Multiple Human Tumor Cell Lines Research Articles

Overcoming Solubility Challenges: Liposomal isoCoQ-Carbazole as a Promising Anti-Tumor Agent for Inoperable and Radiation-Insensitive cancers.

This study evaluated the potential of isoCoQ-Carbazole, a diheterocyclic analog of isoCA-4, as an anti-tumor agent. To overcome its low aqueous solubility, liposomes were developed as a delivery system for the compound. In vitro experiments showed that loaded liposomes exhibited similar activity to the free form on multiple human tumor cell lines. In vivo experiments using a palliative intratumoral injection chemotherapy approach further demonstrated that isoCoQ-Carbazole loaded liposomes significantly reduced tumor growth in a CA-4-resistant HT29 cell model, without inducing any observable toxicity or weight loss in the treated mice. These findings suggest that liposomal isoCoQ-Carbazole may hold promise as a potential therapeutic agent for the treatment of inoperable, radiation-insensitive cancers.

Abstract 2620: BCG016, a first-in-class bispecific antibody-drug conjugate targeting 5T4 and MUC1, demonstrates potent preclinical anti-tumor activity

Abstract 5T4 is a highly expressed tumor-associated antigen associated with adverse clinical outcomes in solid tumors, and is an attractive therapeutic target due to low expression on normal adult tissues. While several therapeutic agents targeting 5T4 antigen are currently being evaluated in human clinical trials, none have yet entered the market. Another target, MUC1, is currently under clinical investigation, with most drugs in clinical trials showing limited efficacy due to shedding of the target antigen MUC1-N. 5T4 and MUC1 are commonly co-expressed in various solid tumors, including lung, breast, colorectal and pancreatic cancers, suggesting that targeting both antigens with a single drug could be a promising therapeutic strategy. We previously identified and evaluated antibodies targeting human 5T4 and MUC1-C (cleaved MUC1) using RenLite® fully human common light chain mice. Here, selected antibodies were constructed into 5T4 × MUC1 bispecific antibodies (bsAb). The 5T4-MUC1 bsAb demonstrated high avidity in multiple human tumor cell lines by flow cytometry. Notably, the parental 5T4 antibody also binds strongly to many tumor cell lines. Internalization assays demonstrate that the internalization of 5T4-MUC1 bsAb in breast cancer cells was enhanced compared to the parental anti-5T4 and anti-MUC1 monoclonal antibodies, as measured by Incucyte® live cell imaging. Subsequently, the 5T4-MUC1 bsAb was then conjugated with monomethyl auristatin E (MMAE) to generate 5T4 x MUC1 bsADC (BCG016). Cytotoxicity assays indicate that BCG016 improved tumor cell killing in vitro compared to benchmark ADCs, and in vivo efficacy studies showed superior anti-tumor efficacy of BCG016 in patient-derived NSCLC xenografts compared to benchmark ADCs and parental ADC combination therapy. In summary, we have generated a novel bispecific ADC targeting 5T4 and the cleaved MUC1-C protein, which remains membrane-bound on tumor cells. The 5T4-MUC1 bsADC showed superior anti-tumor efficacy in PDX models and has the potential to be a novel therapeutic for 5T4 and MUC1 co-expressing tumors. Citation Format: Zhenyan Han, Chengzhang Shang, Yifu Zhang, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. BCG016, a first-in-class bispecific antibody-drug conjugate targeting 5T4 and MUC1, demonstrates potent preclinical anti-tumor activity [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 2620.

Anticancer Potential of a Synthetic Quinoline, 9IV-c, by Inducing Apoptosis in A549 Cell and In vivo BALB/c Mice Models.

In a previous work from the author of this study, the compound of 9IV-c, ((E)-2-(3,4- dimethoxystyryl)-6,7,8-trimethoxy-N-(3,4,5-trimethoxyphenyl)quinoline-4-amine) was synthesized, and the effects of potent activity on the multiple human tumor cell lines were evaluated considering the spindle formation together with the microtubule network. Accordingly, cytotoxic activity, apoptotic effects, and the therapeutic efficiency of compound 9IV-c on A549 and C26 cell lines were investigated in this study. The compound 9IV-c demonstrated high cytotoxicity against A549 and C26 cell lines with IC50 = 1.66 and 1.21 μM, respectively. The flow cytometric analysis of the A549 cancer cell line treated with compound 9IVc showed that This compound induced cell cycle arrest at the G2/M phase and apoptosis. Western blotting analysis displayed that compound 9IV-c also elevated the Bax/Bcl-2 ratio and increased the activation of caspase-9 and -3 but not caspase-8. These data presented that the intrinsic pathway was responsible for 9IV-c -induced cell apoptosis. In vivo studies demonstrated that treatment with the compound of 9IV-c at 10 mg/kg dose led to a decrease in tumor growth compared to the control group. It was found that there was not any apparent body weight loss in the period of treatment. Also, in the vital organs of the BALB/c mice, observable pathologic changes were not detected.

EXTH-15. A NOVEL IMMUNE-BASED TREATMENT STRATEGY FOR COMBATTING PROTEIN TUMOR HETEROGENEITY

Abstract Cell-based immunotherapy has revolutionized the treatment of many hematological malignancies but has mostly fail against solid tumors, including glioblastoma (GBM). The reason for this failure is multi-fold, but one of the most significant barriers is the extensive intratumoral heterogeneity that characterizes these tumors. Unfortunately, this problem has not been eliminated by personalized medicine strategies aimed at sequencing individual tumors and targeting the uncovered neoantigens. Thinking “outside the box” led us to examine other types of tumor-specific alterations that might more ubiquitously characterize tumor cells, while still being targetable by an immune-based platform. Our investigations led us to the phospholipid phosphatidylserine (PS). PS is typically present on the cytoplasm-facing side of the plasma membrane, where it remains out of view to the immune system. Cellular stresses can lead to dysregulation of processes that keep PS facing internally and instead promote PS exposure on the cell surface. We show here that tumor cells frequently lose the capacity to regulate their plasma membrane and exhibit detectable levels of PS on their surface. Moreover, inducing cell stress through radiation and chemotherapy of tumor cells further enhanced PS exposure. In this study, we aimed to target PS with an immune-based therapy. To this end, we engineered a bi-specific T cell engager (BiTE) consisting of the natural high-affinity PS receptor TIM4 linked to an anti-CD3scFv. This TIM4 BiTE is hypothesized to redirect all T cells to PS, resulting in T cell-mediated killing of tumor cells with exposed PS. Indeed, TIM4 BiTE facilitated potent killing of multiple human tumor cell lines in vitro, including in models of protein heterogeneous tumors. When TIM4 BiTE was delivered intratumorally to a heterogeneous glioma-bearing mouse, survival was significantly extended. These data provide evidence that targeting PS with an immune-based platform represents a potential novel treatment strategy for combatting tumor heterogeneity.

Tryptophan depletion sensitizes the AHR pathway by increasing AHR expression and GCN2/LAT1-mediated kynurenine uptake, and potentiates induction of regulatory T lymphocytes

BackgroundIndoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan-dioxygenase (TDO) are enzymes catabolizing the essential amino acid tryptophan into kynurenine. Expression of these enzymes is frequently observed in advanced-stage cancers and is associated...

LncRNA LINC00960 promotes apoptosis by sponging ubiquitin ligase Nrdp1-targeting miR-183-5p.

The ubiquitin ligase Nrdp1/RNF41 promotes the ubiquitin-dependent degradation of multiple important substrates, including BRUCE/BIRC6, a giant ubiquitin-conjugating enzyme inhibiting both apoptosis and autophagy. miR-183-5p is associated with various malignancies potentially by targeting dozens of genes. Here, we show that the lncRNA LINC00960 binds to the Nrdp1-targeting miR-183-5p and promotes apoptosis. Compared to other known miR-183-5p targets, Nrdp1 mRNA is among the few with top scores to complement miR-183-5p. miR-183-5p binds to the 3'UTR of Nrdp1 mRNA and downregulates Nrdp1 at both the mRNA and protein levels. The miR-183-5p mimics inhibit DNA damage-induced apoptosis probably by upregulating BRUCE level, whereas the miR-183-5p inhibitor suppresses the effects of miR-183-5p. LINC00960 is the noncoding RNA with the highest score to complement miR-183-5p. LINC00960 overexpression reduces, but its knockdown increases, the level of miR-183-5p, whereas LINC00960 overexpression increases, but its knockdown decreases, the level of Nrdp1 and apoptosis. Importantly, the expression of LINC00960, which is associated with multiple types of tumors, positively correlates with that of Nrdp1 in several tumors but inversely correlates with that of miR-183-5p in multiple human tumor cell lines, as analysed by quantitative PCR. Thus, miR-183-5p downregulates Nrdp1 expression and inhibits apoptosis, whereas LINC00960 upregulates Nrdp1 and promotes apoptosis by inhibiting miR-183-5p. These results may provide new ideas for the prevention, diagnosis and treatment of apoptosis-related diseases, such as tumors and neurodegenerative diseases.

Abstract C059: Targeting tumor intrinsic p38 MAPK signaling to block IL-1α-mediated inflammatory tumor-stromal crosstalk in pancreatic cancer

Abstract Introduction: Pancreatic cancer (PDAC) remains a major therapeutic challenge due to its innate and acquired chemoresistance. Activating KRAS mutations, a hallmark of PDAC, mediate autocrine effects and crosstalk within the tumor microenvironment (TME), by inducing cytokines and chemokines that promote a pro-inflammatory and immunosuppressive stroma. We have identified KRAS-driven interleukin-1α (IL-1α) as a critical mediator of the inflammatory response due to its pleiotropic effects on cancer-associated fibroblast (CAF) activation and immune evasion, however the mechanisms that regulate IL-1α production remain poorly understood. In our efforts to identify targetable kinase pathways downstream of KRAS that are involved in IL-1α expression, we identified p38 stress-associated MAPK α (p38α MAPK) as a key regulatory pathway involved in IL-1α production in PDAC tumor cells. Methods: KRAS-mutant tumor cells were treated with pharmacologic inhibitors to pathways downstream KRAS and IL1A levels determined in response by qPCR. Mutant KRAS G12D plasmid was overexpressed in mouse embryonic fibroblasts, and IL-1α levels were determined in response by qPCR and ELISA with and without p38 inhibition. Inhibition of phosphorylated p38 MAPK was achieved pharmacologically with ARRY-614 and genetically with an shRNA lentiviral system in human and murine PDAC cell lines. ChIP-qPCR was performed on a human PDAC cell line with and without p38 MAPK inhibition. Tumor cells pre-treated with ARRY-614 were cocultured with human pancreatic stellate cells, and inflammatory CAF genes were measured by qPCR. Ptf1aCre/+;LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice, a highly aggressive PDAC genetically engineered mouse model (GEMM), were treated daily with ARRY-614, chemotherapy, or combination therapy for downstream analysis. Results: Both pharmacologic and genetic inhibition of p38 MAPK significantly reduces IL1A transcription and protein levels in multiple human and murine PDAC tumor cell lines. Furthermore, p38 MAPK inhibition reduces binding of NF-kB to the IL1A promoter and prevents IL-1α-mediated activation of CAFs, characterized by a reduction in pro-inflammatory genes. Lastly, ARRY-614 in combination with chemotherapy significantly reduces overall tumor burden and favorably remodels the tumor microenvironment in a PDAC GEMM. Conclusions: These findings provide a compelling rationale to explore p38 MAPK inhibition in tumor cells as a novel treatment strategy to suppress IL-1α-mediated stromal activation and to combine p38 MAPK inhibition with chemotherapy to overcome therapeutic resistance through modulation of the stromal and immune microenvironment in PDAC. Citation Format: Samara P. Singh, Austin R. Dosch, Siddharth Mehra, Iago de Castro Silva, Anna Bianchi, Vanessa Tonin-Garrido, Nilesh U. Deshpande, Zhiqun Zhou, Nagaraj Nagathihalli, Jashodeep Datta, Nipun B. Merchant. Targeting tumor intrinsic p38 MAPK signaling to block IL-1α-mediated inflammatory tumor-stromal crosstalk in pancreatic cancer [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 C059.

HOA-DN30: a highly effective humanized single-arm MET antibody inducing remission of ‘MET-addicted’ cancers

BackgroundThe tyrosine kinase receptor encoded by the MET oncogene is a major player in cancer. When MET is responsible for the onset and progression of the transformed phenotype (MET-addicted cancers), an efficient block of its oncogenic activation results in potent tumor growth inhibition.MethodsHere we describe a molecular engineered MET antibody (hOA-DN30) and validate its pharmacological activity in MET-addicted cancer models in vitro and in vivo. Pharmacokinetics and safety profile in non-human primates have also been assessed.ResultshOA-DN30 efficiently impaired MET activation and the intracellular signalling cascade by dose and time dependent removal of the receptor from the cell surface (shedding). In vitro, the antibody suppressed cell growth by blocking cell proliferation and by concomitantly inducing cell death in multiple MET-addicted human tumor cell lines. In mice xenografts, hOA-DN30 induced an impressive reduction of tumor masses, with a wide therapeutic window. Moreover, the antibody showed high therapeutic efficacy against patient-derived xenografts generated from MET-addicted gastric tumors, leading to complete tumor regression and long-lasting effects after treatment discontinuation. Finally, hOA-DN30 showed a highly favorable pharmacokinetic profile and substantial tolerability in Cynomolgus monkeys.ConclusionshOA-DN30 unique ability to simultaneously erase cell surface MET and release the ‘decoy’ receptor extracellular region results in a paramount MET blocking action. Its remarkable efficacy in a large number of pre-clinical models, as well as its pharmacological features and safety profile in non-human primates, strongly envisage a successful clinical application of this novel single-arm MET therapeutic antibody for the therapy of MET-addicted cancers.

In Silico Exploration of Novel Tubulin Inhibitors: A Combination of Docking and Molecular Dynamics Simulations, Pharmacophore Modeling, and Virtual Screening.

Microtubules play a critical role in mitosis and cell division and are regarded as an excellent target for anticancer therapy. Although microtubule-targeting agents have been widely used in the clinical treatment of different human cancers, their clinical application in cancer therapy is limited by both intrinsic and acquired drug resistance and adverse toxicities. In a previous work, we synthesized compound 9IV-c, ((E)-2-(3,4-dimethoxystyryl)-6,7,8-trimethoxy-N-(3,4,5-trimethoxyphenyl)quinoline-4-amine) that showed potent activity against multiple human tumor cell lines, by targeting spindle formation and/or the microtubule network. Accordingly, in this study, to identify potent tubulin inhibitors, at first, molecular docking and molecular dynamics studies of compound 9IV-c were performed into the colchicine binding site of tubulin; then, a pharmacophore model of the 9IV-c-tubulin complex was generated. The pharmacophore model was then validated by Güner–Henry (GH) scoring methods and receiver operating characteristic (ROC) analysis. The IBScreen database was searched by using this pharmacophore model as a screening query. Finally, five retrieved compounds were selected for molecular docking studies. These efforts identified two compounds (b and c) as potent tubulin inhibitors. Investigation of pharmacokinetic properties of these compounds (b and c) and compound 9IV-c displayed that ligand b has better drug characteristics compared to the other two ligands.

Effects of substituent pattern on the intracellular target of antiproliferative benzo[b]thiophenyl chromone derivatives.

A new biological scaffold was produced by replacing the 6π-electron phenyl ring-B of a natural flavone skeleton with a 10π-electron benzothiophene (BT). Since aromatic rings are important for ligand protein interactions, this expansion of the π-electron system of ring-B might change the bioactivity profile. One of the resulting novel natural product-inspired compounds, 2-(benzo[b]thiophen-3-yl)-5-hydroxy-7-isopropoxy-6-methoxyflavone (6), effectively arrested the cell cycle at the G2/M phase and displayed significant antiproliferative effects with IC50 values of 0.05-0.08μM against multiple human tumor cell lines, including a multidrug resistant line. A structure-activity relationship study revealed that a 10π-electron system with high aromaticity, juxtaposed 4-oxo and 5-hydroxy groups, and 7-alkoxy groups were important for potent antimitotic activity. Interestingly, two BT-flavonols (3-hydroxyflavone), 16 and 20, with 3-hydroxy and 5-alkoxy groups, induced distinct biological profiles affecting the cell cycle at the G1/S phase by inhibition of DNA replication through an interaction with topoisomerase I.

Abstract PS3-04: Breast tumor targeted fluorescence guidance for intraoperative margin assessment

Abstract BACKGROUND: A majority of the 270,000 women diagnosed with breast cancer in the United States annually are candidates for breast conserving surgery (lumpectomy), however, up to 30% of patients require reoperation due to positive margins. The ideal resolution is to make residual in-breast disease visible at the time of surgery. The goal of this research is to develop a systemically administered fluorescent molecular imaging agent that is targeted to breast tumors for intraoperative margin assessment using fluorescence guided surgery (FGS). METHODS: To identify targetable cell-surface markers for use in FGS, gene expression profiling of publicly available Affymetrix mRNA microarray data from patients with invasive ductal or invasive lobular carcinoma tumors (n=34) and surrounding normal breast tissues (n=32) was performed. Protein expression of select targets was determined by immunohistochemistry (IHC) staining of patient tissue specimens. A humanized CEACAM6 (carcinoembryonic cell adhesion molecule 6) specific antibody fragment (scFv-Fc [IgG4]) was conjugated to the near-infrared IRDye800CW fluorescent dye (LiCor) by NHS-ester linkage to create CEACAM6-800. The conjugate was characterized for in vitro specificity by immunocytochemistry (ICC) and live cell uptake studies. As proof-of-principle, the conjugate (15 µg) was intravenously injected into nude mice bearing orthotopic human MCF-7 breast cancer mammary fat pad (MFP) xenograft tumors with endogenous expression of CEACAM6. FGS was performed using the “SurgVision” clinical imaging platform (approved for clinical use in Europe) at 24 h post-injection.RESULTS: We identified 263 genes with higher mRNA expression in tumor relative to normal. From this list, we selected 9 genes for confirmation of protein expression by IHC. Of the 9 targets, only CEACAM6 had no protein expression in normal breast tissues and robust expression in ~50% of tumors. ICC staining using the CEACAM6-800 conjugate and multiple human ductal carcinoma tumor cell lines confirmed the cell surface localization of CEACAM6. In live-cell uptake studies, CEACAM6-800 accumulated on the cell surface as early as 10 min after addition to the media and was internalized after 90 min. Blocking studies demonstrated specificity. In the proof-of-principal FGS study, we observed high fluorescence in MCF-7 MFP tumors relative to background and the tumors and their residuals were completely removed in the first attempt. CONCLUSION: We have confirmed CEACAM6 protein as a cell-surface adhesion receptor target for breast cancer FGS. The CEACAM6-800, fluorescent-dye to antibody-fragment conjugate was prepared and tested in vitro for sub-cellular localization and specific uptake. In vivo studies demonstrated specific uptake into MFP tumors and FGS for tumor resection. We are currently performing studies to determine the in vivo pharmacokinetics of CEACAM6-800 uptake and clearance in tumor and normal tissues and pre-clinical post-surgery survival using our pre-clinical models. These studies will enable future translational studies. Citation Format: Narges Tafreshi, Mikalai Budzevich, Weihong Sun, Daruka Mahadevan, Marie Catherine Lee, David L Morse. Breast tumor targeted fluorescence guidance for intraoperative margin assessment [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS3-04.

Synthesis of Gelatin-Based Dual-Targeted Nanoparticles of Betulinic Acid for Antitumor Therapy.

Betulinic acid (BA) is a natural antitumor agent and has biological activity against multiple human tumor cell lines with low cytotoxicity to normal cells, while the high hydrophobicity and the short half-life of this compound limit its clinical application. Here, gelatin-based dual-targeted nanoparticles of BA are promising to solve this problem. Hydrophobic BA is loaded in cyclodextrin to increase its solubility and prolong the circulation time in vivo. The nanoscale drug delivery systems can further enhance the bioavailability and the antitumor effect of BA and are passively targeted to the tumor tissue sites by enhanced permeability and retention effect. The RGD sequence of gelatin specifically recognizes tumor cells and brings agents into tumor cells. The nanoparticles were characterized by transmission electron microscopy, Fourier transform infrared, nuclear magnetic resonance, etc. In addition, we observed antitumor activity of the nanoparticles using both cell-based assays and mouse xenograft tumors, which proved that betulinic acid/gelatin-γ-cyclodextrin nanoparticles had a better tumor inhibition effect than betulinic acid/γ-cyclodextrin inclusion compound.

Abstract 1765: A humanized anti-CD47 monoclonal antibody that directly kills human tumor cells and has additional unique functional characteristics

Abstract CD47 is a cell surface glycoprotein that interacts with signal regulatory protein alpha (SIRPα) on macrophages and dendritic cells triggering a “don't eat me” signal that inhibits phagocytosis. Many tumors evade immune surveillance by overexpressing CD47, thereby preventing their recognition by phagocytes. Blocking the interaction of SIRPα/CD47 promotes phagocytosis and tumor cell destruction leading to a reduction in tumor burden. We have developed a humanized anti-CD47 antibody, AO-176, that blocks the interaction between CD47 and SIRPα and exhibits several additional novel functional characteristics. These characteristics include the induction of cell death in multiple human tumor cell lines in a cell autonomous manner (not ADCC), assessed by an increase in phosphatidylserine/7AAD positive staining. A second novel characteristic is enhanced binding to tumor cells at acidic pH. AO-176 binds to human tumor cell lines in the high pM to low nM range at physiologic pH, however, binding is enhanced up to 20-fold at an acidic pH of 6.5. The acidic pH of the tumor microenvironment which ranges from 6.4-7.2 is characteristic of solid tumors and correlates with tumor progression and metastasis. As a result of this enhanced binding at acidic pH, AO-176 has the potential added advantage of tumor-specific targeting. A third novel characteristic exhibited by AO-176 is its selective binding to tumor cells while exhibiting reduced binding to normal cells including red blood cells (cynomolgus monkey and human), endothelial, epithelial and skeletal muscle cells. In addition to these novel characteristics, AO-176 also exhibits dose-dependent efficacy in multiple mouse tumor models. Taken together, the unique combination of functional characteristics of AO-176, including induction of cell-autonomous killing, enhanced binding to tumor cells at acidic pH, significantly reduced binding to normal cells and potent in vivo efficacy provides the preclinical rationale for further development. Citation Format: Robyn Puro, Katherine Liu, Benjamin Capoccia, Michael Donio, Ronald Hiebsch, Myriam Bouchlaka, Alun Carter, Pamela Manning, Kathleen Crowley, Robert Karr. A humanized anti-CD47 monoclonal antibody that directly kills human tumor cells and has additional unique functional characteristics [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 1765.

Tumor cell-intrinsic programmed death protein 1 expression and induction in human cancer cell lines

Abstract The programmed death protein 1 (PD-1) and ligand (PD-L1) axis inhibits T cell activation and reduces anti-tumor immunity. Most studies of the PD-1/PD-L1 axis test extrinsic, immune-dependent effects. However, tumor cell-intrinsic PD-L1 regulates immune-independent tumor mTOR signals, autophagy, and tumor initiating cell generation. Melanoma cell-intrinsic PD-1 regulates melanoma growth and mTOR. We tested PD-1 expression and induction in multiple human tumor cell lines. We detected PD-1 in human cell lines in vitro from ES2 ovarian cancer, VMM122 melanoma, and RT4 bladder cancer by flow cytometry, Western blot and RT-PCR. All lines were negative for the other PD-L1 receptor, CD80, by flow cytometry. Incubating lines with IFNα or IFNγ for 48 hours further augmented PD-1 expression as detected by flow, Western blot and RT-PCR. αPD-1 antibody (50 μg/ml) reduced proliferation of RT4 bladder cancer cells in vitro, demonstrating a direct PD-1 signal effect in these cells, and αPD-L1 similarly reduced in vitro proliferation. Interestingly, anti-PD-1 effects on reducing proliferation were similar: 41.5% reduction; p<0.0001. We used CRISPR/Cas9 to generate PD-L1KO RT4 cells. αPD-L1 did not reduce in vitro proliferation as expected, but unexpectedly, αPD-1 also had no anti-proliferative effect on PD-L1KO RT4 cells, suggesting a PD-L1/PD-1 interaction. In murine B16 melanoma, we generated PD-L1KO and PD-1KO cells using CRISPR/Cas9. Cell-intrinsic PD-L1 and PD-1 downregulate chemokine production. Among chemokines analyzed, PD-L1 suppressed CCL2 and CCL5, PD-1 suppressed CCL4 and CCL7, and both suppressed CCL3 and CXCL2. Understanding intrinsic PD-1 and PD-L1 effects on tumor cells can be exploited to improve cancer immunotherapy

The FDA-approved drug sofosbuvir inhibits Zika virus infection

The rapidly expanding Zika virus (ZIKV) epidemic has affected thousands of individuals with severe cases causing Guillain-Barré syndrome, congenital malformations, and microcephaly. Currently, there is no available vaccine or therapy to prevent or treat ZIKV infection. We evaluated whether sofosbuvir, an FDA-approved nucleotide polymerase inhibitor for the distantly related hepatitis C virus, could have antiviral activity against ZIKV infection. Cell culture studies established that sofosbuvir efficiently inhibits replication and infection of several ZIKV strains in multiple human tumor cell lines and isolated human fetal-derived neuronal stem cells. Moreover, oral treatment with sofosbuvir protected against ZIKV-induced death in mice. These results suggest that sofosbuvir may be a candidate for further evaluation as a therapy against ZIKV infection in humans.

Triethylated chromones with substituted naphthalenes as tubulin inhibitors

Previously synthesized 2-(benzo[b]thiophene-3′-yl)-6,8,8-triethyldesmosdumotin B (1, TEDB-TB) and 2-(naphth-1′-yl)-6,8,8-triethyldesmosdumotin B (2) showed potent activity against multiple human tumor cell lines, including a multidrug-resistant (MDR) subline, by targeting spindle formation and/or the microtubule network. Consequently, ester analogues of hydroxylated naphthyl substituted TEBDs (3–5) were prepared and evaluated for their effects on tumor cell proliferation and on tubulin assembly. Among all new compounds, compound 6, a 4′-acetoxynaphthalen-1′-yl derivative, displayed the most potent antiproliferative activity (IC50 0.2–5.7μM). Selected analogues were confirmed to be tubulin assembly inhibitors in cell-free and cell-based assays using MDR tumor cells. The new analogues partially inhibited colchicine binding to tubulin, suggesting their binding mode would be different from that of colchicine. This observation was supported by computational docking model analyses. Thus, the newly synthesized triethylated chromones with esterified naphthalene groups have good potential for development as a new class of mitotic inhibitors that target tubulin.

Reciprocal positive selection for weakness - preventing olaparib resistance by inhibiting BRCA2.

Human tumor heterogeneity promotes therapeutic failure by increasing the likelihood of resistant cell subpopulations. The PARP-1 inhibitor olaparib is approved for use in BRCA-mutated ovarian cancers but BRCA2-reversion mutations lead to functional homologous recombination repair (HRR) and olaparib resistance. To overcome that resistance and expand use of PARP1 inhibition to cancers with functional HRR, we developed an antisense strategy to render the majority of tumor cells in a population BRCA2-deficient. We predicted that this strategy would render HRR-proficient tumor cells sensitive to olaparib and prevent emergence of resistance in a tumor cell population heterogeneous for HRR proficiency. We report that BRCA2 downregulation sensitized multiple human tumor cell lines (but not non-cancer human kidney cells) to olaparib and, combined with olaparib, increased aneuploidy and chromosomal translocations in human tumor cells. In a mixed HRR-proficient and HRR-deficient cell population, olaparib monotherapy allowed outgrowth of HRR-proficient cells resistant to subsequent olaparib treatment. Combined BRCA2 inhibition and olaparib treatment prevented selection of HRR-proficient cells and inhibited proliferation of the entire population. Treatment with BRCA2 siRNA and olaparib decreased ovarian xenograft growth in mice more effectively than either treatment alone. In vivo use of BRCA2 antisense oligonucleotides may be a viable option to expand clinical use of olaparib and prevent resistance.

Forkhead transcription factor FoxF1 interacts with Fanconi anemia protein complexes to promote DNA damage response.

Forkhead box F1 (Foxf1) transcription factor is an important regulator of embryonic development but its role in tumor cells remains incompletely understood. While 16 proteins were characterized in Fanconi anemia (FA) core complex, its interactions with cellular transcriptional machinery remain poorly characterized. Here, we identified FoxF1 protein as a novel interacting partner of the FA complex proteins. Using multiple human and mouse tumor cell lines and Foxf1+/- mice we demonstrated that FoxF1 physically binds to and increases stability of FA proteins. FoxF1 co-localizes with FANCD2 in DNA repair foci in cultured cells and tumor tissues obtained from cisplatin-treated mice. In response to DNA damage, FoxF1-deficient tumor cells showed significantly reduced FANCD2 monoubiquitination and FANCM phosphorylation, resulting in impaired formation of DNA repair foci. FoxF1 knockdown caused chromosomal instability, nuclear abnormalities, and increased tumor cell death in response to DNA-damaging agents. Overexpression of FoxF1 in DNA-damaged cells improved stability of FA proteins, decreased chromosomal and nuclear aberrations, restored formation of DNA repair foci and prevented cell death after DNA damage. These findings demonstrate that FoxF1 is a key component of FA complexes and a critical mediator of DNA damage response in tumor cells.

Targeting the membrane-anchored serine protease testisin with a novel engineered anthrax toxin prodrug to kill tumor cells and reduce tumor burden.

The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg's native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent.

The clinical development candidate CCT245737 is an orally active CHK1 inhibitor with preclinical activity in RAS mutant NSCLC and Eµ-MYC driven B-cell lymphoma.

CCT245737 is the first orally active, clinical development candidate CHK1 inhibitor to be described. The IC50 was 1.4nM against CHK1 enzyme and it exhibited>1,000-fold selectivity against CHK2 and CDK1. CCT245737 potently inhibited cellular CHK1 activity (IC50 30-220nM) and enhanced gemcitabine and SN38 cytotoxicity in multiple human tumor cell lines and human tumor xenograft models. Mouse oral bioavailability was complete (100%) with extensive tumor exposure. Genotoxic-induced CHK1 activity (pS296 CHK1) and cell cycle arrest (pY15 CDK1) were inhibited both in vitro and in human tumor xenografts by CCT245737, causing increased DNA damage and apoptosis. Uniquely, we show CCT245737 enhanced gemcitabine antitumor activity to a greater degree than for higher doses of either agent alone, without increasing toxicity, indicating a true therapeutic advantage for this combination. Furthermore, development of a novel ELISA assay for pS296 CHK1 autophosphorylation, allowed the quantitative measurement of target inhibition in a RAS mutant human tumor xenograft of NSCLC at efficacious doses of CCT245737. Finally, CCT245737 also showed significant single-agent activity against a MYC-driven mouse model of B-cell lymphoma. In conclusion, CCT245737 is a new CHK1 inhibitor clinical development candidate scheduled for a first in man Phase I clinical trial, that will use the novel pS296 CHK1 ELISA to monitor target inhibition.