Reduced Tumor Cell Proliferation Research Articles

Targeting IL-23 for the interception of obesity-associated colorectal cancer

Inflammation and obesity are two major factors that promote Colorectal cancer (CRC). Our recent data suggests that interleukin (IL)-23, is significantly elevated in CRC tumors and correlates with patient obesity, tumor grade and survival. Thus, we hypothesize that obesity and CRC may be linked via inflammation and IL-23 may be a potential target for intervention in high-risk patients. TCGA dataset and patient sera were evaluated for IL-23A levels. IL-23A [IL-23 p19−/−] knockout (KO) mice were crossed to Apcmin/+ mice and progeny were fed low-fat or high-fat diets. At termination intestines were evaluated for tumorigenesis. Tumors, serum, and fecal contents were analyzed for protein biomarkers, cytokines, and microbiome profile respectively. IL-23A levels are elevated in the sera of patients with obesity and colon tumors. Genetic ablation of IL-23A significantly suppressed colonic tumor multiplicity (76–96 %) and incidence (72–95 %) in male and female mice. Similarly, small-intestinal tumor multiplicity and size were also significantly reduced in IL-23A KO mice. IL-23A knockdown in Apcmin/+ mice fed high-fat diet, also resulted in significant suppression of colonic (50–58 %) and SI (41–48 %) tumor multiplicity. Cytokine profiling showed reduction in several circulating pro-inflammatory cytokines including loss of IL-23A. Biomarker analysis suggested reduced tumor cell proliferation and immune modulation with an increase in tumor-infiltrating CD4+ and CD8+ T-lymphocytes in the IL-23A KO mice compared to controls. Fecal microbiome analysis revealed potentially beneficial changes in the bacterial population profile. In summary, our data indicates a tumor promoting role for IL-23 in CRC including diet-induced obesity. With several IL-23 targeted therapies in clinical trials, there is a great potential for targeting this cytokine for CRC prevention and therapy.

Antioxidant Phytocomplexes Extracted from Pomegranate (Punica granatum L.) Using Hydrodynamic Cavitation Show Potential Anticancer Activity In Vitro.

Hydrodynamic cavitation (HC), as an effective, efficient, and scalable extraction technique for natural products, could enable the affordable production of valuable antioxidant extracts from plant resources. For the first time, whole pomegranate (Punica granatum L.) fruits, rich in bioactive phytochemicals endowed with anti-cancer properties, were extracted in water using HC. Aqueous fractions sequentially collected during the process (M1-M5) were lyophilized (L), filtered (A), or used as such, i.e., crude (C), and analyzed for their biochemical profile and in vitro antioxidant power. The fractions M3 and M4 from the L and C series showed the highest antiradical activity and phytochemical content. While the lyophilized form is preferable for application purposes, sample L-M3, which was produced faster and with lower energy consumption than M4, was used to assess the potential antiproliferative effect on human breast cancer line (AU565-PAR) and peripheral blood mononuclear (PBMC) cells from healthy donors. In a pilot study, cell growth, death, and redox state were assessed, showing that L-M3 significantly reduced tumor cell proliferation and intracellular oxygen reactive species. No effect on PBMCs was detected. Thus, the antioxidant phytocomplex extracted from pomegranate quickly (15 min), at room temperature (30 °C), and efficiently showed potential anticancer activity without harming healthy cells.

HYbrid DegRAding Copolymer (HYDRAC): Heterobifunctional polymers for targeted degradation of undruggable proteins.

22 Background: Of the ~20,000 proteins encoded by the human genome, only a small subset can be modulated via traditional pharmacological approaches. Included in the list of “undruggable” proteins are ones with pivotal roles in cancer. We aim to expand the arsenal of compounds able to affect the activity of these therapeutically relevant targets by introducing a modular proteomimetic platform technology designed to carry side chains consisting of targeting warheads and recruiters of cellular protein degradation machinery. We term these compounds HYbrid DegRAding Copolymers (HYDRACs) in reference to the multiplexing of different protein binders and degradation inducing sequences. Peptides arranged as HYDRACs exhibit favorable emergent properties including resistance to proteolysis, high levels of cell uptake, and payload-specific activity. Initial studies were conducted focusing on a undruggable transcription factor MYC, which is widely dysregulated in human cancers. Methods: A library of HYDRACs and control polymers were synthesized and were cell-penetrating with antiproliferative effects at sub-micromolar IC50s in a formulation- and MYC-dependent manner across multiple cell lines. Results: HYDRAC treatment resulted in significant decreases in MYC protein levels, which was rescued by proteasome inhibitor MG132 and neddylation inhibitor MLN4924, without changes in mRNA levels. RNAseq showed selective overexpression of MYC pathway genes only in HYDRAC treated cells, confirming on-target effects. Circular dichroism showed strong HYDRAC-MYC interactions, which was supported by the presence of MYC following pull-down of biotin-terminated HYDRACs. Removal of the RRRG degron, scrambling the H1 sequence, and disassociation of the two domains on separate polymers all abrogated MYC-degradation. Mice bearing transplanted Myc-CaP tumors showed delayed tumor growth and reduced tumor cell proliferation following HYDRAC treatment. Mice bearing luminescent Luc-MV-411 tumors treated with Cy5.5-labeled HDRACs showed selective tumor accumulation with detectable fluorescent signal in the tumor up to 72 hours following a single IP administration. The versatility of the platform was demonstrated by substituting the degron for recruiters of three different E3 ligases (VHL, KEAP1, and CRBN), which all maintained ability to degrade MYC. HYDRACs were also able to selectively degrade other protein targets including alpha-syn and RAS. Conclusions: We present a platform technology that addresses challenges inherent to small molecule drug design. HYDRACs have the potential to dramatically alter the drug discovery landscape, allowing for development of modulators of "undruggable" targets. We envision the HYDRAC platform as a generalizable approach to designing degraders of proteins of interest, greatly expanding the therapeutic armamentarium for target protein degradation.

Endoglin, a Novel Biomarker and Therapeutical Target to Prevent Malignant Peripheral Nerve Sheath Tumor Growth and Metastasis.

Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive soft-tissue sarcomas that lack effective treatments, underscoring the urgent need to uncover novel mediators of MPNST pathogenesis that may serve as potential therapeutic targets. Tumor angiogenesis is considered a critical event in MPNST transformation and progression. Here, we have investigated whether endoglin (ENG), a TGFβ coreceptor with a crucial role in angiogenesis, could be a novel therapeutic target in MPNSTs. ENG expression was evaluated in human peripheral nerve sheath tumor tissues and plasma samples. Effects of tumor cell-specific ENG expression on gene expression, signaling pathway activation and in vivo MPNST growth and metastasis, were investigated. The efficacy of ENG targeting in monotherapy or in combination with MEK inhibition was analyzed in xenograft models. ENG expression was found to be upregulated in both human MPNST tumor tissues and plasma-circulating small extracellular vesicles. We demonstrated that ENG modulates Smad1/5 and MAPK/ERK pathway activation and pro-angiogenic and pro-metastatic gene expression in MPNST cells and plays an active role in tumor growth and metastasis in vivo. Targeting with ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST growth and metastasis in xenograft models by reducing tumor cell proliferation and angiogenesis. Moreover, combination of anti-ENG therapy with MEK inhibition effectively reduced tumor cell growth and angiogenesis. Our data unveil a tumor-promoting function of ENG in MPNSTs and support the use of this protein as a novel biomarker and a promising therapeutic target for this disease.

Antitumor effects and potential mechanisms of aconitine based on preclinical studies: an updated systematic review and meta-analysis.

Background: Herbs originating from the Aconitum L. (Ranunculaceae), such as Aconitum carmichaelii Debeaux. (Wutou), Aconitum pendulum Busch. (Tiebangchui), and Aconitum kusnezoffii Reichb. (Caowu), etc. are highly valued for their medicinal properties. The roots and tubers of these herbs are commonly used to treat an array of ailments, including joint pain and tumors. The alkaloids present in them are the primary active components, with aconitine being the most notable. Aconitine has gained attention for its exceptional anti-inflammatory and analgesic properties, as well as its potential as an anti-tumor and cardiotonic agent. However, the exact process through which aconitine hinders the growth of cancerous cells and triggers their programmed cell death remains unclear. Therefore, we have undertaken a comprehensive systematic review and meta-analysis of the current research on the potential antitumor properties of aconitine. Methods: We conducted a thorough search of relevant preclinical studies in databases including PubMed, Web of Science, VIP, WanFang Data, CNKI, Embase, Cochrane Library, and National Center for Biotechnology Information (NCBI). The search was conducted up until 15 September 2022, and the data were statistically analyzed using RevMan 5.4 software. The number of tumor cell value-added, tumor cell apoptosis rate, thymus index (TI), and Bcl-2 gene expression level were the main indicators to be analyzed. Results: After applying the final inclusion criteria, a total of thirty-seven studies, comprising both in vivo and in vitro research were analyzed. The results showed that treatment with aconitine led to a significant reduction in tumor cell proliferation, a noteworthy increase in the rate of apoptosis among tumor cells, a decrease in the thymus index, and a reduction in the expression level of Bcl-2. These results suggested that aconitine could inhibit the proliferation, invasion, and migration abilities of tumor cells by regulating Bcl-2 etc., thereby enhancing the anti-tumor effects. Conclusion: In summary, our present study demonstrated that aconitine effectively reduced tumor size and volume, indicating a strong anti-tumor effect. Additionally, aconitine could increase the expression levels of caspase-3, Bax and other targets. Mechanistically, it may regulate the expression levels of Bax and Bcl-2 through the NF-κB signaling pathway, ultimately inhibiting tumor cell proliferation through autophagy.

Modulation of EZH2 Activity Induces an Antitumoral Effect and Cell Redifferentiation in Anaplastic Thyroid Cancer.

Anaplastic thyroid cancer (ATC) is a rare and lethal form of thyroid cancer that requires urgent investigation of new molecular targets involved in its aggressive biology. In this context, the overactivation of Polycomb Repressive Complex 2/EZH2, which induces chromatin compaction, is frequently observed in aggressive solid tumors, making the EZH2 methyltransferase a potential target for treatment. However, the deregulation of chromatin accessibility is yet not fully investigated in thyroid cancer. In this study, EZH2 expression was modulated by CRISPR/Cas9-mediated gene editing and pharmacologically inhibited with EZH2 inhibitor EPZ6438 alone or in combination with the MAPK inhibitor U0126. The results showed that CRISPR/Cas9-induced EZH2 gene editing reduced cell growth, migration and invasion in vitro and resulted in a 90% reduction in tumor growth when EZH2-edited cells were injected into an immunocompromised mouse model. Immunohistochemistry analysis of the tumors revealed reduced tumor cell proliferation and less recruitment of cancer-associated fibroblasts in the EZH2-edited tumors compared to the control tumors. Moreover, EZH2 inhibition induced thyroid-differentiation genes' expression and mesenchymal-to-epithelial transition (MET) in ATC cells. Thus, this study shows that targeting EZH2 could be a promising neoadjuvant treatment for ATC, as it promotes antitumoral effects in vitro and in vivo and induces cell differentiation.

Cell cycle arrest and apoptosis are early events in radiosensitization of EWS::FLI1+ Ewing sarcoma cells by Mithramycin A

Purpose The oncogenic fusion protein EWS::FLI1 is an attractive therapeutic target in Ewing sarcoma (ES). Mithramycin A (MithA) is a potent and specific inhibitor of EWS::FLI1 that can selectively radiosensitize ES cells through transcriptional inhibition of DNA double-strand break (DSB) repair. Here, we evaluate temporal changes in cell cycle progression and apoptosis in ES cells treated with MithA and/or ionizing radiation (RTx), testing the hypothesis that combining MithA with ionizing radiation would synergistically impair cell cycle progression and enhance apoptotic elimination to a greater extent than either agent alone. Materials and Methods Four EWS::FLI1 + ES cell lines TC-71, RD-ES, SK-ES-1, and A673, and one EWS::ERG cell line (CHLA-25) were exposed to 10nM MithA or vehicle and followed 24 h later by exposure to 2 Gy x-radiation or sham irradiation. Reactive oxygen species (ROS) activity was evaluated by cytometric assay, and assay of antioxidant gene expression by RT-qPCR. Cell cycle changes were evaluated by flow cytometry of nuclei stained with propidium iodide. Apoptosis was assessed by cytometric assessment of Caspase-3/7 activity and by immunoblotting of PARP-1 cleavage. Radiosensitization was evaluated by clonogenic survival assay. Proliferation (EdU) and apoptosis (TUNEL) were evaluated in SK-ES-1 xenograft tumors following pretreatment with 1 mg/kg MithA, followed 24 h later by a single 4 Gy fraction of x-radiation. Results MithA-treated cells showed reduced levels of ROS, and were associated with increased expression of antioxidant genes SOD1, SOD2, and CAT. It nonetheless induced persistent G0/G1 arrest and a progressive increase of the sub-G1 fraction, suggesting apoptotic degeneration. In vitro assays of Caspase-3/7 activity and immunoblotting of Caspase-3/7 dependent cleavage of PARP-1 indicated that apoptosis began as early as 24 h after MithA exposure, reducing clonogenic survival. Tumors from xenograft mice treated with either radiation alone, or in combination with MithA showed a significant reduction of tumor cell proliferation, while apoptosis was significantly increased in the group receiving the combination of MithA and RTx. Conclusions Taken together, our data show that the anti-proliferative and cytotoxic effects of MithA are the prominent components of radiosensitization of EWS::FLI1+ ES, rather than the result of acutely enhanced ROS levels.

Abstract 3695: Mitochondrial uncoupling reverses reductive carboxylation in cancer

Abstract When electron transport chain (ETC) activity is inhibited upon hypoxia or when cultured in suspension, reductive carboxylation plays an essential role in supporting tumor growth through converting α-ketoglutarate (α-KG) to citrate, which is converted to acetyl-coenzyme A (acetyl-CoA) for lipogenesis. However, there is no effective way to target reductive carboxylation for tumor therapy. In this study, we use the mitochondrial uncoupler niclosamide ethanolamine (NEN) and BAM15 to reverse reductive carboxylation in cancer cells. Uncoupler treatment activates ETC and increases the cellular NAD+/NADH ratio. Using U-13C-glutamine and 1-13C-glutamine tracers, we discovered that uncoupler treatment accelerates the oxidative TCA cycle and blocks reductive carboxylation, particularly under hypoxia and spheroid culture condition. Uncoupler treatment significantly reduces tumor cell proliferation, survival and sphere formation. In addition, the intracellular acetyl-CoA levels are significantly decreased by NEN treatment. Furthermore, lipidomics analysis showed that NEN treatment reduced fatty acid abundance and altered the lipid composition under both normoxia and hypoxia. Together, these data demonstrate mitochondrial uncoupling redirects the α-KG flux from reductive carboxylation back to the oxidative TCA cycle, highlighting that the ETC activity and NAD+/NADH ratio is the key switch that determines the metabolic fate of α-KG. Reversing reductive carboxylation could be one key mechanism that mitochondrial uncoupler inhibits tumor growth. Citation Format: Haowen Jiang, Clifford Jiajun He, Albert M. Li, Bo He, Yang Li, Jiangbin Ye. Mitochondrial uncoupling reverses reductive carboxylation in cancer. [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 3695.

LncRNA AC093850.2 predicts poor outcomes in patients with triple-negative breast cancer and motivates tumor progression by sponging miR-4299.

Accumulating evidence displays that non-coding RNAs (ncRNAs) are involved in the progression of triple-negative breast cancer (TNBC). This study aimed to investigate the role of lncRNA AC093850.2 in TNBC. The AC093850.2 levels were compared using RT-qPCR in TNBC tissues and corresponding normal tissues. The Kaplan-Meier curve method was conducted to assess the clinical significance of AC093850.2 in TNBC. Bioinformatic analysis was used to predict potential miRNA. Cell proliferation and invasion assays were carried out to explore the function of AC093850.2/miR-4299 in TNBC. lncRNA AC093850.2 expression is raised in TNBC tissues and cell lines, which is related to the shorter overall survival of patients. AC093850.2 is directly bound to miR-4299 in TNBC cells. Downregulation of AC093850.2 reduces tumor cell proliferation, migration, and invasion abilities, while miR-4299 silence attenuated AC093850.2 silencing induced inhibition of cellular activities in TNBC cells. In general, the findings suggest that lncRNA AC093850.2 was closely related to the prognosis and progression of TNBC by sponging miR-4299, which might be a prognosis predictor and potential target for treating TNBC patients.

Iodine-125 brachytherapy suppresses tumor growth and alters bone metabolism in a H1299 xenograft mouse model.

The present study aimed to investigate the efficacy of Iodine-125 (I-125) brachytherapy in a mouse model of non-small cell lung cancer, to further explore the efficacy and appropriate method of implantation of the I-125 radioactive seed. This study also aimed to determine the impact of brachytherapy on bone metabolism. A total of 18 mice were used to establish H1299 xenograft models, and were randomly assigned to three groups. These included non-radioactive seed implantation (Sham IM), fractionated I-125 seed implantation (Fractionated IM) and single I-125 seed implantation (Single IM) groups. Mice were euthanized after 28days of implantation. H&E staining, Ki67 immunohistochemistry, CD31 morphometric analysis and TUNEL immunofluorescence assays were respectively used to determine the histopathological changes, proliferation, micro-angiogenesis and apoptosis of tumors. In addition, bone volume and microstructure were evaluated using trabecular bone area (Tb.Ar), trabecular thickness (Tb.Th), trabecular number (Tb.N) and cortical thickness. Bone metabolic status was analyzed using histomorphometric staining of tartrate-resistant acid phosphate (TRAP) and alkaline phosphatase (ALP) expression in the femur, and using an ELISA assay to determine the expression of C-telopeptide of type 1 collagen (CTX-1) and procollagen type 1 n-terminal propeptide (P1NP) in the serum. Moreover, reverse transcription-quantitative PCR and western blotting were carried out for the analysis of bone remodeling-related gene expression in the bone tissue. Results of the present study demonstrated that compared with the Sham IM group, both the I-125 seed implantation groups, including Fractionated IM and Single IM, demonstrated significant therapeutic effects in both tumor volume and weight. More specifically, the most significant therapeutic effects on tumor inhibition were observed in the Fractionated IM group. Results of Ki67 and CD31 immunohistochemical staining suggested a notable reduction in tumor cell proliferation and micro-angiogenesis, and results of the TUNEL assay demonstrated an increase in tumor cell apoptosis. Although the cortical bone appeared thinner and more fragile in both I-125 seed implantation groups, no notable adverse changes in the morphology of the cancellous bone were observed, and the index of Tb.Ar, Tb.Th and Tb.n was not significantly different among Sham IM and I-125 implantation groups. However, alterations in bone metabolism were characterized by a decrease in CTX-1 and P1NP expression, accompanied by an increase in TRAP activity and a decrease in ALP activity. Results of the present study also demonstrated the notable suppression of osteocalcin and runt-related transcription factor 2. I-125 seed implantation may be an effective and safe antitumor strategy. Moreover, the use of fractionated implantation patterns based on tumor shape exhibited improved therapeutic effect on tumor suppression when the total number of I-125 seeds was equivalent along with reduced complications associated with bone loss.

CNSC-28. IDENTIFICATION OF EAG2-KVß2 POTASSIUM CHANNEL COMPLEX IN NEURON-GBM COMMUNICATIONS REVEALS GBM VULNERABILITY TO DESIGNER INTERFERENCE PEPTIDE

Abstract Glioblastomas (GBMs) are the most aggressive brain tumors. GBM cells form extensive tumoral networks to communicate with each other and with surrounding neurons. Neuronal activity promotes GBM cell proliferation by secreting protumorigenic factors and triggering neuronal-activity-dependent Ca2+ transients, which are persisted and transmitted via tumor networks to promote overall tumor growth and therapy resistance. While how these processes are regulated is largely unknown. Here we show the GBM networks and Ca2+ transients are regulated by a voltage-gated potassium channel complex comprised of EAG2 and Kvβ2. GBM cells selectively overexpress Kvβ2 isoform 4 to facilitate tumor-specific Kvβ2-EAG2 interaction, which regulates neuron-GBM contact-dependent localization of EAG2. Rationally designed interfering peptide K90-114TAT blocks EAG2-Kvβ2 interaction, leading to reduced tumor cell proliferation, increased apoptosis, and prolonged survival of patient-derived xenograft mouse models with no evidence of toxicity to normal tissue. Single-cell RNA sequencing revealed a subgroup of GBM cells is highly sensitive to K90-114TAT treatment. These cells exhibit neuronal signatures and are highly associated with TMZ resistance and worse prognosis. In accordance with the notion, we found neurons promote TMZ resistance of GBM cells, which is regulated by the EAG2-Kvβ2 complex. Treating TMZ-resistant GBM xenograft mice with K90-114TAT yielded significant tumor burden reduction and prolonged survival. Together, our findings revealed the EAG2-Kvβ2 channel complex as a key regulator of neuron promoted GBM progression and designed an interfering peptide with anti-GBM efficacy and low general toxicity, which may have significant clinical impact.

Environmental enrichment counteracts the effects of glioma in primary visual cortex

Experience-dependent neuronal changes and brain plasticity occur throughout life as animals adapt to their environment. Structural, morphological, and cellular modifications promoted by exposure to environmental enrichment (EE) have been reported to improve neuronal functions, increase hippocampal neurogenesis, ameliorate memory tasks and cognitive performance, and have beneficial effects on several brain diseases, including cancer. We specifically addressed the role of the EE in counteracting neuronal dysfunction in mice bearing glioma in the primary visual cortex. By recording spontaneous and evoked currents with patch clamp techniques in acute slices obtained from standard and enriched-housed mice, we found that the presence of glioma globally reduced the excitatory and inhibitory transmissions in the peritumoral area. The exposure to an enriched environment counteracts the tumor-mediated depression of both excitatory and inhibitory neuronal activities, with a more pronounced impact on evoked transmission. The effect of EE on glioma was also associated with reduced tumor cell proliferation. These results elucidate the impact of EE on excitatory and inhibitory neurotransmission of the primary visual cortex in control and glioma-bearing mice.

347 (PB127) - ON203: A new antibody targeting the oxidized form of macrophage migration inhibitory factor demonstrates antitumorigenic activity in preclinical models

Background: The pleiotropic cytokine macrophage migration inhibitory factor (MIF) is associated with tumor aggressiveness, metastasis, and disease progression. Due to its ubiquitous nature, MIF is considered an unsuitable target for therapeutic intervention. Oxidized MIF (oxMIF) is the disease-related, tumor-specific isoform of MIF, which undergoes a structural change enabling selective oxMIF-targeting by antibodies. We now developed ON203, a bioengineered anti-oxMIF antibody with highly improved biophysiochemical and biological properties, designed to exert better tumor retention and effector functions. Material and Methods: After physicochemical characterization of the optimized anti-oxMIF antibody ON203, cell-based assays were performed to evaluate antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. Tumor penetration was assessed by infrared-labeled ON203 injected in tumor-bearing mice. Human cancer cell-line (PC3) xenografted mice and human colorectal carcinoma 3D tumoroids were treated with ON203, and efficacy was assessed either by tumor volume measurements and immunohistochemical analysis or by tumor-cell killing and immune cell composition assessment. Results: ON203 demonstrated strong immune cell effector functions in reporter- and PBMC-mediated tumor cell killing assays. In xenograft models, ON203’s accumulation and retention in the tumor tissue was proven and tumor volumes were significantly reduced upon treatment. Tumor cell proliferation (assessed by Ki67 staining quantification) and tumor vessel density (CD31 staining quantification) were strongly decreased in the ON203-treated tumors compared to the isotype control. Tumor-infiltrating immune cells provided further insights on the immunomodulatory therapeutic effects of ON203. Strikingly, freshly isolated tumoroids from colorectal carcinoma patients responded not only with tumor cell death, but a clear immunomodulatory effect on the tumor microenvironment in responding tumoroids was detected: ON203 activated NK and NKT cells and supported M1 polarization. Conclusions: Our results demonstrate direct antitumorigenic effects of the anti-oxMIF antibody ON203 (i) by blocking the biologic function of oxMIF thereby reducing tumor cell proliferation and angiogenesis and (ii) by immunomodulation of the tumor microenvironment. In the upcoming Phase 1 trial we will evaluate ON203’s safety, tolerability, pharmacokinetics, and pharmacodynamics in patients with solid tumors. ON203 bears a high potential as a standalone therapy or in rational combinations with immune checkpoint inhibitors or antiangiogenic agents in the treatment of solid tumors. Conflict of interest: Ownership: Alexander Schinagl, Michael Thiele

Abstract B010: The NuRD subunit CHD4 is essential for ewing sarcoma cell survival as it regulates global chromatin architecture

Abstract Ewing sarcoma tumorigenesis is tightly linked to epigenetic deregulation. Indeed, the aberrant fusion transcription factor and tumor driver EWS-FLI1 produces extensive rewiring of the cancer cell epigenome. At enhancers containing canonical ETS motifs, the fusion protein represses gene expression but, when binding to GGAA repeats, EWS-FLI1 induces transcription by recruiting activating epigenetic regulators such as the acetyltransferase p300 and the BAF chromatin remodeling complex. The nucleosome remodeling and deacetylase (NuRD) complex is an ATP-dependent multi-subunit complex that modulates chromatin architecture and regulates DNA damage repair, genome stability and gene expression. In Ewing sarcoma, NuRD subunits, such as LSD1, have been suggested to interfere with EWS-FLI1 activity and prevent tumor progression. Hence, we here aimed to comprehensively study the role of NuRD in Ewing sarcoma pathogenesis. First, we performed a NuRD-centered, negative selection, CRISPR/Cas9 screen and identified the chromatin remodeling helicase CHD4 as essential for Ewing sarcoma tumor cell survival. Validation assays using two doxycycline-inducible shRNAs targeting CHD4 demonstrated that silencing of this remodeler in fact drastically reduces tumor cell proliferation and completely prevents colony formation. This cell proliferation impairment was caused by an induction of cell death by apoptosis and not by cell cycle arrest. Surprisingly, this cell death phenotype was not linked to impaired fusion protein activity. CHD4 and NuRD, despite primarily locating to enhancers and super-enhancers in Ewing sarcoma cells, did not preferentially localize to GGAA repeats, unlike EWS-FLI1. Moreover, RNA sequencing assays performed upon CHD4 silencing showed that this ATPase does not regulate the EWS-FLI1 signature. As CHD4 is a chromatin remodeler able to move nucleosomes along the DNA, we performed ATAC sequencing experiments to investigate changes in chromatin status that could explain the dependency of Ewing sarcoma cells on CHD4 for survival. We observed that CHD4 depletion from Ewing sarcoma cells causes a drastic and global chromatin relaxation which renders tumor cells prone to DNA damage and increasingly sensitive to DNA damaging agents. Interestingly, augmented sensitivity to DNA damage was not observed by silencing CHD4 in non-tumorigenic human fibroblasts. Finally, CHD4 depletion also reduced Ewing sarcoma tumor growth in vivo and prolonged mice survival. In conclusion, we demonstrate for the first time that CHD4 is crucial for Ewing sarcoma cell survival and highlight this helicase as a promising therapeutic target for Ewing sarcoma with potential for combination therapy with drugs inducing DNA damage. Importantly, this work has initiated ongoing efforts to develop first-in-class small molecules specifically targeting CHD4 which will be crucial for the future validation of this ATPase as a viable target with clinical application. Citation Format: Joana Graca Marques, Blaz Pavlovic, Quy Ai Ngo, Gloria Pedot, Michaela Roemmele, Larissa Volken, Marco Wachtel, Beat W. Schäfer. The NuRD subunit CHD4 is essential for ewing sarcoma cell survival as it regulates global chromatin architecture [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B010.

Therapeutic Potential of Fingolimod and Dimethyl Fumarate in Non-Small Cell Lung Cancer Preclinical Models

New therapies are required for patients with non-small cell lung cancer (NSCLC) for which the current standards of care poorly affect the patient prognosis of this aggressive cancer subtype. In this preclinical study, we aim to investigate the efficacy of Fingolimod, a described inhibitor of sphingosine-1-phosphate (S1P)/S1P receptors axis, and Dimethyl Fumarate (DMF), a methyl ester of fumaric acid, both already approved as immunomodulators in auto-immune diseases with additional expected anti-cancer effects. The impact of both drugs was analyzed with in vitro cell survival analysis and in vivo graft models using mouse and human NSCLC cells implanted in immunocompetent or immunodeficient mice, respectively. We demonstrated that Fingolimod and DMF repressed tumor progression without apparent adverse effects in vivo in three preclinical mouse NSCLC models. In vitro, Fingolimod did not affect either the tumor proliferation or the cytotoxicity, although DMF reduced tumor cell proliferation. These results suggest that Fingolimod and DMF affected tumor progression through different cellular mechanisms within the tumor microenvironment. Fingolimod and DMF might uncover potential therapeutic opportunities in NSCLC.

Identification of A2BAR as a potential target in colorectal cancer using novel fluorescent GPCR ligands

G-protein coupled receptors (GPCRs) have been largely targeted in a wide range of diseases, but few therapies have been directed against GPCRs in the field of cancer, partly because of the lack of effective target identification strategies. Here, using colorectal cancer (CRC) as a model, we explored the gene expression of a panel of GPCRs in tumor and stromal cells, identifying specific gene sets defining each cellular compartment. We selected the adenosine receptor 2B (A2BAR), specifically expressed in cancer cell lines compared with stromal cells, to explore the use of fluorescent ligands that can be used for target visualization. Fluorescent probes allowed semi-quantitative receptor mapping in living cells and validated the specific expression of A2BAR in CRC cell lines. As well, fluorescent ligands were effective at monitoring real-time A2BAR receptor labeling using live-imaging modalities, and displayed high efficiency when used to label complex 3D cellular systems such as tumor spheroids. Finally, we validated A2BAR as a potential pharmacological tool in CRC, using selective antagonists, finding a reduction in tumor cell proliferation. This proof-of-concept study suggests the use of fluorescent ligands for GPCR characterization through imaging, and as possible new tools used for target validation in drug screening methodologies.

Abstract 711: γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple-negative breast cancer (TNBC) MDA-MB-453 cells is associated with a reduction in cellular proliferation and viability

Abstract Targeted therapy for triple-negative breast cancer (TNBC) remains challenging due to poor understanding of its molecular etiology. Previous studies have shown that androgen receptors (AR) are widely expressed in approximately one-third of TNBC, and are referred to as luminal androgen receptors (LAR). AR has recently emerged as a prognostic and treatment-predictive biomarker in breast cancer. However, the role of AR in TNBC remained elusive. Treatment with AR-antagonist have been shown to suppress AR activation and signaling in TNBC and results in a corresponding reduction in tumor cell proliferation and survival due to the initiation of apoptosis in these tumor cells. γ-Tocotrienol (γT3) is a natural isoform of vitamin E that displays potent anticancer activity. Results show that treatment with 0-7 μM γT3 induces a dose-responsive significant decrease, whereas treatment with 35 nM dihydrotestosterone (DHT) results in a significant increase of AR expression and corresponding increase in growth of MDA-MB-453 TNBC cells. Combined treatment of 7 μM γT3 with 35 nM DHT significantly inhibits DHT-induced proliferation and AR activation in MDA-MB-453 cells. Western blot analysis shows that combined treatment with γT3 and DHT induced a significant decrease in phosphorylated ERK1/2 (active form) expression, but had no effect on total ERK1/2 levels in MDA-MB-453 TNBC cells. Flow cytometry analysis shows that γT3 treatment was also found to induce G0/G1 cell cycle arrest and significantly increased the number of apoptotic and necrotic cells, as compared to MDA-MB-453 TNBC cells in the vehicle-treated control group. Western blot analysis shows that γT3 treatment also inhibited DHT-induced elevations in CDK2, CDK4, CDK6 and cyclin D1 expression (cell cycle progression biomarkers) and induces a significant increase in the expression of tumor suppressor protein, p53, in MDA-MB-453 TNBC cells. Similar treatment with 7 µM γT3 induces a corresponding significant increase in apoptosis biomarker expression of cleaved caspase-3 and Bax in MDA-MB-453 TNBC cells. In summary, results from these studies demonstrated that γT3 treatment inhibits AR expression, activation and signaling, as well as DHT-dependent cell proliferation and viability in TNBC. These finding also suggest that the LAR may be a potential therapeutic target for the treatment TNBC. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Tasmin A. Sultana, Nayef Aldabaan, Paul W. Sylvester. γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple-negative breast cancer (TNBC) MDA-MB-453 cells is associated with a reduction in cellular proliferation and viability [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 711.

Abstract 1108: Evaluation of heme inhibitory therapy in combination with chemotherapy drugs on SCLC progression

Abstract Lung cancer is the leading cause of cancer-related death in the US. It is mainly divided in two types: small cell lung cancer and non-small cell lung cancer. About 85-90% of cases are classified as non-small cell lung cancer (NSCLC). Small cell lung cancer (SCLC) is an aggressive neuroendocrine lung tumor representing 15% of lung cancers. Despite the advent of targeted therapies and chemotherapies, effective therapies for lung cancer are an unlikely outcome for most patients. SCLC is initially highly responsive to cisplatin and etoposide chemotherapy but, in almost every case, becomes rapidly chemo resistant. However, the mechanisms underlying initial sensitivity and subsequent resistance are not well investigated. Thus, alternative approaches and combination therapy is required for treating SCLCs. Recent studies in our lab show that NSCLC cells demonstrate elevated levels of heme via increased heme uptake and synthesis, and elevated oxidative phosphorylation (OXPHOS) and ATP generation which fuels NSCLC proliferation. Also, limiting heme via inhibition of heme uptake using heme sequestering peptides (HeSP2) can delay growth and progression of NSCLC tumors. This HeSP2 can also effectively inhibit angiogenesis, normalize tumor vasculature, and alleviate tumor hypoxia in NSCLC tumors and thereby inhibit lung tumor growth and progression. Tumor heterogeneity is critical for tumor growth, metastasis, and acquired therapeutic resistance. To understand if heme can effectively reduce tumor cell proliferation and growth and improve tumor microenvironment, we treated chemo resistant SCLCs with HeSP2 and in combination with commonly used chemo drugs cisplatin and etoposide. Our data shows that HesP2 can strongly inhibit cell proliferation and decreases oxygen consumption rate along with ATP generation in SCLCs. To gain insights into mechanism by which the heme sequestering peptides limit tumor growth and progression, we implanted subcutaneous or SCLC cell lines in NOD/SCID mice. Mice were treated with saline (control), HeSP2, HeSP2 and cisplatin, HeSP2 and etoposide. Tumor growth was monitored using caliper measurements. Tumors tissues were harvested, processed and paraffin embedded for immunohistochemistry (IHC) and histology experiments. These results indicate that heme sequestration can be a powerful strategy for suppressing lung tumors likely drug-resistant tumors that rely on oxidative phosphorylation for survival. Citation Format: Adnin Ashrafi, Narges Salamat, Li Zhang. Evaluation of heme inhibitory therapy in combination with chemotherapy drugs on SCLC progression [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 1108.

Pancreatic cancer growth promoted by bone marrow mesenchymal stromal cell–derived IL-6 is reversed predominantly by IL-6 blockade

Pancreatic cancer is a highly lethal cancer characterized by local invasiveness, early metastasis, recurrence and high resistance to current therapies. Extensive stroma or desmoplasia is a key histological feature of the disease, and interactions between cancer and stromal cells are critical for pancreatic cancer development and progression. Mesenchymal stromal cells [MSCs] exhibit preferential tropism to primary and metastatic tumor sites and may either suppress or support tumor growth. Although MSCs represent a potential source of pancreatic cancer stroma, their contribution to pancreatic tumor growth remains poorly known. Here, we show that bone marrow MSCs significantly contribute to pancreatic cancer growth in vitro and in vivo. Furthermore, MSCs create a pro-carcinogenic microenvironment through the release of key factors mediating growth and angiogenesis, including interleukin (IL)-6, IL-8, vascular endothelial growth factor and activation of STAT3 signaling in tumor cells. IL-6 released by MSCs was largely responsible for the pro-tumorigenic effects of MSCs. Knockdown of IL-6 expression in MSCs by small interfering RNA (siRNA) abolished the MSC growth-promoting effect in vitro, reducing tumor cell proliferation and clonogenic potential. In addition, in a heterotopic nude mouse model of human pancreatic tumor xenografts, blockade of IL-6 with the anti-IL-6 receptor antibody, tocilizumab, or of its downstream effector STAT3 with the small molecule STAT3 inhibitor S3I-201, abrogated MSC-mediated tumor promotion and delayed tumor formation significantly. Our data demonstrate that MSCs promote pancreatic cancer growth, with IL-6 produced by MSCs playing a pivotal role.

Upregulation of circ_0059961 suppresses cholangiocarcinoma development by modulating miR-629-5p/SFRP2 axis

BackgroundCholangiocarcinoma (CCA) is a dangerous malignancy with a poor prognosis due to inefficient chemotherapy and surgery, and its pathophysiology could be linked to circular RNA (circRNAs) dysregulation. As a result, we wanted to see what role circ_0059961 plays in CCA. MethodsThe quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of circ_0059961, miR-629–5p, and secreted frizzled related protein 2 (SFRP2). Cell counting kit-8 (CCK-8) and 5-Ethynyl-2′-deoxyuridine (Edu) assay were used to determine the role of circ_0059961 in proliferation. The MMP (Δψm) assay was used to assess cell apoptosis. Transwell assay was used to detect cell migration and invasion. The SFRP2 protein and epithelia-mesenchymal transition (EMT) process related proteins expression were measured using Western blot. The putative relationship between miR-629–5p and circ_0059961 or SFRP2 was validated by dual-luciferase reporter assay. The circ_0059961 roles in cholangiocarcinoma were also investigated by tumor xenograft assay. ResultsCirc_0059961 expression was decreased in CCA tissues and carcinoma cells. Overexpressed circ_0059961 reduced tumor cell proliferation, migration, and invasion while inducing apoptosis. Circ_0059961 was proven to be a target of miR-629–5p, while miR-629–5p was confirmed to be a target of SFRP2. Circ_0059961 targeted miR-629–5p to modulate SFRP2 expression. Upregulation of miR-629–5p reversed the tumor-suppressive effects of circ_0059961 overexpression in rescue trials. Furthermore, SFRP2 overexpression restored miR-629–5p enrichment-promoted cell proliferation, migration, and invasion. Upregulated circ_0059961 reduced solid tumor growth in vivo. ConclusionUpregulation of circ_0059961 augmented SFRP2 expression by targeting miR-629–5p, which blocked cholangiocarcinoma tumor cell proliferation, migration, and invasion.