G1 Cell Cycle Checkpoint Research Articles

Evaluating the Mechanism of Cell Death in Melanoma Induced by the Cannabis Extract PHEC-66.

Research suggests the potential of using cannabinoid-derived compounds to function as anticancer agents against melanoma cells. Our recent study highlighted the remarkable in vitro anticancer effects of PHEC-66, an extract from Cannabis sativa, on the MM418-C1, MM329, and MM96L melanoma cell lines. However, the complete molecular mechanism behind this action remains to be elucidated. This study aims to unravel how PHEC-66 brings about its antiproliferative impact on these cell lines, utilising diverse techniques such as real-time polymerase chain reaction (qPCR), assays to assess the inhibition of CB1 and CB2 receptors, measurement of reactive oxygen species (ROS), apoptosis assays, and fluorescence-activated cell sorting (FACS) for apoptosis and cell cycle analysis. The outcomes obtained from this study suggest that PHEC-66 triggers apoptosis in these melanoma cell lines by increasing the expression of pro-apoptotic markers (BAX mRNA) while concurrently reducing the expression of anti-apoptotic markers (Bcl-2 mRNA). Additionally, PHEC-66 induces DNA fragmentation, halting cell progression at the G1 cell cycle checkpoint and substantially elevating intracellular ROS levels. These findings imply that PHEC-66 might have potential as an adjuvant therapy in the treatment of malignant melanoma. However, it is essential to conduct further preclinical investigations to delve deeper into its potential and efficacy.

Role of RPA Phosphorylation in the ATR-Dependent G2 Cell Cycle Checkpoint.

Cells respond to DNA double-strand breaks by initiating DSB repair and ensuring a cell cycle checkpoint. The primary responder to DSB repair is non-homologous end joining, which is an error-prone repair pathway. However, when DSBs are generated after DNA replication in the G2 phase of the cell cycle, a second DSB repair pathway, homologous recombination, can come into action. Both ATM and ATR are important for DSB-induced DSB repair and checkpoint responses. One method of ATM and ATR working together is through the DNA end resection of DSBs. As a readout and marker of DNA end resection, RPA is phosphorylated at Ser4/Ser8 of the N-terminus of RPA32 in response to DSBs. Here, the significance of RPA32 Ser4/Ser8 phosphorylation in response to DNA damage, specifically in the S phase to G2 phase of the cell cycle, is examined. RPA32 Ser4/Ser8 phosphorylation in G2 synchronized cells is necessary for increases in TopBP1 and Rad9 accumulation on chromatin and full activation of the ATR-dependent G2 checkpoint. In addition, our data suggest that RPA Ser4/Ser8 phosphorylation modulates ATM-dependent KAP-1 phosphorylation and Rad51 chromatin loading in G2 cells. Through the phosphorylation of RPA Ser4/Ser8, ATM acts as a partner with ATR in the G2 phase checkpoint response, regulating key downstream events including Rad9, TopBP1 phosphorylation and KAP-1 phosphorylation/activation via the targeting of RPA32 Ser4/Ser8.

ATR activation by Cr-DNA damage is a major survival response establishing late S and G2 checkpoints after Cr(VI) exposure

Inhalation exposure to hexavalent chromium is known to cause lung cancer and other pulmonary toxicity. Cellular metabolism of chromium(VI) entering cells as chromate anion produces different amounts of reactive Cr(V) intermediates and finally yields Cr(III). Direct reduction of Cr(VI) by ascorbate (Asc), the dominant metabolic reaction in vivo but not in standard cell cultures, skips production of Cr(V) but still permits extensive formation of Cr-DNA damage. To understand the importance of different forms of biological injury in Cr(VI) toxicity, we examined activation of several protein- and DNA damage-sensitive stress responses in human lung cells under Asc-restored conditions. We found that Asc-restored cells suppressed upregulation of oxidant-sensitive stress systems by Cr(VI) but showed a strong activation of the apical DNA damage-responsive kinase ATR. ATR signaling was triggered in late S phase and persisted upon entry of cells into G2 phase. Inhibition of ATR prevented the establishment of late-S and G2 cell cycle checkpoints and did not lead to a compensatory activation of a related kinase ATM. Inactivation of ATR also strongly impaired viability of Cr(VI)-treated lung cells including stem-like cells and revealed a significant formation of toxic Cr-DNA damage at low Cr(VI) doses. Our findings identified a major Cr(VI) resistance mechanism involving sensing of Cr-DNA damage by ATR in late S phase and a subsequent establishment of protective cell cycle checkpoints.

Inactivation of GSK3β by Ser 389phosphorylation prevents thymocyte necroptosis and impacts Tcrβand Tcrαrepertoire diversity

Abstract Double negative (DN) thymocytes go through consecutive rounds of Tcrβ and Tcrα gene recombination that involves the generation of DNA double-strand breaks (DSB), the addition of N-nucleotides between coding ends, and DNA repair with the ultimate goal of enhancing TCR repertoire diversity. How DN thymocytes can overcome such genomic instability in order to generate functional Tcrβ and Tcrα genes remains unclear. We performed scRNA-seq analyses of isolated DN3 and DN4 thymocytes to demonstrate a clear trajectory from DN3 through the DN4 stage that is delineated by G1 and G2M cell cycle checkpoints to facilitate DNA repair during recombination of Tcrβ and Tcrα prior to cell proliferation. Inactivation of GSK3β by phosphorylation on Ser 389in response to V(D)J recombination DSB is required to prevent thymocyte death by necroptosis. Failure to inactivate GSK3β through this pathway alters the Tcrβ and Tcrα V segments usage. We also show that inactivation of GSK3β by phosphorylation on Ser 389is required for expression of the cytidine deaminase Apobec3 in DN3 thymocytes to increase N-nucleotide diversity within the Tcrβ N2-region. Thus, G1 and G2M cell cycle checkpoints to ensure proper DNA repair during V(D)J recombination and unique GSK3β-dependent survival pathways to prevent necroptosis during this process are critical to generate a large diversity of pre-selection TCR repertoire. Double negative (DN) thymocytes go through consecutive rounds of Tcrβ and Tcrα gene recombination that involves the generation of DNA double-strand breaks (DSB), the addition of N-nucleotides between coding ends, and DNA repair with the ultimate goal of enhancing TCR repertoire diversity. How DN thymocytes can overcome such genomic instability in order to generate functional Tcrβ and Tcrα genes remains unclear. We performed scRNA-seq analyses of isolated DN3 and DN4 thymocytes to demonstrate a clear trajectory from DN3 through the DN4 stage that is delineated by G1 and G2M cell cycle checkpoints to facilitate DNA repair during recombination of Tcrβ and Tcrα prior to cell proliferation. Inactivation of GSK3β by phosphorylation on Ser389 in response to V(D)J recombination DSB is required to prevent thymocyte death by necroptosis. Failure to inactivate GSK3β through this pathway alters the Tcrβ and Tcrα V segments usage. We also show that inactivation of GSK3β by phosphorylation on Ser389 is required for expression of the cytidine deaminase Apobec3 in DN3 thymocytes to increase N-nucleotide diversity within the Tcrβ N2-region. Thus, G1 and G2M cell cycle checkpoints to ensure proper DNA repair during V(D)J recombination and unique GSK3β-dependent survival pathways to prevent necroptosis during this process are critical to generate a large diversity of pre-selection TCR repertoire. Supported by NIH R01 AI051454

Manipulation of Redox Metabolism Using Pharmacologic Ascorbate Opens a Therapeutic Window for Radio-Sensitization by ATM Inhibitors in Colorectal Cancer

Ataxia telangiectasia mutated kinase (ATM) inhibitors are potent radiosensitizers that regulate DNA damage responses and redox metabolism, but they have not been translated clinically because of the potential for excess normal tissue toxicity. Pharmacologic ascorbate (P-AscH-; intravenous administration achieving mM plasma concentrations) selectively enhances H2O2-induced oxidative stress and radiosensitization in tumors while acting as an antioxidant and mitigating radiation damage in normal tissues including the bowel. We hypothesized that P-AscH- could enhance the therapeutic index of ATM inhibitor-based chemoradiation by simultaneously enhancing the intended effects of ATM inhibitors in tumors and mitigating off-target effects in adjacent normal tissues. Clonogenic survival was assessed in human (human colon tumor [HCT]116, SW480, HT29) and murine (CT26, MC38) colorectal tumor lines and normal cells (human umbilical vein endothelial cell, FHs74) after radiation ± DNA repair inhibitors ± P-AscH-. Tumor growth delay was assessed in mice with HCT116 or MC38 tumors after fractionated radiation (5 Gy×3) ± the ATM inhibitor KU60019 ± P-AscH-. Intestinal injury, oxidative damage, and transforming growth factor β immunoreactivity were quantified using immunohistochemistry after whole abdominal radiation (10 Gy) ± KU60019 ± P-AscH-. Cell cycle distribution and ATM subcellular localization were assessed using flow cytometry and immunohistochemistry. The role of intracellular H2O2 fluxes was assessed using a stably expressed doxycycline-inducible catalase transgene. KU60019 with P-AscH- enhanced radiosensitization in colorectal cancer models in vitro and in vivo by H2O2-dependent oxidative damage to proteins and enhanced DNA damage, abrogation of the postradiation G2 cell cycle checkpoint, and inhibition of ATM nuclear localization. In contrast, concurrent P-AscH- markedly reduced intestinal toxicity and oxidative damage with KU60019. We provide evidence that redox modulating drugs, such as P-AscH-, may facilitate the clinical translation of ATM inhibitors by enhancing tumor radiosensitization while simultaneously protecting normal tissues.

9 Oral - NCI10329: Phase Ib Sequential Trial of Agents against DNA Repair (STAR) Study to investigate the sequential combination of the Poly (ADP-Ribose) Polymerase inhibitor (PARPi) olaparib (ola) and WEE1 inhibitor (WEE1i) adavosertib (ada) in patients (pts) with DNA Damage Response (DDR)-aberrant advanced tumors, enriched for BRCA1/2 mutated and CCNE1 amplified cancers

Background: Combination PARPi plus WEE1i (PARPi + WEE1i) induces replication stress (RS), DNA damage, and abrogates the G2 cell cycle checkpoint. Concurrent PARPi + WEE1i administration effectively inhibits tumor growth but is poorly tolerated. Sequential PARPi+WEE1i administration in vivo mirrors concurrent dosing in cells with high basal RS, while normal cells with low basal RS are protected from DNA damage, improving tolerability while preserving efficacy (Fang et al., Cancer Cell 2019). Based on these compelling preclinical data, the NCI CTEP-sponsored STAR study investigated sequential ola+ada in pts with DDR aberrant advanced tumors (NCT04197713).

Sensitization of osteosarcoma to irradiation by targeting nuclear FGFR1

Over the past 25 years, chemotherapy regimens for osteosarcoma have failed to improve the 65–70% long-term survival rate. Radiation therapy is generally ineffective except for palliative care. We here investigated whether osteosarcoma can be sensitized to radiation therapy targeting specific molecules in osteosarcoma. Large-scale RNA sequencing analysis in osteosarcoma tissues and cell lines revealed that FGFR1 is the most frequently expressed receptor tyrosine kinase in osteosarcoma. Nuclear FGFR1 (nFGFR1) was observed by subcellular localization assays. The functional studies using a FGFR1IIIb antibody or small molecule FGFR1 inhibitors showed that nFGFR1, but not membrane-bound FGFR1, induces G2 cell-cycle checkpoint adaptation, cell survival and polyploidy following irradiation in osteosarcoma cells. Further, the activation of nFGFR1 induces Histone H3 phosphorylation at Ser 10 and c-jun/c-fos expression to contribute cell survival rendering radiation resistance. Furthermore, an in vivo mouse study revealed that radiation resistance can be reversed by the inhibition of nFGFR1. Our findings provide insights into the potential role of nFGFR1 to radiation resistance. Thus, we propose nFGFR1 could be a potential therapeutic target or a biomarker to determine which patients might benefit from radiation therapy.

Increased Replication Stress Determines ATR Inhibitor Sensitivity in Neuroblastoma Cells.

Simple SummaryNeuroblastoma is a childhood cancer with poor survival and new therapies are urgently needed, especially for high-risk disease. Here, we demonstrate that novel drugs targeting a protein called ATR can specifically kill a type of high-risk neuroblastoma associated with MYCN expression. We show that the mechanism by which this occurs is via increasing the stress on cells when they replicate their DNA. Further, we show that by targeting ATR in combination with other drugs that cause replication stress, we can increase killing of both high-risk MYCN amplified and non amplified neuroblastoma.Despite intensive high-dose multimodal therapy, high-risk neuroblastoma (NB) confers a less than 50% survival rate. This study investigates the role of replication stress in sensitivity to inhibition of Ataxia telangiectasia and Rad3-related (ATR) in pre-clinical models of high-risk NB. Amplification of the oncogene MYCN always imparts high-risk disease and occurs in 25% of all NB. Here, we show that MYCN-induced replication stress directly increases sensitivity to the ATR inhibitors VE-821 and AZD6738. PARP inhibition with Olaparib also results in replication stress and ATR activation, and sensitises NB cells to ATR inhibition independently of MYCN status, with synergistic levels of cell death seen in MYCN expressing ATR- and PARP-inhibited cells. Mechanistically, we demonstrate that ATR inhibition increases the number of persistent stalled and collapsed replication forks, exacerbating replication stress. It also abrogates S and G2 cell cycle checkpoints leading to death during mitosis in cells treated with an ATR inhibitor combined with PARP inhibition. In summary, increased replication stress through high MYCN expression, PARP inhibition or chemotherapeutic agents results in sensitivity to ATR inhibition. Our findings provide a mechanistic rationale for the inclusion of ATR and PARP inhibitors as a potential treatment strategy for high-risk NB.

RFWD3 Participates in the Occurrence and Development of Colorectal Cancer via E2F1 Transcriptional Regulation of BIRC5.

Objectives: Colorectal cancer (CRC) is one of the most common human malignancies. It was reported that the alterations in the DNA damage response (DDR) pathways are emerging as novel targets for treatment across different cancer types including CRC. RFWD3 plays a critical role in replication protein A (RPA)-mediated DNA damage in cancer cells. More importantly, RFWD3 can response to DNA damage by positively regulating p53 stability when the G1 cell cycle checkpoint is activated. However, the functional significance of RFWD3 in CRC has not been reported in the existing documents.Materials and Methods: Here, we revealed high expression of RFWD3 in CRC tissues by IHC analysis and The Cancer Genome Atlas (TCGA) database. Besides, overexpression of RFWD3 in CRC cell lines was also confirmed by qRT-PCR and western blot assay. The Celigo cell counting method and wound-healing/transwell migration assay were applied to evaluate CRC cell proliferation and migration. The tumor growth indicators were quantified in nude mice xenografted with shRFWD3 and shCtrl RKO cells.Results: The results indicated that RFWD3 knockdown restricted CRC development in vitro and in vivo. In exploring the downstream mechanism of RFWD3’s action, we found that RFWD3 could transcriptionally activate BIRC5 by interacting with E2F transcription factor 1 (E2F1). Accordingly, we identified BIRC5 as a downstream gene of RFWD3 regulating CRC. Subsequent loss- and gain- of function experiments demonstrated that upon overexpressing BIRC5 in RKO cells with down-regulated RFWD3, the inhibitory effects of cell proliferation, migration and colony formation could be reversed, while the capacity of cell apoptosis was ameliorated, suggesting that the effects of RFWD3 depletion was mainly due to BIRC5 suppression.Conclusion: Taken together, this study revealed that RFWD3 participates in the occurrence and development of colorectal cancer via E2F1 transcriptional regulation of BIRC5.

Sexually dimorphic DNA damage responses and mutation avoidance in the mouse germline.

Germ cells specified during fetal development form the foundation of the mammalian germline. These primordial germ cells (PGCs) undergo rapid proliferation, yet the germline is highly refractory to mutation accumulation compared with somatic cells. Importantly, while the presence of endogenous or exogenous DNA damage has the potential to impact PGCs, there is little known about how these cells respond to stressors. To better understand the DNA damage response (DDR) in these cells, we exposed pregnant mice to ionizing radiation (IR) at specific gestational time points and assessed the DDR in PGCs. Our results show that PGCs prior to sex determination lack a G1 cell cycle checkpoint. Additionally, the response to IR-induced DNA damage differs between female and male PGCs post-sex determination. IR of female PGCs caused uncoupling of germ cell differentiation and meiotic initiation, while male PGCs exhibited repression of piRNA metabolism and transposon derepression. We also used whole-genome single-cell DNA sequencing to reveal that genetic rescue of DNA repair-deficient germ cells (Fancm-/- ) leads to increased mutation incidence and biases. Importantly, our work uncovers novel insights into how PGCs exposed to DNA damage can become developmentally defective, leaving only those genetically fit cells to establish the adult germline.

Abstract 92: Increasing TTFields treatment efficacy by targeting G2 cell cycle checkpoint

Abstract Introduction; Tumor Treating Fields (TTFields) is a novel non-invasive anticancer treatment modality utilizing alternating electric fields. TTFields is FDA approved for glioblastoma patients after demonstrating a 5 months increase in overall survival, and for unresectable mesothelioma patients after the STELLAR trial showed an overall survival of 18 months without increasing the toxicity. While the effectiveness of TTFields is initially attributed to the effects of TTFields during cell division, studies are ongoing analysing its interphase effects and impact on the tumor (micro)environment. Our aim is to further exploit the working mechanism of TTFields by analysing its effect on the cell cycle and identify targets to increase its efficacy. Material and Methods; To analyse the effect of TTFields on the cell cycle, A172 glioblastoma cells were synchronized in S-phase by double thymidine block. Six hours post-release, cells were subjected to TTFields treatment (200kHz; 4,5V/cm). Cell cycle analysis was performed by flow cytometry (PI, α-phospho-histone H3Ser10). Efficacy of combination strategies were tested in 3 different glioblastoma cells lines (A172, U251 and SNB-19). Efficacy was quantified by colony formation assay (CFA) testing the cell reproductive death after treatment (i.e. only viable cells after treatment are replated to analyse the colony forming capacity). Results: TTFields treatment of synchronized A172 cells showed that TTFields causes an accumulation of cells in G2 phase and delayed entry into mitosis with less than 1% of the cells in mitosis 4 hours after TTFields exposure (versus 5% of mitotic cells in the control arm, p<0.001). This arrest can be abrogated by Wee1 inhibition (AZD1775) increasing the mitotic population to more than 35% at 4 hours after TTFields exposure (p<0.001). To test the efficacy of targeting the G2 checkpoint during TTFields treatment, cells were treated with TTFields and a Wee1 (AZD1775, PD0166285) or Chk1 (AZD7762) inhibitor for 72 hours and then replated for CFA. We observed an impressive synergistic effect between TTFields and any of these G2 cell cycle interfering drugs in all three cell lines (p<0.001). Conclusion: The underlying mechanism of the G2 checkpoint activation and the synergistic effect of TTFields plus G2 checkpoint targeting agents is subject of ongoing research. By any means, the combination of TTFields and G2 checkpoint targeting agents causes a synergistic treatment effect and is a very promising strategy. Citation Format: Paul Slangen, Mariska van Geldorp, Mark de Gooijer, Olaf van Tellingen, Gerben Borst. Increasing TTFields treatment efficacy by targeting G2 cell cycle checkpoint [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 92.

Albumin promotes the progression of fibroblasts through late G1 into S-phase in the absence of growth factors

ABSTRACT G1 cell cycle progression is controlled largely by growth factors in early G1 indicating that it is appropriate to divide and by nutrients in late G1 indicating sufficient raw material for cell division. We previously mapped a late G1 cell cycle checkpoint for lipids upstream from a mammalian target of rapamycin complex 1 (mTORC1)-mediated checkpoint and downstream from a mid-G1 checkpoint known as the Restriction point. We therefore investigated a role for lipids in progression through late G1 into S-phase. Quiescent BJ-hTERT human fibroblasts were primed with 10% fetal bovine serum (FBS) for 3.5 h at which time, cells were treated with a mixture of lipids and carrier bovine serum albumin (BSA) along with [3 H]-thymidine deoxyribose ([3 H]-TdR) to monitor progression into S-phase. Surprisingly, BSA by itself was more effective than FBS in promoting progression to S-phase – the lipids had no impact on progression. While insulin strongly stimulated mTORC1 activity, it did not impact on [3 H]-TdR incorporation. Although BSA modestly elevated mTORC1 activity, rapamycin strongly inhibited BSA-induced progression to S-phase. BSA treatment promoted mitosis, but not progression through a second G1. Thus, after priming quiescent cells with FBS, albumin was sufficient to promote progression into S-phase. The BSA was not simply a source of amino acids in that amino acids were present in the culture media. We propose that the presence of albumin – the most abundant protein in serum – reflects a broader availability of essential amino acids needed for cell growth.

Wee1 Kinase Inhibitor AZD1775 Effectively Sensitizes Esophageal Cancer to Radiotherapy.

Esophageal cancer is a deadly malignancy with a 5-year survival rate of only 5% to 20%, which has remained unchanged for decades. Esophageal cancer possesses a high frequency of TP53 mutations leading to dysfunctional G1 cell-cycle checkpoint, which likely makes esophageal cancer cells highly reliant upon G2-M checkpoint for adaptation to DNA replication stress and DNA damage after radiation. We aim to explore whether targeting Wee1 kinase to abolish G2-M checkpoint sensitizes esophageal cancer cells to radiotherapy. Cell viability was assessed by cytotoxicity and colony-forming assays, cell-cycle distribution was analyzed by flow cytometry, and mitotic catastrophe was assessed by immunofluorescence staining. Human esophageal cancer xenografts were generated to explore the radiosensitizing effect of AZD1775 in vivo. The IC50 concentrations of AZD1775 on esophageal cancer cell lines were between 300 and 600 nmol/L. AZD1775 (100 nmol/L) as monotherapy did not alter the viability of esophageal cancer cells, but significantly radiosensitized esophageal cancer cells. AZD1775 significantly abrogated radiation-induced G2-M phase arrest and attenuation of p-CDK1-Y15. Moreover, AZD1775 increased radiation-induced mitotic catastrophe, which was accompanied by increased γH2AX levels, and subsequently reduced survival after radiation. Importantly, AZD1775 in combination with radiotherapy resulted in marked tumor regression of esophageal cancer tumor xenografts. Abrogation of G2-M checkpoint by targeting Wee1 kinase with AZD1775 sensitizes esophageal cancer cells to radiotherapy in vitro and in mouse xenografts. Our findings suggest that inhibition of Wee1 by AZD1775 is an effective strategy for radiosensitization in esophageal cancer and warrants clinical testing.

The response of Plasmodium falciparum to isoleucine withdrawal is dependent on the stage of progression through the intraerythrocytic cell cycle

BackgroundA previous study reported that the malaria parasite Plasmodium falciparum enters an altered growth state upon extracellular withdrawal of the essential amino acid isoleucine. Parasites slowed transit through the cell cycle when deprived of isoleucine prior to the onset of S-phase.MethodsThis project was undertaken to study at higher resolution, how isoleucine withdrawal affects parasite growth. Parasites were followed at regular intervals across an extended isoleucine deprivation time course across the cell cycle using flow cytometry.ResultsThese experiments revealed that isoleucine-deprived parasites never exit the cell cycle, but instead continuously grow at a markedly reduced pace. Moreover, slow growth occurs only if isoleucine is removed prior to the onset of schizogony. After S-phase commenced, the parasite is insensitive to isoleucine depletion and transits through the cell cycle at the normal pace.ConclusionsThe markedly different response of the parasite to isoleucine withdrawal before or after the onset of DNA replication is reminiscent of the nutrient-dependent G1 cell cycle checkpoints described in other organisms.

Abstract P3-10-08: A phase II study of cisplatin and the Wee1 inhibitor adavosertib in metastatic triple-negative breast cancer (mTNBC)

Abstract Introduction: mTNBC progresses rapidly on first-line chemotherapy. For instance, in the TNT trial, median progression-free survival (PFS) was 3 months on first-line carboplatin. Selective inhibition of Wee1, a negative regulator of the G2 cell cycle checkpoint, may enhance the efficacy of DNA-damaging agents by reducing DNA damage repair. We report the results of the first phase II study assessing the efficacy of the Wee1 inhibitor adavosertib (AZD-1775) with cisplatin in mTNBC. Methods: mTNBC patients (pts) with 0-1 prior lines of chemotherapy in the metastatic setting were eligible. Pts received cisplatin 75 mg/m2 IV followed by combination therapy 21 d later with cisplatin plus adavosertib 200 mg oral twice daily x 5 doses over 2.5 d every 21 d. Tumor biopsies were mandatory for patients with safely accessible tumors, and occurred 5-48 h after monotherapy and 5-8 h after the last adavosertib dose in the first combination therapy cycle. The study used a Simon optimal two-stage design that had 90% power to detect the difference between null (20%) and alternative (40%) response rates with a one-sided type I error of 0.1. The primary endpoint was objective response rate (ORR): an ORR > 30% would identify the regimen as worthy of further study. Key secondary endpoints were PFS and overall survival (OS). Exploratory analyses evaluated the association of response, defined as clinical benefit rate (CBR) > 6 months (CR + PR + SD > 6 mos), with transcriptional and immunostaining profiles of on-treatment tumors (n = 16), as well as with targeted panel genomic alterations in archival tissue. Tumor immune cell composition was assessed by CIBERSORT. Results: 34 pts initiated protocol therapy; median age was 56 yrs, 2 (6%) pts had known BRCA2 mutations, and 14 (41%) had 1 prior line of chemotherapy. Median follow-up was 13 mos. ORR was 29% (3 CR + 7 PR), median PFS 4.9 months (95% CI, 2.3-5.8 mos), CBR 35%, and preliminary OS 14.0 mos (95% CI, 11.8-21.8 mos). All-cause AEs occurred in 100% of pts (G3-4, 56%; most commonly diarrhea, 21%), including one death due to sepsis possibly related to study therapy. The primary tumor of the longest responding patient had a possible biallelic loss-of-function alteration (missense mutation and loss of heterozygosity) in the homologous recombination-related gene FANCM. In Hallmark gene set enrichment analyses of 26 on-treatment biopsies across 16 pts, responding tumors (n = 4) demonstrated enriched expression of immune response gene sets (allograft rejection FDR q < 0.001, IL2-STAT5 signaling FDR q = 0.001, inflammatory response FDR q = 0.003), while non-responding tumors (n = 12) showed enrichment of cell cycle gene sets (E2F targets FDR q < 0.001, G2M checkpoint FDR q < 0.001). In the same on-treatment biopsies, responding tumors (n = 4) had higher tumor infiltrating lymphocytes (46% v. 29%, Mann-Whitney p = 0.04) and lower M0 macrophages (3% v. 17%, Mann-Whitney p = 0.04) than non-responding tumors (n =12). Updated data, including OS and tissue immunostaining results, will be presented. Conclusions: Among mTNBC pts, the combination of adavosertib and cisplatin was associated with a 29% ORR, failing to meet the pre-determined ORR of > 30% required to support further investigation. Responses were associated with immune-related gene expression and TILs detected by RNA sequencing. Citation Format: Tanya E. Keenan, Zhenying Tan-Wasielewski, Lorenzo Trippa, Bose Kochupurakkal, Jennifer L. Guerriero, Rie K. Tahara, Grace Winship, Wafa Osmani, Chelsea Andrews, Jake R. Conway, Meng X He, Ricardo Pastorello, Adam Tracy, Robert E. Godin, Beth A. Overmoyer, Eric P. Winer, Elizabeth A. Mittendorf, Geoffrey I Shapiro, Eliezer M Van Allen, Sara M Tolaney. A phase II study of cisplatin and the Wee1 inhibitor adavosertib in metastatic triple-negative breast cancer (mTNBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-08.

Combining ERBB family and MET inhibitors is an effective therapeutic strategy in cutaneous malignant melanoma independent of BRAF/NRAS mutation status

Current treatment modalities for disseminated cutaneous malignant melanoma (CMM) improve survival; however, relapses are common. A number of receptor tyrosine kinases (RTKs) including EGFR and MET have been reported to be involved in CMM metastasis and in the development of resistance to therapy, targeting the mitogen-activated protein kinase (MAPK pathway). IHC analysis showed that patients with higher MET protein expression had a significantly shorter overall survival. In addition, silencing of MET caused an upregulation of EGFR and p-AKT, which was abrogated by concomitant silencing of MET and EGFR in CMM cells resistant to MAPK-targeting drugs. We therefore explored novel treatment strategies using clinically approved drugs afatinib (ERBB family inhibitor) and crizotinib (MET inhibitor), to simultaneously block MET and ERBB family RTKs. The effects of the combination were assessed in cell culture and spheroid models using established CMM and patient-derived short-term cell lines, and an in vivo xenograft mouse model. The combination had a synergistic effect, promoting cell death, concomitant with a potent downregulation of migratory and invasive capacity independent of their BRAF/NRAS mutational status. Furthermore, the combination attenuated tumor growth rate, as ascertained by the significant reduction of Ki67 expression and induced DNA damage in vivo. Importantly, this combination therapy had minimal therapy-related toxicity in mice. Lastly, the cell cycle G2 checkpoint kinase WEE1 and the RTK IGF1R, non-canonical targets, were altered upon exposure to the combination. Knockdown of WEE1 abrogated the combination-mediated effects on cell migration and proliferation in BRAF mutant BRAF inhibitor-sensitive cells, whereas WEE1 silencing alone inhibited cell migration in NRAS mutant cells. In summary, our results show that afatinib and crizotinib in combination is a promising alternative targeted therapy option for CMM patients, irrespective of BRAF/NRAS mutational status, as well as for cases where resistance has developed towards BRAF inhibitors.

Abstract 3725: Numerical simulation of tumor treating fields effects on cell structures: Mechanism and signaling pathway candidates

Abstract Tumor Treating Fields (TTFields) have become a fourth modality for cancer treatment. Mild electric fields (~1-4 V/cm) produce few side effects and significantly extend overall survival of glioblastoma patients, and TTFields are in clinical trials for a variety of tumor cell types. Our goal is to uncover TTFields’ mechanism and cell signaling pathways by numerically modeling their effects on sub-cellular structures, such as microtubules (MTs) and their interactions with motor proteins. METHODS: We have built finite element models in COMSOL Multiphysics (tm) of the MT and its micro-environment to test hypotheses on TTFields’ mechanism of action by predicting effects on sub-cellular structures. RESULTS: One model prediction is that current density induced in the MT counter-ion layer by TTFields essentially shunts electric current within them. The strongest current flows through the counter-ion layer surrounding the MT’s C-termini and energy density in this layer likely exceeds the level to disrupt motor protein ‘walk’ along the MT. The energy density is predicted at 10-20 Joules when both the field and the MTs are aligned with the cell axis. A second mechanism examined by our model is disruption of the ‘foot’ of kinesin, released from its C-terminus contact by ATP (10-19 Joules). The final phase of the walk is driven by thermal buffeting of the forward foot randomly positioning it near enough to the C-terminus for electrostatic forces to bind it. A stall force ~10-19 - 10-16 N from TTFields would prevent diffusion and disrupt the kinesin walk. A recent clinical study segregating patient cohorts treated vs. not treated with dexamethasone found overall survival indefinitely for the non-dexamethosone cohort, leading us to hypothesize that TTFields activate the intrinsic Bcl2-mediated apoptotic signaling pathway. Future modeling will seek to tie disruption of motor protein transport along MTs to activating intrinsic apoptosis, e.g. via failure to silence the G2 cell cycle checkpoint. CONCLUSION: Our modeling predicts that TTFields in cytosol induce electric currents along MTs that are strong enough to disrupt key cellular functions such as the kinesin walk and C-termini transitions, both of which are crucial for motor protein transport. Hence, TTFields disrupt the most delicate mechanisms involved in the carefully-orchestrated succession of steps in mitosis. Citation Format: Kristen W. Carlson, Nirmal Paudel, Jack A. Tuszynski, Zeev Bomzon. Numerical simulation of tumor treating fields effects on cell structures: Mechanism and signaling pathway candidates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3725.

Leucine zipper and ICAT domain containing (LZIC) protein regulates cell cycle transitions in response to ionizing radiation

ABSTRACT Common hallmarks of cancer include the dysregulation of cell cycle progression and the acquisition of genome instability. In tumors, G1 cell cycle checkpoint induction is often lost. This increases the reliance on a functional G2/M checkpoint to prevent progression through mitosis with damaged DNA, avoiding the introduction of potentially aberrant genetic alterations. Treatment of tumors with ionizing radiation (IR) utilizes this dependence on the G2/M checkpoint. Therefore, identification of factors which regulate this process could yield important biomarkers for refining this widely used cancer therapy. Leucine zipper and ICAT domain containing (LZIC) downregulation has been associated with the development of IR-induced tumors. However, despite LZIC being highly conserved, it has no known molecular function. We demonstrate that LZIC knockout (KO) cell lines show a dysregulated G2/M cell cycle checkpoint following IR treatment. In addition, we show that LZIC deficient cells competently activate the G1 and early G2/M checkpoint but fail to maintain the late G2/M checkpoint after IR exposure. Specifically, this defect was found to occur downstream of PIKK signaling. The LZIC KO cells demonstrated severe aneuploidy indicative of genomic instability. In addition, analysis of data from cancer patient databases uncovered a strong correlation between LZIC expression and poor prognosis in several cancers. Our findings suggest that LZIC is functionally involved in cellular response to IR, and its expression level could serve as a biomarker for patient stratification in clinical cancer practice.

Inhibition of ATR Potentiates Acute Myeloid Leukemia Cells to the RNA Pol I Transcription Inhibitor CX-5461

Inhibition of ATR Potentiates Acute Myeloid Leukemia Cells to the RNA Pol I Transcription Inhibitor CX-5461

The lysine acetyltransferase GCN5 contributes to human papillomavirus oncoprotein E7-induced cell proliferation via up-regulating E2F1.

General control nondepressible 5 (GCN5), the first identified transcription‐related lysine acetyltransferase (KAT), is an important catalytic component of a transcriptional regulatory SAGA (Spt‐Ada‐GCN5‐Acetyltransferase) and ATAC (ADA2A‐containing) complex. While GCN5 has been implicated in cancer development, its role in cervical cancer is not known. The human papillomavirus (HPV) oncoprotein E7 abrogates the G1 cell cycle checkpoint and induces genomic instability, which plays a central role in cervical carcinogenesis. In this study, we observed that GCN5 was up‐regulated in HPV E7‐expressing cells, knockdown of GCN5 inhibited cell cycle progression and DNA synthesis in HPV E7‐expressing cells. Notably, GCN5 knockdown reduced the steady‐state levels of transcription factor E2F1. Depletion of E2F1 caused G1 arrest while overexpression of E2F1 rescued the inhibitory effects of GCN5 knockdown on G1/S progression in HPV E7‐expressing cells. Results from chromatin immunoprecipitation (ChIP) assays demonstrated that GCN5 bound to the E2F1 promoter and increased the extent of histone acetylation within these regions. GCN5 also acetylated c‐Myc and increased its ability to bind to the E2F1 promoter. Knockdown of c‐Myc reduced the steady‐state levels of E2F1 and caused G1 arrest. These results revealed a novel mechanism of E7 function whereby elevated GCN5 acetylates histones and c‐Myc to regulate E2F1 expression and cell cycle progression.