Premature Entry Research Articles

Visual and Surgical Outcomes of Outreach Cataract Surgeries in Ilam District of Nepal: An Observational Study

Introduction: Cataracts are the leading cause of preventable blindness worldwide. Although cataract blindness is reversible, its service coverage remains poor, particularly in rural and hilly areas of Nepal. The study aimed to evaluate visual outcomes of outreach cataract surgeries and associated intraoperative and post-operative complications. Methods: This cross-sectional study was conducted at a district of Eastern Nepal after ethical clearance was taken from the Institutional Review Committee (Reference number: 88). Total 131 subjects had cataract surgeries at the surgical camp, and their pre-operative, one-day and one-month post-operative visual acuities were compared, and intraoperative and post-operative surgical complications were recorded. Visual acuity ≥ 6/12 was considered normal. Data was collected in excel and analyzed in SPSS. Results: The mean age of participants was 71.8±9.51 years, with 70 (53.43%) male. A total of 156 eyes from 131 subjects underwent cataract surgeries and among them 25 (19.08%) cases underwent bilateral cataract surgeries. At the day one of surgery, visual acuity improved to normal in 137 (87.82%) of cases, while after one-month, visual acuity improved to normal in 150 (96.15%) of cases with best-corrected glasses. There were 4 (2.56%) intraoperative complications, which included surgery-induced subluxated bag, premature entry, iris trauma, and posterior capsule rent. Conclusions: The visual and surgical outcomes of cataract surgeries performed at an outreach surgical camp were excellent with minimal surgical complications.

"Mitotic" kinesin-5 is a dynamic brake for axonal growth.

During neuronal development, neurons undergo significant microtubule reorganization to shape axons and dendrites, establishing the framework for efficient wiring of the nervous system. Previous studies from our laboratory demonstrated the key role of kinesin-1 in driving microtubule-microtubule sliding, which provides the mechanical forces necessary for early axon outgrowth and regeneration in Drosophila melanogaster. In this study, we reveal the critical role of kinesin-5, a mitotic motor, in modulating the development of postmitotic neurons. Kinesin-5, a conserved homotetrameric motor, typically functions in mitosis by sliding antiparallel microtubules apart in the spindle. Here, we demonstrate that the Drosophila kinesin-5 homolog, Klp61F, is expressed in larval brain neurons, with high levels in ventral nerve cord (VNC) neurons. Knockdown of Klp61F using a pan-neuronal driver leads to severe locomotion defects and complete lethality in adult flies, mainly due to the absence of kinesin-5 in VNC motor neurons during early larval development. Klp61F depletion results in significant axon growth defects, both in cultured and in vivo neurons. By imaging individual microtubules, we observe a significant increase in microtubule motility, and excessive penetration of microtubules into the axon growth cone in Klp61F-depleted neurons. Adult lethality and axon growth defects are fully rescued by a chimeric human-Drosophila kinesin-5 motor, which accumulates at the axon tips, suggesting a conserved role of kinesin-5 in neuronal development. Altogether, our findings show that at the growth cone, kinesin-5 acts as a brake on kinesin-1-driven microtubule sliding, preventing premature microtubule entry into the growth cone. This regulatory role of kinesin-5 is essential for precise axon pathfinding during nervous system development.

The timing and location of entry in growing markets: subgame perfection at work

AbstractWe develop and analyze a dynamic model in which firms decide when and where to enter a growing market. We do not pre‐specify the order of entry, allowing instead for the leader and follower to be determined endogenously. We characterize the subgame perfect equilibria of the dynamic game and show the times and locations of entry are governed by the threat of preemption, which leads to premature entry, less extreme locations, and the dissipation of rents. Using data on gas stations, restaurants, and hotels in isolated markets, we find results consistent with subgame perfection for gas stations and three‐star hotels.

Abstract CT195: First-in-human phase 1 study of WEE1 inhibitor APR-1051 in patients with advanced solid tumors harboring cancer-associated gene alterations

Abstract Background: WEE1 tyrosine kinase plays a key role in cell cycle regulation and DNA damage responses, acting as a principal gatekeeper of the G1-S and G2-M cell cycle checkpoints. Thus, inhibition of WEE1 can result in high cyclin-dependent kinase 1 and 2 activities, leading to aberrant cell cycle progression. Increasing premature entry into mitosis and subsequent apoptosis via WEE1 inhibition is one potential approach to killing cancer cells effectively. APR-1051 is an orally bioavailable, highly potent, small molecule inhibitor of WEE1 that has demonstrated in vivo anti-proliferative activity in multiple cancer cell lines and high selectivity for WEE1 versus polo-like kinases 1, 2, and 3. Increased selectivity for WEE1 may reduce the myelosuppressive toxicity (e.g., limiting Grade 3 toxicities) reported with other WEE1 inhibitors. Methods: The primary aim of this multi-center, open-label Phase I study is to characterize the safety, dose-limiting toxicities (DLTs), maximum tolerated dose (MTD)/maximum administered dose (MAD) and recommended Phase 2 dose (RP2D) of APR-1051 in patients with advanced solid tumors harboring cancer-associated gene alterations. Secondary objectives include evaluating the pharmacokinetic (PK) properties of APR-1051, assessing the effect of food on its PK, and its preliminary efficacy per RECIST version 1.1. The exploratory objective is to assess the pharmacodynamic effects of APR-1051. For dose escalation, an accelerated titration followed by a BOIN design will be utilized to determine the MAD/MTD. Upon completion of the dose escalation, two suitable doses and schedules of single-agent APR-1051 will be determined for further investigation in dose selection optimization. Key inclusion criteria are age at least 18 years with advanced solid tumor and cancer-associated gene alterations (e.g., amplification/overexpression of CCNE1 or CCNE2, deleterious mutations in FBXW7 or PPP2R1A), ECOG PS 0 or 1, and adequate organ function. Key exclusion criteria are cancer therapy within 3 weeks or at least 5 half-lives, prior radiation therapy at the target lesion except if evidence of disease progression, unresolved therapy-related AEs, investigational agent within 5 half-lives or 30 days of study drug, prior WEE1 inhibitor, CNS metastases or unstable involvement, secondary malignancies that are active and/or require therapy, and concomitant moderate/strong inhibitors/inducers of CYP3A4/5, MDR1, or BCRP. Up to 79 patients will be enrolled at 3 to 5 sites in the United States. Citation Format: Timothy A. Yap, Crystal Miller, David Stenehjem, Eric J. Brown, Mike Carleton, Nadeem Q. Mirza. First-in-human phase 1 study of WEE1 inhibitor APR-1051 in patients with advanced solid tumors harboring cancer-associated gene alterations [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 CT195.

An improved B-L model for dynamic stall prediction of rough-surface airfoils

The Beddoes-Leishman (B-L) semi-empirical model is a classical method for predicting the dynamic stall of wind turbine airfoils. However, the model performs poorly for rough-surface airfoils due to the premature entry of turbulence and stall condition under the roughness effect. In this study, an improved dynamic stall model is developed for rough-surface airfoils. Two corrections are involved in the modifications. First, the calculation of flow separation points is corrected considering the difference between the normal and tangential separation points, and then an attenuation coefficient is introduced for the effective angle of attack to account for the rough-surface effect. Subsequently, the proposed model is extensively verified by experimental data of S-series airfoils with rough surfaces. Results show that the proposed model can well predict the dynamic stall characteristics of rough-surface airfoils with a higher accuracy than that of the original B-L model under various mean angle of attacks, oscillating amplitudes and reduced frequencies. At last, the model is utilized to predict the operating loads of a wind turbine using the Blade-Element Momentum theory. Significant difference between the dynamic loads predicted by smooth and rough models is observed. This indicates that the roughness effect on the dynamic stall model as well as the prediction of the operating loads on wind turbines cannot be ignored.

Abstract 6487: The selective WEE1 inhibitor azenosertib shows synergistic anti-tumor activity with KRASG12C inhibitors in multiple KRASG12C models

Abstract KRASG12C is a potent oncogenic driver which results in downstream hyperactivation of MAPK signaling and unchecked oncogenic growth, while simultaneously increasing replication stress (RS) and accumulation of DNA damage. KRASG12C-specific inhibitors are approved and/or recommended in metastatic non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), and while responses are robust, some patients are inherently resistant, and most responders acquire resistance. Resistance mechanisms include reactivation of MAPK signaling, and cancer cells which escape MAPKi are reported to have high levels of genomic instability. WEE1 is a kinase involved in cell cycle progression, allowing cells to repair damaged DNA before entering the next cell cycle phase. Consequently, inhibition of WEE1 leads to premature cell cycle entry and RS, thereby driving cancer cells through the cell cycle with unchecked accumulation of DNA damage, ultimately resulting in mitotic catastrophe and cell death. We hypothesized that high levels of genomic instability and RS in KRASG12C mutant and KRASG12C inhibitor-resistant tumors present a vulnerability to WEE1 inhibition and provide rationale for combining azenosertib, a novel, selective, and orally bioavailable WEE1 inhibitor, with KRASG12C inhibitors. Here, we report azenosertib in combination with KRASG12C inhibitors as an effective therapeutic strategy for KRASG12C tumors. In vitro combination of azenosertib with multiple KRASG12C inhibitors demonstrated synergistic cell growth inhibition across a panel of KRASG12C cell lines in both 2D and 3D assays. In vivo treatment with azenosertib in KRASG12C inhibitor-sensitive cell line-derived xenograft (CDX) models of NSCLC (NCI-H358, NCI-H2122, NCI-H1792), CRC (SW837, SW1463), and pancreatic cancer (MIA PaCa-2) demonstrated significant monotherapy activity as well as synergistic tumor growth inhibition (TGI) when combined with KRASG12C inhibitors, including tumor regression in some models. Importantly, the KRASG12C inhibitor-resistant SW1573 NSCLC CDX model demonstrated synergistic TGI in the combo arm (84%) when compared against monotherapy azenosertib (31%) or adagrasib (58%). Similarly, treatment of a KRASG12C inhibitor-resistant patient-derived xenograft (PDX) model of CRC resulted in synergistic TGI in the combination arm (61%) relative to monotherapy azenosertib (17%) or adagrasib (2%). Finally, analysis of biomarkers from both in vitro and in vivo tumor samples demonstrated synergistic increases in protein markers of RS, DNA damage, and apoptosis with combination therapy. Taken together, our results suggest that the combination of azenosertib with KRASG12C inhibitors enhances tumor growth inhibition over single agent therapy and may be an effective treatment option for patients with KRASG12C tumors. Citation Format: Nathan M. Jameson, Gabriel Kim, Catherine Lee, Blake Skrable, Alexandra Shea, Monah Abed, Olivier Harismendy, Jianhui Ma, Doris Kim, Mark R. Lackner. The selective WEE1 inhibitor azenosertib shows synergistic anti-tumor activity with KRASG12C inhibitors in multiple KRASG12C 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 6487.

The dynamic role of nucleoprotein SHCBP1 in the cancer cell cycle and its potential as a synergistic target for DNA-damaging agents in cancer therapy

BackgroundMalignant tumours seriously threaten human life and health, and effective treatments for cancer are still being explored. The ability of SHC SH2 domain-binding protein 1 (SHCBP1) to induce cell cycle disturbance and inhibit tumour growth has been increasingly studied, but its dynamic role in the tumour cell cycle and corresponding effects leading to mitotic catastrophe and DNA damage have rarely been studied.ResultsIn this paper, we found that the nucleoprotein SHCBP1 exhibits dynamic spatiotemporal expression during the tumour cell cycle, and SHCBP1 knockdown slowed cell cycle progression by inducing spindle disorder, as reflected by premature mitotic entry and multipolar spindle formation. This dysfunction was caused by G2/M checkpoint impairment mediated by downregulated WEE1 kinase and NEK7 (a member of the mammalian NIMA-related kinase family) expression and upregulated centromere/kinetochore protein Zeste White 10 (ZW10) expression. Moreover, both in vivo and in vitro experiments confirmed the significant inhibitory effects of SHCBP1 knockdown on tumour growth. Based on these findings, SHCBP1 knockdown in combination with low-dose DNA-damaging agents had synergistic tumouricidal effects on tumour cells. In response to this treatment, tumour cells were forced into the mitotic phase with considerable unrepaired DNA lesions, inducing mitotic catastrophe. These synergistic effects were attributed not only to the abrogation of the G2/M checkpoint and disrupted spindle function but also to the impairment of the DNA damage repair system, as demonstrated by mass spectrometry-based proteomic and western blotting analyses. Consistently, patients with low SHCBP1 expression in tumour tissue were more sensitive to radiotherapy. However, SHCBP1 knockdown combined with tubulin-toxic drugs weakened the killing effect of the drugs on tumour cells, which may guide the choice of chemotherapeutic agents in clinical practice.ConclusionIn summary, we elucidated the role of the nucleoprotein SHCBP1 in tumour cell cycle progression and described a novel mechanism by which SHCBP1 regulates tumour progression and through which targeting SHCBP1 increases sensitivity to DNA-damaging agent therapy, indicating its potential as a cancer treatment.Graphical

Rac1 promotes kidney collecting duct repair by mechanically coupling cell morphology to mitotic entry.

Prolonged obstruction of the ureter, which leads to injury of the kidney collecting ducts, results in permanent structural damage, while early reversal allows for repair. Cell structure is defined by the actin cytoskeleton, which is dynamically organized by small Rho guanosine triphosphatases (GTPases). In this study, we identified the Rho GTPase, Rac1, as a driver of postobstructive kidney collecting duct repair. After the relief of ureteric obstruction, Rac1 promoted actin cytoskeletal reconstitution, which was required to maintain normal mitotic morphology allowing for successful cell division. Mechanistically, Rac1 restricted excessive actomyosin activity that stabilized the negative mitotic entry kinase Wee1. This mechanism ensured mechanical G2-M checkpoint stability and prevented premature mitotic entry. The repair defects following injury could be rescued by direct myosin inhibition. Thus, Rac1-dependent control of the actin cytoskeleton integrates with the cell cycle to mediate kidney tubular repair by preventing dysmorphic cells from entering cell division.

Experiências no combate ao abandono e evasão escolar no Brasil

This article aims to understand the phenomenon of school dropout and evasion in basic education and its main consequences. The research sought to answer the following questions: What are the main reasons that lead students to drop out of basic school? What are the consequenses of dropping out or evading from the basic school? The study was conducted through bibliographic research, based on authors such as Arroyo (2010), Ávila (2015), Branco (2020), Moura (2020), among others. It was found the main causes of school dropout and evasion are teenage pregnancy, alcohol and substance use, premature entry into the labor market, and social inequality. It was noticed that the problem of school dropout and evasion is not linear, but rather a cycle where most causes are also consequences. Its tacking requires more efficient public policies, political commitment, and social organization.

Abstract B144: Discovery and preclinical evaluation of novel oral WEE1 degraders

Abstract Background: WEE1 is a key component of cell cycle checkpoints as it inhibits cell cycle progression through phosphorylation of CDK1. Inhibition of WEE1 could induce premature mitotic entry of cells with damaged DNA, leading to mitotic catastrophe and cell death. WEE1 small molecule inhibitors (SMIs) have demonstrated clinical benefits particularly in gynecological malignancies. However, limited antitumor activity and multifaceted toxicity profile from SMIs warranted the discovery of novel WEE1-targeting modalities to afford better therapeutic index towards a greater patient population. Here, we report the discovery of a novel oral, heterobifunctional WEE1 degrader NXD01 and its preclinical evaluation. Methods: We discovered NXD01 through our AI-empowered, prediction-guided compound optimization platform neoDegrader. We quantified NXD01-mediated WEE1 protein degradation by western blot and evaluated the anti-proliferative activity of NXD01 by CellTiter-Glo assay in multiple human cancer cell lines. We further assessed downstream effects of WEE1 degradation on phosphorylation of its target CDK1, and markers of cell cycle and apoptosis by western blot and flow cytometry. We studied the in vivo antitumor activity of NXD01 by oral gavage in the SK-UT-1 (human uterine leiomyosarcoma cell) xenograft model, followed by PK/PD analysis of the tumor tissues. Results: In SK-UT-1 cells, NXD01 potently degraded WEE1 protein at a DC50 of 1.4 nM with no obvious hook effect and achieved complete degradation within 4 h of treatment (DC90 = 4.5 nM). Marked reduction in CDK1-Y15 phosphorylation and increased γ-H2AX levels (a marker for DNA damage) and PARP cleavage (apoptosis) were all detected for NXD01 at 100 nM, one-tenth the effective concentration for the WEE1 SMI azenosertib (ZN-c3). Treatment of NXD01 (but not ZN-c3) at 100 nM greatly increased the proportion of cells in the G2/M phase indicating mitotic arrest. These results translated into a more potent anti-proliferative effect by NXD01 compared to ZN-c3 in vitro: the IC50 was 1.3 nM vs 37 nM for MOLT4, a T cell leukemia line, as well as across 10 gynecological cancer cell lines (NXD01 IC50: 10-210 nM; ZN-c3 IC50: 100-975 nM). NXD01 exhibited good exposure after oral administration in mice (dose normalized AUC0-t: 765 mg∙h/mL, t1/2: 3 h, F%: 15.7%). NXD01 showed strong in vivo antitumor activity in the SK-UT-1 xenograft model, achieving 96% tumor growth inhibition (TGI) at 30 mg/kg BID, as compared to 81% TGI for ZN-c3 at 60 mg/kg QD. Analysis of tumor samples suggested that 85% of in-tumor WEE1 protein had been degraded by NXD01 at 12 h post dose. Conclusion: We have discovered a structurally-novel, orally bioavailable, cereblon-mediated, heterobifunctional degrader of WEE1 protein NXD01. NXD01 displayed stronger in vitro and in vivo antitumor activity than currently the most advanced WEE1 small molecule inhibitor azenosertib, making NXD01 a differentiated candidate with strong potential for clinical development. Citation Format: Weiqiang Xing, Meixin Tao, Fan Huang, Liuge Gu, Min Cao, Chunhao Wang, Jianxiong Diao, Chunming Du, Xinyu Bai, Mingchen Chen, Xin Zheng, Ying Lei, Yang Xie, Taylor B. Guo. Discovery and preclinical evaluation of novel oral WEE1 degraders [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 B144.

Abstract B057: Preclinical development of PKMYT1 and ATR inhibitor combinations

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

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.

Targeting WEE1 kinase as a p53-independent therapeutic strategy in high-risk and relapsed acute lymphoblastic leukemia

BackgroundOutcomes for patients with relapsed acute lymphoblastic leukemia (ALL) are poor and there is a need for novel therapies to improve outcomes. Targeted inhibition of WEE1 with small-molecule inhibitor adavosertib (AZD1775) has emerged as a therapeutic strategy to sensitize cancer cells to DNA-damaging chemotherapeutics, particularly in the context of TP53-mutated tumors. However, WEE1 inhibition as a potential therapeutic strategy for patients with high-risk and relapsed ALL, including those with TP53 mutations, has not been definitively evaluated.MethodsAnti-leukemic effects of adavosertib were investigated using a relapsed TP53 isogenic cell model system, primary patient, and patient-derived ALL samples (n = 27) in an ex vivo co-culture model system with bone marrow-derived mesenchymal stem cells. Combination effects with drugs currently used for relapsed ALL were quantified by Excess over Bliss analyses. Investigations for alterations of cell cycle and apoptosis as well as related proteins were examined by flow cytometry and Western blot, respectively.ResultsOur study demonstrates the potent anti-leukemic activity of the clinically advanced WEE1 inhibitor adavosertib in a large majority (n = 18/27) of high-risk and relapsed ALL specimens at lower than clinically attainable concentrations, independent of TP53 mutation status. We show that treatment with adavosertib results in S-phase disruption even in the absence of DNA-damaging agents and that premature mitotic entry is not a prerequisite for its anti-leukemic effects. We further demonstrate that WEE1 inhibition additively and synergistically enhances the anti-leukemic effects of multiple conventional chemotherapeutics used in the relapsed ALL treatment setting. Particularly, we demonstrate the highly synergistic and cytotoxic combination of adavosertib with the nucleoside analog cytarabine and provide mechanistic insights into the combinational activity, showing preferential engagement of apoptotic cell death over cell cycle arrest. Our findings strongly support in vivo interrogation of adavosertib with cytarabine in xenograft models of relapsed and high-risk ALL.ConclusionsTogether, our data emphasize the functional importance of WEE1 in relapsed ALL cells and show WEE1 as a promising p53-independent therapeutic target for the improved treatment of high-risk and relapsed ALL.

Numerical Calculation of Magnetic Field Enhancement and Impact of Surface Defects on Premature Entry of Magnetic Field in Thin Nb Films for SRF Cavities

In this work, we have investigated two series of Nb/Cu samples deposited by HiPIMS technique, and differing in Nb deposition conditions, Nb film thickness and Cu substrate polishing techniques. All the films were additionally irradiated by Nd:YAG laser to smooth their surfaces. The impact of the magnetic field enhancement at the surface defects on the premature start of magnetic field penetration into the superconducting film was studied, combining experiments and numerical calculations. Compared to previous study, improved numerical calculations by using the Finite Element Method (FEM) served to calculate the maximum field enhancement factor β <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">m</sub> , reflecting impact of the most crucial surface defects found in the samples. Magnetization measurements at 4.2 K in DC magnetic field, oriented parallel to the film, were employed to determine the start of the field penetration <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">H</i> <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">en</sub> . The SEM and AFM analyses served to investigate the Nb surface morphology. In some samples, deviations from the <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">H</i> <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">en</sub> (β <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">m</sub> ) dependence were observed. It was found that the magnetic field penetration could start from the Nb/Cu interface rather than from the free Nb surface due to visibly better quality of the free Nb surface observed by SEM analysis, and that could lead to the deterioration of <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">H</i> <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">en</sub> (β <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">m</sub> ) dependence.

DNA replication stress and mitotic catastrophe mediate sotorasib addiction in KRASG12C-mutant cancer

BackgroundSotorasib is the first KRASG12C inhibitor approved by the US Food and Drug Administration for treating KRASG12C-mutant non-small-cell lung cancer (NSCLC). Clinical trials on the therapeutic use of sotorasib for cancer have reported promising results. However, KRASG12C-mutant cancers can acquire resistance to sotorasib after treatment. We incidentally discovered that sotorasib-resistant (SR) cancer cells are addicted to this inhibitor. In this study, we investigated the mechanisms underlying sotorasib addiction.MethodsSotorasib-resistant cells were established using KRASG12C-mutant pancreatic cancer and NSCLC cell lines. Cell viability in the presence or absence of sotorasib and in combination with multiple inhibitors was assessed through proliferation assay and annexin V/propidium iodide (PI) flow cytometry assays. The mechanisms underlying drug addiction were elucidated through 5-bromo-2′-deoxyuridine (BrdU) incorporation assay, immunofluorescence staining, time-lapse microscopy, and comet assay. Furthermore, a subcutaneous xenograft model was used to demonstrate sotorasib addiction in vivo.ResultsIn the absence of sotorasib, the sotorasib-resistant cells underwent p21Waf1/Cip1-mediated cell cycle arrest and caspase-dependent apoptosis. Sotorasib withdrawal resulted in robust activation of mitogen-activated protein kinase (MAPK) pathway, inducing severe DNA damage and replication stress, which activated the DNA damage response (DDR) pathway. Persistent MAPK pathway hyperactivation with DDR exhaustion led to premature mitotic entry and aberrant mitosis, followed by micronucleus and nucleoplasmic bridge formation. Pharmacologic activation of the MAPK pathway with a type I BRAF inhibitor could further enhance the effects of sotorasib withdrawal on sotorasib-resistant cancer cells both in vitro and in vivo.ConclusionsWe elucidated the mechanisms underlying the sotorasib addiction of cancer cells. Sotorasib addiction appears to be mediated through MAPK pathway hyperactivity, DNA damage, replication stress, and mitotic catastrophe. Moreover, we devised a therapeutic strategy involving a type I BRAF inhibitor to strengthen the effects of sotorasib addiction; this strategy may provide clinical benefit for patients with cancer.

TRLS-14. TARGETING REPLICATIVE STRESS IN BRAIN TUMORS BY COMBINING ATR AND PARP INHIBITION

Abstract Pediatric brain tumors like MYC-amplified Medulloblastomas (MB) and Atypical Teratoid and Rhabdoid Tumors (AT/RT) continue to have poor survival rates despite recent advances in understanding tumor biology. MYC is an oncogenic transcription factor often expressed in MB and AT/RT at high levels. While direct inhibition of MYC by small molecules is an active area of research, such approaches lack clinical validation and are not yet actionable. Strategies to target MYC-driven brain tumors that are immediately translatable are urgently needed. Replication stress has been identified as a prominent source of tumorigenesis over the past decade and is a potential target for cancer therapy. Specific oncogenes induce elevated levels of replicative stress through aberrant activation of transcription and dysregulation of DNA replication. In these instances, replicative stress could be rescued by either metabolic supplementation or MYC expression. The activation of ATR and PARP are critical events in response to replicative stress. PARP inhibitors stall replication forks and promote the generation of single-strand DNA, which activates ATR. Genetic and pharmacological evidence suggests the sensitization of MYC-induced tumors to PARP inhibitors. Preclinical studies in diverse tumor backgrounds have demonstrated that the combination of ATR and PARP inhibition is synergistic and leads to premature entry into mitosis, enhanced replicative stress and cell death, and tumor regression. Additionally, a reduction in ATR activity can be lethal in oncogene-induced replication stress. Using high throughput drug screening across MB and AT/RT cell lines, we found that the CNS penetrant PARP inhibitor AZD9754 significantly potentiated the cytotoxicity of ATR inhibition. This emerging synergistic combination therapy reveals new therapeutic targets in MYC-driven replicative stress. Studies are ongoing to validate in vitro findings in vivo.

Abstract 950: Targeting PKMYT1 kinase is an effective treatment strategy in triple negative breast cancers with low molecular weight cyclin E (LMW-E) expression

Abstract Background: Cyclin E is post-translationally modified by neutrophil elastase mediated proteolytic cleavage to generate the low molecular weight isoforms of cyclin E (LMW-E) that are detected in various human cancers. We previously reported that 70% of triple negative breast cancers (TNBC) examined overexpress LMW-E, and these patients have a poor prognosis. Expression of LMW-E promotes genomic instability by causing DNA replication stress. PKMYT1 prevents premature mitotic entry by catalyzing CDK1 phosphorylation at T14, essential for preventing DNA damage and cell death when cyclin E, including LMW-E, is overexpressed. In this study, we tested the hypothesis that LMW-E positive status can be used as a biomarker of response in selecting TNBC patients who are likely to respond to RP-6306, a first in-class and selective inhibitor of PKMYT1 kinase. Results: Assessment of pre-treatment breast biopsies from TNBC patients (n=40) enrolled in a neoadjuvant chemotherapy prospective study for LMW-E and CDK1-pT14 revealed significant positive correlation between these two proteins. Furthermore, positivity of both biomarkers was associated with lack of pathologic complete response (pCR) to neoadjuvant chemotherapy. We next examined the mechanism of response to RP-6306 in vitro and in vivo using TNBC cell lines, patient-derived xenograft (PDX) models and transgenic mouse mammary tumor virus (MMTV) models expressing human LMW-E (hLMW-E). In vitro results using 7 different TNBC cell lines, revealed that high LMW-E levels are significantly predictive of response to RP-6306 (R2=0.78, p= 0.008), while LMW-E knockdown resulted in a 7X increase in IC50 values of RP-6306 (p&amp;lt;0.001). In high LMW-E cells, treatment with RP-6306 resulted in significant (i) downregulation of CDK1-pT14, PKMYT1, WEE1, cyclin B and pRb, (ii) accumulation of sub-G1 and polyploid cell population, (iii) apoptosis, (iv) accumulation of chromosomal breakage, (v) increased DNA damage (increase in γ-H2AX and 53BP1 foci/cell) and lack of DNA repair (downregulation of Rad51), and (vi) premature mitotic entry. Treatment of both breast cancer PDX models and hLMW-E transgenic tumors with RP-6306 revealed that only in animals with high LMW-E tumors, treatment results in significant decrease in tumor volume. However, RP-6306 was ineffective in reducing tumor volume in low cyclin E in vivo models. Immunohistochemical analysis revealed that in vivo efficacy of RP-6306 (in both PDX and transgenic models) was concomitant with increase in γ-H2AX and decrease in CDK1-pT14 and Ki67. Conclusion: Collectively, our results show that overexpression of LMW-E and CDK1-pT14 in TNBC can be used to stratify patients whose tumors are likely to respond to RP-6306. Mechanistically, LMW-E overexpressing TNBC cells activate CDK1 (in vitro and in vivo) to accelerate premature mitotic entry, leading to DNA damage and apoptosis. Citation Format: Amriti Lulla, Tuyen D. Nguyen, Mi Li, Sofia Mastoraki, Yan Wang, Tuyen Bui, Marc Pina, Spiridon Tsavachidis, Gary Marshall, Kelly K. Hunt, Khandan Keyomarsi. Targeting PKMYT1 kinase is an effective treatment strategy in triple negative breast cancers with low molecular weight cyclin E (LMW-E) expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 950.

Abstract 2132: Tumor heterogeneity of CCNE1 copy number assessed by fluorescence in situ hybridization (FISH) in ovarian and uterine cancers and correlation with cyclin E protein expression

Abstract Amplification of CCNE1 is a recurrent genetic alteration that has been associated with chemoresistance and poor prognosis in gynecological malignancies. We identified Protein Kinase, Membrane Associated Tyrosine/Threonine 1 (PKMYT1) as a novel synthetic lethal target in CCNE1 amplified cancers. In this genomic setting, PKMYT1 inhibition causes premature mitotic entry, mitotic catastrophe and, in turn, cell death. In pre-clinical models, correlation between CCNE1 copy number, cyclin E1 expression and sensitivity to PKMYT1 inhibition is imperfect. Thus, a deep understanding of CCNE1 amplification as a predictive biomarker for PKMYT1 inhibition is critical for refining patient selection strategies and strengthening our understanding of the mechanism of action. These learnings will be applied to correlative biomarker analyses in clinical trials evaluating RP-6306, a first-in-class, potent, and highly selective PKMYT1 inhibitor, in tumors with CCNE1 amplifications (NCT04855656, NCT05147272, NCT05147350). In this study, multiple features of CCNE1 were examined using a combination of whole genome and targeted next generation sequencing (NGS), FISH, and immunohistochemistry (IHC). Cyclin E1 levels were evaluated across tumor tissue microarrays spanning 5 tumor indications: triple-negative breast (n=100), colorectal (n=100), gastric (n=100), uterine (n=49), ovarian (n=100) cancers. The highest cyclin E1 protein levels were found in ovarian and uterine malignancies, consistent with indications with high prevalence of CCNE1 amplification (median H-score ovarian = 125; uterine = 100; gastric = 45; TNBC = 48 and CRC = 24). To explore CCNE1 amplification on a single cell level, we established a novel FISH assay using a subtelomeric q-arm control probe. The subtelomeric q-arm band is more frequently diploid than other regions on chromosome 19. Analytical validation of the FISH assay demonstrated sensitivity of 95%, specificity of 95.2%, and overall concordance of 95.1% when compared to a targeted sequencing panel (SNIPDx®). Within the uterine and ovarian cohorts, cancers harboring CCNE1 amplification were found to display higher cyclin E1 protein levels than non-amplified tumors (p-val=0.0002) and cyclin E1 expression was positively correlated with CCNE1 copy number (Spearman ρ=0.67; p-value=4.1e-05). Substantial intratumoral spatial heterogeneity of CCNE1 copy number when measured by FISH was observed across ovarian and uterine tumors. The potential impact of CCNE1 amplification heterogeneity, cyclin E1 protein levels and CCNE1 amplification fragment size on response to RP-6306 in preclinical models are currently being investigated and will be reported. Citation Format: Adam Petrone, Elia Aguado-Fraile, Sunantha Sethuraman, Adrienne Johnson, Ian Silverman, Gary Marshall, Jorge S. Reis-Filho, John Iafrate, Artur Veloso, Victoria Rimkunas. Tumor heterogeneity of CCNE1 copy number assessed by fluorescence in situ hybridization (FISH) in ovarian and uterine cancers and correlation with cyclin E protein expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2132.

Abstract 6107: Novel roles for AMBRA1 in regulating DNA double-strand breaks

Abstract AMBRA1 (Activating Molecule in Beclin-1-Regulated Autophagy) is a tumor suppressor protein whose expression is downregulated in several human malignancies. AMBRA1 was recently found to function as a substrate receptor for the cullin-4-based ubiquitin ligase (CRL4AMBRA1) that promotes the ubiquitin-dependent proteolysis of D-type cyclins (cyclin D1, D2 and D3); this serves to guard against premature S-phase entry and replication stress, providing a potential mechanism for its tumor suppressive activity. Cyclin D1 protein, which is stabilized, along with cyclin D2 and D3, in AMBRA1-deficient cells, has been shown to promote homologous recombination (HR) repair of DNA double-strand breaks (DSBs), suggesting that AMBRA1 may be additionally involved in the repair of DSBs. Using our recently published CRISPR-Cas9-based dual fluorescent reporter assay system, we show that while the loss of AMBRA1 increased D-type cyclins and Rb hyperphosphorylation as expected, it did, surprisingly, inhibit HR-mediated repair and stimulated error-free non-homologous end-joining (NHEJ), and that both of these new activities are independent of cyclin D1 or RB1. High-throughput sequencing of DSB repair junctions confirm these results and shows significant depletion of indels with reduction in microhomology-mediated end-joining in AMBRA1-deficient cells, suggesting that AMBRA1 plays a role in promoting resection at DSBs. In support of this hypothesis, we found that the loss of AMBRA1 reduces RAD51 recruitment to DSBs in cells exposed to ionizing radiation. Consistent with its role in promoting HR, we found that AMBRA1-deficient cells are significantly more sensitive to PARP inhibitors compared to AMBRA1-proficient cells. In summary, we show that AMBRA1 is a novel regulator of DSBs, and that its downregulation in cancer cells and tumors render cancer cells susceptible to inhibition by PARP inhibitors. Citation Format: Yuning Jiang, Tarek Abbas. Novel roles for AMBRA1 in regulating DNA double-strand breaks. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6107.

Abstract 5511: Investigating Wee1 and Myt1 combined inhibition as a potential cancer therapeutic strategy

Abstract Introduction: Cell cycle is under the surveillance of checkpoints to repair any damage before the cell transits between phases. Wee1 and Myt1 kinases prevent premature entry into mitosis by monitoring the G2/M checkpoint by adding an inhibitory phosphorylation on Cdk1. Wee1 is overexpressed in various tumors and MK-1775, a Wee1 small molecule inhibitor, is currently in phase I/II cancer clinical trials. Although MK-1775 was shown to potentiate the effect of other genotoxic therapies, yet clinical resistance has emerged towards it. In examining the mechanism of MK-1775-mediated cytotoxicity, we identified Myt1 as a resistant factor. Emerging evidence suggests that Myt1 is an important cancer therapeutic target. Hence, we are examining a novel Myt1 kinase small molecule inhibitor, RP-6306 in combination with MK-1775 as a potential synthetic lethal cancer therapy. We hypothesize that a combination of MK-1775 and RP-6306, inhibitors against two partially redundant kinases important for adaptation to genotoxic stress, will achieve synthetic lethality while circumventing the issue of resistance development following monotherapies. Model Systems Used: 1) a cervical cancer cell line that is tetracycline inducible for Myt1 expression; 2) cancer cell lines (breast and cervical) that are resistant to MK-1775 through upregulation of Myt1. Methods: We established the IC50 of RP-6306 in a panel of tumorigenic and non-tumorigenic cell lines using standard crystal violet viability assay. Following that, we tested the combination effect of MK-1775 and RP-6306 in model cell lines listed above using zero potency model of synergy. The effect of the combination treatment on the clonogenicity was evaluated using clonogenic assay. Timelapse microscopy on a High Content Imaging system was used to determine the effect of RP-6306 and MK-1775 on mitotic duration and cell fates. Results: We found that MK-1775 and RP-6306 combination treatment shows synergistic cell killing in a panel of cancer cell lines. Wee1 inhibition shows synergistic cell killing with Myt1 inhibition especially in inducible Myt1 overexpressing cells. Wee1 and Myt1 combined inhibition resists the increase in clonogenic potential of the cancer cells transiently overexpressing Myt1. RP-6306 and MK-1775 combination treatment promotes mitotic arrest leading to cell death in Myt1 overexpressing cells. We also found that the mechanism of cell death in the cells treated with the combination treatment is centromere fragmentation leading to mitotic catastrophe. Conclusions: The combined Wee1 and Myt1 inhibition leads to synthetic lethality. Hence, our findings strongly suggest that the combined MK-1775 and RP-6306 treatment has promising potential to mitigate MK-1775 resistance. Our research contributes to the development of novel potential combination therapy while optimizing and improving the efficacy of MK-1775 treatment for potential clinical use. Citation Format: Sargun Sokhi, Joanne D. Hadfield, Jeremy Fung, Gordon K. Chan. Investigating Wee1 and Myt1 combined inhibition as a potential cancer therapeutic strategy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5511.