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Combinatorial methods enable the synthesis of chemical libraries on scales of millions to billions of compounds, but the ability to efficiently screen and sequence such large libraries has remained a major bottleneck for molecular discovery. We developed a novel technology for screening and sequencing libraries of synthetic molecules of up to a billion compounds in size. This platform utilizes the Fiber-optic Array Scanning Technology (FAST) to screen bead-based libraries of synthetic compounds at a rate of 5 million compounds per minute (~83,000 Hz). This ultra-high-throughput screening platform has been used to screen libraries of synthetic “self-readable” non-natural polymers that can be sequenced at femtomole scale by chemical fragmentation and high-resolution mass spectrometry. The versatility and throughput of the platform was demonstrated by screening two libraries of non-natural polyamide polymers with sizes of 1.77M and 1B compounds against the protein targets K-Ras, asialoglycoprotein receptor 1 (ASGPR), IL-6, IL 6 receptor (IL-6R) and TNFα. Hits with low nanomolar binding affinities were found against all targets, including competitive inhibitors of K-Ras binding to Raf and functionally active uptake ligands for ASGPR facilitating intracellular delivery of a non-glycan ligand.

Exon 20 genomic insertions of both epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) are oncogenic drivers and are most commonly found in non-small cell lung cancer (NSCLC). NSCLC patients with exon 20 insertions have a worse prognosis compared to those with other activating EGFR mutations. Moreover, approximately one-third of patients with exon 20 insertion mutations develop central nervous system (CNS) metastases over the course of their disease. Unfortunately, current therapeutics lack sufficient brain exposure for treating this patient population. ORIC-114 is a brain penetrant, orally bioavailable, irreversible small molecule inhibitor designed to target exon 20 insertions in EGFR and HER2. Notably, ORIC-114 is highly selective for the EGFR family of receptors, with excellent kinome selectivity compared to other reported exon 20 inhibitors, reducing the risk of off-target kinase liabilities. The superior brain penetration and free unbound exposure of ORIC-114 in preclinical studies also differentiates it from comparator EGFR and HER2 exon 20 targeted agents. To further characterize ORIC-114, in vivo studies were undertaken to assess activity in both subcutaneous and intracranial NSCLC tumor patient-derived xenograft (PDX) models. Consistent with in vitro potency and selectivity, once daily oral administration of 3 mg/kg ORIC-114 induced robust tumor regressions with greater than 100% tumor growth inhibition in the absence of significant body weight loss in an EGFR exon 20 insertion H773_V774insNPH NSCLC PDX model. In this subcutaneous model, ORIC-114 was superior to CLN-081 in efficacy and tolerability, and superior to BDTX-189 in efficacy. To investigate whether the brain-penetrant attributes of ORIC-114 translated into antitumor activity in the CNS, we utilized an intracranial PC-9 luciferase-labeled EGFR del 19 mutant cell line model. Once daily oral administration of ORIC-114 significantly regressed established intracranial NSCLC tumors and demonstrated greater efficacy than TAK-788, commensurate with the superior brain exposure of ORIC-114. We further explored dosing regimens in this intracranial model and found that ORIC-114 demonstrated equivalent regressions at 1.5 mg/kg twice daily and 3 mg/kg once daily, and strong efficacy with 1.5 mg/kg once daily dosing. Taken together, these data confirm ORIC-114 as a potent, selective, irreversible, brain penetrant exon 20 inhibitor, and a promising therapeutic candidate, including for patients with CNS metastases. Based upon these data, ORIC-114 is entering a Phase 1/1b clinical trial in genetically defined cancers. Citation Format: Jason E. Long, Soochan Kim, Ha Yeong Kim, Dong Guk Shin, Dong Hyun Park, Robert Warne, Akash Das, Ganapati Hegde, Padmini Narayanan, Lidia Sambucetti, Brenda Chan, Xi Chen, Jae H. Chang, Paul Gibbons, Jessica Sun, Matthew Panuwat, Lori S. Friedman, Melissa R. Junttila. ORIC-114, an orally bioavailable, irreversible kinase inhibitor, has superior brain penetration and antitumor activity in subcutaneous and intracranial NSCLC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3335.

19 pags., 7 figs., 2 tabs., Taspase1 is an Ntn-hydrolase overexpressed in primary human cancers, coordinating cancer cell proliferation, invasion, and metastasis. Loss of Taspase1 activity disrupts proliferation of human cancer cells in vitro and in mouse models of glioblastoma. Taspase1 is synthesized as an inactive proenzyme, becoming active upon intramolecular cleavage. The activation process changes the conformation of a long fragment at the C-terminus of the α subunit, for which no full-length structural information exists and whose function is poorly understood. We present a cloning strategy to generate a circularly permuted form of Taspase1 to determine the crystallographic structure of active Taspase1. We discovered that this region forms a long helix and is indispensable for the catalytic activity of Taspase1. Our study highlights the importance of this element for the enzymatic activity of Ntn-hydrolases, suggesting that it could be a potential target for the design of inhibitors with potential to be developed into anticancer therapeutics., This project has been funded in whole with Federal funds from the National Cancer Institute (NCI), National Institutes of Health (NIH), under Chemical Biology Consortium contract no. HHSN261200800001E.

Proteases can play essential roles in severe human pathology, ranging from degenerative and inflammatory illnesses to infectious diseases, with some, such as Taspase1, involved in growth and progression of tumors at primary and metastatic sites. Taspase1 is a N-terminal nucleophile (Ntn)-hydrolase overexpressed in primary human cancers, coordinating cancer cell proliferation, invasion, and metastasis. Loss of Taspase1 activity disrupts proliferation of human cancer cellsin vitroand in mouse xenograft models of glioblastoma, thus this protein has the potential to become a novel anticancer drug target. It belongs to the family of Ntn-hydrolases, a unique family of proteins synthesized as enzymatically inactive proenzymes that become activated upon cleavage of the peptide bond on the N-terminal side of a threonine residue, which then becomes the catalytic site nucleophile. The activation process simultaneously changes the conformation of a long domain at the C-terminus of the alpha-subunit for which no full-length structural information exists and its function is poorly understood. Here we present a novel cloning strategy to generate a fully active, circularly permuted form of Taspase1 to determine the crystallographic structure of catalytically active human Taspase1 to 3.04Å. We discovered that this region forms a long helical domain and is indispensable for the catalytic activity of Taspase1. Together, our study highlights the importance of this element for the enzymatic activity of Ntn-hydrolases and suggests that this long domain could be a novel target for the design of inhibitors with the potential to be developed into anticancer therapeutics.

A set of genetically engineered isogenic cell lines is developed to express either folate receptor alpha or mesothelin, and a control cell line negative for both antigens. These cell lines also express fluorescent and bioluminescent reporter transgenes. The cell lines are used to authenticate specificity and function of a T-cell biofactory, a living vector that is developed to express proportionate amounts of engineered proteins upon engaging with disease cells through their specific antigenic biomarkers. The engineered cell lines are also used to assess the cytolytic function and specificity of primary T cells engineered with chimeric antigen receptors; and the specificity of monoclonal antibodies. The strategy described can be used to generate other cell lines to present different disease-specific biomarkers for use as quality control tools.

An artificial cell-signaling pathway is developed that capitalizes on the T-cell's innate extravasation ability and transforms it into a vector (T-cell Biofactory) for synthesizing calibrated amounts of engineered proteins in vivo. The modularity of this pathway enables reprogramming of the T-cell Biofactory to target biomarkers on different disease cells, e.g. cancer, viral infections, autoimmune disorders. It can be expected that the T-cell Biofactory leads to a "living drug" that extravasates to the disease sites, assesses the disease burden, synthesizes the calibrated amount of engineered therapeutic proteins upon stimulation by the diseased cells, and reduces targeting of normal cells.

Here we describe a new approach for tumor targeting in which augmented concentrations of Fe(II) in cancer cells and/or the tumor microenvironment triggers drug release from an Fe(II)-reactive prodrug conjugate. The 1,2,4-trioxolane scaffold developed to enable this approach can in principle be applied to a broad range of cancer therapeutics and is illustrated here with Fe(II)-targeted forms of a microtubule toxin and a duocarmycin-class DNA-alkylating agent. We show that the intrinsic reactivity/toxicity of the duocarmycin analog is masked in the conjugated form and this greatly reduced toxicity in mice. This in turn permitted elevated dosing levels, leading to higher systemic exposure and a significantly improved response in tumor xenograft models. Overall our results suggest that Fe(II)-dependent drug delivery via trioxolane conjugates could have significant utility in expanding the therapeutic index of a range of clinical and preclinical stage cancer chemotherapeutics.

Background and Aims Recognition of abnormal glycosylation in virtually every cancer type has raised great interest in exploration of the tumor glycome for biomarker discovery. Identifying glycan markers of circulating tumor cells (CTCs) represents a new development in tumor biomarker discovery. The aim of this study was to establish an experimental approach to enable rapid screening of CTCs for glycan marker identification and characterization. Methods We applied carbohydrate microarrays and a high-speed fiber-optic array scanning technology (FAST scan) to explore potential glycan markers of breast CTCs (bCTCs) and targeting antibodies. An anti-tumor monoclonal antibody, HAE3-C1 (C1), was identified as a key immunological probe in this study. Results In our carbohydrate microarray analysis, C1 was found to be highly specific for an O-glycan cryptic epitope, gp C1 . Using FAST-scan technology, we established a procedure to quantify expression levels of gp C1 in tumor cells. In blood samples from five stage IV metastatic breast cancer patients, the gp C1 positive CTCs were detected in all subjects; ∼40% of bCTCs were strongly gp C1 positive. Interestingly, CTCs from a triple-negative breast cancer patient with multiple sites of metastasis were predominantly gp C1 positive (92.5%, 37/40 CTCs). Conclusions Together we present here a practical approach to examine rare cell expression of glycan markers. Using this approach, we identified an O-core glyco-determinant gp C1 as a potential immunological target of bCTCs. Given its bCTC-expression profile, this target warrants an extended investigation in a larger cohort of breast cancer patients.

Identification of mutation-specific targeted therapies is a critical challenge in precision medicine. Synthetic lethality provides the basis for an approach to identify new therapeutic targets for mutations that are difficult to target directly, since in synthetic lethal (SL) interactions, an alteration in one gene leads to dependency on a second gene. Neither alteration by itself is essential for survival, but together these alterations lead to cancer cell death. We previously developed a new computational method, Mining Synthetic Lethals (MiSL), that can infer relationships from primary human tumor genomic and transcriptomic data and enable the identification of SL interactions in the context of native human tumors. The underlying assumption of MiSL is that SL partners of a mutation will be selectively amplified or never deleted in primary tumor samples harboring the mutation. Here, we used MiSL to identify novel SL partners of KRAS mutations in lung cancer. KRAS activating mutations are highly prevalent in lung cancer and aside from early clinical data on a novel covalent inhibitor for KRAS G12C mutations, there is no reported treatment to date for patients with KRAS mutations. We applied MiSL to identify SL interactions with KRAS G12 mutations in non-small cell lung cancer (NSCLC) and identified 220 SL partner genes. Some of these genes were foreseeable dependencies, such as MAPK, growth factor and inflammatory signaling associated genes; while others were novel SL candidates and included genes associated with splicing. Since we have access to a splicing-modulating drug in preclinical development, sudemycin D6 (SD6) which targets SF3B1, a spliceosome subunit, we focused on testing the SF3B1 interactors identified by MiSL, SLU7 and SRRM2. To enable validation studies, we first verified KRAS growth dependency in a panel of NSCLC cell lines using siRNA knock-down (KD). Next, we tested siRNA KD of predicted MiSL candidates on growth of KRAS-dependent (H358 and H441 – KRAS mutant) and KRAS-independent (H2228 – KRAS WT) cell lines. We confirmed that the KD of SLU7 expression reduced viability of KRAS mutant (75 to 85% reduced) but not KRAS wild-type (WT) cells lines. Similar differential viability was also observed with siRNA KD of SF3B1. Pharmacological inhibition of SF3B1 using SD6 also demonstrated that KRAS mutant cell lines were more sensitive to SD6 compared to a KRAS WT line (>5-30x). Finally, we validated these findings in an in vivo tumor xenograft model by treating KRAS WT and KRAS mutant tumors with SD6 and showed that the KRAS mutant tumors were significantly more inhibited by SD6 than KRAS WT tumors. Studies are ongoing to further confirm the SL interaction in lung cancer cell lines that are isogenic except for the KRAS mutation. In conclusion, SL predictions based on primary KRAS G12 mutant tumors indicated that splicing modulation is a vulnerability of KRAS tumors. Finally, synthetic lethal targets identified by primary tumor data mining can provide new therapeutic targets for KRAS specific mutant cancers, which can lead to personalized treatment options for NSCLC cancer patients. Citation Format: Claire E Repellin, Puja Patel, Yihui Shi, Helena Gong, Leena Shewade, Thomas Webb, Lidia Sambucetti, Subarna Sinha. Primary tumor data analysis reveals novel synthetic lethal dependencies between KRAS mutation and the spliceosome [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C018. doi:10.1158/1535-7163.TARG-19-C018

Early detection and intervention are crucial for long-term survival of either primary or recurrent lung cancer patients. Circulating tumor cells (CTCs) could be powerful biomarkers for the diagnosis, prognosis, and treatment planning of cancer. In this work we applied our FAST cytometer, a fiber-optic array scanning technology (FAST) to develop a sensitive diagnostic blood tests (“liquid biopsies”) for NSCLC. This technology is based on the ultra-rapid, sensitive and accurate detection of rare circulating cells expressing the biomarker(s) of interest. By applying the FAST cytometer with a unique NSCLC specific cocktail, we were able to detect CTC in NSCLC setting with higher sensitivity as compared with traditional cytokeratin-based detection. In a pilot cohort of 51 NSCLS patients, we have detected CTC in 74.2% using our unique panel for NSCLC CTC, this has raised our sensitivity from 48.8% using traditional cytokeratin-based detection. In addition, we have also interrogated circulating cancer associated fibroblasts (cCAF) as an additional liquid biopsy marker for NSCLC. We detected cCAF in 67% NSCLC patients. When combined with our enhanced CTC detection method, addition of cCAF further increased our sensitivity to 88.5%. Moreover, using clinically validated PD-L1 antibody (Ventana SP263) as reference, we have demonstrated our capacity to analyze PD-L1 expression on CTC at protein level, we have shown detection of PD-L1+ CTC in 80% of the patients. To summarize, we have developed an unique panel to detect CTC with higher sensitivity using combinational detection and we have demonstrated that by combining CTC detection with cCAF detection, liquid biopsy sensitivity for cancer detection can be further increased without significant increase in false positive rates. Lastly, we developed a method to analyze the PD-L1 expression on CTC using clinically validated Ventana PD-L1 (SP263) rabbit monoclonal antibody as reference which can potentially serve as a companion diagnostics tool for NSCLC immunotherapy Citation Format: Xiaohe Liu, Zheng Ao, Jordan S. Preiss, Joel Neal, Lisa Ya Zhou, Lidia Sambucetti, Heather A. Wakelee. Characterization of circulating tumor cells and its PD-L1 expression with high-sensitivity liquid biopsy panel in non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-238.

Identification of predictive biomarkers for targeted drugs is an active research area in precision oncology. Usually, a genetic alteration in the drug target is used to identify responders, which can limit the use of the drug in other responsive cancers. Alternatively, cell line-based drug sensitivity data are used to build machine-learning models to identify predictive biomarkers. These methods require large amounts of data and the black-box models developed are often hard to interpret. Synthetic lethality provides an alternate approach for predictive biomarker identification of targeted drugs. In synthetic lethal (SL) interactions, a defect in one gene leads to dependency on a second gene. Neither defect by itself is essential for survival, but together they lead to cell death. Thus, the inhibition of the drug target in the presence of a cancer-specific genetic alteration would result in cancer cell death. The genetic alterations can be used as biomarkers for the drug. We developed a novel computational pipeline for predictive biomarker identification based on our tool, Mining Synthetic Lethals (MiSL), that mines SL interactions from pan-cancer primary tumor data (such as TCGA). Since MiSL utilizes primary human tumor data, it enables the identification of SL interactions in the native context of human tumors and is more likely to find relationships relevant to in vivo tumor biology than shRNA/CRISPR screens. To discover biomarkers of a targeted drug, we used MiSL to identify genetic alterations that are SL with targets of the drug in a specific cancer type. As proof of principle, we applied our method to identify biomarkers of sudemycin-D6 (SD6), a potent splicing modulator, in non-small cell lung cancer (NSCLC). First, we identified putative drug targets of SD6. Since SD6 is known to inhibit SF3B1, any gene belonging to the same protein complex as SF3B1 was termed a SD6 drug target. Using these SD6 drug targets, our MiSL-based pipeline identified KRAS mutations, present in ~30% of NSCLC patients, as a SD6 biomarker. To confirm our predictions, we treated a panel of KRAS-mutant-and-dependent NSCLC lines (H358, H23 and H441) and a KRAS-WT NSCLC line (H2228) with SD6 and measured cell viability. All KRAS-mutant lines tested were significantly more sensitive to SD6 (> 5-30X lower IC50) than the KRAS-WT line. Furthermore, the KRAS-mutant lines were also more sensitive (5-20X lower IC50), compared to the KRAS-WT line, to a splicing modulator of a different class – a kinase inhibitor that phosphorylates components of the spliceosome. In vivo studies are underway to further validate our in vitro findings. This study identifies KRAS mutation as a novel predictive biomarker for splicing modulators in NSCLC, which could lead to new therapeutic options for KRAS-mutated NSCLC. Furthermore, our work demonstrates the feasibility of a synthetic lethality-based pipeline to predict biomarkers for targeted drugs. Citation Format: Claire E. Repellin, Puja Patel, Yihui Shi, Helena Gong, Thomas R. Webb, Lidia Sambucetti, Subarna Sinha. Synthetic lethality-based predictive biomarker identification of splicing modulators in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4015.

The splicing of pre-mRNA is a critical process in normal cells and is deregulated in cancer. Compounds that modulate this process have recently been shown to target a specific vulnerability in tumors. We have developed a novel cell-based assay that specifically activates luciferase in cells exposed to SF3B1 targeted compounds, such as sudemycin D6. This assay was used to screen a combined collection of approved drugs and bioactive compounds. This screening approach identified several active hits, the most potent of which were CGP-74514A and aminopurvalanol A, both have been reported to be cyclin-dependent kinases (CDKs) inhibitors. We found that these compounds, and their analogs, show significant cdc2-like kinase (CLK) inhibition and clear structure-activity relationships (SAR) at CLKs. We prepared a set of analogs and were able to ‘dial out’ the CDK activity and simultaneously developed CLK inhibitors with low nanomolar activity. Thus, we have demonstrated the utility of our exon-skipping assay and identified new molecules that exhibit potency and selectivity for CLK, as well as some structurally related dual CLK/CDK inhibitors.

The orphan nuclear receptor estrogen-related receptor α (ERRα) has been implicated in the development of various human malignancies, including breast, prostate, ovary, and colon cancer. ERRα, bound to a co-activator protein (e.g., peroxisome proliferator receptor γ co-activator-1α, PGC-1α), regulates cellular energy metabolism by activating transcription of genes involved in various metabolic processes, such as mitochondrial genesis, oxidative phosphorylation, and fatty acid oxidation. Accumulating evidence suggests that ERRα is a novel target for solid tumor therapy, conceivably through effects on the regulation of tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. This report describes a novel steroidal antiestrogen (SR16388) that binds selectively to ERRα, but not to ERRβ or ERRγ, as determined using a time-resolved fluorescence resonance energy transfer assay. SR16388 potently inhibits ERRα's transcriptional activity in reporter gene assays, and prevents endogenous PGC-1α and ERRα from being recruited to the promoters or enhancers of target genes. Representative in vivo results show that SR16388 inhibited the growth of human prostate tumor xenografts in nude mice as a single agent at 30 mg/kg given once daily and 100 mg/kg given once weekly. In a combination study, SR16388 (10 mg/kg, once daily) and paclitaxel (7.5 mg/kg, twice weekly) inhibited the growth of prostate tumor xenografts in nude mice by 61% compared to untreated xenograft tumors. SR16388 also inhibited the proliferation of diverse human tumor cell lines after a 24-h exposure to the compound. SR16388 thus has utility both as an experimental antitumor agent and as a chemical probe of ERRα biology.

Identification of mutation-specific targeted therapies is a critical challenge in precision medicine, in part because many cancer mutations are loss-of-function and are not druggable. Even when a mutation-specific targeted therapy exists, resistance invariably develops necessitating alterative therapeutic options. Synthetic lethality can help identify new therapeutic targets for these mutations. In synthetic lethal (SL) interactions, an alteration in one gene leads to dependency on a second gene. Neither alteration by itself is essential for survival, but together these alterations lead to cancer cell death. We have recently developed a new computational method (Mining Synthetic Lethals, MiSL) that mines SL interactions from pan-cancer primary tumor data (such as TCGA). The underlying assumption of MiSL is that SL partners of a mutation will be selectively amplified or never deleted and overexpressed in primary tumor samples harbouring the mutation. Since MiSL mines relationships from primary human tumor data, it enables the identification of SL interactions in the native context of human tumors and is more likely to find relationships relevant to in vivo tumor biology than shRNA screens. Here, we applied MiSL to identify SL partners of the BRCA1 mutation in triple-negative breast cancer (TNBC). BRCA1 is mutated in 15-20% of TNBC and is a known cancer susceptibility gene in breast cancer. MiSL identified 22 SL partners of the BRCA1 mutation in TNBC: these were enriched for DNA repair genes consistent with the known SL relationship between BRCA1 and the DNA repair gene PARP. Notably, XRCC6, a gene involved in non-homologous end joining, was predicted to be a SL partner of the BRCA1 mutation in TNBC. MiSL selected XRCC6 because XRCC6 deletions (which were associated with lowered expression of XRCC6) were mutually exclusive with BRCA1 mutations in pan-cancer analysis (p = 0.01), and XRCC6 was upregulated in BRCA1-mutated TNBC compared to BRCA1-WT TNBC (p = 0.02). To test the prediction, we examined the effect of XRCC6 knockdown (KD) in the BRCA1-mutated TNBC cell line SUM149. Consistent with our prediction, we found that XRCC6 KD significantly increased apoptosis (37.3%) and reduced viability (50% reduction, p < 0.0001) in SUM149 cells compared to controls. XRCC6 KD in a BRCA1-WT breast cancer cell line, T47D, resulted in significantly lower reduction in cell viability compared to the BRCA1-mutated cell line (p Thus, we have identified a novel SL partner of the BRCA1 mutation in TNBC that can lead to the development of new therapeutic options for BRCA1-mutated breast cancers and to new chemoprevention strategies for individuals carrying germline BRCA1 mutations. Citation Format: Yihui Shi, Xiaohe Liu, Jia Li, Lidia Sambucetti, Subarna Sinha. Primary tumor data mining identifies a novel synthetic lethal partner of the BRCA1 mutation in breast cancer [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 2974.

Current protein-based cancer therapies have several disadvantages, which include general toxicity, lack of response at the administered dose and differing responses from patients to patient. To circumvent these issues, we propose a platform in which engineered T-cells specifically recognize tumors and secrete therapeutic peptides directly at the disease site leading to targeted cancer cell killing. This approach will increase the specificity of the therapy and decrease toxicity to healthy cells. We transformed acute T-cell lymphoma cells into biofactories for site-specific synthesis of therapeutic proteins upon stimulation by antigen-presenting disease cells. The effector T-cell line was engineered by stably introducing a chimeric T-cell receptor to recognize Folate-Receptor alpha (FRα) or Mesothelin (MSLN) protein. Upon binding of the effector T-cells to the receptor, an intracellular cascade directed expression of non-human proteins is induced. Specific T-cell binding to human ovarian cancer cell lines and signaling was measured by in vitro co-culture using luciferase production as a surrogate for therapeutic peptide secretion. We demonstrated that T-cells can be genetically programed to synthesize and secrete proportionate amounts of engineered proteins upon engaging the tumor-associated antigens (FRα or MSLN) on a human ovarian cancer cell line, OVCAR3 (FRα+MSLN+). A FRα-MSLN- ovarian cancer cell line, A2780cis, was used as the non-targeted negative control. The difference in protein secretion following stimulation by the two cell lines, as measured by luciferase activity, was statistically significant within 1 hour. It reached ~35-fold within 1 to 3 days, and we observed stable expression for at least 10 days. The luminescent signal was proportionate to the number of OVCAR3 cells. To further validate the specificity of target engagement, we generated A2780cis-FRα positive and A2780cis-MSLN positive cell lines and demonstrated selective binding and activation of the corresponding effector cells in co-culture assays. No binding was detected to the A2780cis-vector control cells. In vivo results for T-cell biofactories targeting OVCAR3 tumors 24 hours post-stimulation validated the in vitro results. Currently, we are engineering T-cell biofactories to release cytotoxic peptides and are assessing their therapeutic efficacy against cancer cells in vitro using co-culture assays and supernatant transfer. Our results show that T-cells can be genetically reprogrammed to serve as biofactories for the synthesis of therapeutic proteins upon stimulation by antigen-presenting disease cells. Importantly, these studies demonstrate the feasibility of developing the next generation of adoptively transferred T-cell therapies to target tumors that express FRα (e.g., ovarian, breast, lung) and/or MSLN (e.g. ovarian, lung, pancreatic) on their cell surfaces for cancer therapy. Citation Format: Claire E. Repellin, Puja Patel, Lucia Beviglia, Harold Javitz, Lidia C. Sambucetti, Parijat Bhatnagar. Assessing the therapeutic efficacy of disease-specific T-cell biofactories [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 2557.

Mesothelin is a tumor-associated antigen overexpressed in ovarian cancer and normally expressed by mesothelial linings. While mesothelin function remains to be fully elucidated, the lack of phenotype of mesothelin-knockout mice makes it plausible that mesothelin can safely serve as a vaccine target. We designed a new vaccination strategy based on mesothelin targeting with activation of type I IFN signaling via cyclic dinucleotides (CDN). Vaccines first combined human mesothelin protein with alum- vs. CDN-based adjuvants. C57Bl/6 mice received a priming immunization followed by 2 boosts; two weeks later, immunized mice were injected orthotopically with luciferase-transduced mouse ovarian cancer cells (Luc-ID8). More than 2/3 of the mesothelin/CDN-immunized mice were protected against invasive cancer and presented increased cytotoxic T cells (CTL) and plasmacytoid dendritic cells in spleens. Effector memory T cells were also increased and MDSC were profoundly decreased in peritoneal lavages after immunization with mesothelin +/− CDN compared with all the other groups. IFNγ ELISPOT assays performed with protected animal splenocytes stimulated with mesothelin peptides identified 2 immunogenic regions. We next tested a peptide-based subunit vaccine derived from mouse mesothelin sequence. Immunization and 2 boosts of 25-mer peptides with CDN were sufficient to mount an immune response against 2 overlapping mouse mesothelin peptides. Immunizations with peptide pools mapping in the immunogenic regions are currently assessed for protection against Luc-ID8 growth and progression. Peptide-based prophylactic vaccination against ovarian cancer may be possible in combination with adjuvants that trigger cellular immunity.

A major goal in HIV eradication research is characterizing the reservoir cells that harbor HIV in the presence of antiretroviral therapy (ART), which reseed viremia after treatment is stopped. In general, it is assumed that the reservoir consists of CD4 + T cells that express no viral proteins. However, recent findings suggest that this may be an overly simplistic view and that the cells that contribute to the reservoir may be a diverse population that includes both CD4 + and CD4 − cells. In this study, we directly infected resting CD4 + T cells and used fluorescence-activated cell sorting (FACS) and fiber-optic array scanning technology (FAST) to identify and image cells expressing HIV Gag. We found that Gag expression from integrated proviruses occurred in resting cells that lacked surface CD4, likely resulting from Nef- and Env-mediated receptor internalization. We also extended our approach to detect cells expressing HIV proteins in patients suppressed on ART. We found evidence that rare Gag + cells persist during ART and that these cells are often negative for CD4. We propose that these double-negative α/β T cells that express HIV protein may be a component of the long-lived reservoir. IMPORTANCE A reservoir of infected cells persists in HIV-infected patients during antiretroviral therapy (ART) that leads to rebound of virus if treatment is stopped. In this study, we used flow cytometry and cell imaging to characterize protein expression in HIV-infected resting cells. HIV Gag protein can be directly detected in infected resting cells and occurs with simultaneous loss of CD4, consistent with the expression of additional viral proteins, such as Env and Nef. Gag + CD4 − cells can also be detected in suppressed patients, suggesting that a subset of infected cells express proteins during ART. Understanding the regulation of viral protein expression during ART will be key to designing effective strategies to eradicate HIV reservoirs.

The spliceosome has recently emerged as a new target for cancer chemotherapy and novel antitumor spliceosome targeted agents are under development. Here, we describe two types of novel pharmacodynamic assays that facilitate drug discovery and development of this intriguing class of innovative therapeutics; the first assay is useful for preclinical optimization of small-molecule agents that target the SF3B1 spliceosomal protein in animals, the second assay is an ex vivo validated, gel-based assay for the measurement of drug exposure in human leukocytes. The first assay utilizes a highly specific bioluminescent splicing reporter, based on the skipping of exons 4-11 of a Luc-MDM2 construct, which specifically yields active luciferase when treated with small-molecule spliceosome modulators. We demonstrate that this reporter can be used to monitor alternative splicing in whole cells in vitro. We describe here that cell lines carrying the reporter can be used in vivo for the efficient pharmacodynamic analysis of agents during drug optimization and development. We also demonstrate dose- and time-dependent on-target activity of sudemycin D6 (SD6), which leads to dramatic tumor regression. The second assay relies on the treatment of freshly drawn human blood with SD6 ex vivo treatment. Changes in alternative splicing are determined by RT-PCR using genes previously identified in in vitro experiments. The Luc-MDM2 alternative splicing bioluminescent reporter and the splicing changes observed in human leukocytes should allow for the more facile translation of novel splicing modulators into clinical application.

We have synthesized a histone deacetylase inhibitor, NVP-LAQ824, a cinnamic hydroxamic acid, that inhibited in vitro enzymatic activities and transcriptionally activated the p21 promoter in reporter gene assays. NVP-LAQ824 selectively inhibited growth of cancer cell lines at submicromolar levels after 48–72 h of exposure, whereas higher concentrations and longer exposure times were required to retard the growth of normal dermal human fibroblasts. Flow cytometry studies revealed that both tumor and normal cells arrested in the G2-M phase of the cell cycle after compound treatment. However, an increased sub-G1 population at 48 h (reminiscent of apoptotic cells) was observed only in the cancer cell line. Annexin V staining data supported our hypothesis that NVP-LAQ824 induced apoptosis in tumor and transformed cells but not in normal cells. Western blotting experiments showed an increased histone H3 and H4 acetylation level in NVP-LAQ824-treated cancer cells, suggesting that the likely in vivo target of NVP-LAQ824 was histone deacetylase(s). Finally, NVP-LAQ824 exhibited antitumor effects in a xenograft animal model. Together, our data indicated that the activity of NVP-LAQ824 was consistent with its intended mechanism of action. This novel histone deacetylase inhibitor is currently in clinical trials as an anticancer agent.

The splicing of pre-mRNA is a critical process in normal cells and is deregulated in cancer. Compounds that modulate this process have recently been shown to target a specific vulnerability in tumors. We have developed a novel cell-based assay that specifically activates luciferase in cells exposed to SF3B1 targeted compounds, such as sudemycin D6. This assay was used to screen a combined collection of approved drugs and bioactive compounds. This screening approach identified several active hits, the most potent of which were CGP-74514A and aminopurvalanol A, both have been reported to be cyclin-dependent kinases (CDKs) inhibitors. We found that these compounds, and their analogs, show significant cdc2-like kinase (CLK) inhibition and clear structure-activity relationships (SAR) at CLKs. We prepared a set of analogs and were able to ‘dial out’ the CDK activity and simultaneously developed CLK inhibitors with low nanomolar activity. Thus, we have demonstrated the utility of our exon-skipping assay and identified new molecules that exhibit potency and selectivity for CLK, as well as some structurally related dual CLK/CDK inhibitors. Citation Format: Yihui Shi, Jaehyeon Park, Chandraiah Lagisetti, Wei Zhou, Lidia C. Sambucetti, Thomas R. Webb. A triple exon-skipping luciferase reporter assay identifies a new CLK inhibitor scaffold [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4193. doi:10.1158/1538-7445.AM2017-4193

A major challenge to the discovery and development of novel cancer prevention strategies is the availability of reliable animal models. Key elements for chemoprevention testing in animal include relevance to human cancers, with tumors of similar pathology and genetic abnormalities, the presence of intermediate lesions that resemble human cancer development both histologically and molecularly; and consistent tumor incidence of >60% within less than 6 months. To our knowledge no established animal model responds to these requirements for ovarian cancer prevention. We sought to adapt the Dicerflox/flox-Ptenflox/flox double knockout (DKO) ovarian cancer mouse model (Kim, Matzuk PNAS 2012) for cancer prevention studies, by synchronized induction of the disease with intra-ovary injection of an adenovirus encoding CRE protein (Ad-mCherry-Cre). Premalignant lesions in DKO AdCRE mice were visible by H&E staining after 12 weeks, and evolved into typical serous ovarian cancer by 24 weeks. Histology demonstrated that tumors overexpressed cytokeratin 8, cyclin D, CA125, and mesothelin. In addition, CA125, the main biomarker for ovarian cancer in woman, was consistently elevated in the serum of DKO AdCRE mice 15 weeks after CRE induction, but not in age-matched DKO mice. To assess whether mesothelin-targeted imaging could help monitor cancer development in DKO AdCRE mice, we used an anti-mesothelin recombinant antibody derived from llama (G3a nanobody) that we previously identified and in vitro characterized (Prantner, Scholler, JBN 2015). G3a nanobody is conveniently secreted by yeast with an N-terminal biotinylation (Scholler, JIM 2006) for streptavidin coupling. We addressed here whether G3a nanobody could detect mesothelin-expressing tumors using in vivo imaging (IVIS). Ni-purified G3a nanobody was incubated with fluorescently-labelled streptavidin (IRB680W) for 30 min and injected retro orbitally just before imaging. We found that G3a nanobody generated a robust and mesothelin-specific signal that peeked 4 hours after injection, and detected DKO AdCRE tumors as early as 8 weeks after CRE induction. G3a nanobody specific detection of mesothelin was confirmed in other mouse models of cancer, including in syngeneic models of ovarian cancer induced by orthotopic or intraperitoneal injection of mesothelin+ Luc-ID8 cells, and in xenograft models of human lung cancer obtained by IV injection of mesothelin high EKVX, mesothelin int H460, and mesothelin lo A549 cells. In summary, our results indicate that G3a nanobody is a valuable reagent for early detection of mesothelin-expressing cancer, and that DKO mice induced for Pten and Dicer double knockout by intra ovarian injection of CRE adenovirus develop serous ovarian cancer that express CA125 and mesothelin. We propose this model for in vivo monitoring of cancer development, as a novel system model for ovarian cancer prevention studies. Citation Format: Khushboo Sharma, Catherine Yin, Kalika Kamat, Claire Repellin, Lidia C. Sambucetti, Nathalie Scholler. New cancer prevention model using imaging for early detection of mesothelin-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-135. doi:10.1158/1538-7445.AM2017-LB-135

Rapid tumor growth can establish metabolically stressed microenvironments that activate 5'-AMP-activated protein kinase (AMPK), a ubiquitous regulator of ATP homeostasis. Previously, we investigated the importance of AMPK for the growth of experimental tumors prepared from HRAS-transformed mouse embryo fibroblasts and for primary brain tumor development in a rat model of neurocarcinogenesis. Here, we used triple-negative human breast cancer cells in which AMPK activity had been knocked down to investigate the contribution of AMPK to experimental tumor growth and core glucose metabolism. We found that AMPK supports the growth of fast-growing orthotopic tumors prepared from MDA-MB-231 and DU4475 breast cancer cells but had no effect on the proliferation or survival of these cells in culture. We used in vitro and in vivo metabolic profiling with [(13)C]glucose tracers to investigate the contribution of AMPK to core glucose metabolism in MDA-MB-231 cells, which have a Warburg metabolic phenotype; these experiments indicated that AMPK supports tumor glucose metabolism in part through positive regulation of glycolysis and the nonoxidative pentose phosphate cycle. We also found that AMPK activity in the MDA-MB-231 tumors could systemically perturb glucose homeostasis in sensitive normal tissues (liver and pancreas). Overall, our findings suggest that the contribution of AMPK to the growth of aggressive experimental tumors has a critical microenvironmental component that involves specific regulation of core glucose metabolism.

A series of N-substituted heteroaromatic compounds structurally related to clotrimazole was synthesized, and the effects of these compounds on ethosuximide clearance in rats were determined as a measure of their abilities to induce cytochrome P4503A (CYP3A) activity. Ethosuximide clearance and in vitro erythromycin N-demethylase activity were shown to correlate. In this series, imidazole or other related heteroaromatic “head groups” were linked to triphenylmethane or other phenylmethane derivatives. Within the series, it was found that 1-triphenylmethane-substituted imidazoles elicited the greatest increase in CYP3A activity, and that among the triphenylmethyl-substituted imidazoles, the highest activities were achieved by the substitution of F- or Cl- in either the meta or para position of one of the phenyl rings. Diphenylmethyl-substituted pyridine was effectively devoid of activity. Compounds eliciting the largest increase in CYP3A activity (viz. 1-[(3-fluorophenyl)diphenylmethyl]imidazole, 1-[(4-fluorophenyl)diphenylmethyl]imidazole, and 1-[tri-(4-fluorophenyl)methyl]imidazole) produced little or no increase in ethoxyresorufin O-dealkylase (EROD) activity (i.e. CYP1A), whereas benzylimidazole, which elicited only a small increase in CYP3A activity, produced an almost 9-fold increase in CYP1A activity. For a series of eleven compounds exhibiting a wide range of influence on CYP3A activity, a positive correlation was found between ethosuximide clearance and hepatic CYP3A mRNA levels.

Survival of cancer patients with high-grade serous carcinoma of the ovary (HGSCs) remains less than 30% five years after diagnosis, despite initial chemosensitivity of most HGSCs. We hypothesized that a tumor antigen-targeted vaccine capable of eliciting a robust cell-mediated immune response may help prevent ovarian cancer. Mesothelin (meso) is overexpressed by ovarian, lung and pancreatic cancers and is 57% conserved at the protein level between mouse and human. To elicit cell-mediated immune responses directed against mesothelin, we combined a human mesothelin recombinant protein with various adjuvants including: aluminum hydroxide gel (alum, for Th2 immune response); TLR4 ligand (synthetic lipid MPL, for Th1 response); squalene-oil-in-water (AddaVaxTM, for both Th1 and Th2 responses); and STING (stimulator of interferon genes) ligand (CDN) that activates innate immunity and triggers type 1 IFN response and NF-κB-dependent cytokine secretion. We detected robust anti-meso antibody (Ab) responses after 1 prime and 2 boosts with meso/alum/MPL or meso/CDN/AddaVax. The highest and most stable Ab titers were obtained with CDN/AddaVax-based adjuvants. Four groups of female C57BL/6 mice were immunized with alum/MPL or CDN/AddaVax with or without meso (n = 12 per group). Six weeks after the prime immunization, cells from a luciferase-transduced syngeneic mouse ovarian cancer cell line, ID8 (Luc-ID8) were injected orthotopically in the left ovary of the mice. In vivo bioluminescent imaging (BLI) suggested a 92% take rate of tumors 3 weeks after ID8-Luc injections in mice immunized with meso/alum/MPL or adjuvants only. However, in the group immunized with meso/CDN/Addavax only 30% of the mice showed a detectable signal 3 weeks after ID8-Luc implantation. After 10 weeks the average BLI signal was still significantly lower than in all the other groups. Furthermore, the majority of mice immunized with alum/MPL +/- meso developed ascites by 12 weeks; no significant difference of peritoneal leukocyte numbers or phenotype was observed between the groups immunized with alum/MPL vs. meso/alum/MPL. In contrast, 7 out 11 mice immunized with meso/CDN/Addavax did not develop ascites through 14 weeks post tumor challenge; most of the tumors were unilateral, smaller, with more T and B cells present in the peritoneal lavages compared to the other groups. Immunizations with CDN/AddaVax or meso/CDN/AddaVax strongly increased the frequency of CD62L-CD44+ effector memory T cells, stimulated innate immunity with M1 polarization, profoundly decreased MDSC levels in peritoneal lavages, and correlated with a less aggressive tumor phenotype (EpCAM+ PD-L1-). H&E staining of the harvested tumors and fallopian tubes showed that tumors in animals immunized with meso/CDN/AddaVax were confined to the left ovary and/or fad pad for the majority of the animals as compared to metastatic disease for most animals of the other groups. In addition, tumor-infiltrating lymphocytes (TILs) were visible by H&E in animals immunized with meso/CDN/AddaVax. Further characterization is ongoing. These results support the hypothesis that a vaccine regimen combining mesothelin with CDN and Addavax can protect against aggressive forms of ovarian cancer. A similar vaccination study is in progress implementing a new model of inducible ovarian cancer in mice carrying floxed alleles of Dicer and PTEN. Prevention of ovarian cancer may be possible by vaccination against mesothelin tumor antigen in combination with adjuvants that stimulate type I IFN response. Citation Format: Nathalie Scholler, Paul Stein, Khushboo Sharma, Claire Repellin, Kalika Kamat, Travis Harrison, Robert H. Shoemarker, Shizuko Sei, Lidia Sambucetti. Preclinical development of a preventive vaccine against ovarian cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A041.

Introduction: Biomarker-guided personalized targeted therapies have improved lung cancer treatment; however, non-small cell lung cancer (NSCLC) targets such as KRAS — where activated mutations occur in ∼30% of cases — have so far eluded attempts at therapeutic targeting. There continues to be an urgent need to identify molecular mechanisms driving the disease and new targeted therapies. Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) overexpression promotes migration and invasion in several cancer types and correlates with poor prognosis in lung cancer. We investigated the role of MAP4K4 in NSCLC and tested whether MAP4K4 could serve as a biomarker on circulating tumor cells (CTCs) to monitor pre-metastatic and late stage disease. Methods: We used a novel class of highly selective small molecule inhibitor of MAP4K4, represented by SRI-28731, as a tool to further elucidate the role of MAP4K4 in NSCLC. We measured the expression of MAP4K4 and signaling changes following compound inhibition in lung cancer cell lines by immunoblotting and immunofluorescence. Clonogenic and wound healing assays were performed using our selective compound to assess proliferation and migration. To determine if MAP4K4 could be used as a biomarker in liquid biopsies we used FASTcell™, a non-enrichment optical scanning technology that enables identification and characterization of 1 tumor cell in 25 million blood cells. Results: We confirmed high expression of MAP4K4 in 4 out of 7 lung cancer cell lines, including A549 cells, and 3 of these harbored KRAS mutations. By clonogenic assay, A549 cell proliferation was inhibited by 51% and 99% with 2.5 nM and 5 nM of SRI-28731, respectively. In addition, lung cancer cell migration was impaired in the presence of 10 or 20 nM SRI-28731. Smad 1/5/8 phosphorylation was decreased in cells treated with the MAP4K4 inhibitor. Compound treated cells displayed a change in their actin cytoskeleton compared to DMSO treated cells. To explore whether MAP4K4 could be used as a biomarker on CTCs of metastatic NSCLC patients, we utilized the FASTcell™ platform and detected MAP4K4 expression on A549 cells mixed into normal blood. We are in the process of testing the assay with additional lung cancer cell lines and also testing MAP4K4 expression on CTCs from lung cancer patients. Conclusions: Our preliminary data suggest a role for MAP4K4 in proliferation, migration and signaling in lung cancer. Inhibition of MAP4K4 may be a valid therapeutic approach and the expression of MAP4K4 may serve as a biomarker in liquid biopsies. We showed that high levels of MAP4K4 in human lung cancer cell lines was concomitant to KRAS mutations, suggesting a possible correlation between the two markers. This novel biomarker could provide an opportunity to detect KRAS-positive cancers earlier and our new selective inhibitor could lead to life-saving, personalized treatment options for NSCLC cancer patients. Citation Format: Claire E. Repellin, Xiaohe Liu, Mary R. Stofega, Janey C.L. Snider, Zheng Ao, Ling Jong, Lidia C. Sambucetti. MAP4K4 as a therapeutic target and novel biomarker for NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-344.

Survival of ovarian cancer patients with high-grade serous carcinoma (HGSC) remains low. We hypothesized that a tumor antigen-targeted vaccine capable of eliciting a robust cell-mediated immune response may substantially improve women’s health. Mesothelin is a glycosylphosphatidylinisotol (GPI)-anchored, conserved protein that is overexpressed by ovarian, lung and pancreatic cancers. We designed mesothelin vaccines containing cyclic dinucleotide (CDN)-based adjuvants or alum-based adjuvants to trigger CD8 T cell activation. Anti-mesothelin antibody titers were stable or slightly increased after 2 vaccine boosts, and the highest and most stable titers were obtained with CDN-based adjuvants (meso/CDN). In addition, splenocytes from all mice receiving CDN-containing vaccines secreted mesothelin-specific IFNγ. Next, immunized mice were injected orthotopically with luciferase-transduced mouse ovarian cancer cells (ID8). In vivo imaging performed 3 weeks after tumor injections revealed that most of the mice from the groups immunized with Alum-based vaccines (92%) presented a positive signal of bioluminescence, which corresponds to the routine take rate of the ID8 tumors. In contrast, only 1/3 of the mice in the meso/CDN group showed a positive signal compared with 2/3 of the mice immunized with the control CDN-only vaccine. These results suggest that the meso/CDN vaccine regimen delayed tumor establishment. Longer observation of the mice is ongoing to determine whether the vaccination regimen can prevent tumor development. We tentatively conclude that ovarian cancer prevention may be possible through vaccination against mesothelin tumor antigen in combination with adjuvants that trigger cellular immunity.

Angiogenesis is one of the major processes controlling growth and metastasis of tumors. Angiogenesis inhibitors have been targeted for the treatment of various cancers for more than 2 decades. We have developed a novel class of steroidal compounds aimed at blocking the angiogenic process in cancerous tissues. Our lead compound, SR16388, is a potent antiangiogenic agent with binding affinity to estrogen receptor-α (ER-α) and -β (ER-β) at the nanomolar range. This compound inhibited the proliferation of human microvascular endothelial cells (HMVEC) and various types of human cancer cells in vitro. SR16388 inhibited embryonic angiogenesis as measured in the chick chorioallantoic membrane (CAM) assay. The blood vessel density in the CAM was greatly reduced after the embryos were treated with 3 μg/CAM of SR16388 for 24 h. SR16388 at a dose of 2 μM prevented tube formation in Matrigel after HMVEC cells were treated for 8 h. In a modified Boyden chamber assay, SR16388 inhibited the migration of HMVECs by 80% at 500 nM. Using a novel in vivo Fibrin Z-chamber model, we demonstrated that SR16388 at a single daily oral dose of 3 mg/kg for 12 days significantly inhibited the granulation tissue (GT) thickness and the microvessel density of the GT as compared to control. More importantly, SR16388 down-regulated the pro-angiogenic transcription factors, hypoxia inducible factor 1α (HIF-1α) and signal transducer and activator of transcription 3 (STAT3) in non-small cell lung cancer (NSCLC) cells. Together, these effects of SR16388 can lead to the reduction of vascularization and tumor growth in vivo.

The orphan nuclear receptor estrogen-related receptor alpha (ERRalpha) has been implicated in the development of various human malignancies, including breast, prostate, ovary, and colon cancer. ERRalpha, bound to a co-activator protein (e.g., peroxisome proliferator receptor gamma co-activator-1alpha, PGC-1alpha), regulates cellular energy metabolism by activating transcription of genes involved in various metabolic processes, such as mitochondrial genesis, oxidative phosphorylation, and fatty acid oxidation. Accumulating evidence suggests that ERRalpha is a novel target for solid tumor therapy, conceivably through effects on the regulation of tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. This report describes a novel steroidal antiestrogen (SR16388) that binds selectively to ERRalpha, but not to ERRbeta or ERRgamma, as determined using a time-resolved fluorescence resonance energy transfer assay. SR16388 potently inhibits ERRalpha's transcriptional activity in reporter gene assays, and prevents endogenous PGC-1alpha and ERRalpha from being recruited to the promoters or enhancers of target genes. Representative in vivo results show that SR16388 inhibited the growth of human prostate tumor xenografts in nude mice as a single agent at 30mg/kg given once daily and 100mg/kg given once weekly. In a combination study, SR16388 (10mg/kg, once daily) and paclitaxel (7.5mg/kg, twice weekly) inhibited the growth of prostate tumor xenografts in nude mice by 61% compared to untreated xenograft tumors. SR16388 also inhibited the proliferation of diverse human tumor cell lines after a 24-h exposure to the compound. SR16388 thus has utility both as an experimental antitumor agent and as a chemical probe of ERRalpha biology.

We previously identified metal-responsive transcription factor-1 (MTF-1) as a positive contributor to mouse fibrosarcoma growth through effects on cell survival, proliferation, tumor angiogenesis and extracellular matrix remodeling. In the present study, we investigated MTF-1 protein expression in human tissues by specific immunostaining of both normal and tumor tissue samples. Immunohistochemical (IHC) staining of a human tissue microarray (TMA), using a unique anti-human MTF-1 antibody, indicated constitutive MTF-1 expression in most normal tissues, with liver and testis displaying comparatively high levels of expression. Nevertheless, MTF-1 protein levels were found to be significantly elevated in diverse human tumor types, including breast, lung and cervical carcinomas. IHC analysis of a separate panel of full-size tissue sections of human breast cancers, including tumor and normal adjacent, surrounding tissue, confirmed and extended the results of the TMA analysis. Taken with our previous findings, this new study suggests a role for MTF-1 in human tumor development, growth or spread. Moreover, the study suggests that MTF-1 could be a novel therapeutic target that offers the opportunity to manipulate metal or redox homeostasis in tumor cells.

DNA-damaging agents are widely used for the treatment of human malignancies. Agents containing the multifunctional alkylating moiety tetrakis(2-chloroethyl)phosphorodiamidic acid are currently under development as cancer therapeutics.TLK58747, a phophorodiamidate-based prodrug, was tested in vivo for antitumor efficacy and safety. The in vitro responses of tumor cells to TLK58747 were examined by cytotoxicity assays, cell cycle analysis, immunoblots and microscopy.TLK58747 was efficacious in xenograft models of human breast, pancreas, and prostate cancer, as well as in leukemia and glioma. It caused less bone marrow suppression in rats than did cyclophosphamide. In vitro, TLK58747 inhibited the growth of a wide variety of cancer cells and activated the DNA damage-response pathway, leading to G(2)/M cell cycle arrest and subsequent premature senescence or apoptosis.TLK58747 is a promising new alkylating agent with broad antitumor activity and superior safety that warrants further development.

Recognition of abnormal glycosylation in virtually every cancer type has raised great interest in exploration of the tumor glycome for biomarker discovery. Identifying glycan markers of circulating tumor cells (CTCs) represents a new development in tumor biomarker discovery. The aim of this study was to establish an experimental approach to enable rapid screening of CTCs for glycan marker identification and characterization. We applied carbohydrate microarrays and a high-speed fiber-optic array scanning technology (FASTcell) to explore potential glycan markers of breast CTCs (bCTCs) and targeting antibodies. An anti-tumor monoclonal antibody, HAE3-C1 (C1), was identified as a key immunological probe in this study. In our carbohydrate microarray analysis, C1 was found to be highly specific for an O-glycan cryptic epitope, gpC1. Using FASTcell technology, we established a procedure to quantify expression levels of gpC1 in tumor cells. In blood samples from five Stage IV metastatic breast cancer patients, the gpC1 positive CTCs were detected in all subjects; approximately 40% of bCTCs were strongly gpC1 positive. Interestingly, the CTCs from a triple-negative breast cancer patient with multiple sites of metastasis were predominantly gpC1 positive (92.5%, 37/40 CTCs). Taken together, we present here a practical approach to examine rare cell expression of glycan markers. Using this approach, we identified an O-core glyco-determinant gpC1 as a potential immunological target of bCTCs. Given its bCTC-expression profile, this target warrants an extended investigation in a larger cohort of breast cancer patients. Citation Format: Lidia C. Sambucetti, Xiaohe Liu, Ben Hsieh, Richard Bruce, George Somlo, Jiaoti Huang, Denong Wang. Exploring glycan markers for immunotyping and precision-targeting of breast circulating tumor cells (CTCs). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C08.

The spliceosome is an important emerging target for cancer therapy that has only recently been uncovered that represents an exciting new opportunity for therapeutic intervention in cancer. Two naturally occurring bacterial products show striking selective toxicity to tumors and have recently been found to act by targeting the SF3b subunit of the spliceosome, thus modulating pre-mRNA splicing in tumor cells. However, these natural products, and analogs derived from them, are synthetically quite complex. We have designed and optimized a class of novel synthetic small molecules, called sudemycins, which also modulate alternative splicing and have substantial antitumor activity. Previous work in our lab has shown that our lead compound, sudemycin D6 has significant in vivo antitumor activity. We now report the development of a high-throughput cell-based luciferase reporter system that has multiple applications in facilitating splicing modulatory drug discovery and development. Citation Format: Yihui Shi, Chandraiah Lagisetti Lagisetti, Amanda S. Joyner, Lidia C. Sambucetti, Thomas R. Webb. Targeting the SF3B1 spliceosome protein: Development of a reporter for HTS screen and pharmacodynamic profiling of small molecule drug leads. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4365. doi:10.1158/1538-7445.AM2015-4365

The GADD45 (growth arrest and DNA damage-inducible) family of genes is involved in the regulation of cell cycle progression and apoptosis. To study signaling pathways affecting GADD45beta expression and to examine systematically in vivo the GADD45beta expression in tissues following various toxic stresses, we created a transgenic mouse by fusing the GADD45beta promoter to firefly luciferase (Gadd45beta-luc). In vivo GADD45beta expression was assessed by measuring the luciferase activity in the Gadd45beta-luc transgenic mouse using a non-invasive imaging system (IVIS Imaging System, Xenogen Corporation). We found that a number of agents that induce oxidative stress, such as sodium arsenite, CCl4, lipopolysaccharide (LPS), or tumor necrosis factor-alpha, are able to induce luciferase expression throughout the entire animal. In liver, spleen, lung, intestine, kidney, and heart, we observed an induction of luciferase activity after LPS treatment, which correlates with an increase of GADD45beta mRNA in these tissues. Processes that induce DNA damage activate the NF-kappaB signaling pathway. Several inhibitors of the NF-kappaB signaling pathway, including dexamethasone, thalidomide, and a proteasome inhibitor, bortezomib, showed inhibitory effects on LPS-induced GADD45beta expression as indicated by a decrease of the luciferase activity. Northern blot analysis confirmed a broad inhibitory effect of bortezomib on LPS-induced GADD45beta mRNA expression in spleen, lung, and intestine. In liver of bortezomib-treated mice, we observed a reverse correlation between the luciferase activity and the GADD45beta mRNA level. We speculate that such a discrepancy could be due to severe liver toxicity caused by bortezomib and LPS co-treatment. MAPK inhibitors had transient and inconsistent effects on LPS-induced luciferase expression. Our data are consistent with the notion that NF-kappaB, but not the MAPK signaling pathways, is involved in the in vivo regulation of GADD45beta expression. Thus, NF-kappaB signaling involves induction of GADD45beta expression, which supports the proposed role of GADD45beta in protecting cells against DNA damaged under various stress conditions.

Orphan nuclear receptors (ONRs) have been implicated in the regulation of lipids. Several clinical studies conducted either prospectively or epidemiologically have pointed to a link between the regulation of hepatic CYP enzymes and HDL-cholesterol (HDL-C) and/or apolipoprotein A1 (apoA1). The treatment of rats with a series of imidazole inducers of CYP3A yielded correlations between in vitro CYP3A activity measured as erythromycin demethylase activity and plasma HDL-C and hepatic apoA1 mRNA. Similarly, a correlation was established between in vivo CYP3A activity, measured as ethosuximide clearance, and plasma HDL-C and hepatic apoA1 mRNA. The treatment of wild-type (WT) mice with PXR agonists elicited increases in serum HDL-C and serum apoA1 levels. On the other hand, the treatment of PXR-knockout mice (PXR-KOs) with the same PXR agonists failed to elicit increases in either serum HDL-C or serum apoA1 levels. Superposition of the structures of three imidazoles known to be active CYP3A inducers in rats with the human PXR pharmacophore demonstrated a partial fit and predicted EC 50 values typical of weak-moderate hPXR inducers in humans. These imidazoles have been shown to increase apoA1 and HDL-C in rats and mice. Taken together, these data suggest that PXR plays an important role in the regulation of apoA1 and HDL-C in rodents.

MAP4K4, a Ser/Thr kinase, was identified as an important pro-migratory kinase in an siRNA screen, targeting 5,234 human genes for modulators of tumor cell motility. MAP4K4 siRNA potently suppressed cell invasion and migration of multiple cancer cell lines, indicating a broad role in cell motility. There are no drugs in the clinic that are known to specifically target MAP4K4 for cancer therapy. We have successfully developed an orally active, highly effective and selective MAP4K4 inhibitor (SRI-28731) with potent in vitro and in vivo anticancer activity. SRI-28731 is more potent than Paclitaxel (Taxol) against most of the breast cancer cell lines tested. SRI-28731 exhibits more potent activities against triple negative (MDA-MB-231, BT549 and Hs578T) than estrogen-dependent (T47D and MCF-7) breast cancer cell lines, and its potency is positively correlated with MAP4K4 expression in cancer cell lines. SRI-28731 is also more potent than Docetaxel against both androgen-dependent (LNCaP) and -independent (PC-3 and DU-145) prostate cancer cell lines. In vitro mechanistic studies showed that SRI-28731 induced apoptosis and a time-dependent M phase arrest. Treatment with SRI-28731 (12.5, 25 and 50 mg/kg/day) caused a significant dose-dependent growth reduction of PC-3 tumors (30%, 61% and 88% growth inhibition, respectively), while Docetaxel at its MTD (7.5 mg/kg; Q3Dx2) produced only 10-15% growth inhibition. At the end of PC-3 tumor xenograft studies, we conducted an ex vivo invasion assay using PC-3 tumor cells isolated from tumor-bearing mice. SRI-28731 significantly reduced ex vivo tumor cell invasion by ∼80%. Pharmacokinetic studies showed that SRI-28731 could be detected in plasma up to 8 hours after oral dosing, and drug plasma concentrations remained above the IC50 values needed to inhibit prostate or breast cancer proliferation. To quantitatively define the kinase selectivity of SRI-28731, we tested the interaction of SRI-28731 with 456 kinase protein kinases (KINOMEscan), followed by in vitro pharmacology studies. Our data indicated that SRI-28731 is a highly selective Type-II MAP4K4 inhibitor. Type II kinase inhibitors bind to both the ATP site and an adjacent hydrophobic site exposed in the non-activated kinase state. Generally, type II inhibitors show higher selectivity for targets, and act primarily by locking the equilibrium switch between conformational states in a way that prevents kinase activation, rather than directly inhibiting it. Elevated MAP4K4 expression is strongly associated with higher rate of metastasis, and is regarded as an independent predictor of overall survival in cancer patients. Since MAP4K4 is overexpressed in many human cancer cell lines but is undetectable in non-transformed epithelial cells, targeting MAP4K4 may provide effective anti-metastatic therapy with limited side effects on normal tissues. Citation Format: Chih-Tsung Chang, Jaehyeon Park, Wei Zhou, Xiaohe Liu, Barbara Sato, Dominic Dinh, Anna Furimsky, Lucia Beviglia, Lidia Sambucetti, Ling Jong. SRI-28731, a highly potent and selective MAP4K4 (HGK) inhibitor for cancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 731. doi:10.1158/1538-7445.AM2014-731

More predictive in vivo animal tumor models are urgently needed to test efficacy of newly developed cancer therapies and predict clinical response. At SRI we have developed highly innovative and predictive models initiated with single tumor cells isolated from cancer patient specimens, including colon, lung, and breast tumor xenografts. These tumors propagated in immunocompromised mice recapitulate primary tumor heterogeneity as shown by histology. In our models, the tumor cells are implanted subcutaneously to evaluate inhibition of primary tumor, or at orthotopic sites to recapitulate advanced metastatic disease and analyze circulating tumor cells (CTCs) or disseminated tumor cells (DTCs). Tumor cells are also implanted in organs, e.g. kidney capsule, to mimic the vascularized tumor microenvironment or in the human skin graft to analyze the tumor interactions with human stroma and vasculature, and the effect of treatment on angiogenesis. The cellular suspension method offers several advantages over the widely employed tumor fragments, including the ability to assess the phenotype of tumor cell populations, tumorigenicity, and tumor initiating cells. Cells isolated from tumors of mice in control and treatment groups are mouse-lineage depleted prior to evaluation in a number of in vitro or ex vivo assays, including migration and invasion through extracellular matrix proteins. To determine whether treatment affects frequency of tumor initiating cells (TICs) and metastasis initiating cells (MICs), we will perform in vivo limiting dilution assay (LDA). Moreover, to evaluate the ability of our tumor models to induce angiogenesis we utilized the chicken embryo chorioallantoic membrane (CAM) assay and scored the vascularity of mouse lineage-depleted tumor cells using an established CAM scoring guide with 5 as the highest score and 0 as the lowest score. One patient derived ER- invasive ductal carcinoma breast cancer (SRI-B1) with high growth rate demonstrated strong pro-angiogenic activity, inducing blood vessels in the CAM assay with a score of 4 compared to 5 for a VEGF positive control and 0 of negative control. The high vascular density induced by SRI-B1 tumor cells correlated with in vivo metastatic dissemination to distant organs upon orthotopic implantation in NOD/SCID mice, suggesting that tumor models can be screened in the CAM assay to evaluate their metastatic potential. We plan to further use these models to test the anticancer activity of experimental therapies that target metastasis. Citation Format: Jun Li, Jessica Kalra, Dominic Dinh, Wan-ru Chao, Xiaohe Liu, Lidia Sambucetti, Lucia Beviglia. In vivo animal models with patient-derived tumor xenografts for better clinical prediction. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1205. doi:10.1158/1538-7445.AM2014-1205

Histone acetylation is emerging as a major regulatory mechanism thought to modulate gene expression by altering the accessibility of transcription factors to DNA. In this study, treatment of human tumor cells with the histone deacetylase inhibitor, trapoxin (TPX), resulted in selective changes in genes that control the cell cycle. TPX activated p21(waf1) transcription that led to elevated p21(waf1) protein levels in three human tumor cell lines without altering the protein levels of cdk2, cdk4, or cyclin B. In addition, TPX increased cyclin E transcription without increasing the levels of Rb, E2F, dihydrofolate reductase, or glyceraldehyde-3-phosphate dehydrogenase. The elevated levels of p21(waf1) protein led to decreased Rb phosphorylation and cdk2 activity. These effects resulted in G(1) and G(2) cell cycle arrest in H1299 human lung and MDA-MB-435 breast carcinoma cells and apoptosis in A549 lung carcinoma cells. Chromatin immunoprecipitation assays revealed that TPX increased the level of chromatin acetylation associated with histone H3 in the trapoxin-responsive region of the p21(waf1) promoter. This study demonstrates that inhibition of HDAC by TPX increases acetylation of H3-associated chromatin and alters gene expression with marked selectivity.

Circulating tumor cell (CTC) analysis uses a “liquid biopsy” to provide critical information regarding diagnosis, prognosis, response to therapy and risk of relapse. To date, CTC analysis in non-small cell lung cancer (NSCLC) patients using epithelial cell adhesion molecule (EpCAM) immunomagnetic enrichment techniques has met with limited success. This is thought to be because the majority of CTCs detected in NSCLC patients demonstrate the mesenchymal phenotype to various extents, and epithelial markers on CTCs are lost during the epithelial-mesenchymal transition (EMT). We report here that the non-enrichment, FAST platform can be used for rapid detection and characterization of CTCs with a hybrid EMT phenotype. We developed a model using whole blood from healthy donors spiked with human NSCLC cell lines that have been classified as having varying levels of EMT. These included “more epithelial, high EpCAM type” (A549 and EKVX), and “more mesenchymal, low EpCAM type” (H2228 and Calu-1) cell lines. A multiplexed assay was developed to simultaneously evaluate cellular and molecular markers of the hybrid phenotypes. FAST was used to develop an optimized assay with high yields (more than 75% retention, detection performance as high as 1 CTC/5 ml blood) of CTCs with hybrid phenotype. 100% of NSCLC cells detected by the FAST platform co-expressed mesenchymal/stem cell markers such as Vimentin, N-Cadherin and ALDH1. FACS analysis of NSCLC cells confirmed levels of EpCAM expression ranging from negative to high. CTCs harboring anaplastic lymphoma kinase (ALK) translocations also demonstrated mesenchymal-like phenotypes. Finally, a high incidence of CTC clusters (circulating tumor microemboli; CTM) that have been previously reported with the hybrid phenotype, was detected from Stage IV NSCLC patients. These data illustrate that the FAST platform is suitable for high-sensitivity detection and comprehensive analyses of the full spectrum of EMT phenotypes in NSCLC CTCs. We have also developed assays to multiplex cellular and molecular biomarkers on single CTCs to guide personalized therapy, including the detection of ALK to select patients appropriate for Crizotinib therapy. Further work is planned to analyze the dynamic change of EMT components in CTCs during disease progression to correlate EMT biomarkers in CTCs with patient outcomes and provide new diagnostic tools for lung cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A199. Citation Format: Xiaohe Liu, Janey C. Ly, Laurie Kara, Heather Wakelee, Nathan Collins, Keith Laderoute, Lidia C. Sambucetti. Using Fiber Array Scanning Technology (FAST) Platform for sensitive detection and analysis of circulating tumor cells with a hybrid epithelial/mesenchymal phenotype from non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A199.

p97 (also called VCP in metazoans and CDC48 in yeast) is a highly conserved, ubiquitously expressed, and essential AAA ATPase. p97 plays a key role in endoplasmic reticulum-associated degradation of misfolded secretory and membrane proteins as well as ubiquitin-dependent turnover of a subset of cytoplasmic substrates of the ubiquitin-proteasome system. p97 also plays a critical role in specific cellular processes including Golgi membrane reassembly, membrane fusion, autophagy and DNA repair mechanisms. p97 expression is elevated in a number of cancer types including prostate cancer, non-small-cell lung carcinoma and leukemia. Given its cellular functions in protein homeostasis and the successful clinical development of proteasome inhibitors, p97 has emerged as an exciting new target for cancer therapy. We used p97 siRNA and a recently identified small molecule p97 inhibitor, N2,N4-dibenzylquinazoline- 2,4-diamine (DBeQ) as tools to develop functional assays suitable for further evaluating the activity of potential p97 inhibitors. A panel of unfolded protein response marker genes that are consistently modulated by p97 siRNA or DBeQ treatment has been identified. In addition, methods to monitor autophagic activity have been developed, including high content analysis of LC3B-GFP puncta formation. Cell viability assays were used to determine the susceptibility of various cancer cell lines to p97 inhibition. These assays will be used to evaluate the effects of potential p97 inhibitors on p97-dependent pathways and anti-proliferative activity. Acknowledgement: Funded by NExT Chemical Biology Consortium, NCI Contract No. HHSN261200800001E. Citation Format: Yihui Shi, Xiaohe Liu, Joy Calaoagan, Steven Samuelsson, Tsui-Fen Chou, Raymond J. Deshaies, Lidia C. Sambucetti. Development of functional assays for p97/VCP inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2146. doi:10.1158/1538-7445.AM2013-2146

SR16388 is a novel amino steroid that targets estrogen-binding proteins including genomic estrogen receptors (ERs). Preclinical studies have demonstrated that SR16388 is a well-tolerated, orally active compound with compelling properties as an experimental antitumor agent: SR16388 has been demonstrated to 1) inhibit the proliferation and viability of diverse human cancer cell lines in vitro; 2) have antiangiogenic activity in vivo; and 3) reduce the growth of human tumor xenografts in mice. SR16388, which binds to and potently inhibits ERα and ERβ in breast cancer cells, also inhibits the widely expressed orphan nuclear receptor ERRα that has been implicated in the development of various human malignancies. ERRα is thought to regulate tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. We have been exploring potential mechanisms that could explain SR16388’s broad anti-proliferative effects toward human tumor cell lines with different genotypes and tissues of origin. In these studies, we observed that SR16388 can initiate a cell-cycle arrest at G1 or G2 and ultimately cause cytotoxicity (e.g., by apoptosis) even in cells that do not express ERs or ERRα. We recently observed that SR16388 can induce persistent macroautophagy (autophagy) in diverse tumor cells, including cells from hematopoietic malignancies (e.g., multiple myeloma/MM cells) in addition to carcinomas such as prostate and breast cancers. We demonstrated induction of autophagy by using established assays (e.g., LC3I/II processing; fluorescent reporters for acidic vacuole formation; long-lived protein degradation). Here we present results demonstrating that SR16388 can rapidly and potently induce autophagy in cultures of MM cells (U266, RPMI-8226) and PC3 prostate cancer cells. Induction of autophagy in PC3 cells, in particular, was detected in tumor xenografts as early as 3 hours after dosing of mice with a therapeutic dose level of SR16388. We will present data supporting the hypothesis that SR16388 exerts its anti-proliferative and anti-tumor effects in part through the induction of stress pathways modulated by perturbations of protein homeostasis. These effects could yield therapeutic benefits that derive from targets within solid tumor microenvironments. Citation Format: Lidia C. Sambucetti, Wei Zhou, Joy Calaoagan, Laurie Kara, Xiaohe Liu, Barbara Sato, Steve Samuelson, Nathan Collins, Keith Laderoute. SR16388 impairs protein homeostasis and induces autophagy in diverse human cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1692. doi:10.1158/1538-7445.AM2013-1692

Synergistic combinations of anti-cancer compounds can provide therapeutic benefits by producing better efficacy at potentially lower doses. SR16388, a novel steroidal compound that inhibits estrogen and estrogen-related receptors, has previously been shown to inhibit the proliferation and viability of diverse human cancer cell lines in vitro and to reduce the growth of several types of human xenograft tumors in mice, including, NSCLC and androgen-independent prostate cancer. We examined the two-drug combination effects of SR16388 in cell culture systems using a panel of approved cancer drugs with different mechanisms-of-action. Sixty cancer cell lines were treated with compounds alone or in combination for three days and CellTiter-Glo® Luminescent Cell Viability Assay (Promega Corp.) was used as the endpoint to measure cell viability. Combinations of SR16388 with anti-mitotic drugs Paclitaxel or Vincristine demonstrated synergistic effects on cell killing in multiple cell lines. These synergistic effects were further evaluated by calculating combination-index values using CalcuSyn software (Biosoft, Cambridge, UK)”. Studies are ongoing to elucidate the mechanisms that may lead the synergistic interaction of SR16388 and these antimitotic drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C178.

Multiple myeloma (MM) is a B-cell malignancy that is characterized by accumulation of clonal plasma cells in the bone marrow. The bone marrow microenvironment plays a critical role in MM cell pathogenesis and progression. In the bone marrow milieu, MM cells adhere to the bone marrow stromal cells and trigger the secretion of growth factors such as vascular endothelial growth factor (VEGF). VEGF in turn upregulates the secretion of interleukin-6 (IL-6), the major cytokine that mediates MM cell growth and survival in part through the activation of Janus kinase (Jak)/signal transducers and activators of transcription 3 (STAT3). VEGF also stimulates the proliferation of endothelial and stromal cells and induces angiogenesis via its receptors. Thus, targeting both myeloma cells and bone marrow microenvironment is a valid approach for the development of therapeutic agents for MM. At SRI International, we have developed an antiestrogen, SR16388, that binds to ER-α, ER-β and ERR-α with potent antitumor and antiangiogenic properties. We have previously reported that SR16388 inhibited MM cell proliferation in vitro and MM tumors in vivo. In the current study, we are presenting data to show that the inhibitory effect of SR16388 on MM cell proliferation was achieved by inducing apoptosis, and arresting cells at the G2 phase. Furthermore, at nanomolar concentrations SR16388 inhibited IL-6-induced cell proliferation and blocked the activation of STAT3 induced by IL-6 in RPMI-8226 human MM cells. In human endothelial cells, SR16388 inhibited VEGF-induced cell proliferation and VEGF-induced phosphorylation of STAT3. In a RPIM-8226 tumor xenograft model, the microvessel density in the tumor tissue was markedly reduced by treatment with SR16388. We are currently investigating the effect of SR16388 in combination with known therapeutic agents on the growth of MM tumors in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3532. doi:10.1158/1538-7445.AM2011-3532

SR13668, an orally active cancer therapeutic agent, exhibits potent antitumor activity against various human cancer xenografts (e.g., breast, prostate, lung and ovarian cancer) without significant toxicity. An exploratory IND study of SR13668 has been completed with a two-stage design to compare the bioavailability of five SR13668 formulations, and the preferred formulation will be subjected to investigation in subsequent phase I testing. SR13668 inhibits growth factor-stimulated phosphorylation of Akt and its downstream target GSK3β in PC-3 prostate and high pAkt expressing MDA-MB-468 breast cancer cells in a dose-dependent manner. SR13668 was also shown to block FGF- and IGF-stimulated Akt activation. Screening a broad selection of kinase targets, including Akt(1,2,3), PI3K, and PDK1, indicated that SR13668 is not a kinase inhibitor. Preliminary mechanistic studies showed that SR13668 selectively inhibits 12-lipoxygenase (12-LOX), but does not affect other lipoxygenases (5-LOX and 15-LOX), or cyclooxygenases (COX-1 and COX-2). 12-LOX plays a major role in promoting cancer progression, invasion and metastasis as well as tumor angiogenesis. 12-LOX has been regarded as a novel target for cancer therapy, since it is generally absent in normal epithelia and is often constitutively expressed in various epithelial cancers. Unlike direct inhibitors of PI3K or Akt, SR13668 has no adverse effects on fasting glucose levels or body weights after 14 days of oral treatment with SR13668 at 500 mg/kg/day, a dose more than 10 times higher than that needed for antitumor activity. Pharmacologic intervention studies with lipoxygenase inhibitors for the prevention or inhibition of cancers have been difficult to interpret, as there are very few isoform-selective LOX inhibitors available. SR13668 is a highly specific 12-LOX inhibitor. No off-target activities were observed, when screened against a broad collection of 40 diverse enzymes and 80 transmembrane and soluble receptors, ion channels and monoamine transporters. The Phase 0 clinical study showed that SR13668 has long half-life (11.2 ± 3.1 hours), and patients did not experience significant adverse events related to SR13668 treatment; thus SR13668 is a promising candidate for further clinical investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4483. doi:10.1158/1538-7445.AM2011-4483