We screened a small library of 160 kinase inhibitors for candidates that could decrease CDK6 expression in a luciferase reporter assay and identified several PI3K inhibitors as well as the PKC inhibitor staurosporine that could significantly decrease CDK6 expression. Interestingly, we demonstrated that staurosporine could modulate the Hippo-YAP signaling pathway and thus modulate CDK6 expression. We further confirmed that when combined to CDK4/6 inhibitors, the addition of these candidates reversed the resistance in YAP or CDK6 overexpressing cells. Incidentally, we discovered that a mutant (D224Y) in CDK6 abolished the resistance in CDK6 overexpressing cells. CDK6-D224Y overexpressing MCF7 cells lost their resistance towards CDK4/6 inhibitors, despite that CDK6-D224Y was indistinguishable from CDK6 in kinase activity and function. D224Y resides in an alpha helix far apart from the activation loop of the CDK6 active site, therefore suggesting that mutations in CDK6 that leads to possible conformation changes could modulate the resistance to CDK4/6 inhibitors. We performed BRET (bioluminescence resonance energy transfer) and confirmed that the D224Y changed the BRET values for a palbociclib luminescent tracer, suggesting that this mutant could modulate the binding affinity of CDK4/6 inhibitors towards CDK6. In summary, we identified several novel candidates as well as a CDK6 mutant (D224Y) that could therapeutically reverse CDK4/6 inhibitor resistance conferred by CDK6 overexpression. Conclusions: We identified the PI3K pathway and the Hippo pathway as potential pathways that could modulate the CDK4/6 inhibitors resistance from CDK6 amplification. We further identified the D224Y mutant as a point mutation that abolished resistance. Our findings highlight the role of CDK6 and conformational variants in modulating CDK4/6 inhibitor resistance and uncover potential pathways that could be exploited for therapeutic development for subsequent treatment. Citation Format: Jiun-I Lai, Yong-Ji Zhuang, Ting-Yi Lin, Ta-Chung Chao, Chun-Yu Liu, Ling-Ming Tseng. A kinase inhibitor library screen reveals novel candidates that reverse CDK4/6 inhibitor resistance in CDK6 amplified HR(+) breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-10.

In vitro cell cycle oscillations exhibit a robust and hysteretic response to changes in cytoplasmic density

Cells control the properties of the cytoplasm to ensure proper functioning of biochemical processes. Recent studies showed that cytoplasmic density varies in both physiological and pathological states of cells undergoing growth, division, differentiation, apoptosis, senescence, and metabolic starvation. Little is known about how cellular processes cope with these cytoplasmic variations. Here, we study how a cell cycle oscillator comprising cyclin-dependent kinase (Cdk1) responds to changes in cytoplasmic density by systematically diluting or concentrating cycling Xenopus egg extracts in cell-like microfluidic droplets. We found that the cell cycle maintains robust oscillations over a wide range of deviations from the endogenous density: as low as 0.2× to more than 1.22× relative cytoplasmic density (RCD). A further dilution or concentration from these values arrested the system in a low or high steady state of Cdk1 activity, respectively. Interestingly, diluting an arrested cytoplasm of 1.22× RCD recovers oscillations at lower than 1× RCD. Thus, the cell cycle switches reversibly between oscillatory and stable steady states at distinct thresholds depending on the direction of tuning, forming a hysteresis loop. We propose a mathematical model which recapitulates these observations and predicts that the Cdk1/Wee1/Cdc25 positive feedback loops do not contribute to the observed robustness, supported by experiments. Our system can be applied to study how cytoplasmic density affects other cellular processes.

Compartmentalized control of Cdk1 drives mitotic spindle assembly.

During cell division, dramatic microtubular rearrangements driven by cyclin B-cdk1 (Cdk1) kinase activity mark the onset of mitosis leading to dismantling of the interphase microtubular cytoskeleton and assembly of the mitotic spindle. During interphase, Cdk1 accumulates in an inactive state, phosphorylated at inhibitory sites by Wee1/Myt1 kinases. At mitosis onset, Cdc25 phosphatase dephosphorylates and activates Cdk1. Once activated, Cdk1 clears cytoplasmic microtubules by inhibiting microtubule-stabilizing and growth-promoting microtubule-associated proteins (MAPs). Nevertheless, some of these MAPs are required for spindle microtubule growth and spindle assembly, creating quite a conundrum. We show here that a Cdk1 fraction bound to spindle structures escapes Cdc25 action and remains inhibited by phosphorylation (i-Cdk1) in mitotic human cells. Loss or restoration of i-Cdk1 inhibits or promotes spindle assembly, respectively. Furthermore, polymerizing spindle microtubules foster i-Cdk1 aggregating with Wee1 and excluding Cdc25. Our data reveal that spindle assembly relies on compartmentalized control of Cdk1 activity.