Proliferation Of Tumor Cell Lines Research Articles

Abstract 3318: GFH375 (VS-7375): An oral, selective KRAS G12D (ON/OFF) inhibitor with potent anti-tumor efficacy

Abstract Background: KRAS is the best-known oncogene but had long been considered ‘undruggable’ until the approval of the first KRAS G12C inhibitor sotorasib in 2021. However, most current clinical-stage KRAS inhibitors target the KRAS G12C mutation. Inhibitors of other KRAS mutants such as KRAS G12D, the most frequent KRAS mutation in human cancer, are needed for patients. We have developed GFH375 (VS-7375), an oral, selective KRAS G12D inhibitor targeting both “ON” (GTP-bound) and “OFF” (GDP-bound) states of KRAS proteins. The in vitro potency, selectivity, and in vivo efficacy of monotherapy and combination therapy with avutometinib, a unique RAF/MEK clamp, were evaluated in preclinical studies. Methods: Biochemical and cellular assays were used to investigate inhibition to KRAS cycling and signaling. CellTiter-Glo assay was performed to determine the effects on proliferation of tumor cell lines. Several KRAS G12D CDX tumor models were employed to study the in vivo pharmacodynamic and anti-tumor effects. Results: GFH375 inhibited both nucleotide exchange on GDP-bound KRAS G12D and interaction between GMPPNP-bound KRAS G12D with RAF1 with single-digit nanomolar IC50 values. GFH375 suppressed phosho-ERK1/2 (p-ERK) level with sub-nanomolar IC50 values and potently inhibited cell proliferation across a panel of KRAS G12D tumor cell lines. GFH375 showed high selectivity for KRAS G12D relative to non-G12D KRAS variants, KRAS wild type cells, and NRAS, HRAS, or BRAF mutated cells. Following a single oral dose, GFH375 produced deep and durable inhibition of p-ERK in KRAS G12D CDX tumors. GFH375 demonstrated dose-dependent anti-tumor activity with tumor regressions at 10 or 30 mg/kg given orally twice daily in multiple KRAS G12D PDAC and CRC CDX tumor models. GFH375 also showed strong anti-tumor efficacy in an intracranial KRAS G12D tumor model starting as low as 10 mg/kg orally twice daily. Strong synergy between GFH375 and avutometinib was observed in vitro and was validated in vivo as anti-tumor activity of GFH375 was further enhanced by avutometinib. Conclusions: GFH375 is a highly potent and selective orally active inhibitor of KRAS G12D (ON/OFF) and demonstrated promising anti-tumor activity in multiple KRAS G12D tumor models. GFH375 was also effective in an intracranial tumor model. GFH375 showed strong synergy with avutometinib in vitro and in vivo. GFH375 is currently in IND-enabling development in preparation for clinical studies of monotherapy and potentially in combination with other agents for patients with KRAS G12D mutant tumors. Citation Format: Feng Yan, Tao Jiang, Tao Liang, Lijian Cai, Leitao Zhang, Xiaoming Xu, Yanhui Zhao, Xiaoling Lan, Xiaohui Zhang, Meng Liu, Qiang Liu, Jinting Gao, Fubo Xie, Xueyan Gao, Li Wang, Jingyang Zhang, Hongcan Ren, Dong Liu, Siyuan Le, Lili Tang, Silvia Coma, Yaofeng Cheng, Nathan Sanburn, Jonathan A. Pachter, Fusheng Zhou, Jiong Lan, Qiang Lu. GFH375 (VS-7375): An oral, selective KRAS G12D (ON/OFF) inhibitor with potent anti-tumor efficacy [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 3318.

Synthesis and Antiproliferative Activity Evaluation of B-norcholesterol-6- amide Compounds.

The structure modification of steroids is commonly used to change the biological activity of steroids in medicinal chemistry. In recent years, it has been found that some derivatives derived from the structural modification of cholesterol display good inhibitory activity against tumor cell proliferation in vitro</i>. Using cholesterol as the starting material, different types of B-norcholesterol-6-amide derivatives were synthesized by the reaction of 6-carboxyl-B-norcholesterol with different alkyl amines or 6-amino-B-norcholesterol with different acyl chlorides. The inhibitory activity of compounds on the proliferation of tumor cell lines was investigated by the MTT method. The results showed that the B-norcholesterol-6-amide compounds displayed distinct cytotoxicity against Sk-Ov-3 cells but caused no obvious damage against HEK-293T cells. Additionally, the steroidal amide derivatives formed from 6-amino-B-norcholesterol showed stronger cytotoxicity than those produced from 6-carboxyl-B-norcholesterol. Specially, compounds with chloroalkyl structure displayed significant inhibitory activity against all tumor cells tested. Among them, compounds 19-21 showed cytotoxicity like 2-methoxyestradiol as a positive control, and the IC</i>₅₀ value of compound 20 on HeLa cells was 3.9 μM. After introducing chloroalkyl acyl groups into 6-position of 6-amino-B-norcholesterol, the cytotoxicity of resulting B-norcholesterol-6-amide compounds can be greatly enhanced.

Inhibition of Notch Signaling Enhances Antitumor Activity of Histone Deacetylase Inhibitor LAQ824.

As a novel histone deacetylase inhibitor (HDACi), LAQ824 (LAQ) effectively inhibits the proliferation of hematological malignancies and solid tumors. However, phase II trials of LAQ in solid tumors were terminated due to dose-dependent toxicity. Furthermore, LAQ has been shown to induce the activation of the Notch signaling pathway in hematopoietic stem cells, which is associated with tumor progression and drug resistance in colon and breast cancers. Therefore, in this study, we investigated the strategy of LAQ combined with a Notch signaling pathway inhibitor to treat solid tumors. We used RT-PCR and Western blot methods to demonstrate that LAQ upregulated the Notch signaling pathway in solid tumor cell lines at the molecular level. The combination of LAQ and a Notch signaling pathway inhibitor was shown by a Chou-Talalay assay to have a synergistic effect in inhibiting solid tumor cell line proliferation in vitro. We also demonstrated that the combination of LAQ and a Notch signaling pathway inhibitor significantly inhibited the growth of tumor cells in vivo using an allograft tumor model. This study indicates that inhibition of the Notch signaling pathway provides a valuable strategy for enhancing solid tumor sensitivity to LAQ.

Abstract 6321: HMPL-453, a highly selective inhibitor of fibroblast growth factor receptors 1, 2, and 3, displays potent activity in FGFR-altered tumor models

Abstract Background: Fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate numerous cellular processes. Dysregulation of FGFR signaling due to receptor fusion, mutation or amplification is observed across multiple cancer types, making activated FGFRs an important therapeutic target. Herein, we present the preclinical characterization of HMPL-453, a highly potent and selective inhibitor of FGFR1, 2, and 3, discovered and being currently developed in phase II clinical trial (NCT04353375) by HUTCHMED. Methods: Kinase activity was measured by Transcreener™ Fluorescence Polarization assay or Z’-LYTE kinase assay. In vitro anti-proliferation activity was measured by CellTiter-Glo luminescent or CCK-8 assay. The effect of HMPL-453 on FGFR signaling pathway was detected by western blot. Multiple tumor models with FGFR alteration were applied in Nu/Nu nude mice to determine anti-tumor efficacy of 453 as a single agent. A model in immune-competent BALB/c mice inoculated with the constructed NIH/3T3 cells carrying FGFR2-AHCYL1 fusion was chosen to investigate the combination effect of HMPL-453 with anti-PD-1 antibody. Results: HMPL-453 potently inhibited the tyrosine kinase activities of recombinant FGFR 1, 2, and 3 in vitro (IC50 values of 6, 4, and 6 nM, respectively) with weaker activity against FGFR4 (IC50 = 425 nM). HMPL-453 selectively inhibited proliferation of tumor cell lines with dysregulated FGFR signaling (GI50s: 3~105 nM) compared with cell lines lacking FGFR aberrations (GI50s &amp;gt; 1.5 µM). HMPL-453 demonstrated strong inhibition of phosphorylation of FGFR and downstream protein in tumor cell lines harboring FGFR2 fusion. Oral administration of HMPL-453 could induce time- and dose-dependent inhibition of phosphorylation of FGFR and resulted in remarkable and dose-dependent anti-tumor activity in multiple FGFR-altered tumor models. HMPL-453 at the dose of 50 mg/kg/day could induce tumor regression in most tumor models tested. Moreover, HMPL-453 significantly improved anti-tumor activity of anti-PD-1 antibody in a FGFR2 fusion model by priming the immune environment. Conclusion: HMPL-453 is a highly potent and selective inhibitor of FGFR 1, 2, and 3 with strong activity against FGFR-deregulated tumors in preclinical models, supporting continued investigation in patients with FGFR alterations (such as fusion and mutation) either as a single agent or in combination with PD-1 blockade. Citation Format: Jia Hu, Jun Ni, Longxian Jiao, Jinghong Zhou, Shiming Fan, Renxiang Tang, Wei Zhang, Xuelei Ge, Qihang Zhang, Juntao Yu, Ying Yu, Dongxia Shi, Min Cheng, Weifang Xue, Sumei Xia, Zeyu Zhong, Jian Wang, Yang Sai, Weiguo Qing, Guangxiu Dai, Yongxin Ren, Michael Shi, Weiguo Su. HMPL-453, a highly selective inhibitor of fibroblast growth factor receptors 1, 2, and 3, displays potent activity in FGFR-altered tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6321.

Abstract 517: Targeting chromosomally unstable tumors with a selective KIF18A inhibitor

Abstract Chromosome instability (CIN) is an attribute of cancer cells that has yet to be fully exploited therapeutically, despite its prevalence across many tumor types and lack of occurrence in normal cells. To identify genes uniquely essential to CINhigh cells, we mined the Cancer Dependency map (DepMap) for genes required for proliferation of tumor cell lines with high levels of copy number aberrations, an indicator of CIN. This analysis identified KIF18A, a mitotic kinesin also identified by others as a vulnerability of aneuploid and whole genome doubled cells. Genetic disruption of KIF18A with RNAi confirmed its necessity in CINhigh cells, and these findings translated to in vivo reduction in tumor growth, illustrating the potential of KIF18A as a therapeutic target. High-throughput screening of a chemical library for inhibitors of KIF18A motor domain’s ATPase activity identified an ATP-noncompetitive hit that was optimized for potency, selectivity, and pharmacokinetic properties. These optimized inhibitors are orally bioavailable, exhibit sub-nM potency, and are highly selective for KIF18A vs. other kinesins. They also display a long drug-target residence time, a unique property that allows cells to be trapped in mitosis long enough to result in cell death. Mechanistically, inhibition of KIF18A’s ATPase activity renders KIF18A unable to localize to the metaphase plate, instead becoming trapped at the spindle poles. This inability to translocate results in defects in chromosome congression, evidenced by a less compact metaphase plate, and an accumulation of cells in G2/M. Treatment of CIN cancer cells with these inhibitors results in a proliferative defect due in part to induction of programmed cell death. In contrast to the effect in cancer cells, KIF18A inhibition has no effect on rapidly proliferating non-transformed epithelial cells and primary T cells. The differential impact between normal and cancer cells sets KIF18A inhibitors apart from other anti-mitotic therapies, which have had limited clinical benefit due to their detrimental impact on both cancer and highly proliferative normal cells. The therapeutic potential of KIF18A inhibitors was assessed in human tumor xenograft models. Treatment of multiple tumor models resulted in substantial, dose-dependent inhibition of tumor growth. KIF18A inhibition in the tumor was confirmed through evaluation of KIF18A localization changes, chromosome congression and an increase in mitotic index. The strong biological rationale, robust preclinical data, and superior compound properties warrant clinical development of Volastra’s KIF18A inhibitor which is planned for development for the treatment of chromosomally unstable cancers. Citation Format: Aaron F. Phillips, Rumin Zhang, Akanksha Verma, Tamar Feinberg, Mia Jaffe, Marysol Chu Carty, Ryan Schulz, Sarah Bettigole, Celia Andreu, Scott Drutman, Michael Su, Derek Cogan, Christina H. Eng. Targeting chromosomally unstable tumors with a selective KIF18A inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 517.

Fruit Extract of Sechium chinantlense (Lira & F. Chiang) Induces Apoptosis in the Human Cervical Cancer HeLa Cell Line.

Sechium edule (Cucurbitaceae) is a commercial species of chayote and is just one of several species in the genus Sechium, whose extracts inhibit proliferation in tumor cell lines. The capacity of the wild species Sechium chinantlense (SCH) as an antitumor agent is unknown, as is the mechanism of action. In the present study, HeLa cervical cancer and HaCaT normal cell lines were treated with SCH and cell proliferation was inhibited in both cell lines in a dose-dependent manner similar to the effect of the antineoplastic agent cisplatin (Cis). Additionally, SCH arrested cell cycle progression but only in HeLa cells and induced apoptosis, as shown by phosphatidylserine translocation and caspase-3 activation, while Cis did so in both cell lines. Exploration of the mechanism of action of SCH in HeLa cells suggests that apoptosis was mediated by the intrinsic signaling pathway since there was no activation of caspase-8, but there was a release of cytochrome-c. These findings suggest that the SCH extract has the potential to selectively kill tumor cells by promoting apoptosis, without harming nontumor cells.

Investigational cyclin-dependent kinase 4/6 inhibitor GLR2007 demonstrates activity against isocitrate dehydrogenase wild-type glioblastoma and other solid tumors in mice xenograft models.

Cyclin-dependent kinases, CDK4 and CDK6, are essential in regulating the cell cycle, which is disrupted in cancers like isocitrate dehydrogenase wild-type glioblastoma (GBM). Currently marketed CDK4/6 inhibitors, including abemaciclib, have shown preclinical efficacy in solid tumors, but factors such as poor blood–brain barrier (BBB) penetration limit their efficacy in GBM. GLR2007 is an investigational CDK4/6 inhibitor with the potential for improved BBB penetration. In vitro assays were used to assess the potency and inhibition of CDK4/6 enzymatic activity of GLR2007. Using in vivo assays, the distribution of radiolabeled GLR2007 in rats was determined through quantitative whole-body autoradiography. The antitumor efficacy of GLR2007 was evaluated in human GBM and breast cancer orthotopic mice xenograft models, and human lung, colorectal, and liver cancer in a subcutaneous xenograft model. In tumor cell line proliferation assays, GLR2007 inhibited proliferation at lower concentration values than abemaciclib in 19 of 20 GBM, five of seven breast, 20 of 21 lung, and 24 of 24 liver cancer cell lines. Total levels of radiolabeled GLR2007 in the brains of rats exceeded those in plasma by 2.3–4.5-fold from 2–6 hours after dosing. A xenograft model showed that, compared with vehicle control, 50 mg/kg GLR2007 induced 95.9% tumor growth inhibition (TGI) (P<0.001) in GBM orthotopic xenografts, 81.4% TGI (P=0.037) in breast cancer orthotopic xenografts, and 91.5% TGI (P<0.001) in colorectal cancer subcutaneous xenografts. These studies show possible BBB penetration of GLR2007 and demonstrate its potential as a CDK4/6 inhibitor for the treatment of solid tumors, including GBM.

Abstract 2591: Combination of the BCL-2 inhibitor ZN-d5 with the WEE1 inhibitor ZN-c3 shows additive or synergistic anti-tumor activity in acute myeloid leukemia (AML) models

Abstract Background: The Bcl-2 inhibitor venetoclax is approved in combination with hypomethylating agents such as azacitidine for the treatment of newly diagnosed elderly patients with AML; however, most patients eventually relapse, especially those with TP53 mutations who have poor prognosis. Wee1 is a crucial cell cycle checkpoint kinase that regulates the G2/M checkpoint in response to DNA damage, and its inhibition can cause mitotic catastrophe and apoptosis. ZN-d5 and ZN-c3 are highly selective and potent inhibitors of Bcl-2 and Wee1, respectively. We show here that the combination is highly active in preclinical models of AML. Methods: Cell proliferation in tumor cell lines and AML patient’s samples were measured using CellTiter-Glo® (Promega). Relevant markers were measured by Western blotting. Anti-tumor efficacy was determined in cell line derived subcutaneous xenograft models of acute myeloid leukemia (AML) and in an AML patient-derived systemic xenograft (PDX) model. Flow cytometry was used for analysis of the PDX model to measure human AML blast populations. Results: The in vitro combination of ZN-d5 and ZN-c3 was tested in AML cell lines and showed synergistic or additive anti-proliferative activity which was independent of BCL-2 sensitivity in some cases. In addition, ZN-d5 in combination with ZN-c3 had a synergistic anti-tumor activity in vivo in cell line-derived AML models, such as MV4-11 and HL-60. Also, the triple combination of ZN-d5 + ZN-c3 + azacitidine in the HL-60 in vivo model resulted in synergistic anti-tumor efficacy and significant tumor growth inhibition compared to single- and double-agent treatments. The ZN-d5 + ZN-c3 combination was also tested in vitro in 29 AML patient-derived samples. Response to ZN-c3 alone was observed in 23/29 samples (IC50 &amp;lt; 450 nM), independent of TP53 mutation status. Response to ZN-d5 alone was observed in 17/29 samples (IC50 &amp;lt;200 nM). The combination resulted in synergy being observed in 12/29 models and had greater anti-proliferative activity than either single agent alone in all samples including those insensitive to Bcl-2 inhibition (IC50 &amp;gt; 10 μM). Combination of ZN-d5 and ZN-c3 was also highly active in an AML patient-derived PDX model, resulting in a 99.5% decrease in human CD45+ blast population in the bone marrow. Finally, the combination of ZN-d5 and ZN-c3 was active in 3/3 ex vivo experiments of patients derived AML samples who had progressed on venetoclax. Conclusions: These results justify testing the ZN-d5 and ZN-c3 combination or possibly the triple combination including azacitidine in AML patients independently of TP53 status. Due to synergism observed with these combination(s) and activity in samples from patients resistant to venetoclax, activity may be seen in patients with low sensitivity to Bcl-2 inhibitors or even in patients who progressed on venetoclax plus azacitidine. Citation Format: Hooman Izadi, Tiffany Hoang, Noah Ibrahim, Joseph Pinchman, Brant C. Boren, Jianhui Ma, Petrus R. de Jong, Jiali Li, Kevin D. Bunker, Ahmed A. Samatar, Fernando Doñate. Combination of the BCL-2 inhibitor ZN-d5 with the WEE1 inhibitor ZN-c3 shows additive or synergistic anti-tumor activity in acute myeloid leukemia (AML) models [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 2591.

Abstract 2605: BH3 mimetics synergize with the Wee1 inhibitor ZN-c3 by activating caspases which induce DNA damage and degrade key proteins

Abstract Background: Wee1 is a crucial cell cycle checkpoint kinase that regulates the G2/M checkpoint in response to DNA damage, and its inhibition can cause mitotic catastrophe and apoptosis in tumor cells. Inhibitors of BCL-2 have been shown to also induce apoptosis. ZN-d5 and ZN-c3, currently in clinical development for the treatment of cancer, are highly selective and potent inhibitors of Bcl-2 and Wee1, respectively. The combination of ZN-d5 with ZN-c3 induced robust tumor regressions in preclinical models and the mechanism of action of this activity is presented. Methods: Cell proliferation in tumor cell lines was measured using CellTiter-Glo® (Promega). Cleaved caspase-3, cleaved PARP, phosphorylated CDK1 (p-CDK1), γH2AX, Wee1, RRM2 and other markers were measured by Western blotting, IHC and/or flow cytometry. Anti-tumor efficacy was determined in several xenograft models. Results: The combination of ZN-c3 and ZN-d5 resulted in additive or synergistic anti-proliferative activity in several tumor cell lines. This enhancement of anti-proliferative activity was also observed at concentrations of ZN-d5 that induced caspase activation, but minimal apoptosis. Treatment of tumor cell lines with ZN-d5 alone at these sublethal concentrations caused induction of γH2AX, a marker of double-stranded DNA breaks, and degradation of caspase-sensitive proteins relevant to the DNA Damage Response (DDR), such as RRM2 (a ribonucleotide reductase subunit) and Wee1. Degradation of Wee1 correlated with decreases in p-CDK1, a marker of Wee1 inhibition. These effects were mainly reversible and were inhibited by pre-treatment with caspase inhibitors. Mechanistically, the combination of ZN-c3 and ZN-d5 resulted in increased levels of γH2AX, enhanced inhibition of p-CDK1, and increased levels of apoptotic markers compared to single agent treatment. Similar results were obtained when ZN-c3 was combined with inhibitors of other BCL-2 family proteins such as inhibitors of MCL-1 and BCL-xL. Finally, the combination of ZN-c3 with ZN-d5 or other BH3 mimetics showed synergistic anti-tumor activity in several xenograft models further supporting the rationale for combining these agents. Conclusions: ZN-d5 or other BH3 mimetics caused caspase-mediated DNA damage and degraded relevant proteins such as Wee1 and RRM2 resulting in additive or synergistic anti-tumor activity when combined with ZN-c3. These results support a novel use of BH3 mimetics to activate caspase activity, independently of apoptosis induction, to significantly enhance the activity of DDR inhibitors such as ZN-c3 Citation Format: Hooman Izadi, Noah Ibrahim, Tiffany Hoang, Jianhui Ma, Petrus R. de Jong, Joseph Pinchman, Kevin D. Bunker, Ahmed A. Samatar, Fernando Doñate. BH3 mimetics synergize with the Wee1 inhibitor ZN-c3 by activating caspases which induce DNA damage and degrade key proteins [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 2605.

Abstract 2478: Elevating SOX2 inhibits the proliferation of tumor cells independently of DNA binding

Abstract Purpose: The purpose of these studies was to understand how elevated SOX2 affects tumor cell proliferation. The stem cell transcription factor SOX2 has been implicated in ~20 different tumor types. Importantly, several studies have demonstrated that the effects of SOX2 during development and malignancy are dependent on its dosage. High levels of SOX2 in both contexts are associated with a quiescent, stem-like phenotype. SOX2high quiescent tumor cells present a significant clinical challenge as these cells are largely resistant to conventional chemotherapies and, thus, provide a reservoir to drive disease recurrence. Therefore, understanding the mechanisms through which elevated SOX2 inhibits tumor cell proliferation will provide insights into the biology of drug-resistant, quiescent tumor cells. Methods: Tumor cell lines representative of medulloblastoma (MB) and colorectal (CRC) tumor cells were engineered for the doxycycline (Dox)-inducible expression of mutant forms of SOX2 that are unable to bind to DNA. One SOX2 mutant was created by substituting a proline for a glycine in the DNA binding domain of SOX2 [SOX2(G76P)]. A second SOX2 mutant replaces the DNA binding domain of SOX2 with the DNA binding domain of the yeast transcription factor Gal4 (Gal4-SOX2). The effects of elevating mutant forms of SOX2 on cell proliferation and the cell cycle distribution of the cells were compared to the effects of elevating wild-type SOX2. Results: Our studies demonstrate that elevating SOX2, SOX2(G76P) or Gal4-SOX2 decreases the proliferation of MB and CRC cells to similar extents. In contrast, inducible expression of the DNA binding domain of Gal4 had no effect on cell proliferation. We previously reported that while SOX2 elevation inhibits the proliferation of a diverse set of tumor cell lines, it does not significantly alter their cell cycle distribution. Similarly, elevating SOX2(G76P) or Gal4-SOX2 did not significantly alter cell cycle distribution. To extend these findings, we are engineering additional SOX2 mutants to map the location of the domain of SOX2 required for growth inhibition. We recently engineered MB and CRC cells to express a mutant form of SOX2 that lacks the c-terminal R1 transactivation domain [Gal4-SOX2(ΔR1)]. Significantly, removing the R1 transactivation domain eliminated the growth inhibitory effects of Gal4-SOX2 when elevated. Conclusion: Our findings demonstrate that elevating two mutant forms of SOX2 that cannot bind to DNA inhibit the proliferation of tumor cell lines that represent two different forms of cancer, similar to the effects of wild-type SOX2. Equally important, our studies indicate that the c-terminal R1 transactivation domain of SOX2 is necessary, but no sufficient, to inhibit proliferation when SOX2 levels are elevated. Collectively, these findings and our previously published work indicate that elevated levels of SOX2 inhibit cell proliferation independently of DNA binding. Citation Format: Ethan Patrick Metz, Phillip J. Wilder, Angie Rizzino. Elevating SOX2 inhibits the proliferation of tumor cells independently of DNA binding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2478.

SARS-CoV-2 Host Receptor ACE2 Protein Expression Atlas in Human Gastrointestinal Tract.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through interactions with its receptor, Angiotensin-converting enzyme 2 (ACE2), causing severe acute respiratory syndrome and death in a considerable proportion of people. Patients infected with SARS-CoV-2 experience digestive symptoms. However, the precise protein expression atlas of ACE2 in the gastrointestinal tract remains unclear. In this study, we aimed to explore the ACE2 protein expression pattern and the underlying function of ACE2 in the gastrointestinal tract, including the colon, stomach, liver, and pancreas.MethodsWe measured the protein expression of ACE2 in the gastrointestinal tract using immunohistochemical (IHC) staining with an ACE2-specific antibody of paraffin-embedded colon, stomach, liver, and pancreatic tissues. The correlation between the protein expression of ACE2 and the prognosis of patients with gastrointestinal cancers was analyzed by the log-rank (Mantel–Cox) test. The influence of ACE2 on colon, stomach, liver, and pancreatic tumor cell line proliferation was tested using a Cell Counting Kit 8 (CCK-8) assay.ResultsACE2 presented heterogeneous expression patterns in the gastrointestinal tract, and it showed a punctate distribution in hepatic cells. Compared to that in parallel adjacent non-tumor tissues, the protein expression of ACE2 was significantly increased in colon cancer, stomach cancer, and pancreatic cancer tissues but dramatically decreased in liver cancer tissues. However, the expression level of the ACE2 protein was not correlated with the survival of patients with gastrointestinal cancers. Consistently, ACE2 did not affect the proliferation of gastrointestinal cancer cells in vitro.ConclusionThe ACE2 protein is widely expressed in the gastrointestinal tract, and its expression is significantly altered in gastrointestinal tumor tissues. ACE2 is not an independent prognostic marker of gastrointestinal cancers.

Detection of Biological Activity of Curcuma longa Methanol Extract and Its Antibacterial effect on Staphylococcus aureus

Abstract Plants are known to possess enormous profound benefits, which if well incorporated in daily living have the potential to influence our health in ways unexpected. Significant progress has been made concerning the anti-oxidative and anti-cancerous effect of bioactive components in food products, worthy of note in the curcuminoids derived from turmeric (Curcuma longa) rhizomes. Therefore, to this end, this research aims to describe succinctly the phytocomponents, antioxidant activity and anti-microbial activity of turmeric methanol extract. The zones of inhibition were measured after plating on a Mueller Hinton agar plate and compared to the standards penicillin G and chloramphenicol. Chloramphenicol used as a positive control was found to be most effective on Staphylococcus aureus while Curcuma longa methanol extract and penicillin G were found to have the same microbial inhibition due to the negative control. Compared to the IC50 values of Ascorbic acid which was 5 mg/ ml, Curcuma longa methanol extract had a lower IC50 value is 3.2 mg/ml which according to previous literature is a good one given that lower IC50 values indicate great antioxidant activity. It was gathered by the observation that the qualitative extraction of Curcuma longa using methanol shows the presence of varying chemical components such as AR-tumerone, arachinsaeure, alpha-tumerone amongst others. Research data suggest that ar-turmerone has the potential to promote the positive and negative proliferation of tumor cell lines. As a result, Curcuma longa has high potential to be developed into an antibiotic against Methicillin-resistant Staphylococcus aureus and other clinically important bacterial strains in future.

Identification of differentially expressed miRNAs in differentiating benign from malignant pleural effusion

BackgroundTuberculosis pleural effusion (TPE) and malignant pleural effusion (MPE) are very common clinical complications. Considering the totally different prognosis and clinical treatment of TPE and MPE, the accurate and non-invasive diagnosis are very critical for patients with pleural effusion to initiate efficient management and treatment. However, effective clinical biomarkers were rarely explored to distinguish benign from MPE. The purpose of this study is to identify potential miRNAs which can probably be used to differentiate malignant pleural effusion from TPE.ResultsA total of 23 significantly differentially expressed miRNAs were identified in MPE, with 18 up-expressed and 5 down-expressed. And the target genes of the miRNAs mainly involved in the biology process of nervous system, cancer, immune system and metabolic process etc. Three high confident target genes, AGO4, FGF9 and LEF1 can be regulated by miR-195-5p, miR-182-5p and miR-34a-5p respectively. And these genes participate in the canonical pathway of regulation of the Epithelial-Mesenchymal and the biological functions of apoptosis, growth of tumor and cell proliferation of tumor cell lines. Further, RT-PCR validation results based on 64 collected individuals showed that the expression levels of the three miRNAs were 2–5 times higher in MPE samples, which were consistent with the microarray results. In addition, ROC curve analysis demonstrated that the combination of the three miRNAs can achieve higher AUC of 0.93 (p-value< 0.0001) to differentiate MPE from TPE.ConclusionsThe identified miR-195-5p, miR-182-5p and miR-34a-5p can become potential diagnostic biomarkers for MPE with further evidences.

Targeting the EGF-Receptor and CD38 in Solid and Haematological Malignancies with Nanobody-Based Heavy Chain Antibodies and AAV Vectors

Abstract Background Although monoclonal antibodies binding the EGF-receptor (cetuximab, panitumumab) or CD38 (daratumumab) show therapeutic efficacy, many cancer patients still develop resistance. Therefore, new therapies are necessary. Nanobodies are highly soluble immunoglobulin domains derived from heavy chain antibodies (hcAbs) that naturally occur in camelids. Replication defective AAV (adeno associated virus) vectors, when equipped with a tumor targeting ligand, can be used for the delivery of (suicide) genes to tumor cells. Methods We generated recombinant chimeric nanobody-based human IgG1 hcAbs, by fusing EGFR- or CD38-specific nanobodies derived from immunized llamas to the hinge, CH2 and CH3 domains of human IgG1 (1, 2). To promote cytotoxic effector functions we introduced CDC (complement dependent cytotoxicity)-enhancing and ADCC (antibody-dependent cell-mediated cytotoxicity)-enhancing mutations into the CH2 and CH3 domains of human IgG1. As a second strategy to target CD38-overexpressing cells, we inserted CD38-specific nanobodies into the VP1 capsid protein of AAV2 (3). We analyzed the capacity of these constructs to target EGFR and CD38 in tumor cell lines and multiple myeloma (MM) patient bone marrow samples using proliferation, CDC, ADCC and AAV-transduction assays. Results EGFR-specific hcAbs effectively blocked the proliferation of tumor cell lines (HNSCC, head and neck squamous cell carcinoma and mCRC, metastatic colorectal cancer) expressing WT EGFR and cetuximab/panitumumab-escape variants of EGFR. EGFR- and CD38-specific hcAbs mediated effective ADCC towards tumor cell lines (HNSCC, mCRC and MM respectively). HcAbs carrying the hexabody mutation were more potent at inducing CDC than parental hcAbs. Display of CD38-specific nanobodies on the AAV capsid resulted in a 20- to 100-fold enhanced transduction of myeloma cells in patient bone marrow samples. Some of these results have recently been published (1, 2) or accepted for publication (3). We will present updated data on tumor cell lines and patient samples. Conclusion Nanobody-based hcAbs and nanobody-displaying AAV hold promise as novel tools to target solid and hematological tumors. Legal entity responsible for the study Friedrich Koch-Nolte. Funding DFG (German Research Foundation) and SFB (Collaborative Research Centres). Disclosure The author has declared no conflicts of interest.

GPR1 Promotes Breast Cancer Tumorigenesis and Metastasis via PI3K/Akt Pathway

Background: Obesity has been shown to be highly correlated with breast cancer risk and therapy resistance. However, the underlying mechanism has yet been undetermined. The adipokine chemerin was shown to be associated with the prognosis of breast cancer and downregulated in patients. A survey of public whole genome expression datasets found that the receptor GPR1 is upregulated in breast cancers as well as in other human tumors. Moreover, high chemerin expression in tumors was correlated with adverse outcomes in breast cancer. Methods: GPR1 expression was ablated in human breast cancer cell line (MDA-MB-231) by shRNA. Tumor cell line proliferation, migration, colony formation, cell cycle and apoptosis was monitored in MDA-MB-231 cell line, and then the xenograft beared on nude mice was measured. Findings: Here, we report that disruption of GPR1, through shRNA in breast cancer cell MDA-MB-231, significantly inhibited tumor cell growth and invasion in vitro and tumor xenograft in vivo. This inhibition was involved in the suppression of phosphorylation of the PI3K/Akt signaling pathway. Interpretation: These results show that GPR1, as the receptor of adipokine chemerin, can promote the tumor growth and invasion through AKT/PI3K signaling, suggesting that obesity-derived chemerin may have a role in tumor genesis and invasion. Funding Statement: This work was partially supported by National Key Research and Development Program of China (2018YFC1003703), the National Major Basic Research Program of China (2013CB945503 ), NSFC grant (81830041, 81830041, 31671562), China Postdoctoral Science Foundation Funded Project (2018M640843 ), Guangdong grant (2018A030310058), and Shenzhen grant (JCYJ20170815090309586, JCYJ20170412140326739, JCYJ20170817143406822). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: All procedures related to animal were carried out according to the guidelines of the Committee on the Use of Live Animals for Teaching and Research, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.

Oncogenic Activity of Wrap53 in Human Colorectal Cancer In Vitro and in Nude Mouse Xenografts.

BackgroundWD40-encoding RNA antisense to p53 (Wrap53) has been implicated in cancer development. However, the role of Wrap53 remains unknown in colorectal cancer. The aim of this study was to elucidate the function of Wrap53 in colorectal cancer tumorigenesis and development.Material/MethodsThis study analyzed Wrap53 expression in colorectal cancer tissue specimens using The Cancer Genome Atlas data and tumor cell lines and assessed the effects of Wrap53 knockdown on regulation of cancer cell malignant phenotypes in vitro and in nude mouse xenografts.ResultsWrap53 expression was upregulated in colorectal cancer tissue specimens and cell lines. Knockdown of Wrap53 expression induced colorectal cancer cell line apoptosis and cell cycle arrest in the G1 phase, but reduced tumor cell line proliferation and invasion in vitro. Knockdown of Wrap53 in a nude mouse xenograft assay inhibited tumor cell line xenograft formation and growth.ConclusionsWrap53 is likely a potential oncogene or possesses oncogenic activity in colorectal cancer, promoting colorectal tumorigenesis. Targeting Wrap53 expression may represent a novel strategy for the control of colorectal cancer.

Synthesis and Evaluation of the Tumor Cell Growth Inhibitory Potential of New Putative HSP90 Inhibitors.

Background: Heat shock protein 90 (HSP90) is a well-known target for cancer therapy. In a previous work, some of us have reported a series of 3-aryl-naphtho[2,3-d]isoxazole-4,9-diones as inhibitors of HSP90. Methods: In the present work, various compounds with new chromenopyridinone and thiochromenopyridinone scaffolds were synthesized as potential HSP90 inhibitors. Their binding affinity to HSP90 was studied in vitro. Selected compounds (5 and 8) were further studied in various tumor cell lines regarding their potential to cause cell growth inhibition, alter the cell cycle profile, inhibit proliferation, and induce apoptosis. Their effect on HSP90 client protein levels was also confirmed in two cell lines. Finally, the antitumor activity of compound 8 was studied in A431 squamous cell carcinoma xenografts in nude mice. Results: Our results indicated that treatment with compounds 5 and 8 decreased the proliferation of tumor cell lines and compound 8 induced apoptosis. In addition, these two compounds were able to downregulate selected proteins known as “clients” of HSP90. Finally, treatment of xenografted mice with compound 5 resulted in a considerable dose-dependent inhibition of tumor growth. Conclusions: Our results show that two new compounds with a chromenopyridinone and thiochromenopyridinone scaffold are promising putative HSP90 inhibitors causing tumor cell growth inhibition.

5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (PQR309), a Potent, Brain-Penetrant, Orally Bioavailable, Pan-Class I PI3K/mTOR Inhibitor as Clinical Candidate in Oncology.

Phosphoinositide 3-kinase (PI3K) is deregulated in a wide variety of human tumors and triggers activation of protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR). Here we describe the preclinical characterization of compound 1 (PQR309, bimiralisib), a potent 4,6-dimorpholino-1,3,5-triazine-based pan-class I PI3K inhibitor, which targets mTOR kinase in a balanced fashion at higher concentrations. No off-target interactions were detected for 1 in a wide panel of protein kinase, enzyme, and receptor ligand assays. Moreover, 1 did not bind tubulin, which was observed for the structurally related 4 (BKM120, buparlisib). Compound 1 is orally available, crosses the blood-brain barrier, and displayed favorable pharmacokinetic parameters in mice, rats, and dogs. Compound 1 demonstrated efficiency in inhibiting proliferation in tumor cell lines and a rat xenograft model. This, together with the compound's safety profile, identifies 1 as a clinical candidate with a broad application range in oncology, including treatment of brain tumors or CNS metastasis. Compound 1 is currently in phase II clinical trials for advanced solid tumors and refractory lymphoma.

Specific gene expression signatures induced by the multiple oncogenic alterations that occur within the PTEN/PI3K/AKT pathway in lung cancer

Hyperactivation of the phosphatydil-inositol-3' phosphate kinase (PI3K)/AKT pathway is observed in most NSCLCs, promoting proliferation, migration, invasion and resistance to therapy. AKT can be activated through several mechanisms that include loss of the negative regulator PTEN, activating mutations of the catalytic subunit of PI3K (PIK3CA) and/or mutations of AKT1 itself. However, number and identity of downstream targets of activated PI3K/AKT pathway are poorly defined. To identify the genes that are targets of constitutive PI3K/AKT signalling in lung cancer cells, we performed a comparative transcriptomic analysis of human lung epithelial cells (BEAS-2B) expressing active mutant AKT1 (AKT1-E17K), active mutant PIK3CA (PIK3CA-E545K) or that are silenced for PTEN. We found that, altogether, aberrant PI3K/AKT signalling in lung epithelial cells regulated the expression of 1,960/20,436 genes (9%), though only 30 differentially expressed genes (DEGs) (15 up-regulated, 12 down-regulated and 3 discordant) out of 20,436 that were common among BEAS-AKT1-E17K, BEAS-PIK3CA-E545K and BEAS-shPTEN cells (0.1%). Conversely, DEGs specific for mutant AKT1 were 133 (85 up-regulated; 48 down-regulated), DEGs specific for mutant PIK3CA were 502 (280 up-regulated; 222 down-regulated) and DEGs specific for PTEN loss were 1549 (799 up-regulated, 750 down-regulated). The results obtained from array analysis were confirmed by quantitative RT-PCR on selected up- and down-regulated genes (n = 10). Treatment of BEAS-C cells and the corresponding derivatives with pharmacological inhibitors of AKT (MK2206) or PI3K (LY294002) further validated the significance of our findings. Moreover, mRNA expression of selected DEGs (SGK1, IGFBP3, PEG10, GDF15, PTGES, S100P, respectively) correlated with the activation status of the PI3K/AKT pathway assessed by S473 phosphorylation in NSCLC cell lines (n = 6). Finally, we made use of Ingenuity Pathway Analysis (IPA) to investigate the relevant BioFunctions enriched by the costitutive activation of AKT1-, PI3K- or PTEN-dependent signalling in lung epithelial cells. Expectedly, the analysis of the DEGs common to all three alterations highlighted a group of BioFunctions that included Cell Proliferation of tumor cell lines (14 DEGs), Invasion of cells (10 DEGs) and Migration of tumour cell lines (10 DEGs), with a common core of 5 genes (ATF3, CDKN1A, GDF15, HBEGF and LCN2) that likely represent downstream effectors of the pro-oncogenic activities of PI3K/AKT signalling. Conversely, IPA analysis of exclusive DEGs led to the identification of different downstream effectors that are modulated by mutant AKT1 (TGFBR2, CTSZ, EMP1), mutant PIK3CA (CCND2, CDK2, IGFBP2, TRIB1) and PTEN loss (ASNS, FHL2).These findings not only shed light on the molecular mechanisms that are activated by aberrant signalling through the PI3K/AKT pathway in lung epithelial cells, but also contribute to the identification of previously unrecognised molecules whose regulation takes part in the development of lung cancer.

The Antiproliferative Effect of Cyclodipeptides from Pseudomonas aeruginosa PAO1 on HeLa Cells Involves Inhibition of Phosphorylation of Akt and S6k Kinases.

Pseudomonas aeruginosa PAO1, a potential pathogen of plants and animals, produces the cyclodipeptides cyclo(l-Pro-l-Tyr), cyclo(l-Pro-l-Phe), and cyclo(l-Pro-l-Val) (PAO1-CDPs), whose effects have been implicated in inhibition of human tumor cell line proliferation. Our purpose was to investigate in depth in the mechanisms of HeLa cell proliferation inhibition by the PAO1-CDPs. The results indicate that PAO1-CDPs, both purified individually and in mixtures, inhibited HeLa cell proliferation by arresting the cell cycle at the G0–G1 transition. The crude PAO1-CDPs mixture promoted cell death in HeLa cells in a dose-dependent manner, showing efficacy similar to that of isolated PAO1-CDPs (LD50 of 60–250 µM) and inducing apoptosis with EC50 between 0.6 and 3.0 µM. Moreover, PAO1-CDPs showed a higher proapoptotic activity (~103–105 fold) than their synthetic analogs did. Subsequently, the PAO1-CDPs affected mitochondrial membrane potential and induced apoptosis by caspase-9-dependent pathway. The mechanism of inhibition of cells proliferation in HeLa cells involves inhibition of phosphorylation of both Akt-S473 and S6k-T389 protein kinases, showing a cyclic behavior of their expression and phosphorylation in a time and concentration-dependent fashion. Taken together our findings indicate that PI3K–Akt–mTOR–S6k signaling pathway blockage is involved in the antiproliferative effect of the PAO1-CDPs.