Variety Of Tumor Models Research Articles

Phase 2 study to evaluate the novel mitochondrial PRX3 inhibitor, RSO-021, as an intrapleural monotherapy and in combination with IV paclitaxel in patients with malignant pleural effusion due to mesothelioma or another advanced solid tumor.

TPS8124 Background: Tumor cells generate elevated levels of reactive oxygen species (ROS), leading to increased expression and activity of critical ROS scavenging pathways. Peroxiredoxin 3 (PRX3) is the principal peroxide scavenging enzyme in mitochondria. RSO–021 is a novel small molecule PRX3 inhibitor that inactivates PRX3 through direct covalent adduction of active site cysteine residues, in turn inducing oxidative stress that is incompatible with tumor cell survival. Preclinical studies of RSO-021 show that it selectively inhibits mitochondrial PRX3 in vitro and is highly effective in a variety of tumor models, including mesothelioma and many solid tumors. RSO-021 has completed phase 1 evaluation in patients with advanced mesothelioma or solid tumors with predominant pleural disease and malignant pleural effusions. The phase 1 data supported weekly intrapleural dosing of RSO-021 at the 90 mg dose level, with acceptable safety and early signals of efficacy. Some patients were able to receive >30 weeks of treatment. Pre- and post-dose primary prophylaxis for local inflammatory reactions to intrapleural injection was instituted during dose escalation, using corticosteroids, NSAIDS and paracetamol. It also became clear that RSO-021 should not be administered to patients with a dry pleural tap, due to risk of extravasation. Methods: The objectives of this open-label, multi-center, 4-arm phase 2 study (MITOPE) are to confirm the efficacy, safety, tolerability, and pharmacology of weekly intrapleural RSO-021 administration. Exploratory endpoints include target engagement as well as biomarkers of response and inflammation. The study explores 2 different RSO-021 doses (90 mg/wk and 45 mg/wk) per FDA Project Optimus, both as monotherapy and in combination with IV paclitaxel 175 mg/m2 every 3 weeks at until progression or limiting toxicity. Each arm at each dose has a 2-stage Simon design (12-21 patients) requiring 1 response to trigger stage 2 (null/target response rate 3%/20% and type I/type II error 0.05/0.8) for a maximum of 168 patients in the trial. Three of the 4 study arms are investigating the antitumor activity of RSO-021 monotherapy (arms 1-3) and the fourth is assessing the paclitaxel combination. Eligibility (by arm) requires: newly diagnosed treatment-naïve mesothelioma with pleural effusion; mesothelioma with pleural effusion that progressed on ≥1 standard of care (SoC) regimen; solid tumor with predominant pleural/lung disease and effusion that progressed on ≥1 SoC regimen; and breast, epithelial ovarian or non-small cell lung cancer with predominant pleural/lung disease and effusion that progressed on ≥1 SoC regimen. The first patient was treated in 4Q2023 and the trial is actively recruiting to all 4 arms at 9 UK sites. Additional sites are planned. Clinical trial information: NCT05278975 .

Abstract 3133: A novel bispecific antibody-drug conjugate targeting HER2 and Trop-2 displays potent antitumor activity in a variety of tumor models with promising preclinical characteristics

Abstract Antibody-drug conjugates (ADCs) have exhibited considerable advancements in cancer therapy, though their efficacy is often confined to specific patient subsets or results in modest clinical responses due to low and heterogenous expression of drug targets in tumors. Targeting more than one tumor-associated antigens (TAAs) represents a new strategy to broaden treatment applicability and minimize on-target toxicity of ADC. Our analysis of co-expression of HER2 and Trop-2, two clinically validated ADC targets, in tumors of various histological malignancies manifests the great potential for an ADC simultaneously targeting these two antigens. We describe here a novel bispecific ADC (BIO-201) designed to co-target HER2 and Trop-2. This compound comprises a bispecific anti-HER2/Trop-2 antibody conjugated with a novel topoisomerase I inhibitor via a cleavable linker. BIO-201 demonstrates increased or comparable cell binding and internalization compared to parental antibodies. In in vitro cytotoxicity assays, BIO-201 effectively kills cancer cells with an IC50 in the sub nM range, irrespective of whether they co-express HER2 and Trop-2 or express only one antigen. Co-culture studies reveal that BIO-201 possesses a good bystander killing effect on drug target-negative tumor cells. In vivo studies further demonstrate the superior anti-tumor activity of BIO-201 in various tumor xenograft models, including those expressing different levels of HER2 and/or Trop-2, as well as models resistant to HER-2-targeting therapies. Moreover, BIO-201 exhibits favorable plasma stability, preclinical pharmacokinetics (PK), and toxicity profiles. In conclusion, these findings suggest that the anti-HER2/Trop-2 bispecific ADC, BIO-201, holds significant promise as an effective therapy for a diverse range of cancers, especially those expressing heterogenous levels of HER2 or Trop-2, and has the potential to benefit a broad patient population. Citation Format: Haihong (Helen) Zhong, Yan Xiong, Han Huang, Yuming Pan, Na Wang, Bin Sun, Shen Liu, Wei Yuan, Dingguo Liu, Jan Fang, Haifeng Bao. A novel bispecific antibody-drug conjugate targeting HER2 and Trop-2 displays potent antitumor activity in a variety of tumor models with promising preclinical characteristics [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 3133.

Abstract 2717: A potent and highly tumor-selective KRAS inactivator for tumor targeting

Abstract Cancer driver mutations in the RAS-RAF-MEK-ERK pathway occur in 46% of all human cancers. This has inspired the successful development of many small molecule inhibitors of BRAF and MEK. However, development of RAS small molecule inhibitors has not been successful in large part due to the lack of a sufficiently large and deep hydrophobic pocket in RAS proteins to allow for small molecule binding. Notably, in this regard, many bacterial pathogens have evolved potent protein toxins to disrupt the RAS-RAF-MEK-ERK pathway. Fortunately, these naturally designed potent toxins, such as anthrax toxin, Fortunately, these potent, naturally designed toxins, such as anthrax toxin and DUF5, can be structurally modified to achieve high tumor specificity. DUF5 (amino acids Glu3581-Gln4089) is a toxin effector domain of MARTX (a multifunctional-autoprocessing repeats-in-toxin) from Vibrio vulnificus. DUF5 was recently identified to be a specific and potent endopeptidase that cleaves and inactivates RAS. To address the above critical unmet medical needs, in this work, we have generated an anthrax toxin/DUF5-based, highly tumor-selective RAS inactivator and evaluated its anti-tumor activity in a variety of tumor models. This tumor-selective RAS inactivator can specifically bind to the major anthrax toxin receptor CMG2 on tumor cells and tumor stromal cells, and strictly relies on the simultaneous presence of two distinct tumor-associated proteases, MMPs and urokinase, to gain entry into tumor cells and tumor stromal cells to proteolytically inactivate RAS signaling, achieving potent selective targeting. Our data show that our tumor-selective RAS inactivator displays potent cytotoxicity to cancer cells whose survival depends on oncogenic RAS signaling. Our engineered toxin displayed potent in vivo antitumor activity to a range of tumors with oncogenic RAS mutations in syngeneic and human xenograft mouse tumor models. Therefore, we have developed a tumor-selective, safe, and highly potent RAS inactivator that has high potential for targeting tumors with oncogenic RAS mutations. Citation Format: Shihui Liu, Zehua Zuo, Jie Liu. A potent and highly tumor-selective KRAS inactivator for tumor targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2717.

Retroviral Replicating Vector Toca 511 (Vocimagene Amiretrorepvec) for Prodrug Activator Gene Therapy of Lung Cancer.

Therapeutic efficacy of retroviral replicating vector (RRV)-mediated prodrug activator gene therapy has been demonstrated in a variety of tumor models, but clinical investigation of this approach has so far been restricted to glioma and gastrointestinal malignancies. In the present study, we evaluated replication kinetics, transduction efficiency, and therapeutic efficacy of RRV in experimental models of lung cancer. RRV delivering GFP as a reporter gene showed rapid viral replication in a panel of lung cancer cells in vitro, as well as robust intratumoral replication and high levels of tumor transduction in subcutaneous and orthotopic pleural dissemination models of lung cancer in vivo. Toca 511 (vocimagene amiretrorepvec), a clinical-stage RRV encoding optimized yeast cytosine deaminase (yCD) which converts the prodrug 5-fluorocytosine (5-FC) to the active drug 5-fluorouracil (5-FU), showed potent cytotoxicity in lung cancer cells upon exposure to 5-FC prodrug. In vivo, Toca 511 achieved significant tumor growth inhibition following 5-FC treatment in subcutaneous and orthotopic pleural dissemination models of lung cancer in both immunodeficient and immunocompetent hosts, resulting in significantly increased overall survival. This study demonstrates that RRV can serve as highly efficient vehicles for gene delivery to lung cancer, and indicates the translational potential of RRV-mediated prodrug activator gene therapy with Toca 511/5-FC as a novel therapeutic strategy for pulmonary malignancies.

The immunomodulatory role of all-trans retinoic acid in tumor microenvironment.

Retinoids are essential nutrients for human beings. Among them, all-trans retinoic acid (ATRA), considered one of the most active metabolites, plays important roles in multiple biological processes. ATRA regulates the transcription of target genes by interacting with nuclear receptors bonded to retinoic acid response elements (RAREs). Besides its differentiation-inducing effect in the treatment of acute promyelocytic leukemia and some solid tumor types, its immunoregulatory role in tumor microenvironment (TME) has attracted considerable attention. ATRA not only substantially abrogates the immunosuppressive effect of tumor-infiltrating myeloid-derived suppressor cells but also activates the anti-tumor effect of CD8 + T cells. Notably, the combination of ATRA with other therapeutic approaches, including immune checkpoint inhibitors (ICIs), tumor vaccines, and chemotherapy, has been extensively investigated in a variety of tumor models and clinical trials. In this review, we summarize the current understanding of the role of ATRA in cancer immunology and immunotherapy, dissect the underlying mechanisms of ATRA-mediated activation or differentiation of different types of immune cells, and explore the potential clinical significance of ATRA-based cancer therapy.

Preclinical Safety and Biodistribution in Mice Following Single-Dose Intramuscular Inoculation of Tumor DNA Vaccine by Electroporation.

The safety, biodistribution, and pharmacokinetics of any new therapeutic tumor DNA vaccine must be evaluated in preclinical studies. We previously developed the DNA vaccine (CpDV-IL2-sPD1/MUC1 and survivin), which showed excellent antitumor effects in a variety of tumor models. In this study, we demonstrate the safety and biodistribution after immunization with naked DNA vaccine (10 mg/kg) by electroporation in a mice model. All mice reached the end of the study with good body conditions. By established and validated QPCR method, we found high-copy plasmid DNA at the injection site (muscle) on day 1 in all eight animals, followed by a downward trend. By day 49, a small amount of plasmid DNA was still detectable, but only in one mouse. On reproductive safety, no plasmids existed in the ovary at any time point. Also, only two of the 16 testis samples could detect a very small amount of DNA on days 7 and 14. The most important thing was that plasmids were cleared from almost all organs (heart, liver, spleen, lung, kidney, stomach, blood, thymus, intestine) on day 49. In summary, the results of our experiments demonstrate that the DNA vaccine delivered by electroporation was shown to be safe and merits further development for cancer treatment.

Blocking autophagy overcomes resistance to dual histone deacetylase and proteasome inhibition in gynecologic cancer

Histone deacetylase (HDAC) inhibitors and proteasome inhibitors have been approved by the FDA for the treatment of multiple myeloma and lymphoma, respectively, but have not achieved similar activity as single agents in solid tumors. Preclinical studies have demonstrated the activity of the combination of an HDAC inhibitor and a proteasome inhibitor in a variety of tumor models. However, the mechanisms underlying sensitivity and resistance to this combination are not well-understood. This study explores the role of autophagy in adaptive resistance to dual HDAC and proteasome inhibition. Studies focus on ovarian and endometrial gynecologic cancers, two diseases with high mortality and a need for novel treatment approaches. We found that nanomolar concentrations of the proteasome inhibitor ixazomib and HDAC inhibitor romidepsin synergistically induce cell death in the majority of gynecologic cancer cells and patient-derived organoid (PDO) models created using endometrial and ovarian patient tumor tissue. However, some models were not sensitive to this combination, and mechanistic studies implicated autophagy as the main mediator of cell survival in the context of dual HDAC and proteasome inhibition. Whereas the combination of ixazomib and romidepsin reduces autophagy in sensitive gynecologic cancer models, autophagy is induced following drug treatment of resistant cells. Pharmacologic or genetic inhibition of autophagy in resistant cells reverses drug resistance as evidenced by an enhanced anti-tumor response both in vitro and in vivo. Taken together, our findings demonstrate a role for autophagic-mediated cell survival in proteasome inhibitor and HDAC inhibitor-resistant gynecologic cancer cells. These data reveal a new approach to overcome drug resistance by inhibiting the autophagy pathway.

Mass spectrometry imaging revealed alterations of lipid metabolites in multicellular tumor spheroids in response to hydroxychloroquine

Three-dimensional (3D) multicellular tumor spheroids (MCTS) that mimic the complex tumor microenvironment provide a good platform for in vitro study of drug and endogenous metabolites. Hydroxychloroquine (HCQ) has shown anti-tumor activity in a variety of tumor models. However, the effect of the drug on the alteration of lipid metabolism spatial composition and distribution in the MCTS model is not clear. Herein, we utilized matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) in the analysis of A549 lung cancer multicellular spheroids to investigate the in situ spatial distribution of HCQ and its effect on lipid metabolism. We have successfully observed the spatial variations of HCQ in the inner region of the spheroid at different drug-treated time points. The MSI results also demonstrated that HCQ treatment altered the spatial composition of lipids in the inner and outer regions of treated spheroids. Furthermore, the lipidomic results showed that the identified phosphatidylcholines (PC), lysophosphatidylcholines (LPC), phosphatidylethanolamines (PE), lysophosphatidylethanolamines (LPE), phosphatidylinositols (PI), ceramides (Cer), glucosylceramides (CerG), and diglycerides (DG) were significantly up-regulated, and phosphatidylglycerol (PG) and triglycerides (TG) were remarkable down-regulated. MSI method combined with LC-MS/MS profiling of endogenous metabolites can obtain more detailed information about how spheroids respond to drug and spatial distribution information, thus fostering a better understanding of the relationship between drug-altered lipid metabolism and cancer microenvironment.

Abstract 1798: CTLA-4 photoimmunotherapy depletes intratumoral regulatory T cells (Tregs) to augment local and systemic immunity for anticancer activity

Abstract Introduction: Photoimmunotherapy is an investigational cancer treatment platform that utilizes an antibody conjugated to a light activated dye IRDye® 700DX (IR700) combined with illumination with a non-thermal, red light for selective cell killing. CTLA-4 is a lymphocyte exhaustion marker targeted by anti-CTLA-4 checkpoint inhibitors to induce anticancer immune response and is highly expressed on intratumoral regulatory T cells (Tregs). In this study we used anti-CTLA-4-IR700 photoimmunotherapy (CTLA-4 photoimmunotherapy), which involves intratumoral Treg depletion with checkpoint inhibition, in syngeneic mouse models, including models refractory to immune checkpoint inhibition, to assess anticancer activity and immune response. Methods: Mice inoculated with CT26, LL/2, MCA205, or 4T1 tumors were treated with CTLA-4 photoimmunotherapy. Tumor volume was determined by caliper measurements in target (illuminated) and distal (non-illuminated) tumors. Intratumoral immune responses were characterized by flow cytometry. To evaluate the activation of systemic immune responses, cytotoxic T lymphocyte assays were conducted with splenocytes derived from mice treated with CTLA-4 photoimmunotherapy. Tumor re-challenge studies were conducted to assess for tumor-specific immune memory responses. Results: CTLA-4 photoimmunotherapy induced notable anticancer responses including complete responses (CRs), and enhanced survival of mice in multiple tumor models, including tumors resistant to anti-PD-1 or anti-CTLA-4 treatment. CTLA-4 photoimmunotherapy resulted in a reduction of intratumoral Tregs, an increase of the CD8/Treg ratio, and an increase of intratumoral NK and CD8 T cell activation in comparison to control groups. All CR mice (n=7) rejected new tumor inoculations, suggesting enhancement of systemic immune memory. Splenocytes derived from CTLA-4 photoimmunotherapy-treated mice displayed an expansion of tumor antigen-specific lymphocytes in comparison to control groups. In mice bearing bilateral CT26 tumors, tumor regression was observed in target and distal tumors after CTLA-4 photoimmunotherapy, demonstrating systemic anticancer immunity and potential abscopal effects. Conclusion: CTLA-4 photoimmunotherapy elicited anticancer responses in a variety of tumor models, including tumors resistant to anti-PD-1 or anti-CTLA-4 treatment. Depletion of intratumoral Tregs by CTLA-4 photoimmunotherapy resulted in the local activation of CD8 T cell-mediated anticancer activity and systemic tumor specific immune response. These results indicate that treatment with CTLA-4 photoimmunotherapy can induce anticancer responses in checkpoint resistant mouse tumor models by combining rapid Treg depletion with anti-CTLA-4 checkpoint inhibition into a single method of treatment. Citation Format: C. Daniel de Magalhaes Filho, Stephanie M. Okamura, Daniele M. Bergeron, Ahiram Rodriguez, Abram Lozano, Jerry J. Fong, Miguel Garcia-Guzman. CTLA-4 photoimmunotherapy depletes intratumoral regulatory T cells (Tregs) to augment local and systemic immunity for anticancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1798.

Optimisation of estrogen receptor subtype-selectivity of a 4-Aryl-4H-chromene scaffold previously identified by virtual screening

4-Aryl-4H-Chromene derivatives have been previously shown to exhibit anti-proliferative, apoptotic and anti-angiogenic activity in a variety of tumor models in vitro and in vivo generally via activation of caspases through inhibition of tubulin polymerisation. We have previously identified by Virtual Screening (VS) a 4-aryl-4H-chromene scaffold, of which two examples were shown to bind Estrogen Receptor α and β with low nanomolar affinity and <20-fold selectivity for α over β and low micromolar anti-proliferative activity in the MCF-7 cell line. Thus, using the 4-aryl-4H-chromene scaffold as a starting point, a series of compounds with a range of basic arylethers at C-4 and modifications at the C3-ester substituent of the benzopyran ring were synthesised, producing some potent ER antagonists in the MCF-7 cell line which were highly selective for ERα (compound 35; 350-fold selectivity) or ERβ (compound 42; 170-fold selectivity).

Coexpression of Inhibitory Receptors Enriches for Activated and Functional CD8+ T Cells in Murine Syngeneic Tumor Models.

Exhausted T cells have been described in cancer patients and murine tumor models largely based on their expression of various inhibitory receptors. Understanding of the functional attributes of these cells is limited. Here, we report that among CD8+ T cells in commonly used syngeneic tumor models, the coexpression of inhibitory receptors PD-1, LAG3, and TIM3 defined a group of highly activated and functional effector cells. Coexpression of these receptors further enriched for antigen-specific cells with increased T-cell receptor clonality. Anti-PD-L1 treatment increased the number and activation of these triple-positive CD8+ T cells without affecting the density of PD-1- cells. The intratumoral density of CD8+ T cells coexpressing inhibitory receptors negatively correlated with tumor burden. The density ratio and pretreatment phenotype of CD8+ T cells coexpressing inhibitory receptors was positively correlated with response across a variety of tumor models. Our results demonstrate that coexpression of inhibitory receptors is not a signifier of exhausted T cells, but rather can define a group of activated and functional effector cells in syngeneic tumor models. In the cancer setting, these cells could represent a heterogeneous population of not only exhausted but also highly activated cells responsive to treatment.

Capsaicin inhibits glycolysis in esophageal squamous cell carcinoma by regulating hexokinase‑2 expression.

Capsaicin is a principal component of hot red peppers and chili peppers. Previous studies have reported that capsaicin exhibits antitumor functions in a variety of tumor models. Although various mechanisms underlying the capsaicin‑mediated inhibition of tumor growth have been demonstrated, the impact of capsaicin on tumor metabolism has rarely been reported. The present study demonstrated that capsaicin exhibited an inhibitory effect on tumor glycolysis in esophageal squamous cell carcinoma (ESCC) cells. Following treatment with capsaicin, glucose consumption and lactate production in ESCC cells was decreased. Capsaicin resulted in a decrease of hexokinase‑2 (HK‑2) expression, which is known for its important role in tumor glycolysis. Further investigations demonstrated that phosphatase and tensin homolog (PTEN) expression was increased in ESCC cells treated with capsaicin, and that the RAC‑α serine threonine‑protein kinase signaling pathway was downregulated. In PTEN‑knockdown KYSE150 cells, the decrease in HK‑2 and inhibition of glycolysis caused by capsaicin was attenuated, which suggested that the impact of capsaicin on tumor metabolism was associated with its effect on PTEN.

Abstract 3182: Cytoskeletal modulation results in increased tumor survival and drug resistance through attenuation of p53 dependent apoptosis

Abstract One of the major challenges to eradicating cancer involves the evolution of drug resistant clones and persistence of residual disease that escapes our current limit of detection but may proliferate upon cessation of therapy. These microscopic foci of residual diseases often exhibit stem-cell like properties and are intrinsically more resistant to drug therapy. A better understanding of the mechanisms underlying the innate drug resistance of these cellular populations may lead to improved treatment strategies, resulting in more durable remissions and ultimately improved patient survival. We utilize functional genomic platforms to identify mechanisms responsible for the persistence of drug tolerant cells. A genome wide shRNA enrichment screen was performed using the c-Met addicted cell line GTL-16 to identify genes whose knockdown conferred DTP survival. Inhibition of the RhoA-ROCK-myosin pathway promotes drug resistance in a variety of tumor models, including those treated with either targeted therapy or conventional chemotherapy. Furthermore, pharmacological validation using multiple small molecule inhibitors of ROCK1 phenocopied both myosin heavy chain (MYH9) and light chain (MYL12) shRNA knockdown. More recently, utilizing a combination of gene expression and biochemical approaches, we have identified attenuation of p53 induced apoptosis to be a key event in mediating survival of these drug tolerant persisters. Reactivation of p53 using nutlin results in increased cell death. These findings demonstrate that modulation of cytoskeleton is an important, but underappreciated, mechanism of drug resistance across many tumor types. These downstream effectors may serve as novel therapeutic targets for intervention and also biomarkers to stratify patients and their response to treatment. Citation Format: Victoria E. Wang, John Doench, David Root, Rene Bernards, Jeffrey Settleman, Frank McCormick. Cytoskeletal modulation results in increased tumor survival and drug resistance through attenuation of p53 dependent apoptosis [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 3182. doi:10.1158/1538-7445.AM2017-3182

Induction of NKG2D Ligands on Solid Tumors Requires Tumor-Specific CD8+ T Cells and Histone Acetyltransferases.

NKG2D-mediated immune surveillance is crucial for inhibiting tumor growth and metastases. Malignant tumor cells often downregulate NKG2D ligands to escape from immune surveillance. High-profile studies have shown that restoring NKG2D ligand expression via genetic engineering inhibits tumor formation and progression. However, no effective in vivo approaches are available to restore these ligands across different types of solid tumors because the classic stress signal-dependent induction of this ligand in vitro is transient and has rarely been duplicated in solid tumors in vivo We found that coadministration of an immune stimulatory signal (IL12) and chemotherapy (doxorubicin) restored the NKG2D ligand Rae-1 in multiple tumor types, including a human tumor model. The restored expression of NKG2D ligands was associated with tumor cell death and delay of tumor progression in vivo Induction of tumor-specific NKG2D ligands required the engagement of CD8+ T cells and was regulated by the histone acetyltransferases GCN5 and PCAF. The tumor-specific restoration of NKG2D ligands in a variety of tumor models, including a human tumor model, resulted in NKG2D-dependent tumor regression and extended survival time. The elucidation of a CD8+ T cell-dependent mechanism suggests that activated NKG2D+CD8+ T-cell therapy alone may be able to restore the NKG2D ligand in tumors. Cancer Immunol Res; 5(4); 300-11. ©2017 AACR.

BAY 1125976, a selective allosteric AKT1/2 inhibitor, exhibits high efficacy on AKT signaling-dependent tumor growth in mouse models.

The PI3K-AKT-mTOR signaling cascade is activated in the majority of human cancers, and its activation also plays a key role in resistance to chemo and targeted therapeutics. In particular, in both breast and prostate cancer, increased AKT pathway activity is associated with cancer progression, treatment resistance and poor disease outcome. Here, we evaluated the activity of a novel allosteric AKT1/2 inhibitor, BAY 1125976, in biochemical, cellular mechanistic, functional and in vivo efficacy studies in a variety of tumor models. In in vitro kinase activity assays, BAY 1125976 potently and selectively inhibited the activity of full-length AKT1 and AKT2 by binding into an allosteric binding pocket formed by kinase and PH domain. In accordance with this proposed allosteric binding mode, BAY 1125976 bound to inactive AKT1 and inhibited T308 phosphorylation by PDK1, while the activity of truncated AKT proteins lacking the pleckstrin homology domain was not inhibited. In vitro, BAY 1125976 inhibited cell proliferation in a broad panel of human cancer cell lines. Particularly high activity was observed in breast and prostate cancer cell lines expressing estrogen or androgen receptors. Furthermore, BAY 1125976 exhibited strong in vivo efficacy in both cell line and patient-derived xenograft models such as the KPL4 breast cancer model (PIK3CAH1074R mutant), the MCF7 and HBCx-2 breast cancer models and the AKTE17K mutant driven prostate cancer (LAPC-4) and anal cancer (AXF 984) models. These findings indicate that BAY 1125976 is a potent and highly selective allosteric AKT1/2 inhibitor that targets tumors displaying PI3K/AKT/mTOR pathway activation, providing opportunities for the clinical development of new, effective treatments.

Abstract B133: In vivo imaging of innate immune cells to measure drug response

Abstract Background: Tumor-associated macrophages (TAMs) play key roles in the progression of cancers and their response to various treatments, for example through secretion of pro-survival growth factors; contribution to angiogenesis; and modulation of the tumor immune response via antigen presentation and expression of factors such as PD-L1. TAM effects can be highly localized within the tumor microenvironment, for example through cell-cell contact, yet it has been difficult to directly study interactions between TAMs and tumor cells at high resolution, in orthotopic sites of cancer development, and in real-time as cells respond to therapeutic treatment. To understand the biology underlying heterogeneous interactions between TAMs and tumor cells, we used in vivo fluorescence imaging at single-cell resolution to study pharmacodynamic (PD) response of tumor cells and TAMs to chemotherapeutic treatment. Methods: To directly image host leukocytes in a variety of tumor models, we used a combination of fluorescent genetic reporter host animals (CX3CR1GFP/+ reporter mice) and macrophage labeling via well-validated fluorescent and magnetic dextran-coated nanoparticles. For direct visualization of therapeutic response in cancer cells themselves, we used 53BP1-mApple as a transgenic fluorescent reporter to quantify DNA damage responses at the single-cell level. Automated computational segmentation of 3D images enabled precise quantification of statistical correlation between spatial TAM localization and cellular DNA damage responses. We performed studies in multiple human xenograft and syngeneic tumor models, including orthotopic ovarian cancer models and a syngeneic Kras-mutant p53-null lung adenocarcinoma model; histology validated in vivo imaging results. As a proof-of-principle, we tested cellular response to platinum (Pt)-based DNA-damaging agents. Results: Using a novel combination of orthotopic imaging setups, fluorescent reporters for TAMs and cancer cell pharmacodynamic response, along with automated 3D image segmentation and single-cell quantification, here we demonstrate that in vivo imaging can quantify correlations between local TAM concentrations and the simultaneously observed behavior of thousands of cancer cells. As a proof-of-principle, we measure relationships between single-cell DNA damage and local TAM levels in response to Pt-based chemotherapeutic treatment, and find that highly-localized concentrations of tumor-associated immune cells can influence therapeutic response at the single-cell level (and at cellular length-scale gradients). Importantly, we find these relationships to be highly probabilistic (rather than deterministic), and automated classification of thousands of cells across multiple tumors enables robust quantification of the inherent stochasticity involved in the pharmacodynamic responses. We find that single-cell heterogeneity and its explanation by microenvironmental factors such as local TAM concentration and vascular proximity can be heavily dependent on the therapeutic treatment and tumor model. Conclusions: This work presents in vivo imaging technology that should be useful for studying interactions between cancer cells and TAMs in orthotopic sites including intraperitoneal ovarian cancer and can be extended to other cell interactions (for instance by using fluorescent genetic reporters of various lymphocyte populations). Interaction and co-localization between tumor cells and associated immune cells can influence therapeutic response at the single-cell level, and such information will likely be useful for understanding heterogeneous drug responses to chemotherapeutics and immunotherapeutics alike. Citation Format: Miles A. Miller, Mikael Pittet, Ralph Weissleder. In vivo imaging of innate immune cells to measure drug response. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B133.

Abstract B120: Characterization and combination immunotherapy treatment of an inducible autochthonous murine lung cancer model expressing human carcinoembryonic antigen (CEA) as a tumor-associated self-antigen

Abstract Previous work from our lab has demonstrated that a combination of anti-tumor antibody and an IL-2 fusion protein that exhibits extended serum half-life elicits an immune response that can effectively control a wide variety of tumor models. We have since then combined this therapeutic regimen with a vaccine exhibiting efficient lymph node trafficking that can generate an impressive population of tumor-antigen specific CD8+ T-cells but by itself does not provide good anti-tumor efficacy. The efficacy of this combination immunotherapy is further boosted by immune checkpoint blockade, leading to a robust four-component therapy: 1) anti-tumor antigen antibody; 2) IL-2 fusion protein; 3) anti-tumor antigen vaccine; 4) anti-PD-1 antibody. Although this four-pronged approach is demonstrably effective in the syngeneic subcutaneous melanoma model B16F10, we wish to test its efficacy in a more physiological model. To this end, we have turned to a model developed by the Jacks Lab, known as the KP model: an inducible lung tumor model where lentivirus-driven integration and expression of Cre is able to activate oncogenic Kras and completely remove p53 function. Because our therapeutic regimen requires a targetable tumor-associated antigen with respect to both the antibody and vaccine, we chose to induce expression of human carcinoembryonic antigen (CEA) in these tumors, as CEA has a well-studied structure and biology, and frequently expresses aberrantly in many forms of human adenocarcinomas. Additionally, our lab has previously engineered an antibody targeting CEA possessing picomolar affinity. Finally, to remove any endogenous immunological response against human CEA as a foreign antigen in our mouse system, we have crossed the KP model with a mouse model transgenic for human CEA, which in the literature has been described to have identical spatiotemporal expression of CEA as found in humans and should allow for central tolerance of this antigen. In the course of this work, we have successfully introduced human CEA into our lentivirus constructs and shown tumorigenesis by these constructs in the KP model coincides with expression of tumor-associated CEA, as detected by qPCR. On the therapeutic side, we have tailored the vaccine to successfully drive an anti-CEA CD8+ T-cell response. Performing preliminary therapeutic experiments in a transplant model of the KP tumor with our four-component therapy, we saw tumor control compared to untreated tumors. Upon interrogating the CD8+ T-cell response against CEA, we found 1-15% of CD8+ T-cells in the blood respond to CEA stimulation by intracellular cytokine staining. With regards to the lung tumor model, in the course of establishing the system we have also observed that the growth kinetics of tumors expressing CEA lags behind those tumors without CEA, even in the transgenic background. Preliminary immunophenotyping work by flow cytometry suggests that tumors with CEA seem to have a reduced myeloid-derived suppressor cell (MDSC) population and a higher CD8a+ dendritic cell (DC) population compared to tumors without CEA, suggesting that the former may have a less immunosuppressive tumor microenvironment that is better able to prime an anti-tumor CD8+ T-cell response. We will be planning to conduct therapeutic trials in the more physiological lung tumor KP model in the near future, as well as investigate the differences in the immune response with tumors expressing or lacking CEA. Citation Format: Eric F. Zhu, Kavya Rakhra, Naveen Mehta, Kelly D. Moynihan, Cary F. Opel, Darrell J. Irvine, K. Dane Wittrup. Characterization and combination immunotherapy treatment of an inducible autochthonous murine lung cancer model expressing human carcinoembryonic antigen (CEA) as a tumor-associated self-antigen. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B120.

Abstract 4047: CD8+T cell-mediated tumor-specific induction of the NKG2D ligands to trigger NKG2D-dependent tumor cell death in vivo

Abstract Introduction: High profile studies have shown that restoring NKG2D ligand expression greatly promotes antitumor immune surveillance and tumor regression, but malignant tumor cells often down-regulate NKG2D ligands to escape from immune surveillance. Previous studies showed that engineering tumor cells with NKG2D ligand resulted in inhibition of tumor progression. However, it is not feasible to engineer each tumor cell with NKG2D ligand in clinical treatment. Therefore, a therapeutic treatment that can restore NKG2D ligand on tumor cells is required to promote tumor immune surveillance. In this regard, many therapeutic approaches were discovered to induce NKG2D ligands on tumor cells in vitro. Unfortunately, no effective in vivo approaches are available to restore this ligand across different types of tumors because the classic in vitro stress signal-dependent NKG2D ligand induction has rarely been duplicated in solid tumors in vivo. Experimental procedure: This study reveals a unique approach_two independent co-administration of immune stimulatory signal interleukin-12 and doxorubicin_restores the NKG2D ligands in multiple murine and human tumor types. The induction of NKG2D ligands was determined using immunoblotting, flow cytometry and immunohistochemistry staining. The comparison of Rae-1 induction in immune competent mice versus immune deficient mice (nude, Rag2 -/- or Pfp -/-) implied that mature CD8+T cells are the most critical immune subpopulation. The concentration of interferon γ in tumors was measured using ELISA assay. The accumulation of NKG2D positive immune cells in tumor sections was examined using immunofluorescence straining, followed by TUNEL assay to determine tumor cell death. Results: We showed for the first time that a chemoimmunotherapy led to the tumor specific restoration of NKG2D ligands in a variety of tumor models in vivo. Systemic delivery of interleukin-12 gene therapy plus doxorubicin effectively restored murine NKG2D ligand Rae-1 in vivo in two subcutaneous tumor models (LLC and CT26), one orthotopic tumor model (K7M3) and one spontaneous breast carcinoma model. Significantly, such therapeutic approach can also stimulate human NKG2D ligands in xenograft tumor models. Intriguingly, this tumor-specific Rae-1 induction in vivo requires CD8+T cells. This NKG2D ligand-restoring therapy itself can also attract endogenous NKG2D-positive cells to attack tumor cells. Along with the tumor-specific NKG2D ligand induction, the promoted immune surveillance leads to massive tumor cell death. Conclusion: This study provides the first therapeutic treatment for inducing immune surveillance ligand in tumors in vivo. Further, it reveals a CD8+T cell-dependent novel mechanism for the restoration of NKG2D ligand. The revealed in vivo NKG2D ligand restoring approach could be translated for cancer prevention and treatment. Citation Format: Jiemiao Hu, Xueqing Xia, Eugenie S. Kleinerman, Shulin Li. CD8+T cell-mediated tumor-specific induction of the NKG2D ligands to trigger NKG2D-dependent tumor cell death in vivo. [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 4047. doi:10.1158/1538-7445.AM2015-4047

Abstract 4578: A novel polymer-anticancer drug micelle formulation showing enhanced efficacy over Abraxane

Abstract Advances in the development of novel nanocarriers and their applications in the area of drug delivery have inspired and created new opportunities for anticancer formulation development. About 10 anticancer nanomedicines in various dosage forms of liposomes and polymeric micelles have been approved since 1995, with albumin-bound paclitaxel (Abraxane) being a milestone in the treatment of a variety of aggressive cancers. These nanomedicines outperform the low molecular weight anticancer chemotherapeutic agents by their favorable pharmacokinetics and tissue distribution, resulting in improved efficacy and concomitantly decreased systematic side effect. We have developed a novel linear water soluble polymeric nano-carrier, poly-(L-γ-glutamylglutamine) (PGGA) by addition of a hydrophilic glutamic acid layer onto a poly-(L-glutamic acid) backbone. PGGA is proved to be versatile as nanocarrier for making both polymer drug conjugates as well as polymeric micelle formulations. By incorporating an appropriate hydrophobic moiety to PGGA backbone, a novel series of PGGA-based polymer hydrotrope is created and utilized in producing stable paclitaxel-encapsulated polymeric micelles (PMs). A lead candidate, designated as NittoX, has been identified from a matrix of such PMs varying in encapsulated paclitaxel and conjugated hydrophobic moiety contents. For NittoX bioperformance, an outstanding anticancer efficacy is observed in NCI-H460 lung cancer xenograft. Dose response is evident and NittoX is able to completely suppress tumor growth at the dosage of 80 mg/kg. More importantly, NittoX is statistically significantly more efficacious than Abraxane at the same dose levels. Dosages are well tolerated by animals in terms of body weight loss and survival. In addition to NCI-H460, NittoX is also found efficacious in a variety of tumor models, including pancreatic cancer. The improved performance is even more evident when administrated with multiple-dose regimens. The improved efficacy is presumably attributed to its superior plasma and tumor pharmacokinetic profile. NittoX not only enhances circulation stability over Abraxane, but also demonstrates preferential tumor PK profile with three-fold elevation of Cmax and AUC(0-168h) as compared to Abraxane. Meanwhile, the results from pharmacodynamic analysis of tubulin polymerization alteration and consequent tumor cell proliferation inhibition are in accordance with in vivo tumor efficacy data. Strong correlation between tumor PK and PD is observed. These preclinical data undoubtedly suggest that NittoX has potential to become a more potent taxane nanomedicine superior to Abraxane. Citation Format: Wenbin Ying, Jihua Liu, Kwok Yin Tsang, Li Wang, Haiqing Yin, Hao Bai, Yuwei Wang, Liping Wang, Yoshiro Niitsu. A novel polymer-anticancer drug micelle formulation showing enhanced efficacy over Abraxane. [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 4578. doi:10.1158/1538-7445.AM2014-4578

Taurolidine induces epithelial-mesenchymal transition via up-regulation of the transcription factor Snail in human pancreatic cancer cell lines.

The taurine derivative taurolidine (TRD) exerts anti-neoplastic effects in a variety of tumor models. On the other hand, TRD at low doses was shown to reduce cell-cell adhesion, a prerequisite for metastasis. The aim of this study was to elucidate the effects of low-dose TRD on pancreatic cancer. Human pancreatic cancer cell lines representing diverse states of differentiation were exposed to TRD for 24 h. Cell viability was assessed by MTT assay and trypan blue staining, apoptosis by caspase-3/7 activity, and flow-cytometric cell cycle analysis. Expression of Snail and E-cadherin was analyzed by polymerase chain reaction and Western blotting. MTT-tested viability of all pancreatic cancer cell lines decreased dose-dependently up to 50 % of the untreated control. In contrast to staurosporine TRD (100 and 250 μM) did not induce apoptosis but increased the percentage of cells in G1/G0 arrest. Correlation of MTT test and trypan blue staining revealed a decreased adherence of vital tumor cells at 250 μM TRD. This was associated with reduced expression of the adhesion molecule E-cadherin and an increased expression of the transcription factor Snail, a regulator of epithelial-mesenchymal transition (EMT). Low-dose TRD reduces not only viability but also cell-cell adherence and E-cadherin expression of pancreatic cancer cells, whereas the expression of the EMT inducer Snail was increased. By induction of these EMT hallmarks, low-dose TRD may promote metastasis in pancreatic cancer.