Phase Of Cell Cycle Research Articles

Abstract 1637: EPHB4 is a key regulator of G1/S transition in hepatocellular carcinoma

Abstract Effective therapeutic regimens against advanced hepatocellular carcinoma (HCCs) have recently improved but it remains very limited. Targeting receptor tyrosine kinases (RTKs) has proven to one of the main ways of achieving better survival outcome in HCC patients, yet the precise mechanism for this success remains to be elusive. We have systematically investigated the expression profiles of RTKs transcriptomic datasets i.e. The Cancer Genome Atlas - Liver hepatocellular carcinoma (TCGA-LIHC) (n=371) and our in-house cohort of paired i.e. HKU-QMH (n=41), and identified EPHB4 as one of the most abundantly expressed and frequently mutated RTKs in human HCCs. EPHB4 expression was found to be significantly correlated with Alpha Fetoprotein (AFP) expression, a factor commonly used in the diagnosis of HCCs. Furthermore, EphB4 has been demonstrated to play a regulatory role in VEGF/VEGFR-mediated angiogenesis, critical targets for current successful drugs. We confirmed abundant membrane EphB4 expression in HCC clinical specimens and human HCC cell line models using immunohistochemistry. Clinicopathological correlation analysis of EPHB4 mRNA expression revealed that its overexpression is associated with aggressive tumor features including presence of tumor microsatellite, liver invasion, venous invasion, and absence of tumor encapsulation. Functionally, stably knocking down EPHB4 (shEPHB4) resulted in a strong reduction of proliferation and self-renewal rates of multiple HCC cell lines in vitro. It significantly reduced tumor growth rates in subcutaneous and liver orthotopic models in vivo. Furthermore, limiting dilution assay showed EPHB4 plays a role in tumor initiation property of HCC cells. Mechanistically, we performed Gene Set Enrichment Analysis (GSEA) of transcriptome profiles for shEPHB4 with respective controls and found that knocking down EPHB4 causes HCC cells undergo drastic changes in several cell cycle checkpoints including G1/S transition. Using thymidine blockade, we further confirmed that knockdown of significantly hinders the transition of HCC cells from G1 to S phase of cell cycle. Our data suggests a strong link between EphB4 and cell cycle. Conclusion: We found EphB4 plays a functional role in regulating G1/S transition in human HCCs and understanding regulatory role of EphB4 on cell cycle progression may provide mechanistic insights into the how to effectively target HCC in the clinic. Citation Format: Abdullah Husain, Elley Y. Chiu, Hoi-Tang Ma, Daniel W. Ho, Karen M. Sze, Lo-Kong Chan, Irene O. Ng. EPHB4 is a key regulator of G1/S transition in hepatocellular carcinoma [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 1637.

Abstract 4677: Characterizing resistant and metastatic phenotypes to pharmacological ascorbate in pancreatic cancer

Abstract Background and purpose: Pharmacological ascorbate (P-AscH) is a prooxidant that oxidizes to produce high levels of hydrogen peroxide. It is currently being investigated as an anti-cancer neoadjuvant for pancreatic cancer (PC). In phase I clinical trials, P-AscH has generated encouraging results in terms of efficiency and safety. However, 50% of patients do not respond to P-AscH suggesting that resistance occurs in a subset of patients. We have generated PC cell lines resistant to P-AscH to characterize mechanisms of resistance. Methodology: PANC-1 and MIA PaCa-2 PC cell lines were treated with a stepwise progression of increasing concentrations of P-AscH until 10-fold IC50 concentration was fully tolerated (AscR, ascorbate resistant) as determined by clonogenic survival. Once resistance was achieved, various redox enzyme protein expression and activity were measured to determine their role in resistance to P-AscH. Proliferation and cell cycle analysis were conducted on resistant and sensitive cells. An RNA sequencing screen was conducted as an unbiased approach to identify potential candidates crucial for resistance. Finally, phenotypes associated to increased metastatic potential, including collagen invasion and in vivo tumor colonization were explored for characterization. Results: PANC-1 and MIA PaCa-2 AscR cells developed unique mechanisms to tolerate high levels of P-AscH. PANC-1 AscR cells almost exclusively rely on catalase to detoxify H2O2 generated during P-AscH treatments, while MIA PaCa-2 AscR cells do not. It is interesting to note that not every redox enzyme was upregulated in response to P-AscH treatments, including GPx1/4. The AscR cell lines also differed in growth rates. PANC-1 AscR cells developed a faster proliferation rate, whereas MIA PaCa-2 AscR cells significantly decreased their growth. This is consistent with a shortened G1/G0 cell cycle phase and an accumulation in G2/M, respectively. The RNA sequencing screen revealed a metastatic signature in association with resistance to P-AscH, including upregulation of the PI3K/AKT axis. We observed a significant increase in collagen matrix invasion, as well as metastatic colonization in vivo. Additionally, we have observed increased expression of active AKT in both AscR cell lines, indicating a potential mechanism for the increased metastatic phenotype. Conclusion: Our findings demonstrate distinct mechanisms for AcsR cells to tolerate high doses of P-AscH. Although catalase has demonstrated to be a key factor in controlling redox flux, it may not be necessary for all cell types. Not only can P-AscH resistance alter redox enzyme expression, but we have also observed substantial increase in metastatic potential to drive invasion and tumor colonization. In summary, our data indicate a potential route for PC to tolerate high levels of P-AscH and may explain how some patients do not respond to this treatment regimen. Citation Format: Amanda N. Pope, Brianne R. O'Leary, Juan Du, Garry R. Buettner, Michael D. Henry, Joseph J. Cullen. Characterizing resistant and metastatic phenotypes to pharmacological ascorbate in pancreatic cancer [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 4677.

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

Abstract KRASG12C is a potent oncogenic driver which results in downstream hyperactivation of MAPK signaling and unchecked oncogenic growth, while simultaneously increasing replication stress (RS) and accumulation of DNA damage. KRASG12C-specific inhibitors are approved and/or recommended in metastatic non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), and while responses are robust, some patients are inherently resistant, and most responders acquire resistance. Resistance mechanisms include reactivation of MAPK signaling, and cancer cells which escape MAPKi are reported to have high levels of genomic instability. WEE1 is a kinase involved in cell cycle progression, allowing cells to repair damaged DNA before entering the next cell cycle phase. Consequently, inhibition of WEE1 leads to premature cell cycle entry and RS, thereby driving cancer cells through the cell cycle with unchecked accumulation of DNA damage, ultimately resulting in mitotic catastrophe and cell death. We hypothesized that high levels of genomic instability and RS in KRASG12C mutant and KRASG12C inhibitor-resistant tumors present a vulnerability to WEE1 inhibition and provide rationale for combining azenosertib, a novel, selective, and orally bioavailable WEE1 inhibitor, with KRASG12C inhibitors. Here, we report azenosertib in combination with KRASG12C inhibitors as an effective therapeutic strategy for KRASG12C tumors. In vitro combination of azenosertib with multiple KRASG12C inhibitors demonstrated synergistic cell growth inhibition across a panel of KRASG12C cell lines in both 2D and 3D assays. In vivo treatment with azenosertib in KRASG12C inhibitor-sensitive cell line-derived xenograft (CDX) models of NSCLC (NCI-H358, NCI-H2122, NCI-H1792), CRC (SW837, SW1463), and pancreatic cancer (MIA PaCa-2) demonstrated significant monotherapy activity as well as synergistic tumor growth inhibition (TGI) when combined with KRASG12C inhibitors, including tumor regression in some models. Importantly, the KRASG12C inhibitor-resistant SW1573 NSCLC CDX model demonstrated synergistic TGI in the combo arm (84%) when compared against monotherapy azenosertib (31%) or adagrasib (58%). Similarly, treatment of a KRASG12C inhibitor-resistant patient-derived xenograft (PDX) model of CRC resulted in synergistic TGI in the combination arm (61%) relative to monotherapy azenosertib (17%) or adagrasib (2%). Finally, analysis of biomarkers from both in vitro and in vivo tumor samples demonstrated synergistic increases in protein markers of RS, DNA damage, and apoptosis with combination therapy. Taken together, our results suggest that the combination of azenosertib with KRASG12C inhibitors enhances tumor growth inhibition over single agent therapy and may be an effective treatment option for patients with KRASG12C tumors. Citation Format: Nathan M. Jameson, Gabriel Kim, Catherine Lee, Blake Skrable, Alexandra Shea, Monah Abed, Olivier Harismendy, Jianhui Ma, Doris Kim, Mark R. Lackner. The selective WEE1 inhibitor azenosertib shows synergistic anti-tumor activity with KRASG12C inhibitors in multiple KRASG12C models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6487.

Abstract 7107: Clustered DNA double strand breaks for cancer treatment via multi-gRNA CRISPR system

Abstract Radiation therapy is one of the most common cancer treatments. Ionizing radiation generates DNA double-strand breaks (DSBs), which are the most lethal type of DNA damage. The dominant pathway for repairing DSBs is non-homologous end joining (NHEJ), but homologous recombination (HR) is also important. NHEJ is dominant pathway in all cell cycle phases while HR is largely restricted to S and G2 phases. Unrepaired or misrepaired DSBs can lead to cell death. KU70/80 and DNA-PKcs are essential for NHEJ. KU70/80 binds to broken ends, and DNA-PKcs binds to DNA-bound KU70/80, which activates DNA-PKs kinase allowing NHEJ to proceed. Clustered DSBs (two or more DSBs separated by <200 bp) generate small DNA fragments that are efficiently bound by KU70/80 and DNA-PKcs, but fail to activate DNA-PKcs kinase, inhibiting NHEJ and shifting repair toward HR. Thus, clustered DSBs are poorly repaired and more cytotoxic than dispersed DSBs. There are two types of ionization radiation: high LET and low LET. High LET radiation has a higher ionization density and therefore induces more clustered DSBs than low LET, which explains why high LET radiation is more cytotoxic at a given dose. A major limitation of radiotherapy is normal tissue damage. We hypothesize that mimicking high-LET-induced clustered DSBs using CRISPR/Cas9 targeted to cancer-specific DNA sequences (e.g., indel mutations) will effectively kill cancer cells but spare normal cells. In this approach, DSBs are not targeted to specific genes, nor is CRISPR/Cas9 used for gene editing. Instead, cancer cell killing is achieved by inducing poorly repaired DSBs anywhere in cancer cell DNA. Thus, this therapeutic approach is distinct from conventional targeted therapies that are oncogenic pathway-dependent, and this eliminates oncogenic pathway-dependent resistance mechanisms. However, types of resistance are possible, such as upregulated DSB repair. Our data indicate that the cytotoxicity of clustered DSBs depends on the number of small DNA fragments induced, thus we observe similar cytotoxicity with a localized, 4-DSB cluster and with three non-localized 2-DSB clusters, as each generates 3 small fragments per target chromosome. This finding significantly increases available targets as 2-DSB clusters can be induced at indels and flanking (wild-type) sites, generating clustered DSBs in cancer cells but efficiently repaired dispersed DSBs in normal cells lacking the cancer indel. Data also indicates that a single DSB targeted to each amplicon of highly amplified regions is also cytotoxic, and we are exploring the effectiveness of combination clustered DSBs targeted to amplified regions. We are also exploring how DSB repair inhibitors (like ATMi, DNA-PKcsi or RAD51i) can augment cytotoxic DSB effects, potentially mitigating that resistance pathway. These proof-of-principal studies represent the first approach to targeted cancer therapy that is oncogenic pathway-independent. Citation Format: Gamze Badakul, Neelam Sharma, Jac A. Nickoloff. Clustered DNA double strand breaks for cancer treatment via multi-gRNA CRISPR system [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 7107.

Abstract 956: NCX 4040 and napabucasin synergistically inhibits stemness in human prostate cancer (PCa) cellular models and potently targets transgenic murine PCa cells via inducing oxidative stress

Abstract Prostate cancer (PCa) continues to have a substantial impact on men’s wellbeing and quality of life regardless of recent progress in diagnosis and treatments and it continues to have the second highest occurrence rate following lung cancer. The strategy of combination drug therapy has gradually become a valuable choice for treating various cancer types, addressing the heterogeneity within tumors, and surpassing the constraints of existing treatment methods. In our previous work, when we combined two drugs NCX 4040 (an oxidative stress inducer) and napabucasin (a STAT3 signaling inhibitor), we discovered that the combination treatment synergistically works to inhibit the malignant characteristics of human PCa cells. Also, the combination upregulated the cGAS-STING immunogenic pathway in PCa cells which could potentially activate the cancer-targeting cytotoxic T-cells. In the current study, we aimed at investigating the anti-stemness properties of the drug duo which can not only inhibit cancer cell differentiation but also can be effective in preventing cancer relapse. We used two cell lines i.e., BPH-Cd which served as a model of cadmium induced PCa and DU145 as a model of advanced PCa to examine the anti-stemness properties of the drug combination. Based on western-blot analysis, we found the downregulation of transcription factors such as Oct4, Sox2, Nanog, β-catenin and cell surface markers like CD44, CD133 and ALDHA1, which are responsible for stem-cell like properties. In addition, we have tested the combinatory anticancer effects in transgenic mouse derived PCa cell line (Myc-Cap) as a prelude to conduct future studies in FVB mouse syngeneic model to evaluate the tumor suppressive and the antitumor immunogenic responses of combination treatment as it activated cGAS-STING pathway. By performing the cell viability assay, we found that the IC50 value of NCX 4040 is ~10µM and that of napabucasin is ~1μM, however when used in combination, ~90% inhibition is seen at 10µM of NCX and 250nM of napabucasin dual treatment. The drug combination also showed synergy in inhibiting tumorigenicity in clonogenic assay and migratory potential in wound-healing assay. By investigating the ROS production, cell death and cell cycle mechanism using flow cytometric analysis, it was observed that the combination therapy synergistically induces cellular oxidative stress which causes cancer cells to undergo late apoptosis and halts the growth of Myc-Cap cells in G2M phase of cell cycle. In conclusion, our data suggests that combination therapy of NCX 4040 and napabucasin can be potential treatment strategy for PCa warranting further evaluation in in vivo models. Citation Format: Shrushti Shah, Gnanasekar Munirathinam. NCX 4040 and napabucasin synergistically inhibits stemness in human prostate cancer (PCa) cellular models and potently targets transgenic murine PCa cells via inducing oxidative stress [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 956.

Abstract 1878: CTPS1: An unexplored vulnerability in breast and ovarian cancer

Abstract Breast and ovarian cancer represent the most common cancer types in women worldwide and are among the top fatal cancers. Despite the use of cytotoxic chemotherapy and targeted agents, recurrence is frequent and survival rates for patients with advanced metastatic disease are dismal. These realities highlight the need to identify novel biomarkers and therapeutic vulnerabilities in such tumors and to develop alternative treatment strategies. We took an unbiased approach to tackle this problem in which we leveraged publically available (DepMap) and in-house generated genome-wide CRISPR screens conducted in over 1000 cancer cell lines. We initially focused on the identification of essential genes that were unique to triple-negative breast cancer (TNBC) cells and whose expression levels were associated with worse patient outcomes. This interrogation revealed six genes (CTPS1, RHOA, PRKRA, RAD9A, HUS1, and RAD1) meeting these initial criteria. The essentiality of these genes was validated using gene-specific siRNAs in a panel of TNBC cells and knockdown of CTPS1 was shown to elicit the most inhibitory effect. CTPS1 depletion resulted in a rapid S-phase cell cycle arrest followed by apoptosis, not only in TNBC cells but also in highly aggressive ovarian cancer cells which share many molecular features with TNBC. CTPS1 converts uridine triphosphate to cytidine triphosphate, forming an essential nucleic acid required for multiple cellular processes. CTPS1 mRNA expression was substantially elevated in tumor vs. normal tissue, for both breast and ovarian cancer. Using over 40 breast and ovarian cancer cell lines, RT-PCR analyses confirmed increased expression of CTPS1 in TNBC and most ovarian cancer cell lines compared to other disease sub-types and “normal” cell line/tissue controls. Interestingly, chemoresistant and PARP inhibitor-resistant models exhibited the highest levels of CTPS1 among all cells analyzed. We subsequently identified and acquired STP938, a first-in-class highly selective CTPS1 inhibitor being developed by Step-Pharma. STP938 was found to elicit potent anti-cancer effects at nM concentrations across many cell line and patient-derived models, in both 2D and 3D culture systems. RNAseq analysis in aggressive cell line models following STP938 treatment or CTPS1 knockdown revealed significant regulation of genes related to DNA replication and the cell cycle pathways suggesting that combinatorial/sequential treatments of STP938 with replication stress/DNA damage response related drugs may be synergistic. STP938 is currently being developed for the treatment of lymphoma, and our findings support the repurposing of STP938 for breast and ovarian cancer, an avenue that we are currently developing. Citation Format: Xiyin Wang, Michael Emch, Lauren Voll, Rebecca Russell, Esther Rodman, Nicole Pearson, Xiaonan Hou, Scott Kaufmann, Saravut Weroha, Philip Beer, John Hawse. CTPS1: An unexplored vulnerability in breast and ovarian cancer [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 1878.

Abstract 4549: Combinations of ciclopirox olamine and metformin exert synergistic antitumor activity in triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancer (TNBC) represents a challenging subtype of breast cancer because of its limited treatment options. Recent research has highlighted the potential benefits of combining pharmacological agents to enhance therapeutic outcomes. Studies show that Ciclopirox olamine (CPX), an off-patent anti-fungal drug, induces cell cycle G1-phase arrest and activates the intrinsic apoptosis pathway by downregulating the anti-apoptotic proteins. Meanwhile, metformin, a safe and first-line drug for the treatment of type II diabetes, has been shown to induce cell cycle S-phase arrest and activates the extrinsic apoptosis pathway via induction of endogenous TRAIL in TNBC cells. However, whether the combinations of CPX and metformin would exert potent antitumor activity against TNBC remains unclear. Methods: Cell proliferation (MTS) assays and LIVE/DEAD cell imaging were used to determine the combined inhibitory effects on TNBC cells. IC50s and the combination index (CI) were calculated. Flow cytometry assays were carried out to analyze if the combinations are more potent to block cell cycle progression. Apoptotic-specific ELISA and flow cytometry analysis of Annexin V-PI staining were performed to assess if the combinations are significantly more effective to induce apoptosis. Western blot assays were used to examine if the combinations markedly induced the expression of TRAIL and cleavages of PARP and caspases. Tumor xenograft models will be established to test the in vivo antitumor activity of CPX and metformin either alone or in combinations. Results: Data indicated that both CPX and metformin independently inhibited the proliferation of TNBC cells. A synergistic effect was observed when CPX and metformin were used in combination, resulting in a more potent inhibition of TNBC cell proliferation. The combinatorial treatment effectively suppressed colony formation of TNBC cells. Synergy between CPX and metformin was partially linked to the enhanced activation of the AMPK-mTOR pathway, as the combination significantly enhanced the phosphorylation of AMPKα. The combinations also induced enhanced apoptosis in TNBC cells through both the intrinsic and extrinsic pathways. The combinatorial effects of CPX and metformin on in vivo tumor growth are under investigation. Conclusions: Combinations of CPX and metformin show synergistic effects on inhibiting TNBC cell proliferation. Our studies suggest a potential therapeutic strategy for TNBC, with the combinations of these agents exhibiting an enhanced antitumor activity. Further investigation is warranted to validate the results and explore their clinical applications. Key words: Triple-negative breast cancer, Ciclopirox olamine, Metformin, Combination, Apoptosis Citation Format: Yakun Wu, Hui Lyu, Congcong Tan, Margaret E. Larsen, Shile Huang, Bolin Liu. Combinations of ciclopirox olamine and metformin exert synergistic antitumor activity in triple-negative breast cancer [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 4549.

Abstract 6941: Transcriptomic effects of retinoblastoma progression-related somatic copy number alteration (SCNA)

Abstract Retinoblastoma is a pediatric retinal cancer that initiates in response to biallelic RB1 loss or MYCN amplification in a cone precursor cell of origin. After initial emergence, RB1-/- retinoblastomas progress to more aggressive forms by accumulation of single nucleotide variants (SNVs) and highly recurrent somatic copy number alterations including 1q+, 2p+, 6p+ and 16q- (RB SCNAs), resulting in heterogeneous mixtures of subclonal cell populations. SCNAs are enriched in highly evolved and aggressive tumor subtypes and show prognostic value in retinoblastoma liquid biopsies. However, the transcriptomic effects of retinoblastoma SCNAs are poorly understood. Single cell methods may enable comparison of tumor subclones within emerging retinoblastoma tumors and thereby provide insight into tumor progression. We retrieved single cell RNA sequencing data for 25 retinoblastoma tumors and inferred SCNAs and subclone phylogenies from scRNAseq information using a recently published tool, Numbat. We identified tumors with clonal variation in RB SCNAs and excluded samples with poor transcriptome quality or excessive additional SCNAs. We proceeded with analysis of 11 tumors with subclonal heterogeneity for an SCNA of interest, including 10 with 1q+, eight with 2p+, four with 6p+, and six with 16q-. After removal of nontumor cells, we defined cell clusters, cluster marker genes, and cell cycle phase gene signatures, and we compared cell state distributions for subclones with an acquired SCNA relative to their immediate antecedents. These analyses revealed recurrently discriminated transcriptomic clusters corresponding to six discrete cell cycle states (post-mitotic G1, G1, G1/S, S, S/G2, G2/M) and variable cell stress states, including a cluster with hypoxia and heat shock related marker gene expression in all samples. In each tumor, SCNA-defined tumor subclones had a distinct cell state distribution. Tumor subclones with acquired 1q+ were enriched in G2 states and depleted in G1 states. Subclones with 2p+ were enriched in G2 states. Subclones with 6p+ were enriched in S phase states. Subclones with 16q- were diminished in post-mitotic cell states. Tumor clones with 1q+ and 16q- were most strongly enriched for G2 phase cells. Such 1q+/16q- tumor cells were significantly differentially distributed in three tumor replicates. Tumor clones with other RB SCNAs were enriched over diploid clones in proliferating G2 cells without SCNA specific effects. To identify candidate drivers of SCNA-specific transcriptomic effects we measured RB SCNA clonal variation in gene expression within and across transcriptomic states. We identified recurrently differentially expressed in-segment genes in 1q+, 2p+, 6p+, and 16q-, as well as out-of-segment genes. Finally, we propose molecular mechanisms underlying SCNA recurrence that may enable tumor evolution in retinoblastoma. Citation Format: Kevin Stachelek, David E. Cobrinik. Transcriptomic effects of retinoblastoma progression-related somatic copy number alteration (SCNA) [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 6941.

Abstract 4558: A triterpenoid derivative has strong antitumor activities in gemcitabine-resistant advanced bladder cancer models and promotes immune cell infiltration in a high grade murine syngeneic model

Abstract Bladder cancer (BC) presents a formidable challenge in treatment and patient survival, requiring particular attention to advanced stages, such as muscle-invasive stage (MIBC), due to its potential for rapid progression and substantial impact on patient morbidity and mortality. The five-year survival rate for MIBC is a meager 30%, which drops further down to 8% once tumors metastasize. Advanced bladder cancers (High grade T1) are treated with intravesical therapy with Gemcitabine and Cisplatin (G+C) combination and Stage T2 are treated neoadjuvant with G+C and cystectomy, thereby decreasing the quality of life for patients with BC. Furthermore, tumors progress beyond responding to other chemotherapy regimens such as dose-dense treatment with Methotrexate +Vinblastine + Adriamycin and Cis-platin (MVAC), all of which will further decrease the quality of life with persistent adverse toxicity. Our laboratory has developed a novel compound derived from the pentacyclic triterpenoid, Ursolic acid, termed N-methyl piperazine conjugated-Ursolic acid, or UA4, and have demonstrated its antitumor activities against both Gem-Sensitive (Gem-S) and Gem-Resistant (Gem-R) human advanced BC cell lines, T24 and 5637. We tested the combination of Gem, UA4 and Gem + UA4 in a murine non-MIBC model (MB49) to investigate the role of immune cell infiltration during tumor development and treatment response. UA4 treatment was equally effective in chemo-sensitive and chemo-resistant cell lines at dosages between 4-5µM. Previous in vitro mechanistic studies on UA4 and UA4+Gem combination treatments revealed cell death occurred through apoptosis and autophagy, along with a highly selective form of mitochondrial-mediated autophagy (mitophagy). These findings were confirmed with transmission electron microscopy ultrastructural analysis, cell cycle phase analyses, excess, changes in mitochondrial membrane potential and reactive oxygen species measurements. Gem-S and Gem-R xenografts in athymic mice further supported UA4’s efficacy in vivo, demonstrating a significant reduction in both tumor weight and volume following oral gavage (100mg/kg), with mice exhibiting no signs of systemic toxicity. Combination treatment with UA4 and Gem synergistically increased antiproliferative activity. MB49 syngeneic mice model further supported UA4 as an effective therapy, and immunofluorescence assays with immune-related markers, suggested an anti-inflammatory effect in vivo. Overall, we demonstrate UA4 has a potential to be used as an efficacious, non-toxic compound for further adjuvant use in clinical settings. Citation Format: Juliette Rose Seremak, Kunj Bihari Gupta, Siva S. Panda, Bal L. Lokeshwar. A triterpenoid derivative has strong antitumor activities in gemcitabine-resistant advanced bladder cancer models and promotes immune cell infiltration in a high grade murine syngeneic model [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 4558.

Abstract 1912: Targeting Trop-2 with sacituzumab govitecan (IMMU-132) suppresses the tumor growth in diffuse large B cell lymphoma

Abstract Introduction: Sacituzumab govitecan (SG), also termed IMMU-132, is an antibody-drug conjugate, linking an anti-Trop-2 antibody and the topoisomerase I inhibitor, SN-38. Clinically, SG has shown promising therapeutic effects in patients with human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer. However, the efficacy of SG in diffuse large B-cell lymphoma (DLBCL) remains unexplored. In this study, we sought to determine whether SG can efficiently inhibit cell proliferation and tumor growth of DLBCL by using both in vitro and in vivo models. Methods: We examined the cytotoxicity of SG in the activated B-cell like (ABC)-subtype-DLBCL cell lines, Riva (Ri-1), HBL-1, and SU-DHL-2, and the germinal center B-cell (GCB)-subtype-DLBCL cell lines, SU-DHL-4, SU-DHL-5, and SU-DHL-6 by MTT assay. We assessed the distributions of cell cycle phase and apoptosis in the aforementioned DLBCL cell lines treated with SG for 24 h or 48 h using flow cytometry and Annexin V staining. We confirmed apoptosis (cleavage of poly ADP-ribose polymerase [PARP] and caspase-3) and detected DNA damage (γH2AX and 53BP1)-related protein expression in SU-DHL-2 and SU-DHL-6 cell lines treated with SG at various concentrations for 48 h using western blotting, compared to untreated cells. To examine the anti-tumor effect of SG in vivo, NOD-SCID nude mice of Riva-xenograft mouse models were randomized into a control group and an SG-treated group. Results: Flow cytometry and western blotting revealed overexpression of Trop-2 on the surface of untreated ABC-DLBCL and GCB-DLBCL cell lines. In this study, we showed that SG treatment reduced cell viability in all six DLBCL cell lines with nanomolar IC50 values. Exposing SG to DLBCL cells significantly increased cell cycle arrest at the G2/M and sub-G1 phases of all six DLBCL cell lines. Annexin V staining revealed that SG induced apoptosis in all six DLBCL cell lines. Western blotting confirmed apoptosis induction by showing a dose-dependent upregulation of cleaved PARP and cleaved caspases-3 by SG treatment, along with decreased expression of survivin in both SU-DHL-2 and SU-DHL-6 cells. SG also upregulated the expression of γH2AX and 53BP1 in both SU-DHL-2 and SU-DHL-6 cells. Furthermore, we found that tumor growth of Riva cells in vivo was significantly reduced by treatment with SG when compared with untreated control. Conclusion: SG suppresses tumor growth of ABC-DLBCL and GCB-DLBCL cells in both in vitro and in vivo models by inducing cell cycle arrest, apoptosis, and DNA damage. These findings provide a rationale for transitioning SG to clinical trials for treating patients with relapsed or refractory DLBCL. Citation Format: Jakie Ting, Ming-Feng Wei, Hsiao-Wei Lee, Keng-Hsueh Lan, Ann-Lii Cheng, Sung-Hsin Kuo. Targeting Trop-2 with sacituzumab govitecan (IMMU-132) suppresses the tumor growth in diffuse large B cell lymphoma [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 1912.

Abstract 704: Targeting hypoxic survival pathways to overcome radiotherapy-related treatment resistance

Abstract Tumor cells undergo biological adaptations in order to survive stress conditions, such as hypoxia and nutrient deprivation. These biological adaptations include the activation of hypoxic survival pathways that play an important role in acquiring radioresistance which becomes most challenging with treatment regimens using high doses of radiation/fraction (e.g., hypofractionated radiotherapy). Therefore, inhibiting key regulators within these pathways and thereby exploiting these survival mechanisms, gives rise to new potential drug targets for combined treatment modalities. This novel strategy investigated in our laboratory relies on the concept of biological cooperation: while ionizing radiation kills primarily well-oxygenated cells, such inhibitors of survival kinases will drive quiescent tumor cells under hypoxia and/or nutrient deprivation either directly into cell death or into re-entry into a proliferative and thereby more ionizing radiation-sensitive state. One of the survival kinases we are investigating in combination with ionizing radiation (IR) is DYRK1B (dual-specificity tyrosine regulated kinase 1B). We demonstrated that the expression of DYRK1B was upregulated under serum-starvation and hypoxia, but not in response to IR. The small molecule DYRK1B inhibitor AZ191 and shRNA-mediated DYRK1B knockdown significantly reduced proliferative activity and clonogenicity of SW620 tumor cells alone and in combination with IR under serum-starved conditions, which correlated with increased ROS levels and DNA damage. Furthermore, AZ191 successfully targeted the hypoxic core of tumor spheroids while IR preferentially targeted normoxic cells in the rim of the spheroids. A combined treatment effect was also observed in patient-derived colorectal carcinoma organoids but not in healthy tissue organoids. This data shows that inhibition of DYRK1B in quiescent tumor cells could drive tumor cells on their own into crisis or into a more radiosensitive cell cycle phase and thus supports our strategy of biological cooperation. In the continuation of this project, we have performed a drug-screen with approx. 3’000 clinically relevant compounds in colorectal tumor cells lines under normoxic and hypoxic conditions in order to explore additional hypoxia-mediated survival pathways contributing to radioresistance. Thereby we aim to identify novel combined treatment modalities to overcome radiotherapy-induced hypoxia and hypoxia-related resistance mechanisms. Citation Format: Claire Beckers, Lazaros Vasilikos, Alba Sanchez-Fernandez, Lorena Moor, Martin Pruschy. Targeting hypoxic survival pathways to overcome radiotherapy-related treatment resistance [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 704.

Abstract 5906: A new combinatorial therapy that synergistically targets the NuRD complex and proteostasis to eradicate quiescent ovarian cancer cells

Abstract Despite advances in immune and targeted therapies, chemotherapy remains the primary treatment for ovarian cancer (OvCa) patients. Unfortunately, most OvCa patients develop chemotherapy-resistant disease that is inevitably fatal. Quiescence is an important yet poorly understood mechanism underlying chemotherapy resistance. Therefore, we aimed to characterize quiescent ovarian cancer (qOvCa) cells to identify novel therapeutic targets to improve OvCa patient outcomes. Primary patient-derived qOvCa cells and proliferating cells were isolated using a single-cell microfluidics culture device and collected for single-cell RNA-seq. scRNA-seq of qOvCa cells identified (i) downregulation of MBD3 and CHD4, which are components of the Nucleosome Remodeling and Deacetylase (NuRD) complex, and (ii) upregulation of proteasome-associated genes. Knockdown of either MBD3 or CHD4 by shRNA, or NuRD complex pharmacologic inhibition with the HDAC inhibitors such as Vorinostat resulted in significant cell growth arrest without cell death. Standard and Fucci reporter-based cell cycle analysis further confirmed that inhibition of the NuRD complex with Vorinostat induced 94.47% cells in the G0 phase (p<0.0001), suggesting a dense quiescent state. Through cell cycle phase maps produced by iterative immunofluorescence of 30 core cell cycle regulators and manifold learning, we found Vorinostat-treated OvCa cells demonstrated a completely different cell cycle structure from control cells, with most of the cells in an extreme G0 phase. Bulk RNA-seq of Vorinostat-treated cells in three OvCa cell lines validated the downregulation of pathways relating to cell cycle, cell division, and DNA replication (LFC<-1, p<0.05), which is consistent with stem cells. Upregulation of several pathways involved in proteostasis were also noted. We therefore performed a targeted drug screen for combinatorial therapy approaches to eradicate qOvCa. We found that Vorinostat combined with the proteasome inhibitor Carfilzomib demonstrated the most profound synergistic cell death (Loewe index synergy score 23-73). Increased proteasome activity was also found in both primary and Vorinostat-induced qOvCa, suggesting the essential role of the proteasome in qOvCa. Tumor xenograft studies confirmed that Vorinostat alone can delay tumor growth (p<0.05) and combinatorial therapy of Vorinostat and Carfilzomib is more efficacious (p=0.0049). Taken together, NuRD complex inhibition induces quiescence. Combination therapy with Vorinostat to induce quiescence combined with proteasome inhibitors resulted in the profound death of therapy-resistant quiescent cells. Our work suggests this is a novel therapeutic approach to eradicate chemoresistant qOvCa cells and increase cure rates. Citation Format: Qi Jiang, Michelle Ertel, Santiago Panesso- Gómez, Sara Sannino, April Sagan, Alexander J. Cole, Stacy C. McGonigal, Betsy Ann Varghese, Wayne Stallaert, Jeffrey L. Brodsky, Hatice U. Osmanbeyoglu, Ronald J. Buckanovich. A new combinatorial therapy that synergistically targets the NuRD complex and proteostasis to eradicate quiescent ovarian cancer cells [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 5906.

Dynamic Protein Phosphorylation in Streptococcus pyogenes during Growth, Stationary Phase, and Starvation.

Phosphorylation of proteins at serine, threonine, and tyrosine residues plays an important role in physiological processes of bacteria, such as cell cycle, metabolism, virulence, dormancy, and stationary phase functions. Little is known about the targets and dynamics of protein phosphorylation in Streptococcus pyogenes, which possesses a single known transmembrane serine/threonine kinase belonging to the class of PASTA kinases. A proteomics and phosphoproteomics workflow was performed with S. pyogenes serotype M49 under different growth conditions, stationary phase, and starvation. The quantitative analysis of dynamic phosphorylation, which included a subset of 463 out of 815 identified phosphorylation sites, revealed two main types of phosphorylation events. A small group of phosphorylation events occurred almost exclusively at threonine residues of proteins related to the cell cycle and was enhanced in growing cells. The majority of phosphorylation events occurred during stationary phase or starvation, preferentially at serine residues. PASTA kinase-dependent cell cycle regulation processes found in related bacteria are conserved in S. pyogenes. Increased protein phosphorylation during the stationary phase has also been described for some other bacteria, and could therefore be a general feature in the physiology of bacteria, whose functions and the kinases involved need to be elucidated in further analyses.

A fast-acting lipid checkpoint in G1 prevents mitotic defects

Lipid synthesis increases during the cell cycle to ensure sufficient membrane mass, but how insufficient synthesis restricts cell-cycle entry is not understood. Here, we identify a lipid checkpoint in G1 phase of the mammalian cell cycle by using live single-cell imaging, lipidome, and transcriptome analysis of a non-transformed cell. We show that synthesis of fatty acids in G1 not only increases lipid mass but extensively shifts the lipid composition to unsaturated phospholipids and neutral lipids. Strikingly, acute lowering of lipid synthesis rapidly activates the PERK/ATF4 endoplasmic reticulum (ER) stress pathway that blocks cell-cycle entry by increasing p21 levels, decreasing Cyclin D levels, and suppressing Retinoblastoma protein phosphorylation. Together, our study identifies a rapid anticipatory ER lipid checkpoint in G1 that prevents cells from starting the cell cycle as long as lipid synthesis is low, thereby preventing mitotic defects, which are triggered by low lipid synthesis much later in mitosis.

Flow cytometry study of DNA transformation dynamics in ВНК-21/SUSP/ARRIAH cell culture during rabies virus reproduction

The study examines the DNA transformation dynamics of ВНК-21/SUSP/ARRIAH subline cells during rabies virus reproduction. Cells infected with the virus and control intact cells were cultivated under similar conditions. The identification of dependence of the virus infectivity on reproduction time revealed that the virus infectivity titre increased from (3.2 ± 0.2) lg CCID50/cm3 at the time of inoculation to (7.63 ± 0.3) lg CCID50/cm3 after 48 hours of reproduction, with the most intensive increase having been observed within the first 24 hours. The cell concentration changed from 0.5 to 1.9 million/cm3, i.e. increased by a factor of 3.8. After 24 hours, the cell growth rate slowed down. Findings from the examination of cell cycle phases during rabies virus reproduction in the host cell allowed for the estimation of duration and predominance of G1, S, G2 + M phases at different stages of cultivation. The dynamics of changes in the apoptotic cell population in the control and test samples was similar within 36 hours of cultivation. After the said period, the proportion of apoptotic infected cells was 28–42% higher than that of apoptotic control cells. After 9 hours, the proportion of cells undergoing G1 phase increased by 11.7% in the test samples, whereas it decreased by 16.6% in the control samples. Subsequently, the number of G1 phase cells in the control and test samples changed in the same way: a 40% decrease was observed after 15–18 hours, it was followed by a 45–46% growth jump, then again a 39–40% decrease and an increase were observed. After 33 hours of reproduction and till the end of cultivation, the proportion of infected cells undergoing G1 phase was significantly higher (by 12–21%) as compared with control cells. The percentage of S phase cells in the test and control samples was the same during the first day of the virus reproduction, with sharp jump-like 3.4- and 2.4-fold increases having been observed after 15 and 24 hours, respectively. After 24 hours, the infected and control cells began to demonstrate differences, which gradually increased from 8 to 137% by the end of reproduction. After 30 hours of reproduction, the proportion of test sample cells undergoing G2 + M phase began to decrease by 17–28% as compared with the control cells. The cell switch-over to the synthesis of complete rabies virus particles occurred after 24 hours of reproduction. This is indicated by changes in the host cell cycle phases, as well as by the slowing down of ВНК-21/SUSP/ARRIAH cell population growth.

The pancancer overexpressed NFYCAntisense 1 controls cell cycle mitotic progression through in cis and in trans modes of action

Antisense RNAs (asRNAs) represent an underappreciated yet crucial layer of gene expression regulation. Generally thought to modulate their sense genes in cis through sequence complementarity or their act of transcription, asRNAs can also regulate different molecular targets in trans, in the nucleus or in the cytoplasm. Here, we performed an in-depth molecular characterization of NFYCAntisense1 (NFYC-AS1), the asRNA transcribed head-to-head to NFYC subunit of the proliferation-associated NF-Y transcription factor. Our results show that NFYC-AS1 is a prevalently nuclear asRNA peaking early in the cell cycle. Comparative genomics suggests a narrow phylogenetic distribution, with a probable origin in the common ancestor of mammalian lineages. NFYC-AS1 is overexpressed pancancer, preferentially in association with RB1 mutations. Knockdown of NFYC-AS1 by antisense oligonucleotides impairs cell growth in lung squamous cell carcinoma and small cell lung cancer cells, a phenotype recapitulated by CRISPR/Cas9-deletion of its transcription start site. Surprisingly, expression of the sense gene is affected only when endogenous transcription of NFYC-AS1 is manipulated. This suggests that regulation of cell proliferation is at least in part independent of the in cis transcription-mediated effect on NFYC and is possibly exerted by RNA-dependent in trans effects converging on the regulation of G2/M cell cycle phase genes. Accordingly, NFYC-AS1-depleted cells are stuck in mitosis, indicating defects in mitotic progression. Overall, NFYC-AS1 emerged as a cell cycle-regulating asRNA with dual action, holding therapeutic potential in different cancer types, including the very aggressive RB1-mutated tumors.

Lectin: A Molecular Tool in Cancer Diagnosis and Therapy with Special Reference to Reproductive Cancers.

The prevalence of cancer deaths globally and domestically is higher especially due to the deferment of diagnosis and lack of facilities for women's reproductive cancers. The present review focussed to explore the application of lectins in cancer theranostics. Though there is cancer diagnostic and treatment available there is no promising early diagnostic tool and effective treatment available for the cancer which is the major concern. Lectins are cellulose-binding proteins that are strongly determined in saccharide groups of glycans, glycopeptides, or glycolipids. In the concomitance of events in cells, carbohydrates, and proteins, lectins play an important role. Lectins bind superiorly to the cancer cell membrane and their receptors induce the cytotoxic effect, which results in caspase-mediated cell death, and prohibits tumour development. Lectin snuffing also reveals polyamine stocks and impedes the growth of cancerous cells. They affect the cell cycle by non-apoptotic aggregation, seizure of the cell cycle phase G2, M, and the mediation of caspases. It can also adversely affect the action of telomerase and hinder vascularisation. They promote immunomodulation and adversely limit protein synthesis. Their easy availability and its characteristics support its use in cancer diagnosis and therapy, despite their small corollary effects. Future investigations recommend focussing more on the key applications of lectin by reducing its concurrent effects and carrying out more in-vitro investigations. However, the use of lectin formulations for cancer theranostics is a new area in cancer detection and treatment. In this review, plant lectin appears to be a potential target for cancer research in the fields of diagnosis and theranostics.

CDK-independent role of D-type cyclins in regulating DNA mismatch repair

Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins inhibit the proteasomal degradation of p21, which competes with MMR proteins for binding to PCNA, thereby inhibiting MMR. The ability of D-type cyclins to limit MMR is CDK4- and CDK6-independent and is conserved in G0 and G1. At the G1/S transition, the timely, cullin-RING ubiquitin ligase (CRL)-dependent degradation of D-type cyclins and p21 enables MMR activity to efficiently repair DNA replication errors. Persistent expression of D-type cyclins during S-phase inhibits the binding of MMR proteins to PCNA, increases the mutational burden, and promotes microsatellite instability.

Synthesis of Novel Nilotinib Analogues and Biological Evaluation of Their Antiplatelet Activity and Functionality towards Cancer Cell Proliferation In Vitro.

Nilotinib, a second-generation tyrosine kinase inhibitor for the treatment of chronic myelogenous leukemia (CML), inhibits Bcr-Abl tyrosine kinase activity and proliferation of Bcr-Abl-expressing cells, as well as other malignancies. In the present study, new nilotinib analogues were synthesized and fully characterized. A platelet aggregation assay was performed, and the expression of P-selectin and PAC-1, as well as the effect on the proliferation of healthy endothelial cells, were evaluated. The expression and antimetastatic effects of E-cadherin and N-cadherin were assessed. The analogues inhibited platelet aggregation in a statistically significant manner compared to nilotinib, while they exhibited a strong inhibitory effect on P-selectin and PAC-1 expression when activated by AA. All three analogues caused arrest in the mitosis phase of the HepG2 cell cycle, while analogue-1 exhibited the most potent apoptotic effect compared to nilotinib. Interestingly, none of them promoted apoptosis in HUVECs. All the analogues reduced the expression of E- and N-cadherin in different amounts, while the analogues-1 and -3 exhibited similar antimigratory effects on HepG2 cells. The results of this study reveal considerable potential to develop new tyrosine kinase inhibitors with improved antiplatelet and antitumor properties.

In-cell NMR suggests that DNA i-motif levels are strongly depleted in living human cells

I-Motifs (iM) are non-canonical DNA structures potentially forming in the accessible, single-stranded, cytosine-rich genomic regions with regulatory roles. Chromatin, protein interactions, and intracellular properties seem to govern iM formation at sites with i-motif formation propensity (iMFPS) in human cells, yet their specific contributions remain unclear. Using in-cell NMR with oligonucleotide iMFPS models, we monitor iM-associated structural equilibria in asynchronous and cell cycle-synchronized HeLa cells at 37 °C. Our findings show that iMFPS displaying pHT < 7 under reference in vitro conditions occur predominantly in unfolded states in cells, while those with pHT > 7 appear as a mix of folded and unfolded states depending on the cell cycle phase. Comparing these results with previous data obtained using an iM-specific antibody (iMab) reveals that cell cycle-dependent iM formation has a dual origin, and iM formation concerns only a tiny fraction (possibly 1%) of genomic sites with iM formation propensity. We propose a comprehensive model aligning observations from iMab and in-cell NMR and enabling the identification of iMFPS capable of adopting iM structures under physiological conditions in living human cells. Our results suggest that many iMFPS may have biological roles linked to their unfolded states.