Cell Cycle Checkpoint Regulation Research Articles

Role of TRIP13 in human cancer development.

As an AAA + ATPase, thyroid hormone receptor interacting protein 13 (TRIP13) primarily functions in DNA double-strand break repair, chromosome recombination, and cell cycle checkpoint regulation; aberrant expression of TRIP13 can result in chromosomal instability (CIN). According to recent research, TRIP13 is aberrantly expressed in a variety of cancers, and a patient's poor prognosis and tumor stage are strongly correlated with high expression of TRIP13. Tumor cell and subcutaneous xenograft growth can be markedly inhibited by TRIP13 knockdown or TRIP13 inhibitor administration. In the initiation and advancement of human malignancies, TRIP13 seems to function as an oncogene. Based on available data, TRIP13 may function as a biological target and biomarker for cancer. The creation of inhibitors that specifically target TRIP13 may present novel approaches to treating cancer.

A first-in-human trial of selective CDK7 inhibitor Q901, in patients with advanced solid tumors: Interim results of a phase I study (QRNT-009).

3078 Background: Q901 is a potent, covalently binding CDK7 inhibitor with excellent specificity for CDK7 against other protein kinases. Transcriptional regulation by CDK7 inhibition in cancer cells leads to DNA damage repair response inhibition and increased genomic instability. Q901 also regulates cell cycle checkpoints by inhibiting T-loop phosphorylation and transcriptionally downregulating CDK1, 2 and 4. In preclinical in vivo models, intravenous (iv) administration of Q901 once a week and once every three-week dosing regimen was well tolerated and showed significant antitumor activity. The safety profile, pharmacokinetics (PK), pharmacodynamics (PD) and efficacy data for 18, 36 and 60 mg/m2 are presented. Methods: QRNT-009 (NCT05394103) is an ongoing Phase 1/2 multicenter, open-label, dose escalation and expansion study. Q901 is administered iv once weekly for four weeks, then once every two weeks thereafter in patients with advanced or metastatic ovarian cancer, castration-resistant prostate cancer (CRPC), breast cancer, endometrial cancer, colorectal cancer, small cell lung cancer (SCLC), or pancreatic cancers. Results: As of data cutoff (12Jan2024), 17 patients received Q901 across 3 dose levels: 5 patients at 18 mg/m2, 4 patients at 36 mg/m2, and 8 patients at 60 mg/m2. The median prior lines of systemic therapy were 4 (range 2 - 17). Of 17 patients who received Q901 across all doses, there were no treatment related SAEs, treatment discontinuations, or dose reductions. Most common TRAEs ≥ 15% were nausea (29.4%), anemia (23.6%) and fatigue (23.5%). As of data cutoff, no DLTs were observed. All plasma PK analyses across dose levels showed dose dependent increase of AUC0-last and Cmax. No major metabolite was observed. POLR2A, which is a CDK7 target engagement marker showed biologically effective change starting from 18 mg/m2. 1 patient with pancreatic cancer who received 18 mg/m2 had a partial response with CA19-9 reduction from 4,632 to 219. 2 patients at 18 mg/m2 and 2 patients at 36 mg/m2 had stable disease. Additional safety and efficacy data will be presented. Conclusions: Preliminary data from QRNT-009 study showed that selective CDK7 inhibitor Q901 is well tolerated. Preliminary antitumor activity and pharmacodynamic observations are encouraging. Dose escalation is ongoing to identify MTD or recommended dose for further studies. Clinical trial information: NCT05394103 .

Increased Prevalence of Germline Pathogenic CHEK2 Variants in Individuals With Pituitary Adenomas.

CHEK2 is a cell cycle checkpoint regulator gene with a long-established role as a clinically relevant, moderate risk breast cancer predisposition gene, with greater risk ascribed to truncating variants than missense variants. To assess the rate and pathogenicity of CHEK2 variants amongst individuals with pituitary adenomas (PAs). We assessed 165 individuals with PAs for CHEK2 variants. The study population comprised a primary cohort of 29 individuals who underwent germline and tumor whole-exome sequencing, and a second, independent cohort of 136 individuals who had a targeted next-generation sequencing panel performed on both germline and tumor DNA (n = 52) or germline DNA alone (n = 84). We identified rare, coding, nonsynonymous germline CHEK2 variants amongst 3 of 29 (10.3%) patients in our primary cohort, and in 5 of 165 (3.0%) patients overall, with affected patients having a range of PA types (prolactinoma, thyrotropinoma, somatotropinoma, and nonfunctioning PA). No somatic variants were identified. Two variants were definitive null variants (c.1100delC, c.444 + 1G > A), classified as pathogenic. Two variants were missense variants (p.Asn186His, p.Thr476Met), classified as likely pathogenic. Even when considering the null variants only, the rate of CHEK2 variants was higher in our cohort compared to national control data (1.8% vs 0.5%; P = .049). This is the first study to suggest a role for the breast cancer predisposition gene, CHEK2, in pituitary tumorigenesis, with pathogenic/likely pathogenic variants found in 3% of patients with PAs. As PAs are relatively common and typically lack classic autosomal dominant family histories, risk alleles-such as these variants found in CHEK2-might be a significant contributor to PA risk in the general population.

Exploring the mechanism of action of spirooxindoles as a class of CDK2 inhibitors: a structure-based computational approach.

Cyclin-dependent kinase 2 (CDK2) regulates cell cycle checkpoints in the synthesis and mitosis phases and plays a pivotal role in cancerous cell proliferation. The activation of CDK2, influenced by various protein signaling pathways, initiates the phosphorylation process. Due to its crucial role in carcinogenesis, CDK2 is a druggable hotspot target to suppress cancer cell proliferation. In this context, several studies have identified spirooxindoles as an effective class of CDK2 inhibitors. In the present study, three spirooxindoles (SOI1, SOI2, and SOI3) were studied to understand their inhibitory mechanism against CDK2 through a structure-based approach. Molecular docking and molecular dynamics (MD) simulations were performed to explore their interactions with CDK2 at the molecular level. The calculated binding free energy for the spirooxindole-based CDK2 inhibitors aligned well with experimental results regarding CDK2 inhibition. Energy decomposition (ED) analysis identified key binding residues, including I10, G11, T14, R36, F82, K89, L134, P155, T158, Y159, and T160, in the CDK2 active site and T-loop phosphorylation. Molecular mechanics (MM) energy was identified as the primary contributor to stabilizing inhibitor binding in the CDK2 protein structure. Furthermore, the analysis of binding affinity revealed that the inhibitor SOI1 binds more strongly to CDK2 compared to the other inhibitors under investigation. It demonstrated a robust interaction with the crucial residue T160 in the T-loop phosphorylation site, responsible for kinase activation. These insights into the inhibitory mechanism are anticipated to contribute to the development of potential CDK2 inhibitors using the spirooxindole scaffold.

Abstract B028: WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is a poorly immunogenic, high-grade neuroendocrine carcinoma arising in the lung. Immune checkpoint blockade (ICB) added to chemotherapy is now the standard upfront therapy for SCLC but leads to a modest increase in overall survival and progression-free survival. These modest benefits underscore the critical need to identify pathways and targets that can durably enhance the antitumor responses of ICB in SCLC. SCLC is characterized by a ubiquitous loss of TP53 which disrupts the G1-S cell cycle checkpoint and as a result, most SCLC is dependent on G2-M cell cycle checkpoint regulators including WEE1. We and others have shown that DNA damage response (DDR) components are overexpressed in SCLC and targeting DDR could be an effective therapeutic strategy in SCLC. Methods: In this study, we have performed WEE1 inhibition either alone or in combination with PD-L1 blockade in a panel of SCLC models. Results: We demonstrate that inhibition of WEE1 induces G2/M cell cycle arrest, DNA damage, and cytosolic DNA accumulation in SCLC models. We further show that WEE1 targeting activates the STING-TBK1-IRF3 pathway which increases type I interferons (IFN-α and IFN-β) and pro-inflammatory chemokines (CXCL10 and CCL5), facilitating an immune response via CD8+ cytotoxic T-cell infiltration. We further show that WEE1 inhibition concomitantly activates the STAT1 pathway, increasing IFN-γ and PD-L1 expression. Consistent with these findings, combined WEE1 inhibition (AZD1775) and PD-L1 blockade caused remarkable tumor regression, activation of type I and II interferon pathways, and infiltration of cytotoxic T-cells in multiple immunocompetent SCLC genetically engineered mouse models, including an aggressive model with stabilized MYC. Conclusions: Our study demonstrates the cell-autonomous and immune-stimulating activity of WEE1 inhibition in SCLC. Given the increasing importance of immunotherapy for the management of SCLC and that WEE1 inhibitors are already in clinical trials, combining a WEE1 inhibitor with PD-L1 blockade may offer a particularly attractive strategy for the treatment of SCLC and contribute to the rapid translation of this combination into the clinic. Citation Format: Triparna Sen. WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B028.

Pioglitazone as a Treatment Option for Ataxia-Telangiectasia

Abstract Introduction/Objective The ataxia-telangiectasia mutated (ATM) gene product regulates cell cycle checkpoints, the cellular redox state, and DNA break repair. ATM loss causes the rare disorder ataxia-telangiectasia (A-T), characterized by progressive ataxia, telangiectasias, immune dysfunction, and an elevated cancer risk. The clinical course of A-T is relentless, causing death by the early twenties. Methods/Case Report A-T cells show extreme sensitivity to oxidants and ionizing radiation, impaired glutathione (GSH) synthesis, genomic instability, poor cell cycle checkpoint initiation, and increased reactive oxygen species. A-T cells also show unusual iron responses, with iron chelators increasing A-T cell growth, oxidant exposure resistance, and genomic stability, while labile iron exerts toxic effects not seen in ATM wild-type cells. Since GSH is dysregulated in A-T cells and much of the cellular iron is bound up in iron-sulfur clusters, we examined the cellular sulfur pool in A-T cells with and without an ATM expression vector, and in ATM wild-types cells. Free hydrogen sulfide and protein- bound sulfide levels were unchanged with ATM expression. However, iron-sulfur clusters (the acid-labile sulfur faction) were low in the A-T cells and dramatically increased with ATM expression. ATM expression also increased the expression of NFS1, NFU1, and several mitochondrial complex I iron-sulfur proteins. As NFS1 and NFU1 play central roles in the synthesis of iron-sulfur clusters, and ATM expression increases these clusters, our data reveals a role for ATM in iron metabolism. Interestingly, the type II diabetes drug pioglitazone increased the acid-labile sulfur and protein bound sulfur factions in both cell types. It also increased the A-T cell reduced GSH, cell viability following oxidative stress, and decreased oxidative stress-induced double-stranded DNA break formation. Results (if a Case Study enter NA) Our finding that pioglitazone increased both cellular acid-labile and protein-bound sulfur fractions, indicates that these sulfur pools may be a therapeutic target for some type II diabetes drugs. Our data also indicates that pioglitazone may have value in treating A-T Conclusion We propose that pioglitazone could be a promising A-T treatment drug. More clinical research and trials are needed to determine the potential advantages and side effects of using pioglitazone in A-T.

Novel CDK4/6 lnhibitor, WXJ-8 -Exerts Anti-effects Through Cell Cycle in Breast Cancer

The novel Abemaciclib derivative WXJ-8 may be a promising anti-tumour drug; however, its mechanism of action and clinical value need to be further verified in research. Cell cycle regulating systems are critical in parthenogenesis and progression. Because of its involvement in regulating cell cycle checkpoints, the CDK4/6-CyclinD1-Rb-E2F1 signalling pathway has been proposed as a possible therapeutic target for breast cancer. As a result, WXJ-8, a new and highly specific CDK4/6 inhibitor, was developed and synthesized in this investigation. The activity of WXJ-8 against breast cancer cells in vitro was detected by MTT method. The main subjects were MDA-MB-231 and MCF-7 cells. Plate cloning, wound healing, invasion and adhesion experiments revealed that WXJ-8 had strong anti-proliferation, migration, invasion, and adhesion effects on breast cancer cells. Protein blotting revealed significant down regulation of CDK4, CDK6, p-Rb, E2F1 and other cell cycle factors in the CDK4/6-CyclinD1-Rb-E2F1 pathway, as well as activation of Bcl-2, Caspase-3, and Cleaved Caspase-3 apoptotic factor expression, resulting in MDA-MB-231 cell cycle arrest and apoptosis. Finally, our research implies that WXJ-8 might be a potential anti-triple-negative breast cancer medication candidate.

Development of effective immunotherapy for triple negative breast cancer by WEE1 kinase inhibition

Abstract Treatment options for triple negative breast cancer (TNBC) are limited because they are usually insensitive to most available hormonal or targeted therapeutic agents owing to their triple negative nature. Exciting clinical results using immune checkpoint inhibitors (ICI) directed against PD-1/PD-L1 have been reported. However, only a subset of patients (~20%) respond to ICI. Therefore, identifying rational combinations is needed to enhance the efficacy of immunotherapy in TNBC. We demonstrated there that inhibition of WEE1, a G2/M cell cycle checkpoint regulator induced by DNA damage, increased expression levels of MHC-I, and pro-inflammatory chemokines (CXCL9 and CXCL10) together with PD-L1 in human and mouse TNBC cells. Consequently, WEE1 inhibition enhanced cytotoxic T-lymphocyte (CTL)-mediated killing of tumor cells, and CTL infiltration into the tumor, making tumors susceptible to anti-PD-1 mAb immune checkpoint blockade in mouse TNBC models in a CD8+ T cell–dependent manner. Mechanistically, WEE1 inhibition triggered the cellular damage stress and interferon response through the double-stranded DNA/RNA (dsDNA/RNA) by activating the TLR9/IRF7 pathway. Furthermore, combining WEE1 inhibition and TLR9 agonist CpG DNA resulted in tumor regression, facilitating an effective antitumor T cell response. Our findings have rapid translational implications for the exploration of WEE1 inhibitors alongside agents that stimulate CD8+ T cell activity, including ICI and TLR9 agonists, which are both clinically available to treat TNBC patients. We acknowledge the funding support from NCI R01CA222963 and DoD W81XWH-19-1-0106.

Small molecule ‘4ab’ induced autophagy and endoplasmic reticulum stress-mediated death of aggressive cancer cells grown under adherent and floating conditions

Metastasis is the leading cause of death in cancer patients and a major challenging aspect of cancer biology. Various adaptive molecular signaling pathways play a crucial role in cancer metastasis and later in the formation of secondary tumors. Aggressive cancer cells like triple negative breast cancer (TNBCs) are more inclined to undergo metastasis hence having a high recurrence rate and potential of micro-metastasis. Tumor cells in circulation known as circulating tumor cells (CTCs) offer an attractive drug target to treat metastatic disease. Cell cycle regulation and stress response of CTCs in blood has a crucial role in their survival and progression and thus may be considered therapeutically active hotspots. The cyclin D/cyclin-dependent kinase (CDK) pathway regulates cell cycle checkpoints, a process that is frequently dysregulated in cancer cells. Selective CDK inhibitors can limit the phosphorylation of cell cycle regulatory proteins by inducing cell cycle phase arrest, and thus may be an effective therapeutic strategy for aggressive cancer cells in their dividing phase at the primary or secondary site. However, during the floating condition, cancer cells halt their multiplication process and proceed through the various steps of metastasis. Current study showed that a novel CDK inhibitor 4ab induced autophagy and endoplasmic reticulum (ER) stress inagressive cancer cells grown under adherent and floating conditions resulting in paraptosis. Further, our results showed that 4ab efficiently induced cell death in aggressive cancer cells through ER stress-mediated activation of JNK signaling. Additionally,was observed that treatment of 4ab in tumor-bearing mice displayed a significant reduction in tumor burden and micro-metastasis. The outcome of these studies showed that 4ab can be a potential anti-tumor and anti-metastatic agent. Graphical representation of 4ab: image representing the effect of 4ab on death-inducing pathways in aggressive cancer cells. 4ab induces ER stress and activates autophagy leading to vacuolation of thereby causing apoptosisin aggressive cancer cells.

Heterogenous Differences in Cellular Senescent Phenotypes in Pre-Eclampsia and IUGR following Quantitative Assessment of Multiple Biomarkers of Senescence

Premature ageing of the placenta in pregnancy outcomes is associated with the persistent presence of oxidative stress and placental insufficiency reducing its functional capacity. In this study, we investigated cellular senescence phenotypes of pre-eclampsia and IUGR pregnancies by simultaneously measuring several biomarkers of senescence. Maternal plasma and placental samples were collected at term gestation from nulliparous women undergoing pre-labour elective caesarean section with pre-eclampsia without intrauterine growth restriction (PE; n = 5), pre-eclampsia associated with intrauterine growth restriction (n = 8), intrauterine growth restriction (IUGR < 10th centile; n = 6), and age-matched controls (n = 20). Placental absolute telomere length and senescence gene analysis was performed by RTqPCR. The expression of cyclin-dependent kinase inhibitors (p21 and p16) was determined by Western blot. Senescence-associated secretory phenotypes (SASPs) were evaluated in maternal plasma by multiplex ELISA assay. Placental expression of senescence-associated genes showed significant increases in CHEK1, PCNA, PTEN, CDKN2A, and CCNB-1 (p < 0.05) in pre-eclampsia, while TBX-2, PCNA, ATM, and CCNB-1 expression were evident (p < 0.05) and were significantly decreased in IUGR compared with controls. Placental p16 protein expression was significantly decreased in pre-eclampsia only compared with controls (p = 0.028). IL-6 was significantly increased in pre-eclampsia (0.54 pg/mL ± 0.271 vs. 0.3 pg/mL ± 0.102; p = 0.017) while IFN-γ was significantly increased in IUGR (4.6 pg/mL ± 2.2 vs. 2.17 pg/mL ± 0.8; p = 0.002) compared with controls. These results provide evidence of premature senescence in IUGR pregnancies, and while cell cycle checkpoint regulators are activated in pre-eclampsia, the cellular phenotype is one of cell repair and subsequent proliferation rather than progression to senescence. The heterogeneity of these cellular phenotypes highlights the complexity of characterising cellular senescence and may equally be indicative of the differing pathophysiological insults unique to each obstetric complication.

DNA Repair Mechanisms, Protein Interactions and Therapeutic Targeting of the MRN Complex.

Repair of a DNA double-strand break relies upon a pathway of proteins to identify damage, regulate cell cycle checkpoints, and repair the damage. This process is initiated by a sensor protein complex, the MRN complex, comprised of three proteins-MRE11, RAD50, and NBS1. After a double-stranded break, the MRN complex recruits and activates ATM, in-turn activating other proteins such as BRCA1/2, ATR, CHEK1/2, PALB2 and RAD51. These proteins have been the focus of many studies for their individual roles in hereditary cancer syndromes and are included on several genetic testing panels. These panels have enabled us to acquire large amounts of genetic data, much of which remains a challenge to interpret due to the presence of variants of uncertain significance (VUS). While the primary aim of clinical testing is to accurately and confidently classify variants in order to inform medical management, the presence of VUSs has led to ambiguity in genetic counseling. Pathogenic variants within MRN complex genes have been implicated in breast, ovarian, prostate, colon cancers and gliomas; however, the hundreds of VUSs within MRE11, RAD50, and NBS1 precludes the application of these data in genetic guidance of carriers. In this review, we discuss the MRN complex's role in DNA double-strand break repair, its interactions with other cancer predisposing genes, the variants that can be found within the three MRN complex genes, and the MRN complex's potential as an anti-cancer therapeutic target.

HELQ suppresses migration and proliferation of non-small cell lung cancer cells by repairing DNA damage and inducing necrosis.

HELQ plays a key role in DNA damage response and cell-cycle checkpoint regulation. It has been implicated in ovarian and pituitary tumors and may play a role in germ cell maintenance. This study investigated the role of HELQ in lung cancer. The expression of HELQ in patients with non-small-cell lung cancer (NSCLC) was downregulated compared with normal human lungs. Clinical prognostic analysis of Kaplan-Meier plots revealed that patients with NSCLC with low HELQ levels had a reduced overall survival. Further, we found that HELQ depletion enhanced lung cancer cell malignancy. Furthermore, overexpression of HELQ in lung cancer cells reduced cell migration in vitro, while DNA damage repair was inhibited. Both in vitro and in vivo studies have shown that HELQ induces cell death. Mechanistically, we found that cells overexpressing HELQ showed a tendency to induce necrosis. After analyzing the database of HELQ interactors. we found that RIPK3 may interact with it and proved this conclusion by immunoprecipitation. Our findings identified the tumor suppressive role of HELQ in malignant human lung cancer and unraveled a potential therapeutic strategy for cancer treatment through HELQ activation. Moreover, HELQ may also be a predictive biomarker for the clinical predisposition, progression, and prognosis of lung cancer.

SPAG5 Activates PI3K/AKT Pathway and Promotes the Tumor Progression and Chemo-Resistance in Gastric Cancer.

The sperm-associated antigen 5 (SPAG5) is an important protein in mitosis and cell cycle checkpoint regulation, with more attention as a novel oncogene in various cancers. High level of SPAG5 expression has been detected in our clinical gastric cancer (GC) samples and The Cancer Genome Atlas GC data. However, the bio-function and potential mechanism of SPAG5 in GC remain unclear. In this study, we investigated the role of SPAG5 in GC development and the correlation between SPAG5 and 5-fluorouracil (5-FU) treatment. SPAG5 expression was increased in GC samples compared with that in normal tissues (80.8% vs. 22.0%), which was apparently associated with a worse outcome. Biological experiments showed that knockdown of SPAG5 induced apoptosis and suppressed proliferation in cells and animal models. Downregulation of SPAG5 enhanced the sensitivity of 5-FU in GC cells. Gene microarray chip identified 856 upregulated and 787 downregulated genes in SPAG5 silencing cells. Furthermore, 12 significant genes, including CDKN1A, CDKN1B, EIF4E, MAPK1, and HSP90B1, belonged to the PI3K/AKT signaling pathway using ingenuity pathway analysis. Meanwhile, real-time PCR and Western blotting results showed that knockdown of SPAG5 inhibited PI3K/AKT signaling pathway. Collectively, SPAG5 promotes the growth of GC cells by regulating PI3K/AKT signaling pathway, which could be the promising target gene in GC therapy.

Circulating Extracellular Vesicles and Particles Derived From Adipocytes: The Potential Role in Spreading MicroRNAs Associated With Cellular Senescence.

Aging is associated with adipose tissue dysfunction and is recognized as a risk factor for shortened life span. Considering that in vitro findings have shown the involvement of microRNA in extracellular vesicles and particles (EVPs) on senescence, we hypothesized that circulating EVPs derived from adipocytes can be involved in the aging process via their microRNA cargo. We aimed to determine the microRNA profiles of circulating EVPs derived from adipocytes (FABP4+) from aged and young adult animals and to perform in silico prediction of their downstream signaling effects. Plasma was obtained from Wistar rats (3 and 21 months old), and adipocyte-derived EVPs were isolated using the commercially available kit. Fatty acid-binding protein 4 (FABP4) was used for adipocyte-derived EVPs isolation; microRNA isolation and microarray expression analysis were performed. The analysis revealed 728 miRNAs, 32 were differentially between groups (p < 0.05; fold change ≥ |1.1|), of which 15 miRNAs were upregulated and 17 were downregulated in circulating EVPs from aged animals compared to young adults. A conservative filter was applied, and 18 microRNAs had experimentally validated and highly conserved predicted mRNA targets, with a total of 2,228 mRNAs. Canonical pathways, disease and functions, and upstream regulator analyses were performed using IPA-QIAGEN, allowing a global and interconnected evaluation. IPA categories impacted negatively were cell cycle, cellular development, cellular growth and proliferation, and tissue development, while those impacted positively were “digestive system cancer” and “endocrine gland tumor.” Interestingly, the upregulated miR-15-5p targets several cyclins, such as CCND1 and CCND2, and miR-24-3p seems to target CDK4 (cyclin-dependent kinase 4); then potentially inhibiting their expression, both miRNAs can induce a negative regulation of cell cycle progression. In contrast, silencing of negative cell cycle checkpoint regulators, such as p21 and p16, can be predicted, which can induce impairment in response to genotoxic stressors. In addition, predicted targets, such as SMAD family members, seem to be involved in the positive control of digestive and endocrine tumors. Taken together, this exploratory study indicates that miRNA signature in circulating adipocyte-derived EVPs may be involved with the double-edged sword of cellular senescence, including irreversible proliferation arrest and tissue-dependent cancer, and seems to be suitable for further validation and confirmatory studies.

Correlation of CCNE1 copy number levels with molecular profiles and outcomes for high grade serous ovarian and endometrial cancer (121)

<b>Objectives:</b> This study aimed to characterize <i>CCNE1</i> copy number (CN) in relation to other molecular characteristics of tumors, along with patient outcomes, to help guide future clinical trial design and potential novel targeted therapeutics development for these aggressive molecular subtypes of high-grade serous ovarian (HGSOC) and endometrial cancers (EMCA). <b>Methods:</b> Tumor samples were analyzed using Next-Gen sequencing (NGS) of the DNA (NextSeq, 592 genes or NovaSeq, whole-exome sequencing) and RNA (NovaSeq, whole-transcriptome sequencing) and immunohistochemistry (IHC) (Caris Life Sciences, Phoenix, AZ). PD-L1 IHC used SP-142 (cut-off ≥1%). Microsatellite instability (MSI) was tested by fragment analysis, IHC, and NGS. Tumor mutation burden (TMB) was measured by totaling somatic mutations per tumor (high ≥10 mutations/MB). Relative abundance of immune-cell infiltrates was calculated by MCPCounter. <i>CCNE1</i> amplification (<i>CCNE1</i>^Amp) was defined as CN ≥6 and calculated by NGS/WES. Real-world overall survival (OS) was obtained from insurance claims data, and Kaplan-Meier estimates were calculated for molecularly defined patient cohorts. Statistical significance was determined using Chi-square and Wilcoxon rank-sum test, and p-values adjusted for multiple comparisons (q) to be <0.05. <b>Results:</b> <i>CCNE1</i> was amplified in 608/6306 (9.6%) of HGSOC, 214/2098 (10.2%) of serous EMCA, 191/749 (15.3%) of uterine carcinosarcomas (UCS), and 17/2053 (0.80%) of endometrioid EMCA. <i>TP53</i> mutations positively correlated with <i>CCNE1</i>^Amp. <i>CCNE1</i>^Amp HGSOC had fewer mutations in <i>BRCA1/2</i>, <i>ARID1A</i>, and <i>NF1</i> but increased CN amplification in other genes (Table 1). <i>PTEN</i> mutations were negatively correlated with <i>CCNE1</i>^Amp in UCS and endometrioid EMCA. There was no difference in immuno-oncology biomarkers (TMB, dMMR/MSI, PD-L1). However, increased <i>CCNE1</i> levels were associated with decreased fibroblasts in HGSOC and serous EMCA, decreased B cells in serous EMCA, and decreased CD3+ T cells in UCS. In HGSOC, <i>CCNE1</i>^Amp was associated with increased expression of cell-cycle checkpoint regulators, Fanconi anemia, homologous recombination, mismatch repair, and single-strand break repair pathway genes, but decreased expression of these genes in endometrioid EMCA. In tumors with >1 specimen serially submitted, there was a significant increase in <i>CCNE1</i> CN over time in 29% (49/169) of HGSOC and 23% of EMCA (36/158). There was no difference in <i>CCNE1</i> CN in tumors harboring <i>BRCA</i> reversion mutations (<i>n</i>=8) compared to wild-type (<i>n</i>=5110). <i>CCNE1</i>^Amp correlated with mRNA expression in HGSOC and EMCA. <i>CCNE1</i>^Amp was associated with worse OS after carboplatin-based treatment in HGSOC as well as in serous EMCA and UCS. <b>Conclusions:</b> <i>CCNE1</i>^Amp in HGSOC and EMCA is less common than previously identified. Both HGSOC and endometrioid EMCAs have distinct molecular characteristics of <i>CCNE1</i>^Amp tumors. <i>CCNE1</i> CN may increase with the emergence of treatment resistance. Finally, <i>CCNE1</i>^Amp tumors appear overall non-immunogenic.

Abstract 1538: Association of ceRNA dysregulation with clinical Response to Immunotherapy in cutaneous melanoma (CM)

Abstract Cutaneous melanoma (CM) is an aggressive malignant tumor of epidermal melanocytes for which treatment options and survival chances decrease dramatically when metastatic. To date, immunotherapy is becoming a new standard treatment for CM although less than 30% of the patients´ response positively. Therefore, there is a strong interest to identify response predictors and molecular pathways that might lead to therapy failure. Two novel biomarker sources are LncRNA and CircRNA which can function as natural sponges of miRNA as competitive endogenous RNAs (ceRNA) or can associate to RNA-Binding Proteins (RBP) to regulate gene expression. Moreover, several studies have revealed that ceRNA networks are implicated in tumorigenic processes. Considering this, we set to interrogate the role of the ceRNA in the prediction of response to CM immunotherapy. In this pilot study, differential expression was quantified by RNA-seq in 16 FFPE pre-treatment metastatic samples from CM patients treated with Nivolumab. Total RNA isolation was performed from 10 µm slides (4) with RNAeasy FFPE kit. Total RNAseq was done with Truseq Stranded RNA gold kit, and libraries were sequenced on Illumina Nextseq 550. LncRNA were obtained by STARs alignment and explored on LncATLAS. To assess the presence of high confident cirRNA we used 5 different pipelines with a minimum filtering cut-off of 2 junction reads in at least 2 samples and with at least 3 software. Deseq2 pipeline of total mapped reads was used to perform differential expression. We identified 23 circRNA and 74 lncRNA differentially expressed (DE) with a fold change of 1.5 and a p value &amp;lt; 0,01. Ingenuity pathway analysis was carried out to generate DE ceRNA-mRNA networks and associated RBP networks. Interestingly, in the DE ceRNA-mRNA networks, most of the 47 significant canonical pathways comprised immunological pathways such the antigen presentation pathway, Th1 and Th2 activation pathways and PD1-PDL1 cancer immunotherapy pathways. Regarding the DE ceRNA associated RBP networks, HOTAIR regulatory pathway and senescence as well as G1/S cell cycle checkpoint regulator pathways were enriched. Furthermore, we were able to predict the interaction of the DE ceRNA with the activation of key promoters of the immune response such as IL27 (Interleukin 27), EIB3 (Epstein-Barr Virus Induced 3), IL2 (Interleukin 2) and IFNA2 (Interferon Alpha2), as well as the inhibition of negative immune regulators such as SAFB2 (Distal-Less Homeobox 2) and FOXD1 (Forkhead Box D1). In conclusion, we have evidenced for the first time the potential utility of ceRNA as immunotherapy predictors. We have generated a response signature of ceRNA expression and used network analysis to associate them with specific key modulators of the immune response. This study opens a new venue for the investigation of the role of these regulatory ncRNA in the resistance to immunotherapy in cancer. Citation Format: Javier Oliver, Juan Luis Onieva, Maria Garrido-Barros, Alicia Garrido-Aranda, Vanessa De Luque, Martina Alvarez, Alfonso Sanchez, Elisabeth Perez, Patricia Chaves, Maria Jose Lozano, Miguel Berciano, Manolo Cobo, Emilio Alba, Antonio Rueda, Isabel Barragan. Association of ceRNA dysregulation with clinical Response to Immunotherapy in cutaneous melanoma (CM) [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 1538.

Abstract 3708: OMX-0407, a highly potent SIK3 inhibitor, sensitizes tumor cells to cell death and eradicates tumors in combination with PD-1 inhibition

Abstract Post-translational modifications of DNA and histones can improve the intrinsic antitumor capacity of the immune system. Using the iOTarg screening platform, salt-inducible kinase 3 (SIK3) was identified as a novel epigenetic modulator in cancer therapy. SIK3, a serine/threonine kinase of the AMP-activated protein kinase family, is known for regulating the NF-κB driven gene landscape through phosphorylation of class IIa histone deacetylases (HDACs) and CREB-regulated transcriptional coactivators causing the tumor to evade death receptor-mediated killing. Moreover, salt inducible kinases were recently described to regulate cell cycle checkpoints and thereby promote cancer cell proliferation. We demonstrate that SIK3 knockdown abates downstream pro-survival signaling and induces cell death in a distinct panel of tumor cell lines. OMX-0407, an orally available, single-digit nanomolar inhibitor of SIK3 was shown to effectively reduce TNF-induced HDAC4 phosphorylation and downstream NF-κB activity in a dose-dependent manner, thereby enhancing apoptosis in murine and human tumor cell lines. OMX-0407 dose-dependent suppression of intratumoral NF-κB activity was shown in vivo in an MC38 NF-κB-luc reporter cell line. Using OMX-0407 monotherapy, this translated to significant tumor growth inhibition as well as prolonged survival in the highly immune infiltrated syngeneic murine colorectal carcinoma model MC38. Besides its direct inhibitory effects on cancer cells, OMX-0407 repolarizes the tumor microenvironment (TME) by strongly decreasing regulatory T cells (T-regs) and M2-polarized macrophages in the tumor bed, while not affecting the peripheral T cell compartment. Thus, exposure to OMX-0407 leads to a distinct pro-inflammatory TME, characterized by an expansion of activated cytotoxic T lymphocytes (CTL) and an increased CTL-to-T-reg ratio. Using an immune-excluded tumor phenotype as seen in the breast cancer mouse model EMT6, we demonstrated that OMX-0407 and anti-PD-1 act synergistically by combining the sensitization towards cell death with a reduction in immunosuppressive TME and an increase in cytotoxic T cell activity, despite having only minimal anti-tumor efficacy as monotherapy. Thereby, partial or complete tumor remission in 60% of the animals and extension of overall survival were achieved. In summary, OMX-0407, a first-in-class oral SIK3 inhibitor, demonstrates potent monotherapy efficacy in a pro-inflammatory tumor setting by reshaping the immune compartment and accelerating tumor cell death. The ability of OMX-0407 to remodel an immunosuppressed TME in a generally cold tumor setting, harbors great clinical potential for OMX-0407 combination therapy with anti-PD-1/PD-L1 immune checkpoint blockade, specifically in patients with high unmet medical need who are resistant to current immune checkpoint inhibitor monotherapy. Citation Format: Christina Hartl, Ilona-Petra Maser, Tillmann Michels, Ronny Milde, Vanessa Klein, Philipp Beckhove, Nisit Khandelwal, Hannes Loferer, Stefan Bissinger. OMX-0407, a highly potent SIK3 inhibitor, sensitizes tumor cells to cell death and eradicates tumors in combination with PD-1 inhibition [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 3708.

The ataxia-telangiectasia mutated gene product regulates the cellular acid-labile sulfide fraction

The ataxia-telangiectasia mutated (ATM) protein regulates cell cycle checkpoints, the cellular redox state, and double-stranded DNA break repair. ATM loss causes the disorder ataxia-telangiectasia (A-T), distinguished by ataxia, telangiectasias, dysregulated cellular redox and iron responses, and an increased cancer risk. We examined the sulfur pool in A-T cells, with and without an ATM expression vector. While free and bound sulfide levels were not changed with ATM expression, the acid-labile sulfide faction was significantly increased. ATM expression also increased cysteine desulfurase (NFS1), NFU1 iron-sulfur cluster scaffold homolog protein, and several mitochondrial complex I proteins’ expression. Additionally, ATM expression suppressed cystathionine β-synthase and cystathionine γ-synthase protein expression, cystathionine γ-synthase enzymatic activity, and increased the reduced to oxidized glutathione ratio. This last observation is interesting, as dysregulated glutathione is implicated in A-T pathology. As ATM expression increases the expression of proteins central in initiating 2Fe-2S and 4Fe-4S cluster formation (NFS1 and NFU1, respectively), and the acid-labile sulfide faction is composed of sulfur incorporated into Fe-S clusters, our data indicates that ATM regulates aspects of Fe-S cluster biosynthesis, the transsulfuration pathway, and glutathione redox cycling. Thus, our data may explain some of the redox- and iron-related pathologies seen in A-T.

Small Molecule Anticancer Compound Modulates Cell Cycle DNA Damage Response Pathway and Inhibit Tumorigenesis in Triple Negative Breast Cancer

ObjectiveTo investigate the mechanism of action and therapeutic benefits of NSC001, a small molecule anticancer compound, in the treatment of triple negative breast cancer (TNBC), a clinical subtype of breast cancer that is more aggressive, has poor prognosis. In TNBC, patients harbor tumors that lack expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (Her2).HypothesisWe hypothesize that NSC001 will have a beneficial therapeutic effect on TNBC cells through the regulation of specific tumor associated signaling pathways in vitro.Material and methodsWe screened 1360 compounds from the National Cancer Institute (NCI) Diversity and Mechanistic Set library using the cell counting kit‐8 (CCK‐8) reagent from Dojindo Molecular Technologies, Inc. The screening was performed by applying 10µM of each candidate drug upon plates harboring the aggressive TNBC cell line, MDA‐MB‐231. Treatment time for all screening was fixed at 24 hours. The efficacy and potential mechanism of action of one of the potent drug candidates identified (NSC001) was further investigated on three TNBC cell lines (MDA‐MB‐231, MDA231‐LM2‐4175, and MDA‐MB‐468) using mass spectrometry and proteomic analysis. Afterwards, the results from the proteomic analysis were validated using molecular methods including cell viability assays, apoptosis assays, cell cycle assays, and western blotting.ResultsWe identified eight compounds that reduced the viability of MDA‐MB‐231 cells by up to 80% following a 24 hour treatment time point. Next, our in vitrodata show that NSC001 reduces the cell viability of TNBC cells in a dose‐and‐time dependent manner. Cell death data show that NSC001 induces potent apoptosis in a dose dependent manner in TNBC cells. Also, the results from colony formation assay show that 100nM [NSC001] is sufficient to inhibit the proliferative capacity of TNBC cells. Mass spectrometry and proteomic data reveals that NSC001 activates the cell cycle G2/M DNA damage checkpoint regulation in all the three TNBC cell lines used. Furthermore, we confirmed this finding in our cell cycle assay where we observed a clear G2/M cell cycle phase arrest. The arrest was potently induced at a dose significantly lower than the IC50 of NSC001. Lastly, we validated our proteomic results using western blotting, which confirmed that this low dose of NSC001 induces expression of p21, a cell cycle checkpoint regulator and tumor suppressor protein which is known to trigger cell cycle arrest in most cancer cell lines.ConclusionOverall, our data suggest that NSC001 may be a potent small molecule anticancer compound which should be further investigated for its potential to be used clinically in the treatment of TNBC patients.

7MO WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in small cell lung cancer

Small cell lung cancer (SCLC) is a poorly immunogenic, high-grade neuroendocrine carcinoma arising in the lung. Immune checkpoint blockade (ICB) added to chemotherapy is now the standard upfront therapy for SCLC but leads to a modest increase in overall survival and progression-free survival. These modest benefits underscore the critical need to identify pathways and targets that can durably enhance the antitumor responses of ICB in SCLC. SCLC is characterized by a ubiquitous loss of TP53 which disrupts the G1-S cell cycle checkpoint and as a result, most SCLC is dependent on G2-M cell cycle checkpoint regulators including WEE1.