Panel Of Human Cancer Cell Lines Research Articles

Exploratory Study Identifies Matrix Metalloproteinase-14 and -9 as Potential Biomarkers of Regorafenib Efficacy in Metastatic Colorectal Cancer.

In identifying biomarkers for anticancer drugs, the lack of objectivity in selecting candidate factors makes interpretation difficult. We performed preclinical analysis and a translational validation study to identify candidate biomarkers for regorafenib efficacy in metastatic colorectal cancer (mCRC). Using in silico COMPARE analysis with a human cancer cell line panel, JFCR39, we selected candidate biomarkers whose expression correlates with regorafenib sensitivity. We validated predictive values in mCRC patients receiving regorafenib (discovery, n = 53) and FTD/TPI (control, n = 16). Blood samples were obtained at baseline (BL), before the second cycle (2nd), and at progressive disease (PD), and biomarker levels were measured using ELISA. Our analysis showed that high matrix metalloproteinase (MMP)-14 expression was associated with a high sensitivity to regorafenib. In the discovery cohort, high MMP-14 levels at BL and PD were correlated with tumor shrinkage and longer progression-free survival (PFS). A subsequent analysis of other related factors further indicated that the patients with decreased MMP-9 levels at the 2nd had higher disease control rates, tumor shrinkage, longer PFS, and overall survival than those with increased changes. These findings were not observed in the control cohort. Our study suggests MMP-14 and MMP-9 may serve as prognostic markers for regorafenib and provide insights into novel combination therapies with anti-MMP-9 agents or FTD/TPI.

Abstract LB168: A selective, potent, and orally bioavailable CDK7 inhibitor demonstrates superior anti-cancer activity in colorectal cancer models

Abstract Dysregulated cell cycle and gene expression are pivotal factors in cancer development and the regulators of these processes are attractive targets for cancer treatment. The success of CDK4/6 inhibitors such as palbociclib, ribociclib, and abemaciclib underscores the promising role of targeting cell cycle regulators in anti-cancer therapy. On the other hand, a robust correlation has been established between the overexpression of oncogenes and super-enhancer (SE). The genes associated with SE show increased vulnerability to transcription factor disruption compared to genes with typical enhancers. It has been demonstrated in previous studies that inhibiting transcription factors can effectively decrease the expression of SE-associated oncogenes, including c-MYC, thereby highlighting potential anticancer effects for oncogene-addicted cancers. Currently, agents targeting transcription factors are undergoing clinical trials, offering promising therapeutic options for patients with various types of cancers. CDK7 plays a crucial role in orchestrating cell cycle progression and transcription by phosphorylating CDKs, the C-terminal domain of RNA polymerase II, and hormone receptors like estrogen and androgen receptors. The inhibition of CDK7 has been shown to effectively inhibit tumor growth through various mechanisms, including inducing cell cycle arrest and inhibiting transcription, thereby leading to the death of cancer cells. Notably, ongoing clinical studies have demonstrated that small-molecule CDK7 inhibitors effectively control cancer progression as a stand-alone and in combination with other anti-cancer agents. C-16661, a selective, potent, orally bioavailable, and reversible ATP-competitive inhibitor of CDK7, displayed a single-digit nanomolar IC50 value in both biochemical and cellular assays. It exhibited excellent selectivity in a kinase panel assay, and remarkable potency in a human cancer cell line panel assay. C-16661 treatment resulted in strong inhibition of both its direct and indirect downstream targets including pS5-POLII, pT161-CDK1, c-MYC, and MCL1 in HCT116, a human colorectal carcinoma cell line. While C-16661 exhibited good PK profiles in animals, it demonstrated excellent tumor growth inhibition without any tolerability issues in a colorectal cancer CDX mouse efficacy model. Citation Format: Heuijoon Park, Kyoungwan Seo, Jihyun Yu, Jieun Kim, Min Sung Joo, Sungeun Kim, Joonhyung Lee, Jeonghyeon Lee, Eun-Jung Kim, Joonwoo Nam, Wooseok Han. A selective, potent, and orally bioavailable CDK7 inhibitor demonstrates superior anti-cancer activity in colorectal cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB168.

Abstract 6441: Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in ovarian cancer

Abstract Background. HSF1 helps cancer cells cope with multiple stresses caused by oncogene activation. Methods. See details below. Results. We discovered the bisamide NXP800 as an inhibitor of HSF1-mediated transcription through phenotypic screening and med chem optimization. Gene expression microarray analysis of human cancer cell lines and tumor xenografts treated with bisamide inhibitors indicated activation of the ATF4 axis of the ISR as a key mechanism of action. In SK-OV-3 ovarian cancer xenografts, NXP800 showed clear PK/PD relationships with increased expression of ATF4 transcriptional target genes alongside decreased expression of HSF1 transcriptional targets. NXP800 also caused tumor regressions in this model. Expanding in additional human ovarian cancer xenograft models, we observed efficacy in five and a complete lack of response in three others. The sensitive models all had homozygous deleterious mutations in the ARID1A gene, whereas the non-responding xenografts were all wild type (WT). ARID1A is a component of the SWI/SNF chromatin remodelling complex involved in repression and activation of target genes. Subsequent screening of the large Sanger human cancer cell line panel confirmed ARID1A as the most significant common disease-related alteration predicting sensitivity to NXP800 in ovarian cancer cell lines. This predictive relationship was confirmed in an ARID1A isogenic HCT-116 cell line pair; sensitivity was greater in the homozygous ARID1A mutant cells compared to WT, resulting in PARP cleavage in the mutant cells only. In vivo there was no effect in WT HCT-116 xenografts whereas growth inhibition was observed in the homozygous mutant cells, resulting in significantly smaller tumors. In addition, the induction of ATF4 target genes was stronger and more prolonged in the mutant cells. It is known that whereas short term ATF4 activation is adaptive, persistent activation can promote the induction of apoptosis. CHIP-seq analysis confirmed clear relationships between ARID1A status, ATF4 and HSF1 promoter occupancy, and the distribution of BRG1 and RNA pol II at target sites, although these were often gene-specific and complex. A relatively simple example is the regulation of the INHBE gene (expression of which is a PD biomarker). In untreated samples there is no binding of ATF4, BRG1 and RNA pol II at the INHBE promoter. NXP800 treatment results in the recruitment of BRG1, ATF4 and RNA pol II in ARID1A mutant TOV-21G cells but not in the RMGI WT cells, leading to increased expression in the mutant cells only. Conclusions. We propose that ARID1A loss alters the binding and recruitment of ATF4 and HSF1 leading to the altered and prolonged expression of ATF4 target genes and increased sensitivity to NXP800. NXP800 is currently in phase Ib for the treatment of ARID1A mutant platinum resistant ovarian cancer (NCT05226507). Citation Format: Robert H. te Poele, Marissa Powers, Swee Sharp, Emmanuel de Billy, Maria Taskinen, Loredana Pellegrino, Sharon Gowan, Asadh Miah, Angela Hayes, Matthew Cheeseman, Keith Jones, Suzanne Eccles, Florence Raynaud, Paul Clarke, Paul Workman. Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6441.

Quinazoline-chalcone hybrids as HDAC/EGFR dual inhibitors: Design, synthesis, mechanistic, and in-silico studies of potential anticancer activity against multiple myeloma.

Two new series of quinazoline-chalcone hybrids were designed, synthesized as histone deacetylase (HDAC)/epidermal growth factor receptor (EGFR) dual inhibitors, and screened in vitro against the NCI 60 human cancer cell line panel. The most potent derivative, compound 5e bearing a 3,4,5-trimethoxyphenyl chalcone moiety, showed the most effective growth inhibition value against the panel of NCI 60 human cancer cell lines. Thus, it was selected for further investigation for NCI 5 log doses. Interestingly, this trimethoxy-substituted analog inhibited the proliferation of Roswell Park Memorial Institute (RPMI)-8226 cells by 96%, at 10 µM with IC50 = 9.09 ± 0.34 µM and selectivity index = 7.19 against normal blood cells. To confirm the selectivity of this compound, it was evaluated against a panel of tyrosine kinase enzymes. Mechanistically, it successfully and selectively inhibited HDAC6, HDAC8, and EGFR with IC50 = 0.41 ± 0.015, 0.61 ± 0.027, and 0.09 ± 0.004 µM, respectively. Furthermore, the selected derivative induced apoptosis via the mitochondrial apoptotic pathway by raising the Bax/Bcl-2 ratio and activating caspases 3, 7, and 9. Also, the flow cytometry analysis of RPMI-8226 cells showed that the trimethoxy-substituted analog produced cell cycle arrest in the G1 and S phases at 55.82%. Finally, an in silico study was performed to explore the binding interaction of the most active compound within the zinc-containing binding site of HDAC6 and HDAC8.

Synthesis, in silico, in vitro evaluation of furanyl- and thiophenyl-3-phenyl-1H-indole-2-carbohydrazide derivatives as tubulin inhibitors and anticancer agents.

Twenty-one new indole derivatives comprising of seven furanyl-3-phenyl-1H-indole-carbohydrazide derivatives and fourteen thiophenyl-3-phenyl-1H-indole-carbohydrazide derivatives were synthesised and biologically evaluated for their microtubule-destabilising effects, and antiproliferative activities against the National Cancer Institute 60 (NCI60) human cancer cell line panel. Among the derivatives, 6i showed the best cytotoxic activity exhibiting selectivity for COLO 205 colon cancer (LC50 = 71 nM), SK-MEL-5 melanoma cells (LC50 = 75 nM), and MDA-MB-435 (LC50 = 259 nM). Derivative 6j showed the strongest microtubule-destabilising effect. Both 6i and 6j were able to induce G2/M cell cycle arrest and apoptosis in MDA-MB-231 triple-negative breast cancer cells. Molecular docking simulation results suggested that these derivatives inhibit tubulin by binding at the colchicine site. The calculated molecular descriptors showed that the most potent derivatives have acceptable pharmacokinetic profiles and are favourable for oral drug administration.

Characterization of Anticancer Effects of the Analogs of DJ4, a Novel Selective Inhibitor of ROCK and MRCK Kinases.

The Rho associated coiled-coil containing protein kinase (ROCK1 and ROCK2) and myotonic dystrophy-related Cdc-42 binding kinases (MRCKα and MRCKβ) are critical regulators of cell proliferation and cell plasticity, a process intimately involved in cancer cell migration and invasion. Previously, we reported the discovery of a novel small molecule (DJ4) selective multi-kinase inhibitor of ROCK1/2 and MRCKα/β. Herein, we further characterized the anti-proliferative and apoptotic effects of DJ4 in non-small cell lung cancer and triple-negative breast cancer cells. To further optimize the ROCK/MRCK inhibitory potency of DJ4, we generated a library of 27 analogs. Among the various structural modifications, we identified four additional active analogs with enhanced ROCK/MRCK inhibitory potency. The anti-proliferative and cell cycle inhibitory effects of the active analogs were examined in non-small cell lung cancer, breast cancer, and melanoma cell lines. The anti-proliferative effectiveness of DJ4 and the active analogs was further demonstrated against a wide array of cancer cell types using the NCI-60 human cancer cell line panel. Lastly, these new analogs were tested for anti-migratory effects in highly invasive MDA-MB-231 breast cancer cells. Together, our results demonstrate that selective inhibitors of ROCK1/2 (DJE4, DJ-Allyl) inhibited cell proliferation and induced cell cycle arrest at G2/M but were less effective in cell death induction compared with dual ROCK1/2 and MRCKα/β (DJ4 and DJ110).

Syntheses and Preliminary Biological Evaluations of the Dibromopyrrole‐Containing Marine Natural Products Agesasine A, Agesasine B, Longamide E and Various Congeners

AbstractTotal syntheses of the title marine natural products have been achieved and so confirming the structures originally assigned to them. Upon subjecting agesasine A and its corresponding ethyl ester to Mitsunobu conditions, a 1,5‐cyclodehydration reaction takes place to give 2‐oxazolines. In contrast, on subjecting agesasine B to the same Mitsunobu conditions, a simple dehydration reaction occurs to give the corresponding acrylate. A total synthesis of longamide E was achieved by engaging a 1,2‐disubstituted pyrrole in a lactam‐forming reaction and this was followed by a two‐fold and fully regio‐controlled bromination reaction. A distinctly different and possibly biomimetic route was used to synthesize, via the open‐chain natural product nakamurine B, longamide B and its methyl ester. Preliminary biological evaluations of the title alkaloids and various analogues against a small human cancer cell line panel reveals cytotoxic properties that vary significantly with structure.

Abstract 6106: Epigenetic-miRNA regulatory circuit confers ovarian cancer chemoresistance through RAD18-mediated DNA damage tolerance and repair signaling

Abstract Epithelial ovarian cancer (EOC) is the deadliest gynecological malignancy. This cancer typically is not detected, 70-80% of patients, until an advanced stage (stage III-IV). The standard of care OC entails complete surgical resection of all visible abdominal-pelvic tumors, followed by platinum-based chemotherapy combined with a taxane. New recommendations for patients responding to adjuvant therapy is to include a PARP inhibitor with or without bevacizumab for maintenance therapy. Despite initial responses, the majority of stage 3 & 4 more than 70% of OC patients experience recurrent disease. Frequently, individuals become resistant to these treatments. This dreadful circumstance highlights the pressing need to identify the molecular mechanisms underlying the disease's aggressive nature upon recurrence and the development of treatment resistance. Several genetic and epigenetic factors have been linked to the reprogramming of tumor cells by controlling the pattern of transcriptional and post-transcriptional gene expression. Particularly, the miRNA-mediated regulatory circuit plays a significant role in tumor progression and therapeutic responses. Our analysis of miRNA expression signature in human ovarian cancer cell line panel showed little to no expression of miR221_5p and a corresponding increase in DNA damage response and repair gene RAD18 expression. RAD18 plays a critical role in cellular DNA damage tolerance and repair activity against chemotherapeutics, including platinum drugs. Similarly, loss of miRNA221_5p is associated with aggressive tumor cell growth, stemness, chemoresistance to platinum drugs, and poor prognosis in several cancers. Based on this information, we have hypothesized that miR221_5p regulates RAD18-mediated DNA damage tolerance and repair and may offer novel therapeutic intervention to overcome EOC chemoresistance. Our experimental data confers that miR221_5p post-transcriptionally regulates RAD18 by binding to its 3’-UTR region and restores OC cell sensitivity to platinum drugs. Mechanistically, our results demonstrate that miRNA221_5p epigenetically regulates RAD18-mediated DNA damage tolerance and homologous recombination repair and could be a novel therapeutic to overcome OC chemoresistance. Collectively, our studies identify a novel chemotherapy-induced epigenetic modulator in OC therapeutic resistance and offer novel miRNA 221-5p-mediated therapeutic intervention for the treatment of chemoresistant OC and to prevent disease recurrence. Citation Format: Tasmin Rahman Omy, Chinnadurai Mani, Komaraiah Palle, Mark Reedy. Epigenetic-miRNA regulatory circuit confers ovarian cancer chemoresistance through RAD18-mediated DNA damage tolerance and repair signaling. [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 6106.

Design, synthesis and evaluation of 5-chloro-6-methylaurone derivatives as potential anti-cancer agents

A series of novel 5-chloro-6-methylaurone derivatives (6a-p) were synthesized and characterized by various spectroscopic techniques. The synthesized compounds were tested for anticancer activity against 60-human cancer cell line panel derived from nine cancer types at NCI, Bethesda, USA. Among the synthesized compounds, six compounds (6e, 6f, 6h, 6i, 6k and 6 m) exhibited growth inhibition and cytotoxic activity against various human cancer cell lines in one-dose data. The most potent compound among the series, 6i was active against 55 out of 60 human cancer cell lines. Compound 6i showed remarkable % growth inhibition and cytotoxicity against various cancer cell lines exhibiting % GI in the range 36.05–199.03. The compound 6i was further evaluated for five dose assay and exhibited GI50 1.90 µM and 2.70 µM against melanoma and breast cancer cell lines respectively. Further evaluation of 6i for five-dose assay exhibited a diverse spectrum of anti-cancer activity towards all the 60 human cancer cell line panel with the selectivity index ratio ranging 0.854–1.42 and 0.66–1.35 for GI50 and TGI respectively. Based on one-dose and five-dose data compound 6i was further evaluated for cell apoptosis against MDA-MB-468 breast cancer cell line and was found to induce early apoptosis in cells explaining its mode of action. The in-silico studies for the synthesized compounds as LSD1 inhibitors (2H94) have shown better docking score and binding energy comparable to vafidemstat. All the compounds followed Lipinski rule of five. These findings concluded that the compound 6i could lead to the development of a promising therapeutic anticancer agent.

Novel Silver Complexes Based on Phosphanes and Ester Derivatives of Bis(pyrazol-1-yl)acetate Ligands Targeting TrxR: New Promising Chemotherapeutic Tools Relevant to SCLC Management

Bis(pyrazol-1-yl)acetic acid (HC(pz)2COOH) and bis(3,5-dimethyl-pyrazol-1-yl)acetic acid (HC(pzMe2)2COOH) were converted into the methyl ester derivatives 1 (LOMe) and 2 (L2OMe), respectively, and were used for the preparation of silver(I) complexes 3-5. The Ag(I) complexes were prepared by the reaction of AgNO3 and 1,3,5-triaza-7-phosphaadamantane (PTA) or triphenylphosphine (PPh3) with LOMe and L2OMe in methanol solution. All Ag(I) complexes showed a significant in vitro antitumor activity, proving to be more effective than the reference drug cisplatin in the in-house human cancer cell line panel containing examples of different solid tumors. Compounds were particularly effective against the highly aggressive and intrinsically resistant human small-cell lung carcinoma (SCLC) cells, either in 2D and 3D cancer cell models. Mechanistic studies revealed their ability to accumulate into cancer cells and to selectively target Thioredoxin (TrxR), thus leading to redox homeostasis unbalance and ultimately inducing cancer cell death through apoptosis.

56 (PB046) - Discovery and functional characterization of potent, balanced AXL/ MER inhibitors using a novel MER X-Ray crystal structure

The TAM family of kinases consist of AXL, MER and TYRO3. The aim of our program is to design potent inhibitors of this family that demonstrate excellent kinome selectivity and good PK properties for probing the role of these kinases in immune evasion, resistance, tumour cell growth and survival. A novel construct of MER was designed including the juxtamembrane domain which was used for X-ray crystallography. X-rays of MER in the fully active conformation were obtained for the first time. Additionally, contrasting crystal structures of a type 2 inhibitor KIN101222 were generated using both this novel construct and the previously reported truncated construct. This juxtamembrane domain containing construct afforded novel insight into designing optimal kinome selectivity Derived compounds were profiled in vitro in biochemical assays, TAM driven Ba/F3 proliferation, inhibition of pAXL in a murine embryonal fibroblast cell line over-expressing AXL and a 125 cell panel of human cancer cell lines. Finally, preferred compounds were profiled in vivo in a mechanistic model in which AXL and MER expressing Ba/F3 cells were implanted into mice. The compounds collectively evaluated in this study demonstrated potent inhibition of a Ba/F3 cell line derivative expressing a recombinant AXL kinase, with IC50 values <10 nM, 3–5 fold cellular selectivity over a similar recombinant MER assay, and excellent broader kinome selectivity. The activity of a representative molecule from this series shows a markedly different activity profile to the clinical AXL inhibitor Bemcentinib in the human cancer cell line panel. When dosed in vivo in the mechanistic model, a number of the compounds tested induced either stasis or regression at doses which were well tolerated. In some examples e.g. KIN101433, stasis/ regression was observed at doses of 5 mg/kg QD and below. In summary, selective type 2 AXL/MER inhibitors are well tolerated in mice and show a remarkably benign profile in a broad human cancer cell line panel. Further profiling is ongoing oriented towards investigating the role of these molecules in modulating the immune system and more detailed investigation of their toxicological properties. No conflict of interest.

Imidazo[4,5-b]pyridine derived tubulin polymerization inhibitors: Design, synthesis, biological activity in vitro and computational analysis

Imidazo[4,5-b]pyridine derived acrylonitriles were synthesized and explored for their in vitro antiproliferative effect on a diverse human cancer cell line panel. Three compounds, 20, 21 and 33, showed strong activity in the submicromolar range (IC50 0.2–0.6 μM), and were chosen for further biological experiments. Immunofluorescence staining and tubulin polymerization assays confirmed tubulin as the main target, but excluded its colchicine-binding site as a potential interacting unit. This was supported by the computational analysis, which revealed that the most potent ligands act on the extended colchicine site on the surface between interacting tubulin subunits, where they interfere with their polymerization and reveal pronounced antitumor properties. In addition, lead molecule 21 potently inhibited cancer cell migration, while it did not affect the viability of normal cells even at the highest concentration tested (100 µM).

Abstract ND08: NXP800: A first-in-class orally active, small-molecule HSF1 pathway inhibitor

Abstract We report the discovery of NXP800 — a first-in-class orally active HSF1 pathway inhibitor identified in our academic laboratory at the Institute of Cancer Research, London — and its progression into Phase I clinical trials in collaboration with our partner Nuvectis Pharma. HSF1 (Heat Shock Factor 1) is an ancient stress-inducible transcription factor that plays a key role in the transcriptional activation of the eukaryotic heat shock response. This leads to expression of many heat shock proteins, including molecular chaperones, and through this HSF1 acts as a master transcriptional regulator of proteostasis. Importantly, HSF1 is hijacked by cancer cells to activate a set of genes that overlaps with, but is not identical to, the classical heat shock response. The HSF1 pathway has been shown to play a key role in oncogenesis and the hallmark features of malignancy and is important in the initiation and progression of many experimental cancer models. Importantly, while HSF1 has been demonstrated to be important in mouse models of oncogenesis, knockout of the homologue of HSF1 has only modest effects in mice and flies under non-stressed laboratory conditions – supporting the potential for a therapeutic window. In addition, gene amplification, expression and activation of HSF1 has been shown to predict clinical outcome in many human cancers. Despite strong scientific rationale and pre-clinical validation, pharmaceutical exploitation has been limited because analysis of the available molecular structures of HSF1 shows it to be very difficult to drug. Because of this, we set out to discover inhibitors of the HSF1 pathway using an initial phenotypic screen that reports on the activation of the HSF1-regulated gene HSP72. This identified the bisamide series that was followed up by a detailed understanding of structure-activity relationships and medicinal chemistry optimization, particularly to improve pharmaceutical properties, leading to the selection of NXP800 (CCT361814) as the development candidate. NXP800 has good pharmacokinetic properties in mice, including oral bioavailability; also, pharmacodynamic studies show modulation of biomarkers of the HSF1 pathway and stress response in human tumour xenograft models. NXP800 exhibits striking therapeutic activity in xenografts of human ovarian clear cell ovarian cancer (OCCC) and endometrioid ovarian cancer, two serious conditions of high unmet medical need with limited treatment options – including prolonged tumour growth inhibition and regression at doses that show good toleration. The efficacy and tolerability data indicate a clear therapeutic window, supported by selectivity and safety profiting in pharmacology and kinome panels. In addition, we identified deficiency in ARID1A, a component of the SWI/SNF chromatin remodelling complex, as indicative of greater therapeutic responsiveness. This was confirmed through broader evaluation in human cancer cell line panels that also indicates therapeutic potential for NXP800 in additional cancer types. Studies to support mechanism of action and identify patient selection biomarkers will be described. A Phase 1 trial of NXP800 is now open, incorporating validated predictive, pharmacokinetic and pharmacodynamic biomarkers that provide a robust Pharmacological Audit Trail and support the prediction of dose-to-human. Citation Format: Paul Workman. NXP800: A first-in-class orally active, small-molecule HSF1 pathway 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 ND08.

P-250 A novel small molecule inhibits enzyme associated with colorectal cancer

Colorectal cancer (CRC) represents one of the most common malignant tumor with high morbidity worldwide. N6-methyladenosine (m6A) methylation, is the most frequently observed RNA modifications that tremendously contribute to cancer initiation, progression, and resistance. METTL3 (Methyltransferase like 3), is a predominant catalytic enzyme in the m6A methyltransferase system. METTL3 has been implicated in carcinogenesis in various cancers including colon cancer by stabilizing oncogenic mRNAs in an m6A-dependent manner which is why it is considered as an attractive target for cancer therapy. Here, we identified STM2457, as a selective small-molecule METTL3 inhibitor that exerts promising anticancer activity in CRC. We detected the mRNA and protein expression of METTL3 and its target oncogenes in human colorectal cancer cell line panel. The anti-tumor activities of STM2457 in human colorectal cancer cell line panel were investigated by MTT, wound healing, colony formation, western blot assays, and 3D spheroid model. m6A level of mRNA was measured by an m6A-RNA methylation quantification kit. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) was used to screen the target genes of METTL3. We discovered that STM2457 treatment exerted potential anticancer effects in CRC cells. Pharmacological inhibition of METTL3 by STM2457 reduced cellular proliferation, invasion, colony formation, and tumorsphere formation of CRC cells. STM2457 treatment reduced m6A level in CRC cells. Mechanistically, MeRIP-seq illustrated that SLC2A1 (Solute Carrier Family 2 Member 1) as a target of METTL3. The TCGA data analysis showed that SLC2A1 mRNA and protein were highly expressed in colorectal cancer tissues. In addition, the highly expressed SLC2A1 mRNA associated with metastasis and poor survival in CRC patients. Thus, the METTL3/m6A/SLC2A1 axis accelerated colorectal carcinogenesis. The data show that the blockade of METTL3/m6A/SLC2A1 axis by a potent and novel small-molecule inhibitor, STM2457 is a promising approach for treatment of CRC.

Hydroxychloroquine Sensitivity in Human and Canine Cancer Cell Line Panels Reveals Potential Genetic Signatures of Autophagy‐Dependence and Druggable Targets

BackgroundAutophagy is a lysosomal degradation pathway cells use to survive in stressed environments and has been implicated as a mechanism in cancer chemotherapy resistance. Hydroxychloroquine (HCQ) is an established autophagy inhibitor that is safe and well tolerated in humans. Subsets of cancers have also been found to be dependent on autophagy as a main means of survival even in optimal environmental conditions, and these autophagy‐dependent cancers respond favorably to autophagy inhibition alone or with combination treatments. Since subsets of the same cancer‐type are autophagy‐dependent and others are not, it is critical to understand which tumors will benefit most from autophagy inhibition in the clinic. Analyzing a large set of cell line drug responses can be beneficial in determining which tumor types will benefit from treatment with that drug or drugs with similar mechanisms of action. Further, since human and canine cancers have many commonalities including genetic similarities, comparisons between both species are important for providing new insights to cancer treatments across the oncology field. Multiple cell line data sets exist with publicly available data. The NCI60 is a panel of 60 human lines including breast, colon, lung, melanoma, ovarian, renal, prostate, leukemia, and central nervous system cancers. The Flint Animal Cancer Center (FACC) at Colorado State University has a panel of 36 canine lines with tumor types including osteosarcoma, transitional bladder cell carcinoma, melanoma, leukemia/lymphoma, mast cell, hemangiosarcoma, histiocytic sarcoma, mammary carcinoma, a soft tissue sarcoma, and a thyroid carcinoma.MethodsResponse of the canine FACC cell lines to HCQ was assessed by monitoring growth of fluorescently labeled cells. HCQ data for the human lines was obtained from the NCI60 website. A differential gene expression analysis was completed to determine genes most significantly correlated with HCQ sensitivity.ResultsHCQ Dm values (median dose of drug at which half of the cells are affected) at 96 hr in the canine cell lines ranged widely from less than 2 μM to almost 40 μM. Compared to the human cell lines whose data was collected at 48 hr using a 10 μM dose, canine cell lines appear to be more sensitive overall. However, when directly comparing just the human and canine osteosarcoma cell lines at 96 hr post treatment, they have similar differential HCQ sensitivities between the least and most affected cell lines, indicating that HCQ may have the same affect in other cancer types if the data is analyzed four days following drug dosing instead of just two days like the NCI60 data.ConclusionsThis analysis provides potential to produce a gene signature for tumors that respond favorably to HCQ treatment and may infer which tumors are more autophagy‐dependent and therefore will benefit from autophagy inhibition clinically. It also gives potential druggable gene targets for those tumors that will not benefit from autophagy inhibition (the HCQ‐resistant lines) based on which genes are upregulated in that group of tumors.Support or Funding InformationNCI R01CA190170This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of endogenous multidrug resistance 1 and P‐glycoprotein expression on anticancer drug resistance in colon cancer cell lines

P-glycoprotein (P-gp, multidrug resistance 1 (MDR1)) overexpression confers multidrug resistance to cancer cells, and P-gp in cell lines transfected with MDR1 or selected with chemotherapeutics significantly affect the anticancer drug efficacy. Although human cancer cell line panels consisting of defined tumor cell lines expressing endogenous P-gp have been used to screen drugs in pharmaceutical industries, endogenous P-gp affecting in vitro anticancer drug efficacy is unclear. The impact of P-gp expression on anticancer drug efficacy was assessed by using five colon cancer cell lines expressing varying endogenous P-gp levels and by selecting from the Cancer Cell Line Encyclopedia (CCLE). mRNA expression of MDR1 was considered as a surrogate of the protein expression of its gene product, P-gp, in CL-11, C2BBe1 and RKO cells, whereas P-gp protein expression in plasma membranes or crude membrane fractions was lower than expected from mRNA expression in CW-2 and CL-40 cells. The EC50 of paclitaxel and vinorelbine decreased in the presence of a P-gp inhibitor in CW-2 and CL-11 cells that highly express P-gp. No significant alterations in EC50 were observed in the CL-40, C2BBe1 and RKO cells, which show lower P-gp expression. Accordingly, the apparent in vitro efficacy of anticancer drugs could be underestimated if the endogenous P-gp expression is higher than in CL-11 cells. The effect of P-gp needs to be carefully evaluated in cell lines that highly express P-gp, which account for 1.5% of cancer cell lines, including all cancer types, and 14.5% of colon cancer cell lines in CCLE, considering the protein expression levels in plasma membranes.

Synthesis and Biological Evaluation of New Madecassic Acid Derivatives Targeting ERK Cascade Signaling.

In the present study, a series of novel madecassic acid derivatives was synthesized and screened against the National Cancer Institute's 60 human cancer cell line panel. Among them, compounds 5, 12, and 17 displayed potent and highly differential antiproliferative activity against 80% of the tumor cells harboring the B-RafV600E mutation within the nanomolar range. Structure-activity analysis revealed that a 5-membered A ring containing an α,β-unsaturated aldehyde substituted at C-23 with a 2-furoyl group seems to be crucial to produce this particular growth inhibition signature. In silico analysis of the cytotoxicity pattern of these compounds identified two highly correlated clinically approved drugs with known B-RafV600E inhibitory activity. Follow-up analysis revealed inhibition of the ERK signaling pathway through the reduction of cellular Raf protein levels is a key mechanism of action of these compounds. In particular, 17 was the most potent compound in suppressing tumor growth of B-RafV600E-mutant cell lines and displayed the highest reduction of Raf protein levels among the tested compounds. Taken together, this study revealed that modifications of madecassic acid structure can provide molecules with potent anticancer activity against cell lines harboring the clinically relevant B-RafV600E mutation, with compound 17 identified as a promising lead for the development of new anticancer drugs.

Girinimbine Inhibits the Proliferation of Human Ovarian Cancer Cells In Vitro via the Phosphatidylinositol-3-Kinase (PI3K)/Akt and the Mammalian Target of Rapamycin (mTOR) and Wnt/β-Catenin Signaling Pathways.

BackgroundWorldwide, ovarian cancer is increasing in prevalence and has a high mortality rate. Girinimbine is a carbazole alkaloid isolated from Murraya koenigii (the curry tree) and is used in Chinese herbal medicine. The aim of this study was to evaluate the effects of girinimbine on cell proliferation, cell migration, and apoptosis in human ovarian cancer cells in vitro.Material/MethodsA human ovarian cancer cell line panel, which included SKOV3 cells and the SV40 immortalized normal human ovarian cell line, were treated with increasing doses of girinimbine. Cell proliferation was evaluated using the MTT assay. Confocal immunofluorescence using 4′,6-diamidino-2-phenylindole (DAPI), Annexin-V, and propidium iodide (PI) were used to measure cell apoptosis. Cell migration and invasion were determined by transwell assays. Protein expression was determined by Western blot.ResultsGirinimbine inhibited SKOV3 ovarian cancer cell proliferation in a dose-dependent manner. The half maximal inhibitory concentration (IC50) of grinimbine was 15 μM for the SKOV3 cells, and 120 μM for the SV40 cells. Grinimbine treatment resulted in apoptosis of SKOV3 cells, from 2.2% in untreated cells to 58.8% at a dose of 30 μM, which was associated with an increase in the Bax/Bcl-2 ratio. Girinimbine inhibited cell migration and invasion of the SKOV3 cancer cells in vitro and inhibited the PI3K/AKT/mTOR and Wnt/β-catenin signaling pathways.ConclusionsGirinimbine, a carbazole alkaloid used in Chinese herbal medicine, inhibited the proliferation and cell migration of human ovarian cancer cells in vitro, in a dose-dependent manner, via the PI3K/Akt/mTOR and Wnt/β-catenin signaling pathways.

Abstract 310: AZD4573, a novel CDK9 inhibitor, rapidly induces cell death in hematological tumor models through depletion of Mcl1

Abstract Cyclin-dependent kinase 9 (CDK9) regulates elongation of transcription through phosphorylation of RNA polymerase II (pSer2-RNAPII), and its short-term inhibition downregulates genes with short-lived transcripts and labile proteins. We developed a novel and selective CDK9 inhibitor, AZD4573, with nanomolar potency and physicochemical properties suitable for IV administration and short exposure. Initial transcriptomic and proteomic analyses were performed on MCF7 breast cancer cells treated with AZD4573 for 4h or 8h to determine which genes were most rapidly modulated by CDK9 inhibition. Of the 1,290 genetically-linked, cancer-associated genes analyzed on these platforms, MCL1 ranked amongst the top as most robustly and sustainably suppressed at both the mRNA and protein level with AZD4573 treatment. MCL1 is an anti-apoptotic member of the BCL2 gene family and is frequently amplified and/or over-expressed in various cancers, conferring a survival advantage to the tumor cell. Mcl1 has also been notoriously difficult to drug with small molecules, although inhibitors are now in early clinical development. Transient inhibition of CDK9 with AZD4573, however, provides a means to indirectly target Mcl1 and induce apoptosis in Mcl1-addicted tumors. In human cancer cell line panel screens, AZD4573 demonstrated the ability to induce rapid caspase activation (6h) and loss of viability (24h) across a diverse set of hematological cancers (median caspase EC50=30nM, GI50=11nM) but with minimal effect on solid tumors (median EC50 &amp; GI50 &amp;gt;30μM). The activity of AZD4573 in the cell line panels displayed a strong correlation with that of the Mcl1 inhibitor, AZD5991 (r2 = 0.837), suggesting transient CDK9 inhibition induces cell death through an Mcl1-dependent mechanism. Furthermore, knockdown of BAK and/or BAX in sensitive cell lines rescued the cells from AZD4573-mediated caspase induction and cell death, underscoring the mechanistic involvement of the intrinsic apoptosis pathway. In the AML cell line, MV411, AZD4573 led to a rapid dose- and time-dependent decrease in pSer2-RNAPII with concomitant loss of Mcl1 mRNA and protein, resulting in caspase induction and loss of cell viability. In contrast, protein levels of the other anti-apoptotic proteins, Bcl2 and BclxL, remained unchanged out to 6h post-AZD4573 treatment of MV411 cells. With intermittent dosing of AZD4573, in vivo efficacy was observed in multiple hematological tumor xenograft models. AZD4573 also exhibited the ability to drive deeper and more durable regressions in combination with multiple targeted agents. Based on our findings, AZD4573 could be an effective treatment option, either as single agent or in combination, for patients with hematological malignancies. Thus, we have initiated a first-time-in-human study with AZD4573 in October 2017 (NCT03263637). Citation Format: Justin Cidado, Theresa Proia, Scott Boiko, Maryann San Martin, Steven Criscione, Douglas Ferguson, Wenlin Shao, Lisa Drew. AZD4573, a novel CDK9 inhibitor, rapidly induces cell death in hematological tumor models through depletion of Mcl1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 310.

Triazolo[4,5-d]pyrimidines as Validated General Control Nonderepressible 2 (GCN2) Protein Kinase Inhibitors Reduce Growth of Leukemia Cells

Cellular stress signals activate adaptive signaling pathways of the mammalian integrated stress response (ISR), of which the unfolded protein response (UPR) is a subset. These pathways converge at the phosporylation of eIF2α. Drug-like, potent and selective chemical inhibitors (valid chemical probes) targeting major ISR kinases have been previously identified, with the exception of GCN2. We synthesized and evaluated a series of GCN2 inhibitors based on a triazolo[4,5-d]pyrimidine scaffold. Several compounds potently inhibited GCN2 in vitro and displayed good selectivity over the related kinases PERK, HRI, and IRE1. The compounds inhibited phosporylation of eIF2α in HEK293T cells with an IC50 < 150 nM, validating them as chemical probes for cellular studies. These probes were screened against the National Cancer Institute NCI-60 human cancer cell line panel. Uniform growth inhibition was observed in the leukemia group of cell lines. Growth inhibition in the most sensitive cell lines coincided with high GCN2 mRNA expression levels. Oncomine analysis revealed high GCN2 expression accompanied by lower asparagine synthetase (ASNS) expression in patient-derived acute lymphoblastic leukemias with B-Cell origins (B-ALL) as well. Notably, asparaginase, which depletes amino acids and triggers GCN2 activity, is a licensed, first-line B-ALL treatment. Thus, we hypothesize that leukemias exhibiting high GCN2 expression and low ASNS expression may be susceptible to pharmacologic GCN2 inhibition.