γ-H2AX Immunofluorescence Staining Research Articles

A novel mechanism of major ginsenosides from Panax ginseng against multiple organ aging in middle-aged mice: Phosphatidylcholine-myo-inositol metabolism based on metabolomic analysis

Aging is a complex, degenerative process associated with various metabolic abnormalities. Ginsenosides (GS) is the main active components of Panax ginseng, which has anti-aging effects and improves metabolism. However, the anti-aging effect and the mechanism of GS in middle-aged mice has not been elucidated. In this study, GS after 3-month treatment significantly improved the grip strength, fatigue resistance, cognitive indices, and cardiac function of 15-month-old mice. Meanwhile, GS treatment reduced the fat content and obviously inhibited histone H2AX phosphorylation at Ser 139 (γ-H2AX), a marker of DNA damage in major organs, especially in the heart and liver. Further, the correlation analysis of serum metabolomics combined with aging phenotype suggested that myo-inositol (MI) upregulated by GS was positively correlated with left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), the main indicators of cardiac function. More importantly, liver tissue metabolomic analysis showed that GS increased MI content by promoting the synthesis pathway from phosphatidylcholine (PC) to MI for the inhibition of liver aging. Finally, we proved that MI reduced the percentage of senescence-associated β-galactosidase staining, γ-H2AX immunofluorescence staining, p21 expression, and the production of reactive oxygen species in H2O2-induced cardiomyocytes. These results suggest that GS can enhance multiple organ functions, especially cardiac function for promoting the healthspan of aging mice, which is mediated by the conversion of PC to MI in the liver and the increase of MI level in the serum. Our study might provide new insights into the potential mechanisms of ginsenosides for prolonging the healthspan of natural aging mice.

Abstract 1987: PARP inhibitor (PARPi) rechallenge shows differential sensitivity in PARPi olaparib-resistant BRCA1-mutated (BRCA1m) high-grade serous ovarian cancer (HGSC)

Abstract Background: PARPis have changed the treatment paradigm in HGSC, particularly for women with BRCA mutations. PARPi, olaparib (O), is a standard of care maintenance therapy following platinum-based chemotherapy in newly diagnosed BRCA-mutated (BRCAm) HGSC. But patients ultimately develop resistance to O. We investigated whether rechallenge with different PARPi monotherapy could cause cytotoxicity in BRCAm HGSC after becoming O-resistant. We also aimed to identify possible mechanisms of actions of PARPi’s differential sensitivity. Methods: We developed a PARPi-resistant line (UWB-OlaJR) from BRCA1m PARPi-sensitive cells (UWB1.289) by gradient exposure of O with initial lower doses to a final concentration of 20 μM over 10 months. Four PARPis including O, niraparib (N), talazoparib (T), and veliparib (V) were tested. Cell growth was assessed using 5-day XTT and 10-day clonogenic assays. DNA damage and homologous recombination (HR) repair activity were evaluated using immunofluorescence (IF) staining of γH2AX and RAD51 foci, respectively. Replication fork (RF) dynamics was measured by DNA fiber assay. All data were repeated in triplicate and analyzed using t-test or one-way ANOVA. Data were shown as mean ± SD and P < 0.05 was considered statistically significant. Results: 5-day XTT assay shows a 53.7-fold increase in resistance to O in UWB-OlaJR relative to UWB1.289 (IC50 2.2 μM vs. 118.1 μM). Cross-resistance was observed with other 3 PARPis, showing increased IC50 values compared to its parental line (V: 23.4-fold, N: 160.5-fold, T: 229.6-fold). However, 10-day clonogenic assay showed no resistance against N (1.09-fold) and T (0.51-fold) in UWB-OlaJR, indicating differential sensitivity to different PARPis by a long-term cytotoxicity measure. Surprisingly, γH2AX foci did not increase in response to PARPis in UWB-OlaJR compared to UWB1.289 (O: 2.58-fold, V: 2.33-fold; N: 2.89-fold, T: 2.58-fold; all P < 0.05), suggesting possible restored DNA repair function. All four PARPis induced replication stress in both O-sensitive and -resistant cell lines as evidenced by decreased RF speed compared to untreated group (O: 0.57- and 0.7-fold, V: 0.68- and 0.74-fold, N: 0.47- and 0.47-fold, T: 0.47- and 0.52-fold; all P < 0.001). Notably, N and T treatments reduced fork speed to a greater extent than O and V in UWB-OlaJR. However, RF protection was not affected when UWB-OlaJR treated with different PARPis compared to UWB1.289 (O: 0.89-fold, V: 0.87-fold, N: 0.71-fold, T: 0.83-fold; all P < 0.05), suggesting that differential sensitivity to different PARPis was unlikely due to modulation of RF stabilization. Conclusions: Our findings demonstrate different PARPis may induce varied levels of cytotoxicity and replication stress in O-resistant BRCA1m HGSC, possibly via modulating alternative RF dynamics besides RF protection. Citation Format: Chi-Ting Shih, Tzu-Ting Huang, Jayakumar R. Nair, Jung-Min Lee. PARP inhibitor (PARPi) rechallenge shows differential sensitivity in PARPi olaparib-resistant BRCA1-mutated (BRCA1m) high-grade serous ovarian cancer (HGSC) [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 1987.

CDK1 promotes the phosphorylation of KIFC1 to regulate the tumorgenicity of endometrial carcinoma.

This study aims to clarify the mechanical action of cyclin-dependent protein kinase 1 (CDK1) in the development of endometrial carcinoma (EMCA), which may be associated with the phosphorylation of kinesin family member C1 (KIFC1) and further activate the PI3K/AKT pathway. The protein and gene expression of CDK1 in EMCA tissues and tumor cell lines were evaluated by western blot, quantitative polymerase chain reaction, and immunohistochemistry staining. Next, Cell Counting Kit-8 and colony formation assay detected cell survival and proliferation. Cell migration and invasion were measured by Transwell assay. Cell apoptosis and cell cycle were tested by flow cytometry. Immunofluorescence staining of γH2AX was used to evaluate DNA damage, respectively. Subsequently, a co-immunoprecipitation assay was used to detect the interaction between CDK1 and KIFC1. The phosphorylated protein of KIFC1 and PI3K/AKT was detected by western blot. Finally, the effect of CDK1 on the tumor formation of EMCA was evaluated in a nude mouse xenograft model. CDK1 was highly expressed in EMCA tumor cell lines and tissues, which contributed to cell survival, proliferation, invasion, and migration, inhibited cell apoptosis, and induced DNA damage of EMCA cells dependent on the phosphorylation of KIFC1. Moreover, the CDK1-KIFC1 axis further activated PI3K/AKT pathway. Finally, CDK1 knockdown repressed tumor formation of EMCA in vivo. We report that increased CDK1 promotes tumor progression and identified it as a potential prognostic marker and therapeutic target of EMCA.

Proliferation and Differentiation Potential of Bone Marrow-Derived Mesenchymal Stem Cells From Children With Polydactyly and Adults With Basal Joint Arthritis.

This study compared the proliferation and differentiation potential of bone marrow-derived mesenchymal stem cells (BMSCs) derived from infants with polydactyly and adults with basal joint arthritis. The proliferation rate of adult and infant BMSCs was determined by the cell number changes and doubling times. The γH2AX immunofluorescence staining, age-related gene expression, senescence-associated β-galactosidase (SA-β-gal) staining were analyzed to determine the senescence state of adult and infant BMSCs. The expression levels of superoxide dismutases (SODs) and genes associated with various types of differentiation were measured using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). Differentiation levels were evaluated through histochemical and immunohistochemical staining. The results showed that infant BMSCs had a significantly higher increase in cell numbers and faster doubling times compared with adult BMSCs. Infant BMSCs at late stages exhibited reduced γH2AX expression and SA-β-gal staining, indicating lower levels of senescence. The expression levels of senescence-related genes (p16, p21, and p53) in infant BMSCs were also lower than in adult BMSCs. In addition, infant BMSCs demonstrated higher antioxidative ability with elevated expression of SOD1, SOD2, and SOD3 compared with adult BMSCs. In terms of differentiation potential, infant BMSCs outperformed adult BMSCs in chondrogenesis, as indicated by higher expression levels of chondrogenic genes (SOX9, COL2, and COL10) and positive immunohistochemical staining. Moreover, differentiated cells derived from infant BMSCs exhibited significantly higher expression levels of osteogenic, tenogenic, hepatogenic, and neurogenic genes compared with those derived from adult BMSCs. Histochemical and immunofluorescence staining confirmed these findings. However, adult BMSCs showed lower adipogenic differentiation potential compared with infant BMSCs. Overall, infant BMSCs demonstrated superior characteristics, including higher proliferation rates, enhanced antioxidative activity, and greater differentiation potential into various lineages. They also exhibited reduced cellular senescence. These findings, within the context of cellular differentiation, suggest potential implications for the use of allogeneic BMSC transplantation, emphasizing the need for further in vivo investigation.

Multilineage differentiation potential in the infant adipose- and umbilical cord-derived mesenchymal stem cells.

This study aims to compare the biological properties of infant adipose-derived mesenchymal stem cells (infant ADSCs) from excised polydactyly fat tissue and umbilical cord-derived mesenchymal stem cells (UCSCs) in terms of proliferation and differentiation capabilities. The proliferation of infant ADSCs and UCSCs was analyzed by determining the fold changes of cell numbers and doubling time periods. The state of senescence and replicative stress was compared by analyzing the expression of age-related genes, senescence-associated β-galactosidase (SA-β-gal) staining, and phosphorylated histone variant H2AX (γH2AX) immunofluorescence staining. The expression levels of superoxide dismutase ( SODs ) and genes related to multilineage differentiation were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Differentiation levels were determined using histochemical staining, immunohistochemical staining, and immunofluorescence staining. Infant ADSCs exhibited higher proliferation rates and expression levels of SOD1 , SOD2 , and SOD3 at passages 3-5 compared with UCSCs. Senescence related genes ( p16 , p21 , and p53 ), SA-β-gal staining, and replicative stress analysis were reduced in infant ADSCs. The expression levels of chondrogenic genes ( COL2 and COL10 ), osteogenic genes ( RUNX2 and ALP ), adipogenic genes ( LPL ), and hepatogenic genes ( ALB and TAT ) in infant ADSC-differentiated cells were significantly higher than those in UCSCs. Histochemical and immunofluorescence staining confirmed these results. Only the expression levels of tenogenic genes ( MMP3 , DCN , and COL3 ) in infant ADSC-differentiated cells were lower than those in UCSCs. Infant ADSCs exhibit higher proliferation rates, reduced cellular senescence and replicative stress, better antioxidative activity, and higher differentiation potential toward chondrogenic, osteogenic, adipogenic and hepatogenic lineages than UCSCs.

Abstract B067: CRM1 inhibitor selinexor induces DNA damage repair-related vulnerability in prostate cancer cells

Abstract Background: Advanced metastatic prostate cancer is the second leading cause of death in men in the United States. Androgen deprivation therapy has been used as a front-line therapy; however, despite initial success, it fails subsequently and results in the development of castration-resistant prostate cancer. Due to these unmet therapeutic needs, there is an urgency to identify novel targets to develop next-generation therapeutics. Chromosome region maintenance 1 (CRM1) is one of the seven members of mammalian export proteins that function to export the proteins using nuclear export signal (NES). Several tumors, including prostate cancer, overexpress CRM1 to efflux tumor suppressor proteins to the cytoplasm and exhibit dysregulated nuclear export. CRM1-mediated export can lead to functional inactivation of p53, cyclinD1, p16, p21, and p27. Essential DNA repair proteins such as Rad51 and BRCA1 exhibit leucine-containing hydrophobic residues rich in NES and are known to be shuttled by CRM1. Based on this, we hypothesize that CRM1 inhibition may affect the expression, kinetics, localization, and stability of DNA repair network proteins. Methods: Androgen receptor-positive and negative prostate cancer cells were treated with Selinexor. The dose of the drug and duration was dependent on the endpoint measurement. Cell death was monitored using MTT assay and western blot measurement of cleaved PARP. Total RNA and cell lysate were collected to measure the relative levels of BRCA-1, RAD51, p53, and other members of the DNA repair pathway (HR and NHEJ). DNA damage was estimated using the number of micronuclei and immunofluorescence staining for γ-H2AX and COMET assay. Results: Selinexor treatment in prostate cancer cells affects the functioning of the DNA repair network and can be combined with inhibitors of DNA repair proteins. Citation Format: Rajendra Kumar, Janet Mendonca, Yuhan Yang, Abhishek Shetty, Lillian Wilson, Kavya Boyapati, Deven Topiwala, Keerti Soundappan, Eleni Panagopoulos, Michael Carducci, Sushant Kachhap. CRM1 inhibitor selinexor induces DNA damage repair-related vulnerability in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B067.

The kinesin light chain-2, a target of mRNA stabilizing protein HuR, inhibits p53 protein phosphorylation to promote radioresistance in NSCLC.

Radioresistance hinders radiotherapy for the treatment of lung cancer. Kinesin light chain-2 (KLC2) has been found to be upregulated in lung cancer and also to be associated with poor prognosis. This study aimed to investigate the effect of KLC2 on radiosensitivity in lung cancer. The radioresistant role of KLC2 was determined by colony formation, neutral comet assay, and γH2AX immunofluorescent staining assay. We further verified the function of KLC2 in a xenograft tumor model. The downstream of KLC2 was identified through gene set enrichment analysis and validated by western blot. Finally, we analyzed clinical data from the TCGA database to reveal the upstream transcription factor of KLC2, which was validated by RNA binding protein immunoprecipitation assay. Here, we found that downregulation of KLC2 could significantly reduce colony formation, increase γH2AX level, and double-stranded DNA breaks in vitro. Meanwhile, overexpressed KLC2 significantly increased the proportion of the S phase in lung cancer cells. KLC2 knockdown could activate P53 pathway, and ultimately promoting radiosensitivity. The mRNA of KLC2 was observed to bind with Hu-antigen R (HuR). The mRNA and protein expression of KLC2 in lung cancer cells was significantly reduced when combined with siRNA-HuR. Interestingly, KLC2 overexpression significantly increased the expression of HuR in lung cancer cells. Taken together, these results indicated that HuR-KLC2 forms a positive feedback loop, which decreases the phosphorylation of p53 and thereby weaken the radiosensitivity of lung cancer cells. Our findings highlight the potential prognosis and therapeutic target value of KLC2 in lung cancer patients treated with radiotherapy.

Chronological Age and DNA Damage Accumulation in Blood Mononuclear Cells: A Linear Association in Healthy Humans after 50 Years of Age.

Aging is characterized by the progressive deregulation of homeostatic mechanisms causing the accumulation of macromolecular damage, including DNA damage, progressive decline in organ function and chronic diseases. Since several features of the aging phenotype are closely related to defects in the DNA damage response (DDR) network, we have herein investigated the relationship between chronological age and DDR signals in peripheral blood mononuclear cells (PBMCs) from healthy individuals. DDR-associated parameters, including endogenous DNA damage (single-strand breaks and double-strand breaks (DSBs) measured by the alkaline comet assay (Olive Tail Moment (OTM); DSBs-only by γH2AX immunofluorescence staining), DSBs repair capacity, oxidative stress, and apurinic/apyrimidinic sites were evaluated in PBMCs of 243 individuals aged 18-75 years, free of any major comorbidity. While OTM values showed marginal correlation with age until 50 years (rs = 0.41, p = 0.11), a linear relationship was observed after 50 years (r = 0.95, p < 0.001). Moreover, individuals older than 50 years showed increased endogenous DSBs levels (γH2Ax), higher oxidative stress, augmented apurinic/apyrimidinic sites and decreased DSBs repair capacity than those with age lower than 50 years (all p < 0.001). Results were reproduced when we examined men and women separately. Prospective studies confirming the value of DNA damage accumulation as a biomarker of aging, as well as the presence of a relevant agethreshold, are warranted.

Abstract 2405: Delineating molecular vulnerabilities of ATM mutant prostate cancers

Abstract Background: Mutations in DNA Damage Response (DDR) genes, including Ataxia, Telangiectasia, Mutated (ATM), are common in advanced castration-resistant prostate cancers (CRPCs). Poly (ADP-ribose) polymerase (PARP) inhibitors are approved in DDR mutant CRPCs, but demonstrate limited clinical efficacy in CRPCs with ATM mutations. In this project, we sought to specifically define the impact of ATM loss on DDR pathways in CRPC, with the goal of identifying alternate therapeutic vulnerabilities. Methods: ATM-KO CRPC cell lines were generated via CRISPR-Cas9 mediated knockout. ATM loss and abolishment of downstream ATM kinase activity was confirmed via western blot. We performed an unbiased phospho-proteomic evaluation of DDR pathways in parental and ATM-KO cells after ionizing radiation (IR). Clonogenic survival assays were performed with either an ATR inhibitor (VX970), the selective DNA-PKcs inhibitor (M3814), or combination therapy. Kinetics of DDR protein recruitment and resolution were interrogated with immunofluorescence (IF) staining for γH2ax, 53BP1, MDC1, and Rad51 foci. Results: ATM-KO cells were able to effectively repair DNA damage following IR, as measured by recruitment and resolution of γH2ax, 53BP1, MDC1, and Rad51 foci. Phospho-proteomic studies demonstrated that ATM-KO cells maintain canonical DDR pathways through ATR and DNA-PKcs kinase activation. Treatment of ATM-KO cells with either VX-970 or M3814 only incrementally affected DDR in ATM-KO cells compared to parental controls, as evidenced by clonogenic survival assays and maintenance of DDR foci. Importantly, combination treatment with VX-970 and M3814 prevented downstream DDR foci recruitment and radio-sensitized ATM-KO CRPC to a greater extent than parental controls. This suggested that activity of any of the trinity of kinases is sufficient to mediate DDR, and that blockade of both ATR and DNA-PKcs is required to effectively prevent DDR in ATM-KO CRPC. We then leveraged a RUVBL1 ATPase inhibitor Compound B, which significantly attenuates levels of these kinases in lung cancer cells. We confirmed that Compound B treatment attenuated ATR and DNA-PKcs protein expression and kinase activity in ATM-KO CRPC cells, and demonstrated sensitivity of ATM-KO cells to Compound B. Conclusions: Our data demonstrates that dual targeting of ATR and DNA-PKcs is necessary in ATM-KO CRPC, as either kinase is independently capable of mediating DDR following IR. Our initial studies indicate that the RUVBL1 ATPase inhibitor Compound B may effectively block DDR in ATM-mutant CRPC, and could be utilized as a novel therapeutic strategy in this molecular subtype. Citation Format: Mia Hofstad, Lan Yu, Andrea Woods, Zoi Sychev, Collin Gilbreath, Xiaofang Huo, Ralf Kittler, Justin M. Drake, Ganesh V. Raj. Delineating molecular vulnerabilities of ATM mutant prostate cancers [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 2405.

Abstract 6173: AKT1 prevents aberrant R-loops accumulation via regulating DHX9 and suppresses transcription-replication collision in PARP inhibitor-resistant ovarian cancer

Abstract Background: Acquired resistance to PARP inhibitors (PARPis) is a pressing problem in high-grade serous ovarian cancer (HGSC), highlighting the development of novel therapeutic options. To address this unmet need, we conducted high-throughput drug combination screens in PARPi-resistant HGSC cells using ATR inhibitor (ATRi), given that ATR/CHK1 signaling activation is one of the main mechanisms of PARPi resistance. In the screens, PI3K/AKT pathway inhibitors were synergistic with ATRi. Mechanistically, ATR signaling plays a major role in R-loop dynamics and replication stress (RS) response pathways, but the role of AKT is unclear. Here, we aim to investigate AKT’s role in R-loop-mediated RS in PARPi-resistant HGSC cells. We also hypothesize that AKT inhibitor (AKTi) would augment ATRi-induced R-loop formation by reducing the expression of helicases. Methods: We used PARPi-resistant variants and parental PEO1 as a negative control. ATRi (ceralasertib) and AKTi (capivasertib) were used for further mechanistic studies. Induction of RS, DNA double-strand break, and R-loop were evaluated using immunofluorescence staining for markers pRPA, γH2AX, and S9.6, respectively. We measured multiple nuclear helicases and in situ DHX9-R-loop interaction by western blot and proximity ligation assay. DHX9-AKT1 interaction was confirmed by co-immunoprecipitation. All data were repeated in triplicate, analyzed using one-way ANOVA test, and shown as mean ± SD. P &amp;lt; 0.05 was considered statistically significant. Results: The ATRi and AKTi combination induced greater RS, as evidenced by a 1.7-12.0-fold increase in pRPA+/γH2AX+ population relative to each drug alone. Adding AKTi to ATRi significantly increased R-loops compared to monotherapy alone (signal intensity 14-19 x 105 vs. 9.9-14.6 x 105, P &amp;lt; 0.001), suggesting that AKT also contributes to R-loop dissolution. Moreover, the combination showed a higher percentage of cells with γH2AX and R-loops relative to monotherapy (54.7-75.4% vs. 33.3-49.0%, P &amp;lt; 0.001) in the presence of pan-caspase inhibitor, indicating the increased DNA damage is unlikely caused by increased apoptosis but by R-loop accumulation. Also, AKTi addition to ATRi significantly reduced the nuclear DHX9 among other helicases. Moreover, we found that AKT1 directly binds with DHX9 via its kinase domain and couples with DHX9 to resolve R-loops. Conclusion: Our data suggest ATRi and AKTi combination could be a new therapeutic combination in HGSC with PARPi resistance. Mechanistically, our data reveal a previously unknown direct role of AKT1 and its interaction with DHX9 in R-loop resolution besides ATR. Citation Format: Tzu-Ting Huang, Chih-Yuan Chiang, Jayakumar R. Nair, Chi-Ting Shih, Kelli Wilson, Ken Chih-Chien Cheng, Jung-Min Lee. AKT1 prevents aberrant R-loops accumulation via regulating DHX9 and suppresses transcription-replication collision in PARP inhibitor-resistant ovarian cancer [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 6173.

Influence of 2-Nitroimidazoles in the Response of FaDu Cells to Ionizing Radiation and Hypoxia/Reoxygenation Stress

Cellular adaptations to hypoxia promote resistance to ionizing radiation (IR). This presents a challenge for treatment of head and neck cancer (HNC) that relies heavily on radiotherapy. Standard radiosensitizers often fail to reach diffusion-restricted hypoxic cells, whereas nitroimidazoles (NIs) [such as iodoazomycin arabinofuranoside (IAZA) and fluoroazomycin arabinofuranoside (FAZA)] can preferentially accumulate in hypoxic tumours. Here, we explored if the hypoxia-selective uptake of IAZA and FAZA could be harnessed to make HNC cells (FaDu) susceptible to radiation therapy. Cellular response to treatment was assessed through clonogenic survival assays and by monitoring DNA damage (immunofluorescence staining of DNA damage markers, γ-H2AX and p-53BP1, and by alkaline comet assay). The effects of reoxygenation were studied using the following assays: estimation of nucleoside incorporation to assess DNA synthesis rates, immunofluorescent imaging of chromatin-associated replication protein A as a marker of replication stress, and quantification of reactive oxygen species (ROS). Both IAZA and FAZA sensitized hypoxic HNC cells to IR, albeit the former is a better radiosensitizer. Radiosensitization by these compounds was restricted only to hypoxic cells, with no visible effects under normoxia. IAZA and FAZA impaired cellular adaptation to reoxygenation; high levels of ROS, reduced DNA synthesis capacity, and signs of replication stress were observed in reoxygenated cells. Overall, our data highlight the therapeutic potentials of IAZA and FAZA for targeting hypoxic HNC cells and provide rationale for future preclinical studies.

EOAI, a ubiquitin-specific peptidase 5 inhibitor, prevents non-small cell lung cancer progression by inducing DNA damage

ObjectiveTargeting deubiquitinases (DUBs) has emerged as a promising avenue for anticancer drug development. However, the effect and mechanism of pan-DUB inhibitor EOAI on non-small cell lung cancer (NSCLC) remains to be studied.Materials and methodsThe expression of ubiquitin-specific peptidase 5 (USP5) in NSCLC was evaluated by immunohistochemistry. The effect of the USP5 inhibitor, EOAI, on NSCLC cell growth and cell cycle was evaluated by CCK-8 and PI staining. Apoptosis was detected by Annexin V-FITC/PI double staining. Autophagy was examined by LC3 immunofluorescence. Comet assay and γ-H2AX immunofluorescence staining were used to detect DNA damage, and Western blotting was used to detect the expression of apoptosis, cycle, autophagy and DNA damage-related proteins. In vivo experiments demonstrated the effect of EOAI on NSCLC.ResultsWe also found that USP5 was significantly upregulated in NSCLC tissues in this study. In addition, we show that EOAI can cause DNA damage in NSCLC cells while modulating the transcriptional activity of P53, thereby inducing cell cycle arrest in NSCLC cells, autophagy and apoptosis. In vivo experiments have shown that EOAI can inhibit tumors and synergistically enhance the anti-tumor effect of cisplatin.ConclusionUSP5-mediated epigenetic regulation of oncogenes promotes the occurrence of NSCLC, which provides ideas for developing potential targeted therapy.

Therapeutic efficacy of cyclin-dependent kinase inhibition in combination with ionizing radiation for lung cancer

Purpose To evaluate the therapeutic efficacy of cyclin-dependent kinase (CDK) inhibition in combination with ionizing radiation for lung cancer. Materials and methods Human lung adenocarcinoma (A549) and squamous cell carcinoma (H520) cells were used to evaluate the therapeutic efficacy of CDK inhibition in combination with ionizing radiation in vitro using colony formation assay, γH2AX immunofluorescence staining, western blotting, and cell cycle phase analysis. We also performed in vivo evaluations of ectopic tumor growth. Results In vitro pretreatment with the CDK inhibitor, seliciclib, before irradiation significantly decreased the survival of A549 and H520 cells in a dose-dependent manner. Although CDK inhibition alone did not increase the intensity of γH2AX foci, its combination with ionizing radiation increased DNA double-strand breaks, as shown by γH2AX immunofluorescence staining and western blotting. The combination of CDK inhibition and ionizing radiation-induced G2/M arrest and increased apoptosis, as evidenced by the increased proportion of cells in G2/M arrest, subG1 apoptotic population, and expression of apoptotic markers (cleaved PARP-1 and cleaved caspase-3). Mechanistic studies showed reduced expression of cyclin A with combined treatment, indicating cell cycle shifting effects. An in vivo xenograft model showed that the combination of CDK inhibition and ionizing radiation delayed xenograft tumor growth, and increased the proportion of cleaved PARP-1- and cleaved caspase-3-positive cells, compared to either treatment alone. Conclusions We provide preclinical tumoricidal evidence that the combination of CDK inhibition and ionizing radiation is an efficacious treatment for lung cancer.

27 Entinostat restores responsiveness to olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines

Poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) reduce disease progression in patients diagnosed with ovarian cancer. The anticancer effects of PARPi are most effective in tumors with BRCA mutations and homologous recombination (HR) deficiency (HRD). One mechanism of PARPi resistance is through restoration of HR repair. Our group has shown that entinostat (an HDACi) potentiates the effects of olaparib (a PARPi). The objective of this study was to investigate mechanisms of intrinsic PARPi resistance by studying the combination of entinostat and olaparib in HR proficient and HR deficient mouse ovarian cancer cell lines. We used the ID8 TP53 null (HR proficient) and ID8 TP53 null/BRCA2 null (HR deficient) mouse ovarian cancer cell lines that harbor molecular features consistent with human high-grade serous ovarian cancer. To recapitulate an ongoing clinical trial using this drug combination, cells were pre-treated with 0.25 μM Entinostat or control for 24 hours, followed by 24 or 72 hours of drug treatment [0.5 μM Entinostat, 10 μM of Olaparib, the combination of both, or control]. Clonogenicity assays were used to assess colony formation and MTS assays were used to measure cell viability and proliferation. We then performed immunofluorescence (IF) staining for RAD51 (a marker for HR repair), γH2AX (a marker of DNA damage) and Ki67 (a marker of proliferation). As expected in the clonogenicity assay, there was no statistically significant difference in cell survival between the olaparib treated ID8 TP53 null (HR proficient) cells and vehicletreated controls (p=0.0993; paired t test). Alternatively, the ID8 TP53 null/BRCA2 null (HR deficient) cell line treated with olaparib alone reduced cell viability compared to control (p=0.0245; paired t test). Clonogenicity assays also demonstrated that the combination of entinostat and olaparib reduced colony formation compared to control in the ID8 TP53 null cell line (p<0.0001; one-way ANOVA). MTS assays corroborated this treatment effect (p<0.0001; two way ANOVA). Preliminary data suggests that compared to controls and each drug alone, IF staining for RAD51 and Ki67 expression was reduced in cells treated with combination therapy. Also preliminarily, there was increased number of γH2AX foci in combination treatment compared to controls and each drug alone. Entinostat restores responsiveness to olaparib in ID8 TP53 null HR proficient mouse ovarian cancer cells that molecularly resemble high-grade serous ovarian carcinoma. These results suggest this drug combination can overcome intrinsic resistance to PARPi and provide additional preclinical support for clinical investigation of this combination for the treatment of HR proficient ovarian cancer.

PIG3 downregulation enhances the radiosensitivity of NSCLC cells by promoting G2/M cell cycle arrest and apoptosis

ObjectiveTo investigate the mechanism of p53-induced gene 3 (PIG3)-regulation of radioresistance in human non-small cell lung cancer (NSCLC) cells, in order to explore new biomarkers and therapeutic targets to combat radioresistance and improve the 5-year survival rate. MethodsThe PIG3 gene was knocked down in A549 ​cells using siRNA, and was overexpressed in H1299 ​cells using a PIG3 expression plasmid. After confirming PIG3 knockdown and overexpression through the Western blot analysis, the radiosensitivity, DNA damage, cell cycle distribution, and apoptosis in these cells were analyzed using colony formation assay, immunofluorescence staining for γH2AX, and flow cytometry, respectively. ResultsPIG3 silencing markedly increased the radiosensitivity of NSCLC cells, with radiosensitization ratios of 1.12 and 1.25. Compared with the corresponding negative control, PIG3 knockdown significantly enhanced G2/M phase arrest (siNC: 26.12 ​± ​2.50, siPIG3#1: 34.98 ​± ​4.19, siPIG3#2: 37.79 ​± ​3.53, P ​< ​0.05), promoted radiation-induced apoptosis (siNC: 14.61 ​± ​1.85, siPIG3#1: 17.26 ​± ​1.14, siPIG3#2: 20.70 ​± ​2.04, P ​< ​0.05), and reduced the number of γ-H2AX foci 0.5, 1, and 2 ​h after radiation (P ​< ​0.05). Conversely, PIG3 overexpression markedly decreased the radiosensitivity of NSCLC cells, as evidenced by the reduction of G2/M phase arrest (NC: 33.18 ​± ​2.11 vs. PIG3: 24.21 ​± ​3.09, P ​< ​0.05) and apoptosis (NC: 15.49 ​± ​0.56 vs. PIG3: 12.79 ​± ​0.29, P ​< ​0.05), and increased DNA damage (P ​< ​0.05). ConclusionsPIG3 downregulation increases the radiosensitivity of NSCLC cells, and PIG3-upregulation leads to the progression in radioresistance. Therefore, PIG3 is a potential target for radiotherapy for NSCLC.

Abstract 5485: Discovery and preclinical evaluation of a novel highly selective and potent CDK12 inhibitor

Abstract Recent research highlights that RNA processing is systematically altered in cancer, demonstrating the pivotal influence of RNA deregulation on tumorigenesis, growth and progression. Based on our expanding knowledge of RNA biology, RNA deregulation has drawn much attention from the perspective of cancer therapy. Indeed, small molecules that attack the RNA maturation processes and produce aberrant RNA are currently under development. The first step in gene expression is transcription, which involves copying a DNA sequence to make an RNA. Transcription is performed by enzymes called RNA polymerase (Pol II) that links nucleotides to form RNA strand. The C-terminal domain (CTD) of Pol II comprises heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 repeats and is dynamically post-translationally phosphorylated to regulate the distinct stages of transcription initiation, elongation and termination steps. Particularly, Ser2 and Ser5 phosphorylation have the closest association with the regulation of transcription. Therefore, we focus on the kinases that carry out this modification. Cyclin-dependent kinase 12 (CDK12) belongs to the cyclin-dependent kinase (CDK) family of serine/threonine protein kinases. CDK12 regulates the elongation step of RNA transcription by phosphorylation of Ser2 on the CTD. CDK12 complexes with cyclin K to promote the elongation of transcripts, such as BRCA1 and BRCA2, involved in DNA damage responses. It is expected that the inhibition of CDK12 have a synergistic effect with PARP inhibitors and chemotherapeutic reagents. Here we present data on CRD-1835439, orally available, selective, and first in class CDK12 inhibitor with optimized drug-properties. Biochemically, CRD-1835439 is more than 300-fold selective over other CDK isoforms such as CDK7 and CDK9. Importantly, cell based assays showed CRD-1835439 has selective inhibitory activity for phosphorylation of Ser2 on the CTD but not for other Ser5 and Ser7 phosphorylation. Comprehensive analysis by PolyA-seq, Chip-seq and RNA-seq revealed that CRD-1835439 inhibits transcriptional elongation on DNA damage response genes including BRCA1 and BRCA2. The effects on DNA damage and repair were assessed by immunofluorescence staining for γH2AX and RAD51 proteins. In vivo, oral treatment with CRD-1835439 in cell line derived xenograft models, resulted in increase of DNA damage biomarkers, induction of apoptosis and tumor regressions in a dose-dependent fashion. Besides the efficacy as a single reagent, the efficacy was augmented when CRD-1835439 was combined with PARP inhibitors in in vitro and in vivo. In summary, the novel small-molecule CDK12 inhibitor CRD-1835439 demonstrated preclinical efficacy along with target engagement. Our results underscore the preclinical therapeutic potential of CRD-1835439 as a single-agent or in combination with PARP inhibitors for the treatment of intractable cancers. Citation Format: Hiroko Yamakawa, Akio Mizutani, Yasuyoshi Arikawa, Shunsuke Ebara, Yoshihiko Satoh, Daisuke Morishita. Discovery and preclinical evaluation of a novel highly selective and potent CDK12 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5485.

Abstract 5487: An auristatin-based peptide-drug conjugate targeting Kita-Kyushu lung cancer antigen 1 for precision chemoradiotherapy in gastric cancer

Abstract Gastric cancer is a worldwide health problem. Concurrent chemoradiotherapy (CRT) represents an important strategy to reduce recurrence and prolong survival for gastric cancer. However, non-targeted cytotoxic drugs capable of enhancing radiosensitization remain the main choice to be combined with radiotherapy, which has limited the anti-tumor efficacy of CRT by off-target side effects. Monomethyl auristatin E (MMAE) is a highly potent anti-tubulin agent and has been reported to act as an effective radiosensitizer recently. Kita-Kyushu lung cancer antigen 1 (KK-LC-1) is a cancer/testis antigen highly and exclusively expressed in multiple types of cancers including gastric cancer, representing a promising target for drug delivery. Previously, we identified a KKLC-1-targeting cyclic nonapeptide (1131 peptide) by screening a phage display peptide library. Here, we described a novel peptide-drug conjugate (PDC) 1131-MMAE by connecting the 1131 peptide with MMAE via a protease-sensitive linker for tumor-selective radiosensitization. We demonstrated that 1131-MMAE could be efficiently internalized by gastric cancer cells through KKLC1-mediating endocytosis and exhibited a comparable cytotoxicity profile with free MMAE. 1131-MMAE could arrest the cell cycle at the most radiosensitive G2/M phase in a dose-dependent manner and selectively enhance the radiation response of KK-LC-1 positive gastric cancer cells by increasing DNA double-strand break as indicated by γH2Ax immunofluorescence staining. Furthermore,1131-MMAE was able to inhibit tumor growth as a monotherapy at 2mg/kg with a good safety profile in vivo. Increased tumor control was achieved when 1131-MMAE was combined with radiotherapy at a relatively low dose of 0.3mg/kg. Overall, we proposed an MMAE-based PDC targeting a novel tumor marker KK-LC-1 and demonstrated its tumor-selective radiosensitization in gastric cancer models for the first time. This anti-KKLC1 PDC may help to promote the clinical progress in precision chemoradiotherapy for multiple types of cancer. Citation Format: Xiaotong Chen, Fangcen Liu, Xiaoxiao Yu, Lin Li, Jiayao Yan, Baorui Liu. An auristatin-based peptide-drug conjugate targeting Kita-Kyushu lung cancer antigen 1 for precision chemoradiotherapy in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5487.

Abstract 2597: Inhibition of BARD1-PLK1 axis enhances PARP inhibitor/platinum sensitivity in homologous repair proficient pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a grim prognosis. Though the overall 5-year survival rates have recently improved to 10%, there is still an unmet need for better therapies. PARP inhibitors (PARPi)/platinum therapies have demonstrated clinical efficacy in BRCA1/2 mutated PDAC and has recently led to the FDA approval of PARPi (olaparib) in the maintenance setting, offering the best personalized treatment approach for such patients. Although promising, the benefits of these therapeutic strategies are currently limited to only a few patients. We have recently established that BARD1 (BRCA1- Associated-Ring-Domain-1), the main binding partner of BRCA1 in regulating homologous recombination repair is upregulated in PDAC cells exposed to PARPi/platinum compounds, by a post-transcriptional RNA binding mechanism. We therefore hypothesized that inhibition of BARD1 will sensitize cells to these agents. We found that inhibition of BARD1 (both by siRNA/shRNA and CRISPR-KO) not only sensitized the cells to olaparib or oxaliplatin but enhanced DNA damage in cells, as analyzed by comet assays and γ-H2AX immunofluorescence staining. BARD1 inhibition led to a decrease in homologous repair efficiency of cells as analyzed by the well established pDRGFP assay. To further understand BARD1 dependent downstream signaling, we performed comparative transcriptomic analysis of BARD1 silenced cells compared to scramble control. RNA seq analysis revealed modulation of several cell cycle checkpoints and DNA damage response pathways. Notably, through gene set enrichment analysis (GSEA), we found that silencing BARD1 causes de-enrichment of pathway regulating Polo-Like-Kinase-1 (PLK1, a serine/threonine kinase) activity (p-val=0.05, FDR=0.07). PLK1 is frequently upregulated in PDAC cells and promotes progression of cells through mitosis. Based on these results, we found that a PLK1 specific inhibitor (onvansertib) synergized with olaparib to reduce cell survival of homologous repair proficient (HR-P) PDAC cells. Importantly, the mechanism of drug synergy involved induction of apoptosis and DNA damage, as assessed by cleaved caspase-3 and γ-H2AX protein analysis. These results are suggestive of targeting the BARD1-PLK1 axis in order to enhance sensitivity of PARPi/platinums in HR proficient cells. Our ongoing and future studies are aimed at understanding the interaction of BARD1 and PLK1 in PDAC cells and analyze the effects of onvansertib and olaparib on tumor growth in vivo. Accomplishment of these studies will establish novel therapeutic targets and synthetic lethal combinations that have not been explored yet for deadly pancreatic cancers, ultimately benefiting patient survival in the long run. Citation Format: Carolyn Coats, Anoohya Arkala, Eric Londin, Avinoam Nevler, Jonathan R. Brody, Charles J. Yeo, Aditi Jain. Inhibition of BARD1-PLK1 axis enhances PARP inhibitor/platinum sensitivity in homologous repair proficient pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2597.

Abstract 795: Entinostat restores responsiveness to Olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines

Abstract Poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) reduce disease progression in patients diagnosed with ovarian cancer. The anticancer effects of PARPi are most effective in tumors with BRCA mutations and homologous recombination (HR) deficiency (HRD). One mechanism of PARPi resistance is through restoration of HR repair. Our group has shown that entinostat (an HDACi) potentiates the effects of olaparib (a PARPi). The objective of this study was to investigate mechanisms of intrinsic PARPi resistance by studying the combination of entinostat and olaparib in HR proficient and HR deficient mouse ovarian cancer cell lines. We used the ID8 TP53 null (HR proficient) and ID8 TP53 null/BRCA2 null (HR deficient) mouse ovarian cancer cell lines that harbor molecular features consistent with human high-grade serous ovarian cancer. To recapitulate an ongoing clinical trial using this drug combination, cells were pre-treated with 0.25 µM Entinostat or control for 24 hours, followed by 24 or 72 hours of drug treatment [0.5 µM Entinostat, 10 µM of Olaparib, the combination of both, or control]. Clonogenicity assays were used to assess colony formation and MTS assays were used to measure cell viability and proliferation. We then performed immunofluorescence (IF) staining for RAD51 (a marker for HR repair), γH2AX (a marker of DNA damage) and Ki67 (a marker of proliferation). As expected in the clonogenicity assay, there was no statistically significant difference in cell survival between the olaparib treated ID8 TP53 null (HR proficient) cells and vehicle-treated controls (p= 0.0993; paired t test). Alternatively, the ID8 TP53 null/BRCA2 null (HR deficient) cell line treated with olaparib alone reduced cell viability compared to control (p=0.0245; paired t test). Clonogenicity assays also demonstrated that the combination of entinostat and olaparib reduced colony formation compared to control in the ID8 TP53 null cell line (p&amp;lt;0.0001; one-way ANOVA). MTS assays corroborated this treatment effect (p&amp;lt; 0.0001; two way ANOVA). Preliminary data suggests that compared to controls and each drug alone, IF staining for RAD51 and Ki67 expression was reduced in cells treated with combination therapy. Also preliminarily, there was increased number of γH2AX foci in combination treatment compared to controls and each drug alone. Entinostat restores responsiveness to olaparib in ID8 TP53 null HR proficient mouse ovarian cancer cells that molecularly resemble high-grade serous ovarian carcinoma. These results suggest this drug combination can overcome intrinsic resistance to PARPi and provide additional preclinical support for clinical investigation of this combination for the treatment of HR proficient ovarian cancer. Citation Format: Tyler Woodard, Viju Gupta, Simona Miceska, Bisiayo Fashemi, Wendy Zhang, Dineo Khabele. Entinostat restores responsiveness to Olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 795.

A Comparative Assessment of Replication Stress Markers in the Context of Telomerase.

Simple SummaryGenetic alterations such as oncogenic- or aneuploidy-inducing mutations can induce replication stress as a tumor protection mechansim. Previous data indicated that telomerase may ameliorate the cellular responses that induce replication stress. However, the mechanisms how this may occur are still unclear. In order to address this question, the accurate evaluation of replication stress in the presence and absence of telomerase is crucial. Therefore, we used telomerase negative normal human fibroblasts, as well as their telomerase positive counterparts to compare the suitability of three protein markers (pRPA2, γ-H2AX and 53BP1), which were previously reported to accumulate in response to harmful conditions leading to replication stress in cells. In summary, we find that pRPA2 is the most consistent and reliable marker for the detection of replication stress. Further, we demonstrated that the inhibition of the DNA-damage activated ATM and ATR kinases by specific small compounds impaired the accumulation of pRPA2 foci in the absence of telomerase. These data suggest that telomerase rescues the cells from replication stress upon supression of DNA damage induction by modulating the ATM and ATR signaling pathways, and may therefore support tumor formation of genetically unstable cells.Aberrant replication stress (RS) is a source of genome instability and has serious implications for cell survival and tumourigenesis. Therefore, the detection of RS and the identification of the underlying molecular mechanisms are crucial for the understanding of tumourigenesis. Currently, three protein markers—p33-phosphorylated replication protein A2 (pRPA2), γ-phosphorylated H2AX (γ-H2AX), and Tumor Protein P53 Binding Protein 1 (53BP1)—are frequently used to detect RS. However, to our knowledge, there is no report that compares their suitability for the detection of different sources of RS. Therefore, in this study, we evaluate the suitability of pRPA2, γ-H2AX, and 53BP1 for the detection of RS caused by different sources of RS. In addition, we examine their suitability as markers of the telomerase-mediated alleviation of RS. For these purposes, we use here telomerase-negative human fibroblasts (BJ) and their telomerase-immortalized counterparts (BJ-hTERT). Replication stress was induced by the ectopic expression of the oncogenic RAS mutant RASG12V (OI-RS), by the knockdown of ploidy-control genes ORP3 or MAD2 (AI-RS), and by treatment with hydrogen peroxide (ROS-induced RS). The level of RS was determined by immunofluorescence staining for pRPA2, γ-H2AX, and 53BP1. Evaluation of the staining results revealed that pRPA2- and γ-H2AX provide a significant and reliable assessment of OI-RS and AI-RS compared to 53BP1. On the other hand, 53BP1 and pRPA2 proved to be superior to γ-H2AX for the evaluation of ROS-induced RS. Moreover, the data showed that among the tested markers, pRPA2 is best suited to evaluate the telomerase-mediated suppression of all three types of RS. In summary, the data indicate that the choice of marker is important for the evaluation of RS activated through different conditions.