Human Solid Tumors Research Articles

Expression and Significance of N-myc downstream regulated gene 2 in the process of Esophageal Squamous Cell Carcinogenesis

ABSTRACT It has been reported that the expression of tumor suppressor gene N-myc downstream-regulated gene 2 (NDRG2) was significantly reduced in human solid tumors, including esophageal squamous cell carcinoma (ESCC). This study aimed to explore whether the difference of NDRG2 expression exists in different stages of ESCC and provides a basis for the early diagnosis and prognosis of ESCC. Immunohistochemical staining was used to investigate the expression level of NDRG2 in samples from 91 patients with mild-to-moderate dysplasia, early ESCC, and advanced ESCC. The relationship between the expression of NDRG2 and clinicopathological characteristics of the patients was analyzed. The results showed that positive expression rates of NDRG2 in tissues adjacent to early ESCC (76.7%), or from mild-to-moderate dysplasia (74.1%), and early ESCC (83.3%) were significantly higher than in tissue from advanced ESCC (55.9%). The positive expression rate in advanced ESCC was significantly lower than in the other three tissue types (p < 0.05). There was a significant difference (p < 0.05) and correlation (Cramer’s V = 0.351, p = 0.019, <0.05) between the expression of NDRG2 and the clinical stage in the 64 patients with ESCC. In conclusion, this study found that the expression of NDRG2 gradually decreased with the progression of esophageal lesions into advanced ESCC. This difference in positive expression rate was more obvious in male patients and patients under 60 years of age. Therefore, the detection of NDRG2 plays an important role in differentiating early ESCC from advanced ESCC.

Evolution of the Solid Human Tumor Cells Properties in Various Experimental Systems in Vitro

Background Human malignant cell models which reflect the structural and physiological complexity of tumor tissue are of great importance for preclinical research in oncology. Spheroids/tumoroids derived from solid tumors are of great interest as cellular models mimicking the first vascular-free growth phase of a tumor node. The fact of the identity between artificially created tumor multicellular aggregates and the real tumor tissue, however, needs to be specified, described and validated in order to see how closely the spheroids are biologically similar to the malignized tissues in vivo compared to the monolayer cell cultures traditionally used. We present here a comparison study of the characteristics of solid tumor cells of different histogenesis (melanomas, soft tissue sarcomas and bone sarcomas, epithelial tumors) cultured in two dimensions (monolayer culture) and three dimensional space (spheroid), namely: spatial organization, multiplication, metabolic activity. Patients and Methods For the creation of 2 D and 3D cell models the cells isolated from the patient's solid tumor fragments obtained intraoperatively were used. 15 samples of skin melanoma, 20 samples of soft tissue and osteogenic sarcomas (STBS), and 9 samples of epithelial tumors (ET). The tumor cells were all cultivated for at least 10 passages. We used phase contrast, confocal microscopy, and immunohistochemistry to investigate spheroids and monolayer cultures. The supernatants of tumor cells grown in 2D and 3D cultures were studied using ELISA and multiplex analysis for the production of a spectrum of chemokines and cytokines supporting the immunosuppression, invasion and metastasis processes. Results Tumor specimens received were predominantly of metastatic origin (75%). In 100% of cases 2D cultures were received, in 88.6% of cases (39 out of 44) we succeeded in obtaining spheroids. There was no direct correlation between the efficiency of tumoroid formation and the tumor's histogenetic origin and the stage of the cancer process (primary tumor, recurrence, metastasis). The median size of spheroids by 4-5 days of cultivation with a starting concentration of 10000 cells per well was 657.14 μm for melanoma (min 400 - max 1000 μm), 571.42 μm (min 400 - max 700 μm), 507.14 μm (min 300 - max 600 μm) for soft tissue sarcomas, 650.0 μm (min 400 - max 900 μm) for osteogenic sarcomas. Immunochemical analysis of Ki-67, GLUT1, and Ecadherin markers was carried out for tumor tissue samples, single-layer tumor cultures, and tumoroids of every patient. The distribution of the stained groups in the spheroids was distinct from the monolayer cultures and more in accordance with the distribution of such in the tissue tumor, the number of Ki-67+ cells was increasing in the spheroids. We detected no dependence of Ki-67+ and GLUT1+ cell localization grade on spheroid size. We identified E-cadherin in tumor tissue and tumoroids of breast carcinoma and one melanoma culture. Monolayer cultures did not express it. The increase in secretory cell activity of the solid tumor cells from 2D to 3D system was observed when CCL2, CCL3, CXCL1, CXCL16, MIF, IL10, MICA (p&lt;0.01) were investigated. Conclusion The presence of patient-specific cells of solid tumors in a 3D environment causes activation of the proliferative and metabolic processes as compared to monolayer cultures, which makes these models approximate the real world clinical picture. The production of chemokines that can attract to the tumor various types of immune system cells, to include their immature versions, as well as production of cytokines and Immunosuppression factors that, when present in the tumor microenvironment in the high concentrations, contribute to the formation of immune cells having suppressive capacities occurs in the 3D cell system. Three-dimensional model of the initial tumor nodule formation stage thus demonstrates the forming process of tumor cells favorable for them microenvironment. Construction of three-dimensional models - spheroids of tumor cells of differing histogenesis demands individual approach and more thorough investigation

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Pancancer cytotoxic T-cell trapping: are neutrophil extracellular traps a viable biomarker for immunotherapy response?†.

In a recent issue of The Journal of Pathology, a study by de Andrea, Ochoa, Villalba-Esparza et al demonstrated the presence of neutrophil extracellular traps (NETs) in a variety of human solid tumor types detected by multiplex immunofluorescence. In addition to NET identification and quantification, they found that there is individual heterogeneity in NET abundance in tumors and in circulation, and that NETs are positively associated with IL-8 and negatively associated with tumor-infiltrating CD8+ T cells. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

In vivo solid tumor targeting with recombinant VEGF-diphtheria immunotoxin.

Objective(s):A variety of signaling molecules have been identified that play a role in angiogenesis, of prime importance, vascular endothelial growth factor (VEGF) and its resceptor (VEGFR), which is highly expressed in most human solid tumors. Targeting VEGF or/and VEGFR with immunotoxin may be a promising approach to directly affect cancer cells. Immunotoxins are for targeted treatment comprising two functional moieties, an antibody that binds to target cells along with toxin that kills molecules. Materials and Methods:In this study, an immunotoxin comprising domain of diphtheria toxin subunit A (DT386) genetically fused to mouse VEGF (mVEGF-DT) was developed. The second construct, which contains the DT386 domain, was made to investigate the action of the DT386 domain on tumor cells. Both gene constructs were cloned, expressed, and were further purified. The biological activity of mVEGF-DT and DT386 proteins was assessed on the TC1 cell line bearing mouse model. Proteins were injected intra-tumoral in mice, in separate groups. Results:Tumors in the mVEGF-DT group started to dwindle after six injections, but tumor size in both control groups (DT386 and PBS), continued to grow. Conclusion:Successful targeting of solid tumor cells by mVEGF-DT immunotoxin demonstrates the therapeutic potential utility of these conjugates for tumor targeting.

Cell membrane-anchored and tumor-targeted IL-12 (attIL12)-T cell therapy for eliminating large and heterogeneous solid tumors

BackgroundAdoptive T-cell transfer has become an attractive therapeutic approach for hematological malignancies but shows poor activity against large and heterogeneous solid tumors. Interleukin-12 (IL-12) exhibits potent antitumor efficacy against solid...

Semi-Automated Computational Assessment of Cancer Organoid Viability Using Rapid Live-Cell Microscopy.

The creation of patient-derived cancer organoids represents a key advance in preclinical modeling and has recently been applied to a variety of human solid tumor types. However, conventional methods used to assess in vivo tumor tissue treatment response are poorly suited for the evaluation of cancer organoids because they are time-intensive and involve tissue destruction. To address this issue, we established a suite of 3-dimensional patient-derived glioma organoids, treated them with chemoradiotherapy, stained organoids with non-toxic cell dyes, and imaged them using a rapid laser scanning confocal microscopy method termed “Apex Imaging.” We then developed and tested a fragmentation algorithm to quantify heterogeneity in the topography of the organoids as a potential surrogate marker of viability. This algorithm, SSDquant, provides a 3-dimensional visual representation of the organoid surface and a numerical measurement of the sum-squared distance (SSD) from the derived mass center of the organoid. We tested whether SSD scores correlate with traditional immunohistochemistry-derived cell viability markers (cellularity and cleaved caspase 3 expression) and observed statistically significant associations between them using linear regression analysis. Our work describes a quantitative, non-invasive approach for the serial measurement of patient-derived cancer organoid viability, thus opening new avenues for the application of these models to studies of cancer biology and therapy.

Comparative study of genomic, immunological and functional features of squamous cell carcinomas in five anatomical locations

Squamous cell carcinomas (SCCs) are the most common human solid tumors and a major cause of cancer mortality. They are a group of aggressive, genetically complex neoplasms with a high risk of recurrence and metastasis. In the Republic of Moldova in 2018 the incidence of head, neck, esophagus, lung, skin and cervical cancers constituted 3242 cases of which about 50% represented the histopathological form of Squamous Cell Carcinoma. Early diagnosis, improving the outcomes of disease management and adaptation of therapies are current and necessary requirements for reducing mortality. Genetic changes, alteration of intracellular signaling pathways, and immune system response in these cancers may be influenced by various factors such as: oncogenic viruses (HPV), local microbiome, tumor localization, etc. In order to extend the understanding and the area of knowledge about these malignancies, to highlight the biological similarities and differences between certain types of SCCs, are required complex, comparative studies of genomic, epigenomic, proteomic, metabolomic, imunological and microbiome events. This complex, comparative approach has not been widely applied in previous studies and will allow the identification of similarities and differences between the multi-omics and non-omics features, the determination of individual and common biomarkers in 5 types of Squamous Cell Carcinomas: skin, head-neck, esophagus, lung and cervical. The goal of the research is the comparative study of SCCs from different anatomical locations in order to improve the screening, early diagnosis, disease management and treatment of these tumors.

The Role of the CREB Protein Family Members and the Related Transcription Factors in Radioresistance Mechanisms.

In the framework of space flight, the risk of radiation carcinogenesis is considered a “red” risk due to the high likelihood of occurrence as well as the high potential impact on the quality of life in terms of disease-free survival after space missions. The cyclic AMP response element-binding protein (CREB) is overexpressed both in haematological malignancies and solid tumours and its expression and function are modulated following irradiation. The CREB protein is a transcription factor and member of the CREB/activating transcription factor (ATF) family. As such, it has an essential role in a wide range of cell processes, including cell survival, proliferation, and differentiation. Among the CREB-related nuclear transcription factors, NF-κB and p53 have a relevant role in cell response to ionising radiation. Their expression and function can decide the fate of the cell by choosing between death or survival. The aim of this review was to define the role of the CREB/ATF family members and the related transcription factors in the response to ionising radiation of human haematological malignancies and solid tumours.

Abstract P222: Combinations of receptor tyrosine kinase inhibitors targeting the tumor and stromal cells of complex spheroids from the National Cancer Institute’s Patient-Derived Models Repository (PDMR; https://pdmr.cancer.gov/)

Abstract Tumor stroma is critical in shaping the tumor microenvironment and promoting tumor growth and metastasis. Six stroma-targeted kinase inhibitors (cediranib, vandetanib, axitinib, pazopanib, cabozantinib, and lenvatinib) were tested both as single agents and in combination with five tumor cell-targeted kinase inhibitors (erlotinib, gefitinib, osimertinib, rociletinib, and erdafitinib)using a complex spheroid cell culture model. Complex spheroids, containing 60% tumor cells, 25% endothelial cells (HUVEC) and 15% mesenchymal stem cells (MSC), serve as a cell culture model of human solid tumors incorporating both malignant and stromal cells. Seventeen tumor cell lines were grown as complex spheroids, including varied sarcoma and non-small cell lung cancer types. Drug sensitivities of the HUVEC and MSC were also evaluated, both individually as monolayers and mixed as monolayers and spheroids. Overall, the HUVEC and MSC were more sensitive to the VEGFR inhibitors than to the EGFR inhibitors. Growth as complex spheroids reduced the sensitivity of HUVEC and MSC to several kinase inhibitors compared to each cell type alone or in mixed monolayer culture. A significant correlation was observed between the EGFR mRNA expression in patient-derived tumor lines and their sensitivity to the EGFR inhibitors. In combination regimens, Erlotinib was most effective when combined with the VEGFR inhibitors pazopanib, vandetanib and cediranib, which produced more than one log of cell killing at concentrations less than the clinical Cmax of each drug. Osimertinib and rociletinib, which irreversibly target EGFR variants, were more cytotoxic towards complex spheroids of NCI-H1975 NSCLC (EGFR L858R, T790M) than NCI-H522 NSCLC (EGFR wildtype). Combinations of the mutant-selective EGFR inhibitors with a VEGFR inhibitor (especially, vandetanib, lenvatinib or cediranib) increased the cytotoxicity in complex spheroids containing EGFR mutant and wildtype tumor lines. Highly effective combinations included erdafitinib with vandetanib and erlotinib with cediranib. The combination of cediranib and erlotinib was also evaluated in nine patient-derived xenograft (PDX) models and resulted in two partial responses in a penile squamous carcinoma and uterine sarcoma, while the remainder showed stable, slowed, or progressive disease. Using &amp;gt;1 log of cell kill at drug concentrations less than the mouse Cmax concentration for each drug as a predictor for in vivo responsiveness, the complex spheroid assay was 88% accurate in predicting response or progressive disease in the PDX models. This criteria for predicting in vivo activity from in vitro model results will be further explored. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261200800001E. Citation Format: Thomas S. Dexheimer, Julie Laudeman, Thomas Silvers, Rene Delosh, Russell Reinhart, Chad Ogle, Eric Jones, Nathan P. Coussens, Beverly A. Teicher, Naoko Takebe, Alice Chen, James H. Doroshow. Combinations of receptor tyrosine kinase inhibitors targeting the tumor and stromal cells of complex spheroids from the National Cancer Institute’s Patient-Derived Models Repository (PDMR; https://pdmr.cancer.gov/) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P222.

Development of a Systematic Review Protocol and a Scoping Review of Ultrasound-Induced Immune Effects in Peripheral Tumors

PurposePublication numbers reporting that ultrasound can stimulate immune reactions in tumors steadily increase. However, the presented data are partially conflicting, and mechanisms are difficult to identify from single publications. These shortcomings can be addressed by a systematic review and meta-analysis of current literature. As a first step, we here present the methodology and protocol for a systematic review to answer the following research question: Does ultrasound alter the immune reaction of peripheral solid tumors in humans and animals compared to control conditions without ultrasound?ProceduresWe designed a protocol to perform a systematic review and meta-analysis. The suitability of the protocol to detect and sort relevant literature was tested using a subset of publications. We extracted study characteristics, ultrasound parameters, and study outcomes to pre-evaluate the differences between publications and present the data as a scoping review.ResultsFrom 6532 publications detected by our preliminary literature search, 320 were selected for testing our systematic review protocol. Of the latter, 15 publications were eligible for data extraction. There, we found large differences between study characteristics (e.g., tumor type, age) and ultrasound settings (e.g., wavelength 0.5–9.5 MHz, acoustic pressure 0.0001–15,000 W/cm2). Finally, study outcomes included reports on cells of the innate (e.g., dendritic cells, macrophages) and adaptive immune system (e.g., CD8-/CD4-positive T cells).ConclusionWe designed a protocol to identify relevant literature and perform a systematic review and meta-analysis. The differences between extracted features between publications show the necessity for a comprehensive search and selection strategy in the systematic review to get a complete overview of the literature. Meta-analyses of the extracted outcomes can then enable evidence-based conclusions.

The Immune Landscape of Human Primary Lung Tumors Is Th2 Skewed.

Tumor-specific T helper (Th) cells have a central role in the immune response against cancer. However, there exist distinct Th cell subsets with very different and antagonizing properties. Some Th subsets such as Th1 protect against cancer, while others (Th2, T regulatory/Treg) are considered detrimental or of unknown significance (T follicular helper/Tfh, Th17). The Th composition of human solid tumors remains poorly characterized. Therefore, we established a four-color multiplex chromogenic immunohistochemical assay for detection of Th1, Th2, Th17, Tfh and Treg cells in human tumor sections. The method was used to analyze resected primary lung tumors from 11 patients with non-small cell lung cancer (NSCLC). Four microanatomical regions were investigated: tumor epithelium, tumor stroma, peritumoral tertiary lymphoid structures (TLS) and non-cancerous distal lung tissue. In tumor epithelium and stroma, most CD4+ T cells identified had either a Th2 (GATA-3+CD3+CD8-) or Treg (FOXP3+CD3+CD8-) phenotype, whereas only low numbers of Th1, Th17, and Tfh cells were observed. Similarly, Th2 was the most abundant Th subset in TLS, followed by Treg cells. In sharp contrast, Th1 was the most frequently detected Th subset in non-cancerous lung tissue from the same patients. A higher Th1:Th2 ratio in tumor stroma was found to be associated with increased numbers of intratumoral CD8+ T cells. The predominance of Th2 and Treg cells in both tumor stroma and tumor epithelium was consistent for all the 11 patients investigated. We conclude that human primary NSCLC tumors are Th2-skewed and contain numerous Treg cells. If human tumors are Th2-skewed, as our data in NSCLC suggest, reprogramming the type of immune response from a detrimental Th2 to a beneficial Th1 may be critical to increase the response rate of immunotherapy.

Sensitive identification of neoantigens and cognate TCRs in human solid tumors

The identification of patient-specific tumor antigens is complicated by the low frequency of T cells specific for each tumor antigen. Here we describe NeoScreen, a method that enables the sensitive identification of rare tumor (neo)antigens and of cognate T cell receptors (TCRs) expressed by tumor-infiltrating lymphocytes. T cells transduced with tumor antigen-specific TCRs identified by NeoScreen mediate regression of established tumors in patient-derived xenograft mice.

IMMU-45. COMBINATION OF EGFRVIII CAR T-CELL THERAPY AND PARACRINE GAM MODULATION FOR THE TREATMENT OF GBM

Abstract The GBM immune tumor microenvironment mainly consists of protumoral glioma-associated microglia and macrophages (GAMs). We have previously shown that blockade of CD47, a ‘don't eat me’-signal overexpressed by GBM cells, rescued GAMs' phagocytic function in mice. However, monotherapy with CD47 blockade has been ineffective in treating human solid tumors to date. Thus, we propose a combinatorial approach of local CAR T cell therapy with paracrine GAM modulation for a synergistic elimination of GBM. We generated humanized EGFRvIII CAR T-cells by lentiviral transduction of healthy donor human T-cells and engineered them to constitutively release a soluble SIRPγ-related protein (SGRP) with high affinity towards CD47. Tumor viability and CAR T-cell proliferation were assessed by timelapse imaging analysis in co-cultures with endogenous EGFRvIII-expressing BS153 cells. Tumor-induced CAR T-cell activation and degranulation were confirmed by flow cytometry. CAR T-cell secretomes were analyzed by liquid chromatography-mass spectrometry. Immunocompromised mice were orthotopically implanted with EGFRvIII+ BS153 cells and treated intratumorally with a single CAR T-cell injection. EGFRvIII and EGFRvIII-SGRP CAR T-cells killed tumor cells in a dose-dependent manner (72h-timepoint; complete cytotoxicity at effector-target ratio 1:1) compared to CD19 controls. CAR T-cells proliferated and specifically co-expressed CD25 and CD107a in the presence of tumor antigen (24h-timepoint; EGFRvIII: 59.3±3.00%, EGFRvIII-SGRP: 52.6±1.42%, CD19: 0.1±0.07%). Differential expression analysis of CAR T-cell secretomes identified SGRP from EGFRvIII-SGRP CAR T-cell supernatants (-Log10qValue/Log2fold-change= 3.84/6.15). Consistent with studies of systemic EGFRvIII CAR T-cell therapy, our data suggest that intratumoral EGFRvIII CAR T-cells were insufficient to eliminate BS153 tumors with homogeneous EGFRvIII expression in mice (Overall survival; EGFRvIII-treated: 20%, CD19-treated: 0%, n= 5 per group). Our current work focuses on the functional characterization of SGRP binding, SGRP-mediated phagocytosis, and on the development of a translational preclinical model of heterogeneous EGFRvIII expression to investigate an additive effect of CAR T-cell therapy and GAM modulation.

Early prediction of clinical response to checkpoint inhibitor therapy in human solid tumors through mathematical modeling.

Checkpoint inhibitor therapy of cancer has led to markedly improved survival of a subset of patients in multiple solid malignant tumor types, yet the factors driving these clinical responses or lack thereof are not known. We have developed a mechanistic mathematical model for better understanding these factors and their relations in order to predict treatment outcome and optimize personal treatment strategies. Here, we present a translational mathematical model dependent on three key parameters for describing efficacy of checkpoint inhibitors in human cancer: tumor growth rate (α), tumor-immune infiltration (Λ), and immunotherapy-mediated amplification of anti-tumor response (µ). The model was calibrated by fitting it to a compiled clinical tumor response dataset (n = 189 patients) obtained from published anti-PD-1 and anti-PD-L1 clinical trials, and then validated on an additional validation cohort (n = 64 patients) obtained from our in-house clinical trials. The derived parameters Λ and µ were both significantly different between responding versus nonresponding patients. Of note, our model appropriately classified response in 81.4% of patients by using only tumor volume measurements and within 2 months of treatment initiation in a retrospective analysis. The model reliably predicted clinical response to the PD-1/PD-L1 class of checkpoint inhibitors across multiple solid malignant tumor types. Comparison of model parameters to immunohistochemical measurement of PD-L1 and CD8+ T cells confirmed robust relationships between model parameters and their underlying biology. These results have demonstrated reliable methods to inform model parameters directly from biopsy samples, which are conveniently obtainable as early as the start of treatment. Together, these suggest that the model parameters may serve as early and robust biomarkers of the efficacy of checkpoint inhibitor therapy on an individualized per-patient basis. We gratefully acknowledge support from the Andrew Sabin Family Fellowship, Center for Radiation Oncology Research, Sheikh Ahmed Center for Pancreatic Cancer Research, GE Healthcare, Philips Healthcare, and institutional funds from the University of Texas M.D. Anderson Cancer Center. We have also received Cancer Center Support Grants from the National Cancer Institute (P30CA016672 to the University of Texas M.D. Anderson Cancer Center and P30CA072720 the Rutgers Cancer Institute of New Jersey). This research has also been supported in part by grants from the National Science Foundation Grant DMS-1930583 (ZW, VC), the National Institutes of Health (NIH) 1R01CA253865 (ZW, VC), 1U01CA196403 (ZW, VC), 1U01CA213759 (ZW, VC), 1R01CA226537 (ZW, RP, WA, VC), 1R01CA222007 (ZW, VC), U54CA210181 (ZW, VC), and the University of Texas System STARS Award (VC). BC acknowledges support through the SER Cymru II Programme, funded by the European Commission through the Horizon 2020 Marie Skłodowska-Curie Actions (MSCA) COFUND scheme and the Welsh European Funding Office (WEFO) under the European Regional Development Fund (ERDF). EK has also received support from the Project Purple, NIH (U54CA210181, U01CA200468, and U01CA196403), and the Pancreatic Cancer Action Network (16-65-SING). MF was supported through NIH/NCI center grant U54CA210181, R01CA222959, DoD Breast Cancer Research Breakthrough Level IV Award W81XWH-17-1-0389, and the Ernest Cockrell Jr. Presidential Distinguished Chair at Houston Methodist Research Institute. RP and WA received serial research awards from AngelWorks, the Gillson-Longenbaugh Foundation, and the Marcus Foundation. This work was also supported in part by grants from the National Cancer Institute to SHC (R01CA109322, R01CA127483, R01CA208703, and U54CA210181 CITO pilot grant) and to PYP (R01CA140243, R01CA188610, and U54CA210181 CITO pilot grant). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

A Phase 2 Multi-Arm Study of Magrolimab Combinations in Patients with Relapsed/Refractory Multiple Myeloma

A Phase 2 Multi-Arm Study of Magrolimab Combinations in Patients with Relapsed/Refractory Multiple Myeloma

ASPP2κ Is Expressed In Human Colorectal Carcinoma And Promotes Chemotherapy Resistance And Tumorigenesis.

Alternative splicing is a common physiologic mechanism to generate numerous distinct gene products from one gene locus, which can result in unique gene products with differing important functional outcomes depending on cell context. Aberrant alternative splicing is a hallmark of cancer that can contribute to oncogenesis and aggressiveness of the disease as well as resistance to therapy. However, aberrant splicing might also result in novel targets for cancer therapy. ASPP2 is a haplo-insufficient tumor suppressor, that functions through both p53-dependent as well as p53-independent mechanisms to enhance cell death after stress. Interestingly, the common human tumor TP53 mutations result in a loss of the binding sites to ASPP2, leading to impaired induction of apoptosis. Vice versa, attenuation of ASPP2 has been described to be associated with high-risk disease, therapy failure and poor clinical outcome especially in tumors harboring the TP53 wildtype (WT) isoform. We have recently identified a novel, dominant-negative splicing variant of ASPP2, named ASPP2κ, with oncogenic potential. Exon-skipping results in a reading-frame shift with a premature translation stop, omitting most of the ASPP2 C-terminus - which harbors the p53-binding domain. Consequently, the ASPP2-p53 interaction is abrogated, which in part impacts on oncogenesis, aggressiveness of disease and response to therapy. Since ASPP2κ has been shown in hematologic malignancies to promote tumorigenesis, we further wished to determine if aberrant ASPP2κ expression plays a role in human solid tumors. In this report, we find that ASPP2κ is frequently expressed in human colorectal tumors (CRC). Using ASPP2κ overexpressing and interference CRC models, we demonstrate a functional role of ASPP2κ in contributing to oncogenesis and resistance to therapy in CRC by 1) enhancing proliferation, 2) promoting cell migration and, 3) conferring resistance to chemotherapy induced apoptosis. Our findings have far-reaching consequences for future diagnostic and therapeutic strategies for ASPP2κ expressing colorectal cancer patients and provide proof-of-principle to further explore ASPP2κ as potential predictive marker and target for therapy in clinical trials.

Conditionally Activated IL-12 or IFNα Indukine™ Molecules Inhibit Syngeneic Lymphoma Tumor Growth in Mice, Induce Anti-Tumor Immune Responses and Are Tolerated in Non-Human Primates

Systemic therapy with proinflammatory immune modulators to activate anti-tumor immunity is a promising approach to treat cancer. However, poor pharmacokinetic properties and dose-limiting toxicities such as inflammation, cytokine release syndrome, and tissue damage have prevented or limited the clinical use of cytokines such as interleukin 12 (IL-12) and interferon α (IFNα). Previous clinical development of rhIL-12 was terminated due to toxicity, and although rIFNα is approved for the treatment of melanoma, lymphoma, and leukemia, its use has been limited by systemic toxicity and modest efficacy.INDUKINE™ molecules, engineered using Werewolf Therapeutics' Predator™ discovery platform, are novel, systemically delivered cytokine pro-drugs which prevent systemic toxicity and deliver cytokines to the tumor microenvironment (TME) where they are activated. Several INDUKINE™ molecules using Werewolf's proprietary linkers have been previously shown to be cleaved by a wide range of human solid tumors but have yet to be tested in primary human lymphoma samples or syngeneic mouse lymphoma models.IL-12 is a potent, pleiotropic cytokine for immune-mediated killing of cancer cells, whose mechanism of action (MOA) includes activation of both cytotoxic T and NK cells. The WTX-330 INDUKINE™ molecule, a wild-type IL-12 pro-drug, contains a half-life extension (HLE) domain to support infrequent dosing and a high affinity anti-IL-12 neutralizing antibody domain to maintain the molecule in its inactive state in the periphery. Both the HLE and blocking domains are tethered to IL-12 via two identical tumor protease-sensitive linkers. Linker cleavage in the TME removes the HLE domain and the blocker, resulting in active IL-12 within the TME with wild-type IL-12 biologic and PK profiles.IFNα is a member of the type-I IFN family and activates innate immune responses either directly by engaging IFNα receptors (IFNAR) ubiquitously expressed on immune cells or indirectly by inducing chemokines that attract myeloid and lymphoid cells to the tumor site. The WTX-613 INDUKINE™ molecule is an inactive IFNα2b pro-drug with two identical HLE domains tethered to IFNα2b via two identical tumor protease-sensitive linkers. The HLE domains sterically block binding of WTX-613 to IFNAR until cleavage of the linkers in the TME releases active IFNα.Since human IL-12 and IFNα2b are not cross reactive in mice, surrogate WTX-330 and WTX-613 INDUKINE™ molecules were created, consisting of a mouse/human chimeric IL-12 or a mouse IFNα1 to explore anti-tumor responses in syngeneic hematologic cancer models. In the subcutaneously (s.c.) A20 B cell lymphoma model, the WTX-330 surrogate showed dose-dependent anti-tumor activity with 4 out of 10 tumor-free mice at the top dose. The WTX-613 surrogate demonstrated tumor stasis lasting beyond the treatment phase. Utilizing the s.c. EG7.OVA T lymphoblast line, tumor growth was efficiently blocked by the WTX-613 surrogate while the WTX-330 surrogate inhibited tumor growth during the dosing period. Both treatments were well tolerated by the mice at active dose levels.The WTX-330 and WTX-613 surrogates strongly activated NK and CD8+ cell responses and induced APC and effector cell markers in the MC38 syngeneic tumor model supporting a MOA as described for wild-type IL-12 and IFNα. Similar studies are on-going in lymphoma models. PK analysis in mice revealed extended half-life (T1/2) for both WTX-330 and WTX-613 surrogates compared to the short T1/2 of native IL-12 or IFNα1. Finally, WTX-330 and WTX-613 were well tolerated in non-human primates (NHP), resulting in plasma exposure levels for INDUKINE™ molecules that exceeded those needed for anti-tumor activity in mice. In addition, plasma levels of free IL-12 after dosing with WTX-330 were very low compared to tolerated levels of wild-type IL-12. Similar studies for WTX-613 are on-going.Preclinical data obtained so far for both programs support the continued development and future evaluation of these innovative and differentiated therapies in hematologic malignancies, both as monotherapies and in multiple combinations with standard of care. DisclosuresIsaacs: Werewolf Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Seidel-Dugan: Werewolf Therapeutics: Current Employment, Other: current shareholder .

Combination Therapy Strategy for Lymphoma Based on Cancer Energy Metabolism of OPB-111077, a Novel Antitumor Agent Inhibiting Mitochondrial Oxidative Phosphorylation

Development of antitumor agents targeting energy metabolism pathways of cancer cells has attracted much attention in recent days. In general, most cancer cells are known to display Warburg effect. Recently, it has been reported that cancer cells exist as heterogeneous groups in the microenvironment surrounding cancer cells and that not only glycolysis but also mitochondrial oxidative phosphorylation (OXPHOS) is important for cancer growth. In addition, many research groups have reported that the acquisition of anticancer drug resistance is caused by metabolic reprogramming from glycolysis to OXPHOS. Therefore, OXPHOS inhibitors could be useful antitumor agents. OPB-111077 is an orally active novel antitumor agent and has the inhibitory effect on mitochondrial OXPHOS. OPB-111077 had the inhibitory effect on mitochondrial respiratory chain complex I which led to the inhibition of energy production and activation of AMPK-mTOR energy stress sensor pathway. In in vitro growth inhibition studies, OPB-111077 showed potent inhibitory effects on the growth of various human blood tumor cell lines derived from leukemia, multiple myeloma and lymphoma with IC 50 values between 18.6 and 525.3 nM, and on the growth of human solid tumor cell lines derived from liver cancer, lung cancer, gastric cancer and breast cancer with IC 50 values ranging from 92.6 to 1727.7 nM. In in vivo studies using SCID mice bearing tumors from human tumor cell lines, daily oral administration of OPB-111077 demonstrated significant antitumor effects against leukemia, lymphoma, liver, gastric and breast cancer cell line-derived tumor, dose-dependently.Here, we report the combination therapy strategy based on the cancer energy metabolism of OPB-111077. We found that OPB-111077 combined with alkylating agent, cyclophosphamide or bendamustine, showed the synergistic effect on xenograft mice model using human diffuse large B‐cell lymphoma (DLBCL) cell line. To clarify this mechanism, we examined the effect of alkylating agents on mitochondrial respiration in vitro using flux analyzer and found that alkylating agents induced OXPHOS nature in the DLBCL cell line. Furthermore, the administration of alkylating agents to DLBCL cell line xenograft model mice followed by mRNA expression analysis of their tumor tissue confirmed that expression of OXPHOS-related markers was induced. These results suggest the administration of alkylating agents induced reprogramming to OXPHOS predominant nature in tumor. The tumor environment is actively reprogrammed to OXPHOS by alkylating agents, and it is changed to a state in which the antitumor effect of OPB-111077 can be more exerted. Thus, alkylating agents enhance the effect of OPB-111077 by tilting glycolytically dominant tumors to OXPHOS dominant. The alkylating agents, cyclophosphamide and bendamustine, are used as standard therapies for lymphoma. OPB-111077 could be treated in a wide range of applications as a combination therapy with the alkylating agents for lymphoma.In clinical, we have confirmed that OPB-111077 has tolerable and controllable toxicity profile of single agent in some advanced cancer patients including DLBCL. Bendamustine and rituximab (BR) therapy is indicated for the treatment of patients who are ineligible for transplantation of relapsed / refractory (R/R) DLBCL. However, there is still a high unmet need for patients with R/R DLBCL due to no standard treatment for transplant‐ineligible patients. We are conducting combination therapy of OPB-111077 with bendamustine and rituximab in Phase 1 clinical trial for R/R DLBCL in Japan. DisclosuresOhi: Otsuka Pharmaceutical Co.,Ltd.: Current Employment. Okuno: Otsuka Pharmaceutical Co.,Ltd.: Current Employment. Tanaka: Otsuka Pharmaceutical Co.,Ltd.: Current Employment. Ohtani: Otsuka Pharmaceutical Co.,Ltd.: Current Employment. OffLabel Disclosure:Compound: OPB-111077 Purpose: Development of novel antitumor agent

The antiproliferative potential of fungi associated with coral and algae collected from a Veracruz Reef System, Gulf of Mexico

Coral reefs are ecosystems with great biodiversity and architectural complexity. Among these, marine fungi constitute one of the least studied groups, despite being potentially an inexhaustible source of bioactive metabolites. For this reason, in this research, we evaluate the antiproliferative activity of 52 fungal strains associated with marine organisms such as algae and coral collected from four islands located in the National Park Sistema Arrecifal Veracruzano, Mexico. One hundred four chloroform:methanol extracts corresponding to the culture broth, and biomass produced from each fungal strain was obtained from these. Subsequently, they were evaluated against six human solid tumor cell lines (HBL-100, HeLa, SW1573, T-47D, WiDr, and A549). The outcome was that six extracts showed activity against at least one cancer cell line. The identity of these fungi corresponded to the genera Nigrospora , Pestalotiopsis , Cladosporium , and Ochroconis according to their morphological and molecular analysis. In this sense, the strain found to have the highest antiproliferative potential [GI 50 (μg mL -1 ) 4.6, 2.5, 8.0, 5.6, 52.0, 4.0] grouped phylogenetically with a species of Nigrospora sp. The preceding confirms that ecosystems provide.