Multiple Human Cancer Cell Lines Research Articles

Abstract 1161: Primary and metastatic tumors exhibit systems-level differences in dependence on mitochondrial respiratory function

Abstract The Warburg effect, aerobic glycolysis, is a hallmark feature of cancer cells grown in culture. However, the relative roles of glycolysis and respiratory metabolism in supporting in vivo tumor growth and processes such as tumor dissemination and metastatic growth remain poorly understood, particularly on a systems level. Using a CRISPRi mini-library enriched for mitochondrial ribosomal protein and respiratory chain genes in multiple human lung cancer cell lines we analyzed in vivo metabolic requirements in xenograft tumors grown in distinct anatomic contexts. While knockdown of mitochondrial ribosomal protein and respiratory chain genes (mito-respiratory genes) has little impact on growth in vitro, tumor cells depend heavily on these genes when grown in vivo as either flank or primary orthotopic lung tumor xenografts. In contrast, respiratory function is comparatively dispensable for metastatic tumor growth. RNA-Seq and metabolomics analysis of tumor cells expressing individual sgRNAs against mito-respiratory genes indicate overexpression of glycolytic genes and increased sensitivity of glycolytic inhibition compared to control when grown in vitro, but when grown in vivo as primary tumors these cells downregulate glycolytic mechanisms. Metabolic shifts associated with silencing mito-respiratory genes can be detected in vivo with metabolic imaging. These studies demonstrate that discrete perturbations of mitochondrial respiratory chain function impact in vivo tumor growth in a context-specific manner with differential impacts on primary and metastatic tumors. Citation Format: Neal K. Bennett, Hiroki J. Nakaoka, Danny Laurent, Ross Okimoto, Yoshitaka Sei, Andrew E. Horvai, Trever G. Bivona, Georgios Batsios, Pavithra Viswanath, Johanna ten Hoeve, Thomas G. Graeber, Ken Nakamura, Jean L. Nakamura. Primary and metastatic tumors exhibit systems-level differences in dependence on mitochondrial respiratory function [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 1161.

Abstract P3-11-06: Chromatin landscape analysis based on ATAC-seq and RNA- seq reveals that GRHL2 is a novel key transcription factor for endocrine therapy resistance

Abstract Current data using gene expression and DNA modification profiles yield limited information for evaluating the potential resistance to therapies. Recent studies show that the chromatin landscape is dynamic and defines cell identity and disease status. Thus, changes in open chromatin sites could provide important information on disease progression and identify potential therapeutic targets. The majority of breast cancers express estrogen receptor (ER) and initially respond to endocrine therapies blocking ER activity. However, endocrine therapy resistance (ETR) occurs de novo or follows an initial response. Recent studies have identified new drivers of ETR, such as mTOR and CDK4/6. Therapy with these inhibitors in combination with endocrine therapy have improved patient survival, but the molecular mechanism of ETR is not fully understood and additional therapeutic options for ETR are needed. To investigate the molecular mechanisms of ETR, we established in vitro Long-Term Estrogen Deprivation (LTED) model using multiple human ER-positive breast cancer cell lines. These cells showed differential time-dependent patterns of ESR1 expression upon LTED with ESR1 upregulation in some cell lines and downregulation in others. LTEDs with ESR1 upregulation were sensitive to fulvestrant, while the others were resistant. We analyzed time-course of chromatin landscape transitions during acquisition of ETR in ESR1 up- and downregulated LTEDs cells by Assay for Transposase Accessible Chromatin (ATAC-seq) combined with RNA-seq. Because transcription factors play a primary key role in gene expression and in cell-fate changes including response to therapies, we performed bioinformatic analysis to uncover differential open chromatin sites, select enhancer signatures and identify digital footprints of transcription factors. Unlike ChIP-seq, which identifies binding sites for a known transcription factor, ATAC-seq permits analysis of all sites potentially accessible to the transcriptional machinery and predict occupancy by ~700 different transcription factors at once. With these algorithms, we found that Grainyhead-like 2 (GRHL2) is associated with development of both ESR1 overexpressed and downregulated types of LTEDs. Furthermore, RNA-seq analysis showed that GRHL2 target genes are upregulated in LTEDs. Furthermore, in the METABRIC database, high GRHL2 expression is significantly associated with poor outcome in ER+ breast cancer patients treated with endocrine therapy. Taken together, these data suggest that GRHL2 may be a novel important transcription factor in endocrine therapy resistant breast cancer regardless of fulvestrant sensitivity. Our studies provide evidence that chromatin landscape analysis, coupled with the transcription factor network algorithm, is capable of identifying novel biomarkers or therapeutic targets, This approach will expand the range of translational research as it is applicable to many cancers and diseases. Citation Format: Saori Fujiwara, Songjoon Baek, Kaustubh Wagh, Diana Stavreva, Lyuba Varticovski, Gordon Hager. Chromatin landscape analysis based on ATAC-seq and RNA- seq reveals that GRHL2 is a novel key transcription factor for endocrine therapy resistance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-06.

Inhibition of Autophagy Increases Cell Death in HeLa Cells through Usnic Acid Isolated from Lichens.

The Western Ghats, India, is a hotspot for lichen diversity. However, the pharmacological importance of lichen-associated metabolites remains untapped. This study aimed to evaluate the cytotoxic potential of lichens of this region. For this, sixteen macrolichens were collected and identified from two locations in the Western Ghats. The acetone extract of Usnea cornuta (UC2A) showed significant cytotoxicity towards multiple human cancer cell lines. Interestingly, co-treatment with chloroquine (CQ), an autophagy inhibitor, increased the cytotoxic potential of the UC2A extract. A gas chromatography mass spectrometry (GCMS) study revealed usnic acid (UA), atraric acid and barbatic acid as the dominant cytotoxic compounds in the UC2A extract. Further, UA was purified and identified from the UC2A extract and evaluated for cytotoxicity in HeLa cells. The monodansyl cadaverine and mitotracker red double staining revealed the autophagy-inducing activities of UA, and the inhibition of autophagy was confirmed via CQ treatment. Autophagy inhibition increased the cytotoxicity of UA by 12-16% in a concentration-dependent manner. It also increased lipid peroxidation, ROS levels and mitochondrial depolarization and decreased glutathione availability. A decrease in zeta potential and a 40% increase in caspase 3/7 activity were also noted after CQ treatment of UA-treated cells. Thus, cytotoxicity of UA can be increased by inhibiting autophagy.

Design, synthesis and biological evaluation of 2-((4-sulfamoylphenyl)amino)-pyrrolo[2,3-d]pyrimidine derivatives as CDK inhibitors

To explore the potential use of CDK inhibitors in pancreatic ductal adenocarcinoma (PDAC) therapy, a series of novel 2-((4-sulfamoylphenyl)amino)-pyrrolo[2,3-d]pyrimidine derivatives was designed, synthesised, and investigated for inhibition on both CDK kinase activity and cellular proliferation of pancreatic cancer. Most of new sulphonamide-containing derivatives demonstrated strong inhibitory activity on CDK9 and obvious anti-proliferative activity in cell culture. Moreover, two new compounds suppressed cell proliferation of multiple human pancreatic cancer cell lines. The most potent compound 2g inhibited cancer cell proliferation by blocking Rb phosphorylation and induced apoptosis via downregulation of CDK9 downstream proteins Mcl-1 and c-Myc in MIA PaCa-2 cells. CDK9 knockdown experiment suggests its anti-proliferative activity is mainly mediated by CDK9. Additionally, 2g displayed moderate tumour inhibition effect in AsPC-1 derived xenograft mice model. Altogether, this study provided a new start for further optimisation to develop potential CDK inhibitor candidates for PDAC treatment by alone or combination use.

Transcriptional activity mediated by β-CATENIN and TCF/LEF family members is completely dispensable for survival and propagation of multiple human colorectal cancer cell lines

Unrestrained transcriptional activity of β-CATENIN and its binding partner TCF7L2 frequently underlies colorectal tumor initiation and is considered an obligatory oncogenic driver throughout intestinal carcinogenesis. Yet, the TCF7L2 gene carries inactivating mutations in about 10% of colorectal tumors and is non-essential in colorectal cancer (CRC) cell lines. To determine whether CRC cells acquire TCF7L2-independence through cancer-specific compensation by other T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) family members, or rather lose addiction to β-CATENIN/TCF7L2-driven gene expression altogether, we generated multiple CRC cell lines entirely negative for TCF/LEF or β-CATENIN expression. Survival of these cells and the ability to propagate them demonstrate their complete β-CATENIN- and TCF/LEF-independence. Nonetheless, one β-CATENIN-deficient cell line eventually became senescent, and absence of TCF/LEF proteins and β-CATENIN consistently impaired CRC cell proliferation, reminiscent of mitogenic effects of WNT/β-CATENIN signaling in the healthy intestine. Despite this common phenotype, β-CATENIN-deficient cells exhibited highly cell-line-specific gene expression changes with little overlap between β-CATENIN- and TCF7L2-dependent transcriptomes. Apparently, β-CATENIN and TCF7L2 independently control sizeable fractions of their target genes. The observed divergence of β-CATENIN and TCF7L2 transcriptional programs, and the finding that neither β-CATENIN nor TCF/LEF activity is strictly required for CRC cell survival has important implications when evaluating these factors as potential drug targets.

Discovery of Potent Inhibitors of Cyclin-Dependent Kinases 7 and 9: Design, Synthesis, Structure-Activity Relationship Analysis and Biological Evaluation.

Cyclin-dependent kinases (CDKs) 7 and 9 are deregulated in various types of human cancer and are thus viewed as therapeutic targets. Accordingly, small-molecule inhibitors of both CDKs are highly sought-after. Capitalising on our previous discovery of CDKI-73, a potent CDK9 inhibitor, medicinal chemistry optimisation was pursued. A number of N-pyridinylpyrimidin-2-amines were rationally designed, chemically synthesised and biologically assessed. Among them, N-(6-(4-cyclopentylpiperazin-1-yl)pyridin-3-yl)-4-(imidazo[1,2-a]pyrimidin-3-yl)pyrimidin-2-amine was found to be one of the most potent inhibitors of CDKs 7 and 9 as well as the most effective anti-proliferative agent towards multiple human cancer cell lines. The cellular mode of action of this compound was investigated in MV4-11 acute myeloid leukaemia cells, revealing that the compound dampened the kinase activity of cellular CDKs 7 and 9, arrested the cell cycle at sub-G1 phase and induced apoptosis.

Structural, Quantum Chemical, Molecular Docking, and Dynamics Studies of Quercetin—A Potent Inhibitor for Colon Cancer

According to several studies, phytochemicals, such as polyphenols, flavones, and flavonoids have significant anticancer properties that can combat various malignancies. Even though its action is highly dependent on the intracellular availability of reduced glutathione, Quercetin is regarded as an excellent free-radical scavenging antioxidant flavonoid. Apart from its antioxidant properties, Quercetin has a direct pro-apoptotic action in tumor cells. It has been shown to inhibit the proliferation of multiple human cancer cell lines at various cell cycle stages. The choice of a suitable chemopreventive agent was based on epidemiological studies that showed that consuming a specific dietary component (e.g. Quercetin) can lower the incidence of specific cancer death. MLK4 (KIAA1804) is the second most often altered kinase in colorectal carcinomas with microsatellite stability (MSS) (CRC). MLK4 regulates various physiological cellular processes, including cell cycle, senescence, and apoptosis, and mechanistic evidence suggests MLK4 is involved in carcinogenesis. In this study, the molecular structure of the Quercetin molecule was optimized using DFT/B3LYP method with a cc-pVTZ basis set, and the structural parameters were calculated. The generated vibrational spectra and the optimized molecular geometry were thoroughly reviewed and compared with experimental findings, resulting in a strong correlation. FMOs analysis was performed, which confirms the molecular reactivity of the Quercetin molecule. The anti-tumor efficacy of Quercetin was examined against the colon cancer target protein through in silico parameters, such as molecular docking and dynamics, ADME studies, MM-GBSA calculations, and the Pharmacophore hypothesis. From the analysis, the docked complex showed the highest docking score and maintained the complex stability and flexibility throughout the simulation period. Thus, the present study concluded that Quercetin could be a potent inhibitor of targeted colon cancer protein.

Abstract C044: MICAL2 expression in pancreatic cancer cells modulates the tumor microenvironment through the TGF beta pathway

Abstract Introduction: Pancreatic cancer is characterized by a desmoplastic, fibroinflammatory stroma. The crosstalk between cancer cells and their surrounding tumor microenvironment (TME) promotes disease progression, metastasis, and chemoresistance. We identified MICAL2 as a super-enhancer associated gene in pancreatic cancer. MICAL2 (molecule interacting with Cas-L) proteins are Flavin monooxygenases that promotes actin depolymerization and indirectly regulate SRF transcription. In other work, we have found that MICAL2 promotes pancreatic cancer growth and progression. In this study, we evaluated how MICAL2 pancreatic cancer cell expression modulates the PDAC tumor microenvironment. Methods: RNA-Seq analysis performed on human pancreas cancer cells (AsPC) before and after MICAL2 knockdown revealed differential regulation of TGF-β and other proinflammatory cytokines. We validated the expression of TGF-β and other potent cytokines in multiple human and mouse pancreas cancer cell lines by q-PCR. We next exposed human and mouse pancreatic stellate cells (PSCs) to conditioned media from cancer cells before and after MICAL2 knockdown and checked the expression of TGF-β responsive genes by qPCR. KrasG12D/+; Trp53R172H/+; Pdx1-cre (KPC) cells with and without MICAL2 were orthotopically injected to assess the in vivo tumor growth and metastasis. We sorted epithelial cells and CAFs from KPC orthotopic tumors with and without MICAL2 by using EPCAM, PDGFR, and PDPN markers. Results: MICAL2 knockdown (KD) resulted in downregulation of TGF-β gene in both human and mouse pancreas cancer cell lines (50% reduction, p<0.05). Human hPSCs and mouse mPSCs co-cultured with pancreatic cancer cells showed a significant downregulation of myofibroblastic and inflammatory CAFs genes including a-SMA, fibronectin, IL1α and IL6 upon MICAL2 KD as compared to hPSCs and mPSCs co-cultured with shcontrol cells. Orthotopic injections of KPC MICAL2 KD cells led to decreased tumor growth as compared to shcontrol (0.26 gm vs. 1 gm, p = 0.008). Our Immunofluorescence revealed less collagen deposition and α-SMA expression and reduced secretion of IL6 by stromal cells in MICAL2 KD tumors. Flow sorting showed less percentage of epithelial and CAF population in MICAL2 KD orthotopic tumors as compared to Shcontrol tumors. A qPCR analysis of the sorted populations showed less expression of TGF-β on epithelial cells and reduced expression of IL6 on PDGFRα/PDPN+ CAFs extracted from MICAL2 KD tumors as compared to control tumors. Conclusion: These data reveal that MICAL2 expression mediates tumor-stromal crosstalk through TGF- β. Ongoing work is focused on dissecting the role of MICAL2 in priming the metastatic niche through remodeling of the TME. Citation Format: Bharti Garg, Shweta Sharma, Sohini Khan, Edgar Esparza, Sarah Sass, Dawn Jacquish, Evangeline Mose, Herve Tiriac, Andrew Lowy. MICAL2 expression in pancreatic cancer cells modulates the tumor microenvironment through the TGF beta pathway [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C044.

CTNI-49. EARLY SIGNAL OF ACTIVITY FROM A PHASE 2 STUDY OF ST101, A FIRST-IN-CLASS PEPTIDE ANTAGONIST OF CCAAT/ENHANCER-BINDING PROTEIN Β (C/EBPΒ), IN RECURRENT GLIOBLASTOMA (GBM)

Abstract BACKGROUND C/EBPβ is a transcription factor that is active during embryofetal development but held in an inactive state in most mature cells (Zahnow 2009). C/EBPβ activates a proliferation/survival gene signature in multiple cancers, where it inversely correlates with disease prognosis and survival. ST101 is a cell-penetrating peptide antagonist of C/EBPβ. ST101 exposure leads to selective tumor cell death in multiple human cancer cell lines, including GBM, without impacting normal cell viability. In vivo, ST101 displays rapid uptake into multiple organs, the ability to cross the blood-brain barrier, and a long plasma half-life due to its resistance to degradation. It has potent anti-tumor activity in multiple GBM models, as a single agent or in combination, which supported moving into clinical development. TRIAL DESIGN: This phase 2 study is enrolling adult patients with GBM that has recurred or progressed after one standard treatment regimen (surgery, radiotherapy +/-temozolomide). Patients require measurable disease at baseline and at least 3 months from prior radiotherapy. Subjects receive the recommended phase 2 dose of ST101 (500mg IV weekly). Recruitment in the phase 2 portion of this trial began in January, 2022. RESULTS As of June 1, 2022, 14 GBM patients were enrolled. One patient has a confirmed mRANO partial response (PR) after 18 weeks of therapy, seven patients have not reached the first on-study assessment and six patients progressed. The median duration of therapy was 5 weeks. ST101 has a favorable safety profile with minor infusion related reactions being the most common adverse event. Based on the confirmed PR, the GBM cohort will be expanded. CONCLUSION This first-in-class C/EBPβ inhibitor, ST101, showed an early signal of activity in recurrent GBM. More extensive follow-up and clinical experience will be presented as this trial expands and matures.

Optimized Transcriptional Signature for Evaluation of MEK/ERK Pathway Baseline Activity and Long-Term Modulations in Ovarian Cancer.

Ovarian cancer is the most aggressive and lethal of all gynecologic malignancies. The high activity of the MEK/ERK signaling pathway is tightly associated with tumor growth, high recurrence rate, and treatment resistance. Several transcriptional signatures were proposed recently for evaluation of MEK/ERK activity in tumor tissue. In the present study, we validated the performance of a robust multi-cancer MPAS 10-gene signature in various experimental models and publicly available sets of ovarian cancer samples. Expression of four MPAS genes (PHLDA1, DUSP4, EPHA2, and SPRY4) displayed reproducible responses to MEK/ERK activity modulations across several experimental models in vitro and in vivo. Levels of PHLDA1, DUSP4, and EPHA2 expression were also significantly associated with baseline levels of MEK/ERK pathway activity in multiple human ovarian cancer cell lines and ovarian cancer patient samples available from the TCGA database. Initial platinum therapy resistance and advanced age at diagnosis were independently associated with poor overall patient survival. Taken together, our results demonstrate that the performance of transcriptional signatures is significantly affected by tissue specificity and aspects of particular experimental models. We therefore propose that gene expression signatures derived from comprehensive multi-cancer studies should be always validated for each cancer type.

Primary and metastatic tumors exhibit systems-level differences in dependence on mitochondrial respiratory function.

The Warburg effect, aerobic glycolysis, is a hallmark feature of cancer cells grown in culture. However, the relative roles of glycolysis and respiratory metabolism in supporting in vivo tumor growth and processes such as tumor dissemination and metastatic growth remain poorly understood, particularly on a systems level. Using a CRISPRi mini-library enriched for mitochondrial ribosomal protein and respiratory chain genes in multiple human lung cancer cell lines, we analyzed in vivo metabolic requirements in xenograft tumors grown in distinct anatomic contexts. While knockdown of mitochondrial ribosomal protein and respiratory chain genes (mito-respiratory genes) has little impact on growth in vitro, tumor cells depend heavily on these genes when grown in vivo as either flank or primary orthotopic lung tumor xenografts. In contrast, respiratory function is comparatively dispensable for metastatic tumor growth. RNA-Seq and metabolomics analysis of tumor cells expressing individual sgRNAs against mito-respiratory genes indicate overexpression of glycolytic genes and increased sensitivity of glycolytic inhibition compared to control when grown in vitro, but when grown in vivo as primary tumors these cells down-regulate glycolytic mechanisms. These studies demonstrate that discrete perturbations of mitochondrial respiratory chain function impact in vivo tumor growth in a context-specific manner with differential impacts on primary and metastatic tumors.

Potent Chlorambucil-Platinum(IV) Prodrugs.

The DNA-alkylating derivative chlorambucil was coordinated in the axial position to atypical cytotoxic, heterocyclic, and non-DNA coordinating platinum(IV) complexes of type, [PtIV(HL)(AL)(OH)2](NO3)2 (where HL is 1,10-phenanthroline, 5-methyl-1,10-phenanthroline or 5,6-dimethyl-1,10-phenanthroline, AL is 1S,2S-diaminocyclohexane). The resultant platinum(IV)-chlorambucil prodrugs, PCLB, 5CLB, and 56CLB, were characterized using high-performance liquid chromatography, nuclear magnetic resonance, ultraviolet-visible, circular dichroism spectroscopy, and electrospray ionization mass spectrometry. The prodrugs displayed remarkable antitumor potential across multiple human cancer cell lines compared to chlorambucil, cisplatin, oxaliplatin, and carboplatin, as well as their platinum(II) precursors, PHENSS, 5MESS, and 56MESS. Notably, 56CLB was exceptionally potent in HT29 colon, Du145 prostate, MCF10A breast, MIA pancreas, H460 lung, A2780, and ADDP ovarian cell lines, with GI50 values ranging between 2.7 and 21 nM. Moreover, significant production of reactive oxygen species was detected in HT29 cells after treatment with PCLB, 5CLB, and 56CLB up to 72 h compared to chlorambucil and the platinum(II) and (IV) precursors.

Dolutegravir Inhibits Proliferation and Motility of BT-20 Tumor Cells Through Inhibition of Human Endogenous Retrovirus Type K.

Increasing evidence points to the role of endogenous retroviruses (ERVs) in driving cancer cell proliferation. The purpose of this study was to explore the possibility of repurposing antiretroviral agents to inhibit ERVs as a new approach to cancer treatment. We found that an integrase strand-transfer inhibitor, dolutegravir (DTG), effectively inhibited the proliferation of multiple cancer cell lines and its antiproliferative potency was positively correlated with the expression levels of the human endogenous retrovirus type K (HERV-K). DTG inhibited the expression of HERV-K in multiple human cancer cell lines and the mouse mammary tumor virus (MMTV) in the murine 4T1 mammary cancer cell line. We chose the fast-growing BT-20 cell line as a model to study the in vitro antiproliferative mechanisms of DTG. BT-20 cells overexpressing both HERV-K env and pol genes became more resistant to DTG than cells transduced with vector alone. Knockdown of HERV-K also increased DTG resistance of BT-20 cells. The antiproliferative effect of DTG correlated with enhanced expression of E-cadherin and reduction in cell motility and invasiveness. Surprisingly, DTG stimulated expression of the env gene of MMTV in vivo and promoted metastasis of 4T1 tumor cells to the lungs. Taken together, our data support the role of ERVs in tumor development and encourage the further search for antiretroviral agents to treat malignancies in which ERVs are active.

SubCellBarCode: integrated workflow for robust spatial proteomics by mass spectrometry.

The molecular functions of a protein are defined by its inherent properties in relation to its environment and interaction network. Within a cell, this environment and network are defined by the subcellular location of the protein. Consequently, it is crucial to know the localization of a protein to fully understand its functions. Recently, we have developed a mass spectrometry- (MS) and bioinformatics-based pipeline to generate a proteome-wide resource for protein subcellular localization across multiple human cancer cell lines ( www.subcellbarcode.org ). Here, we present a detailed wet-lab protocol spanning from subcellular fractionation to MS-sample preparation and analysis. A key feature of this protocol is that it includes all generated cell fractions without discarding any material during the fractionation process. We also describe the subsequent quantitative MS-data analysis, machine learning-based classification, differential localization analysis and visualization of the output. For broad applicability, we evaluated the pipeline by using MS data generated by two different peptide pre-fractionation approaches, namely high-resolution isoelectric focusing and high-pH reverse-phase fractionation, as well as direct analysis without pre-fractionation by using long-gradient liquid chromatography-MS. Moreover, an R package covering the dry-lab part of the method was developed and made available through Bioconductor. The method is straightforward and robust, and the entire protocol, from cell harvest to classification output, can be performed within 1-2 weeks. The protocol enables accurate classification of proteins to 15 compartments and 4 neighborhoods, visualization of the output data and differential localization analysis including treatment-induced protein relocalization, condition-dependent localization or cell type-specific localization. The SubCellBarCode package is freely available at https://bioconductor.org/packages/devel/bioc/html/SubCellBarCode.html .

Abstract 6174: Developing a novel antibody drug conjugate against cervical cancer

Abstract Background: Cervical cancer ranks fourth in incidence and mortality among female tumors, with over 0.6 million patients diagnosed worldwide in 2020. A large percentage of cervical cancer patients will develop advanced diseases (stage IV or recurrent disease) and the 5-year survival rate of these patients was only 15-20% due to the lack of efficacy and adverse effects of first line chemodrugs. Recently, antibody drug conjugates (ADCs) emerge as a novel class of targeted therapeutics that selectively ablate tumor cells without damaging normal organs and tissues, which has shown promising efficacy in treating solid tumors. In this study, we sought to develop a cervical cancer-targeted ADC using a novel molecular target. Methods: Firstly, human cervical cancer and paracancerous tissue were stained by immunohistochemistry (IHC) in order to determine the difference of the expression level of a molecular target. Three human cervical cancer cell lines (C-33A, Si-Ha and Ca-Ski) and one normal cervical cell line (HCerEpiC) were used to determine the subcellular location of an ADC target on the cells by immunofluorescence (IF) staining, flow cytometry and single photon confocal microscopy. Secondly, endocytosis capability of target antibodies was visualized by confocal imaging and was quantified by flow cytometric analysis. Furthermore, a panel of ADC formulations with different chemical linkers and payloads were designed, constructed and characterized as ADC candidates for cervical cancer therapy. We next evaluated in vitro inhibitory activity of these ADC candidates on three cervical cancer cell lines by CCK8 assay. Finally, orthotopic and lung metastasis animal models of cervical cancer were established to determine the in vivo inhibitory activity and biosafety of constructed ADC candidates. Results: IHC staining of clinical specimens showed that the expression level of CD54 in cervical tumor tissues was significantly higher than those of paracancerous tissues. Fluorescence imaging and flow cytometry further determined the overexpression level, plasma membrane location and antigen-mediated endocytosis activity of CD54 in multiple human cervical cancer cell lines. Among several ADC formulations, an optimized CD54-targeted ADC linker and warhead combination was identified by comparing its IC50 with other ADC candidates and standard-of-care chemodrugs for cervical cancer. The optimized CD54 ADC exhibited promising anti-tumor activity on primary and lung metastatic cervical tumor models. Conclusion: In our study, we identified CD54 as a novel ADC target for cervical cancer. An optimized CD54-targeted ADC formulation was determined and its anti-tumor activity and biosafety profile were determined in multiple human cervical cancer cell lines in vitro and orthotopic and lung metastatic cervical tumor models in vivo, providing a promising targeted therapeutic candidate for the targeted treatment of cervical cancer. Citation Format: Shili Yao, Bing Zhu, Xinyan Wang, Rui Xu, Tong Yang, Peng Guo, Huarong Tang, Tao Zhu. Developing a novel antibody drug conjugate against cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6174.

Abstract 2464: In vivo imaging of ovarian and non-small cell lung cancer models hosted in the Sprague-Dawley Rag2 -/-Il2rg -/- SRG rat

Abstract Human cancer xenografts are a vital tool for understanding tumor biology, growth kinetics, and therapeutic efficacy using animal models. While in vivo studies are traditionally done in immunocompromised mice, we have created a Sprague Dawley Rag2 -/-, Il2rg -/- rat (SRG࣪ OncoRat®) that is an excellent host for human xenografts. Lacking B, T, and NK cells, the SRG rat readily supports the growth of multiple human cancer cell lines, including lines that do not engraft well or grow consistently in existing mouse models. In vivo luminescent imaging has routinely been used in mouse models, and is particularly useful for studying orthotopic xenografts or metastatic models. In this study, we validated the SRG rat as a model that can effectively be used for in vivo imaging of human cancers. For the current study, we assessed two tumor models. Ovarian cancer cell line OV81.2-luc is a luciferase positive cell line that was generated from ascites collection from a grade IIIC, serous ovarian carcinoma. Orthotopic tumors were established by inoculating OV81.2-luc cells intraperitoneally into female SRG rats and female NSG mice, which were treated with either vehicle or cisplatin chemotherapy for 28 days. Human non-small cell lung cancer line H358-luc is a luciferase positive cell line that, when injected subcutaneously into the hind flank of SRG rats, metastasizes to the lungs. Tumors were established by inoculating H358-luc cells subcutaneously into male SRG rats, and measured thrice weekly with calipers. For both studies, tumorgenicity was measured via weekly in vivo luciferase imaging using Spectral Instruments AMI HT system. Tumors established rapidly for both OV81.2-luc and H358-luc in both SRG rats and NSG mice, with visible luciferin positive signal starting one week post cell inoculation. In OV81.2-luc hosting animals, upon necropsy, tumors were found on multiple abdominal organs including the peritoneum, ovary, mesentery, intestines, kidneys, liver, and body cavity wall. Metastatic events outside the abdominal cavity were not evident in OV81.2-luc hosting animals. In H358-luc tumor bearing rats, a high metastatic tumor burden was found in the lungs. These data confirm that the SRG rat is an excellent host for studying human cancer when compared to commonly used immunodeficient mouse models. Data demonstrate that the SRG rat has a high utility for studies using both in vivo imaging, such as orthotopic tumor implantation, and studies on metastasis. As the most immunodeficient rat commercially available, the SRG rat retains the ability to establish human tumors while possessing size, physiology, and metabolism-based advantages when compared to mice. Citation Format: Diane Begemann, Fallon Noto. In vivo imaging of ovarian and non-small cell lung cancer models hosted in the Sprague-Dawley Rag2 -/-Il2rg -/- SRG rat [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 2464.

Abstract 3597: Direct targeting of KRASG12X mutant cancers with RMC-6236, a first-in-class, RAS-selective, orally bioavailable, tri-complex RASMULTI(ON) inhibitor

Abstract Mutant RAS is common in pancreatic carcinoma (PDAC), non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) and exists predominantly in the GTP-bound (RAS(ON)) state, leading to excessive downstream oncogenic signaling. KRASG12C(OFF) inhibitors have provided clinical proof of concept for targeting mutant KRAS. Preclinical data suggests inhibition of RAS(ON) may be a superior therapeutic strategy. In addition, KRASG12 mutations such as KRASG12D and KRASG12V remain unserved.RMC-6236 is a first-in-class, potent, oral tri-complex RASMULTI(ON) small molecule inhibitor designed to treat cancers driven by a variety of RAS mutations. RMC-6236 non-covalently binds to an abundant intracellular chaperone protein, cyclophilin A (CypA), resulting in a binary complex that engages RAS(ON) to form a high-affinity, RAS-selective tri-complex that sterically inhibits RAS binding to effectors. Exposure to RMC-6236 suppressed ERK phosphorylation and cell growth, and induced apoptosis in multiple human RAS-addicted cancer cell lines in vitro.RMC-6236 induced dose-dependent, deep, and durable suppression of RAS pathway activation up to 48 hours in preclinical xenograft models in vivo. Prolonged exposure in tumors was observed relative to blood and various healthy tissues, likely mediated by high affinity binding of RMC-6236 to tumor CypA.RMC-6236 at tolerable doses induced profound and durable tumor regressions in multiple cell line-derived (CDX) and patient-derived (PDX) RASMUT xenograft models, including NSCLC, CRC and PDAC. Anti-tumor activity was particularly notable in KRAS position 12 (G12X) mutant tumors, particularly KRASG12D, KRASG12V, and KRASG12R, with significant tumor regressions observed. Tumor growth inhibition was durable even in tumors that did not regress. Intermittent scheduling of RMC-6236 was active and permitted a higher dose intensity than daily dosing.RMC-6236 promoted anti-tumor immunity in vivo and was additive with anti-PD1 antibodies, driving durable complete responses and immunologic memory in a KRAS mutant CRC model. Furthermore, RMC-6236 treatment reversed oncogenic RAS-driven immune evasion mechanisms in a checkpoint blockade refractory KRAS mutant model, significantly transforming the tumor microenvironment in favor of anti-tumor immunity.These preclinical results support the inclusion of NSCLC, PDAC, and CRC patients in our planned clinical trial of RMC-6236 in patients with KRASG12X advanced solid tumors. Citation Format: Elena S. Koltun, Meghan A. Rice, W. Clay Gustafson, David Wilds, Jingjing Jiang, Bianca J. Lee, Zhengping Wang, Stephanie Chang, Mike Flagella, Yunming Mu, Nuntana Dinglasan, Nicole Nasholm, James W. Evans, Yingyun Wang, Kyle Seamon, Yang Liu, Cristina Blaj, John Knox, Rebecca Freilich, Elsa Quintana, Jim Cregg, Alun Bermingham, Adrian L. Gill, Jacqueline Am Smith, Mallika Singh. Direct targeting of KRASG12X mutant cancers with RMC-6236, a first-in-class, RAS-selective, orally bioavailable, tri-complex RASMULTI(ON) inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3597.

Abstract 5471: PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, is active in preclinical models of prostate cancer

Abstract CDK9 is a serine/threonine kinase belonging to the subclass of the transcription associated CDKs. CDK9 complexes with cyclin T and cyclin K, the positive transcription elongation factor b (P-TEFb), and phosphorylates Serine 2 of RNA polymerase II (pSer2 RNAPII) to activate transcription. Consequently, targeting CDK9 could effectively interfere with epigenetic and transcriptional reprogramming and prevent disease progression and treatment resistance in human cancers. Androgen receptor (AR)-dependence in prostate cancer is linked to CDK9 function. CDK9 stabilizes AR-associated proteins, and pharmacological inhibition of CDK9 can inhibit AR, AR variants, and their downstream transcription programs. We evaluated the novel CDK9 inhibitor, PRT2527, in prostate cancer models to evaluate its effects on cell proliferation, stem-like tumor cells, and tumor growth. PRT2527 is a potent inhibitor of CDK9/CyclinT1 complex, and when evaluated at concentration 200 times the biochemical IC50, PRT2527 was highly selective for CDK9 inhibition. We verified in biochemical assays the ability of PRT2527 to suppress pSer2 RNAPII and reduce expression of c-Myc, a common target of CDK9, in a concentration-dependent manner in multiple human prostate cancer cell lines. PRT2527 also inhibited c-Myc-dependent transcription in vitro in luciferase reporter assays. Furthermore, RNA sequencing showed altered expression of several genes with significant enrichment of c-Myc and E2F targets and RNAPII dependent transcription among downregulated genes in PRT2527-treated VCaP cells. In vitro, PRT2527 inhibited the proliferation of androgen-dependent and androgen-independent prostate cancer cell lines (IC50, ≤50 nM). PRT2527 was highly effective (IC50, ≤10 nM) in tumor-spheroid assays in blocking the growth of stem-like tumor cells and significantly suppressed the in vitro growth of tumor organoids from both human cell lines and patient-derived xenografts (PDXs). In mice, PRT2527 IV administration reduced pSer2 RNAPII in tumor xenografts and c-Myc-dependent transcriptional activity in a DU145 luminescence reporter model. PRT2527 administration in mice significantly reduced growth of PDX LuCaP 35 (castration-sensitive, adenocarcinoma) and LuCaP 145.2 (castration-resistant, neuroendocrine) along with the fraction of tumor-initiating stem-like cells in ex vivo assays. PRT2527 reduced pSer2 RNAPII in both PDXs, whereas c-Myc decreased in LuCaP 35 and Sox2 in LuCaP 145.2, relative to basal expression levels. Collectively, our data demonstrate that PRT2527 has potent pharmacodynamic and antitumor activity in multiple models of castration-sensitive and castration-resistant prostate cancer. PRT2527 is advancing into phase 1 studies in solid tumors. Citation Format: Elisa Federici, Gianluca Civenni, Aleksandra Kokanovic, Giada Sandrini, Luca Guarrera, Simone Mosole, Alessia Cacciatore, Valeria Uboldi, Manuel Lessi, Giovanni Papa, Domenico Albino, Elisa Storelli, Jessica Merulla, Andrea Rinaldi, Marco Bolis, Yang Zhang, Kris Vaddi, Peggy Scherle, Bruce Ruggeri, Giuseppina M. Carbone, Carlo V. Catapano. PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, is active in preclinical models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5471.

Abstract OT2-01-01: Phase 1 study of ST101, a first-in-class peptide antagonist of CCAAT/enhancer-binding protein β (C/EBPβ), in patients with advanced solid tumors, with a phase 2 expansion in patients with hormone receptor positive breast cancer (HR+ BC)

Abstract Background: CCAAT/Enhancer Binding Protein Beta (C/EBPβ) is a transcription factor that is active during embryofetal development but held in an inactive state in most mature cells. C/EBPβ is upregulated or overactivated in multiple cancers, where it inversely correlates with disease prognosis. In breast cancer, C/EBPβ drives the expression of factors that promote tumor survival, proliferation and inhibit differentiation. ST101 is a cell-penetrating all D amino acid peptide antagonist of C/EBPβ. ST101 exposure inhibits C/EBPβ target gene expression, leading to selective tumor cell death in multiple human cancer cell lines, including hormone receptor positive breast cancer (HR+ BC) and triple negative breast cancer (TNBC), without impacting normal cell viability. In vivo, ST101 displays rapid uptake into multiple organs, the ability to cross the blood-brain barrier, and a long plasma half-life due to its resistance to proteolytic degradation. Potent ST101 anti-tumor activity, demonstrated by dose-dependent inhibition of tumor growth in subcutaneous HR+ and orthotopic TNBC xenograft models in vivo, supported advancing ST101 into clinical development.Trial design: This phase 1-2 study uses a standard 3+3 design with dose doubling for the first 4 dose levels then 50% escalations thereafter. The recommended phase 2 dose will be used in 4 expansion cohorts in specific tumor types, including HR+ BC. Patients receive intravenous ST101 once weekly.Eligibility criteria: The dose-escalation phase is enrolling patients ≥18 years of age with advanced, unresectable metastatic solid tumors refractory to or intolerant of other therapeutic options. In expansion, patients with HR+ BC must have progressed after 1-3 prior hormone-based therapies. Previous treatment with CDK 4/6 inhibitor, mTOR inhibitor, or chemotherapy is allowed as monotherapy or in combination. Specific aims: The primary objective of phase 1 is to evaluate safety and tolerability of ST101. Secondary objectives include the recommendation of a dose and regimen of ST101 for further evaluation, analysis of pharmacokinetics, assessment of several pharmacodynamic measures, and to assess preliminary efficacy. Statistical design: The recommended phase 2 dose will be used in a 15-30 patient HR+ BC expansion cohort, with a Simon 2-stage design, which requires one response to expand the cohort to 30 patients. Up to 120 patients are planned in a total of four expansion cohorts, which should be enrolling by Q3 2021.Accrual: We began recruitment in August 2020. Enrollment is ongoing, and by July 2021, 18 patients were recruited in five dose-escalation cohorts up to 6 mg/kg; a 6th cohort (9 mg/kg) is ongoing. Dose escalation should be complete by Sept 2021, and the phase 2 portion in the HR+ BC cohort will be underway (n=15-30). Please contact rob.michel@bexonclinical.com if you have a specific interest in this trial. Citation Format: Alice S Bexon, Hendrik-Tobias Arkenau, Jeff Evans, Gerald S Falchook, Stefan N Symeonides, Meredith A McKean, Elisa Fontana, Manojkumar Bupath, Alistair McLaren, Sreenivasa Chandana, Tze-en Ding, Emerson A Lim, Jim Rotolo, Gina Capiaux, Rob Michel, Stephen Kaesshaefer, Nehal J Lakhani. Phase 1 study of ST101, a first-in-class peptide antagonist of CCAAT/enhancer-binding protein β (C/EBPβ), in patients with advanced solid tumors, with a phase 2 expansion in patients with hormone receptor positive breast cancer (HR+ BC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-01-01.

Direct targeting of RAS in pancreatic ductal adenocarcinoma with RMC-6236, a first-in-class, RAS-selective, orally bioavailable, tri-complex RASMULTI(ON) inhibitor.

591 Background: RAS proteins (such as KRAS, NRAS, HRAS) are small GTPases that drive cell proliferation and survival when bound to GTP. Mutant RAS proteins exist predominantly in the GTP-bound (RAS(ON)) state, leading to excessive downstream signaling via interaction with effectors such as RAF kinases. Oncogenic KRAS is required for the initiation, progression, and maintenance of pancreatic ductal adenocarcinoma (PDAC) (Hezel et al, 2006, Ying et al 2012). Although extinction of KRAS expression as well as pharmacological inhibition of RAS effectors clearly abrogate the growth of human PDAC models, clinical trials of drugs targeting key components of the RAS pathway have remained largely unsuccessful. Several factors contribute to these failures including redundancy in signaling surrogates downstream of KRAS and/or tumor complexity driven by co-occurring genomic alterations and intra-tumoral heterogeneity. Methods: RMC-6236 is a small molecule that binds to an intracellular chaperone protein, Cyclophilin A (CypA), resulting in an inhibitory binary complex that binds active, GTP-bound RAS to form a tri-complex and suppresses RAS signaling by disrupting interactions with effectors such as RAF kinases. Results: Here, we demonstrate that single agent RMC-6236, a first-in-class, orally bioavailable, RAS-selective tri-complex inhibitor of multiple RAS mutations and wild-type RAS (RASMULTI inhibitor) is highly efficacious in preclinical models of KRAS mutant PDAC (with marked activity in RAS-mutant colorectal cancer models described in Koltun et al, AACR 2021). RMC-6236 suppresses phosphorylation of ERK kinases, downstream effectors of RAS involved in cell proliferation, and induces growth suppression and apoptosis in multiple human cancer cell lines in vitro. Oral administration of RMC-6236 produces deep, durable, and dose-dependent suppression of tumor RAS pathway activation in vivo. An extended duration of tumor pharmacodynamic activity, relative to plasma exposure, is observed that likely reflects retention of RMC-6236 in tumor tissue due to high affinity binding to CypA. Daily dosing of RMC-6236 drives profound and durable tumor regressions in multiple cell line derived (CDX) and patient derived (PDX) xenograft models of KRAS mutant PDAC at doses that are well-tolerated. Conclusions: These results indicate that direct targeting of mutant and possibly wild-type RAS in PDAC, without inhibition of signaling nodes outside the canonical RAS pathway, has the potential to translate into clinical benefit for patients with pancreatic cancer harboring mutations in KRAS that may be superior to therapies aimed at upstream or downstream signaling elements within the RAS pathway. Our preclinical data strongly support the inclusion of PDAC patients in our planned clinical trial of RMC-6236 in patients with advanced solid tumors.