Prevent Tumor Formation Research Articles

Protein homeostasis maintained by HOOK1 levels promotes the tumorigenic and stemness properties of ovarian cancer cells through reticulum stress and autophagy

BackgroundOvarian cancer has a high mortality rate mainly due to its resistance to currently used therapies. This resistance has been associated with the presence of cancer stem cells (CSCs), interactions with the microenvironment, and intratumoral heterogeneity. Therefore, the search for new therapeutic targets, particularly those targeting CSCs, is important for improving patient prognosis. HOOK1 has been found to be transcriptionally altered in a substantial percentage of ovarian tumors, but its role in tumor initiation and development is still not fully understood.MethodsThe downregulation of HOOK1 was performed in ovarian cancer cell lines using CRISPR/Cas9 technology, followed by growth in vitro and in vivo assays. Subsequently, migration (Boyden chamber), cell death (Western-Blot and flow cytometry) and stemness properties (clonal heterogeneity analysis, tumorspheres assay and flow cytometry) of the downregulated cell lines were analysed. To gain insights into the specific mechanisms of action of HOOK1 in ovarian cancer, a proteomic analysis was performed, followed by Western-blot and cytotoxicity assays to confirm the results found within the mass spectrometry. Immunofluorescence staining, Western-blotting and flow cytometry were also employed to finish uncovering the role of HOOK1 in ovarian cancer.ResultsIn this study, we observed that reducing the levels of HOOK1 in ovarian cancer cells reduced in vitro growth and migration and prevented tumor formation in vivo. Furthermore, HOOK1 reduction led to a decrease in stem-like capabilities in these cells, which, however, did not seem related to the expression of genes traditionally associated with this phenotype. A proteome study, along with other analysis, showed that the downregulation of HOOK1 also induced an increase in endoplasmic reticulum stress levels in these cells. Finally, the decrease in stem-like properties observed in cells with downregulated HOOK1 could be explained by an increase in cell death in the CSC population within the culture due to endoplasmic reticulum stress by the unfolded protein response.ConclusionHOOK1 contributes to maintaining the tumorigenic and stemness properties of ovarian cancer cells by preserving protein homeostasis and could be considered an alternative therapeutic target, especially in combination with inducers of endoplasmic reticulum or proteotoxic stress such as proteasome inhibitors.

Abstract A011: Bladder cancerization is linked to epithelial basalization and is reversed by EGF inhibition

Abstract Introduction: Epithelial changes can extend beyond the boundaries of an incipient tumor, leading to cancer recurrence even after effective excision of the primary tumor. This ‘field cancerization’ and consequent high recurrence rate is especially pronounced in bladder cancer. The purpose of this study is to characterize the urothelium in the context of ‘field cancerization’ in a mouse model of bladder cancer as well as in patients with bladder cancer and to determine if reversal of the field effect will prevent tumor formation. Results: We investigated field cancerization by single cell transcriptomics (scRNA-Seq) in a murine (male FVB mice) bladder carcinogenesis model using N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) and found that prior to overt tumor formation urothelial cells throughout the entire bladder take on a strongly basal phenotype, characterized by reduced expression of differentiation markers (i.e. Upk1a, Upk2, and Upk3a) and increased expression of basal markers (i.e. Krt14, Krt6a, Krt5). Using monocle, we applied single cells from a BBN treated mouse bladder library to a pseudotime trajectory generated from a control mouse bladder library in order to unbiasedly characterize urothelial differentiation in the non-tumor urothelium of BBN treated mouse bladders. scRNA-Seq analysis of non-tumor urothelium from bladder cancer patients revealed a similar ‘basalization’ among non-tumor urothelial cells. In order to apply our single cell findings to bulk RNA-seq data, we developed a basal index from differentially expressed genes in basal clusters versus non-basal clusters from healthy control human urothelium single cell libraries, and we applied this basal index to non-tumor bladder samples from the The Cancer Genome Atlas (TCGA). We found that among a small subset of patients with paired tumor and non-tumor urothelium in the bladder TCGA dataset, the basal index, when applied to the non-tumor urothelium, was prognostic of survival. Finally, we found that basalization in BBN treated mice could be reversed pharmacologically by epidermal growth factor (EGF) pathway antagonism using erlotinib and trametinib and that over a 4-month time course this reversal dramatically decreased tumor incidence and improved survival, suggesting a new, more effective approach using mechanism-based cancer drugs for the prevention of bladder cancer recurrence in patients. Conclusions: The non-tumor urothelium in BBN treated mouse bladders, prior to overt tumor formation, demonstrates a basal phenotype which we characterize with scRNA-Seq and pseudotime analysis. Similarly, in patients with bladder cancer we observe that the non-tumor urothelium also takes on a basal phenotype. This ‘basalization’ of the non-tumor urothelium may potentially be prognostic, and pharmacologic reversal of the basal phenotype in the non-tumor urothelium using EGF pathway inhibitors prevents tumors in BBN treated mice. Citation Format: Aaron M. Kershner, Kris B. Prado, Bernhard M. Kiss, Karim Mrouj, Lolita Penland, Wan-Jin Lu, Jiyun Choi, Edward Diaz, Rovin Lachmansingh, Bertha Chen, Chia-Sui Kao, Joseph C. Liao, Philip A. Beachy. Bladder cancerization is linked to epithelial basalization and is reversed by EGF inhibition [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A011.

Linc-NSC affects cell differentiation, apoptosis and proliferation in mouse neural stem cells and embryonic stem cells in vitro and in vivo

BackgroundStem cell therapy is a promising therapeutic strategy. In a previous study, we evaluated tumorigenicity by the stereotactic transplantation of neural stem cells (NSCs) and embryonic stem cells (ESCs) from experimental mice. Twenty-eight days later, there was no evidence of tumor formation or long-term engraftment in the NSCs transplantation group. In contrast, the transplantation of ESCs caused tumor formation; this was due to their high proliferative capacity. Based on transcriptome sequencing, we found that a long intergenic non-coding RNA (named linc-NSC) with unknown structure and function was expressed at 1100-fold higher levels in NSCs than in ESCs. This finding suggested that linc-NSC is negatively correlated with stem cell pluripotency and tumor development, but positively correlated with neurogenesis. In the present study, we investigated the specific role of linc-NSC in NSCs/ESCs in tumor formation and neurogenesis.MethodsWhole transcriptome profiling by RNA sequencing and bioinformatics was used to predict lncRNAs that are widely associated with enhanced tumorigenicity. The expression of linc-NSC was assessed by quantitative real-time PCR. We also performed a number of in vitro methods, including cell proliferation assays, differentiation assays, immunofluorescence assays, flow cytometry, along with in vivo survival and immunofluorescence assays to investigate the impacts of linc-NSC on tumor formation and neurogenesis in NSCs and ESCs.ResultsFollowing the knockdown of linc-NSC in NSCs, NSCs cultured in vitro and those transplanted into the cortex of mice showed stronger survival ability (P < 0.0001), enhanced proliferation(P < 0.001), and reduced apoptosis (P < 0.05); the opposite results were observed when linc-NSC was overexpressed in ESCs. Furthermore, the overexpression of linc-NSC in ECSs induced enhanced apoptosis (P < 0.001) and differentiation (P < 0.01), inhibited tumorigenesis (P < 0.05) in vivo, and led to a reduction in tumor weight (P < 0.0001).ConclusionsOur analyses demonstrated that linc-NSC, a promising gene-edited target, may promote the differentiation of mouse NSCs and inhibit tumorigenesis in mouse ESCs. The knockdown of linc-NSC inhibited the apoptosis in NSCs both in vitro and in vivo, and prevented tumor formation, revealing a new dimension into the effect of lncRNA on low survival NSCs and providing a prospective gene manipulation target prior to transplantation. In parallel, the overexpression of linc-NSC induced apoptosis in ESCs both in vitro and in vivo and attenuated the tumorigenicity of ESCs in vivo, but did not completely prevent tumor formation.

Abstract 4018: An innovative in vivo model for CAR-T cell therapy development: Tolerability and efficacy evaluation of CD19-targeting CAR-T cells on human lymphoma using the chicken CAM assay

Abstract BACKGROUND Chimeric antigen receptor (CAR) T cell therapy is a revolutionary approach in cellular immunotherapy, where a patient’s T cells are genetically engineered to recognize and attack cancer cells. In the last decade, several CAR-T cell therapies have been approved by the FDA. However, this encouraging outcome was obtained only with certain blood cancers, like B cell leukemias, lymphomas, etc. Many research/clinical trials are now underway worldwide to extend the CAR T-cell therapy benefits to a large spectrum of cancers, using rodents. This model presents several drawbacks, such as the immunodeficiency of humanized models, ethical constraints, time, and cost. An alternative, pertinent, 3Rs-compliant in vivo model for developing CAR-T therapy is urgently needed. Xenografts on the chicken embryo’s ChorioAllantoic Membrane (CAM) have proven extremely valuable for in vivo cancerology studies. In this work, we evaluated in vivo the anti-tumor efficacy of CD19-targeting CAR-T cells on human lymphoma using Inovotion’s CAM assay. METHODS Human lymphoma Raji cells (ATCC) were grafted on the CAM on embryonic development day (EDD) 9. A 2nd generation of CD19-targeting CAR-T cells (CAR-T-(CD3/4-1BB)) developed by Allogenica (France) were co-injected at EDD9 at E/T (effector CAR-T cell vs target Raji cell) ratio 1:1. One group was injected with CAR-T (CD3/4-1BB) only once at EDD9 and a second group received a first injection of CAR-T (CD3/4-1BB) at EDD9 and a second injection at EDD13. Embryonic viability was checked daily. On EDD18. The efficacy of CAR-T therapy was evaluated basing on tumor weight, metastatic invasion in the lower CAM and CAR-T infiltration in tumors detected by qPCR. Activation of the chicken embryo immune system was detected as well. RESULTS The treatment with CAR-T-(CD3/4-1BB) cells was well tolerated in ovo, no significant embryo mortality/abnormality was observed. CAR-T-(CD3/4-1BB) cells prevented tumor formation (tumor weight was 88.67% to 90.62% lower than control, p &amp;lt; 0.001); and prevented metastasis formation in the lower CAM (86.88% to 81.43% lower than control, p &amp;lt; 0.001). CONCLUSION Different to in vitro models and immunodeficient murine models, chicken embryo possesses a progressively mature immune system. Our results show that CAR-T-(CD3/4-1BB) cells can prevent tumor formation and metastatic invasion when co-injected with tumor cells. Inovotion’s CAM assay is a viable alternative in vivo model for developing CAR-T cell therapy on a large spectrum of cancers. It is fully 3Rs-compliant. Citation Format: Yan Wang, Xavier Rousset, Chloé Prunier, Emilien Dosda, Alejandra Gutierrez-Guerrero, Pauline Abrial, Inna Menkova, Jean Viallet. An innovative in vivo model for CAR-T cell therapy development: Tolerability and efficacy evaluation of CD19-targeting CAR-T cells on human lymphoma using the chicken CAM assay [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 4018.

Ascorbic acid predominantly kills cancer stem cell-like cells in the hepatocellular carcinoma cell line Li-7 and is more effective at low cell density and in small spheroids

The development of therapies that target cancer stem cells (CSCs) is an important challenge in cancer research. The antioxidant system is enhanced in CSCs, which may lead to resistance to existing therapies. Ascorbic acid (AA) has the potential to act as both an antioxidant and a pro-oxidant agent, but its effects on CSCs are a subject of current research. Here, we investigated the effect of AA focusing specifically on CSCs with the hepatocellular carcinoma cell line Li-7. The Li-7 cell line is heterogenous consisting of CD166- and CD166+ cells; CD166- cells include CSC-like cells (CD13+CD166- cells) and CD13−CD166- cells that can revert to CD13+CD166- cells. The addition of AA to the culture medium caused cell death in both cell populations in CD166- cells in a concentration dependent manner. In contrast, AA administration had a limited effect on CD166+ non-CSC cells. The level of reactive oxygen species after AA treatment was elevated only in CD166- cells. The effect of AA only occurred at low cell densities in 2D and 3D cultures. In a mouse tumor model injected with Li-7 cells, intraperitoneal administration of AA failed to prevent tumor formation but appeared to delay tumor growth. Our findings shed light on why AA administration has not become a mainstream treatment for cancer treatment; however, they also show the possibility that AA can be used in therapies to suppress CSCs.

Abstract 3389: Intraductal ablative procedure for local preventive intervention in rat models of breast cancer

Abstract There are limited options for primary prevention of breast carcinoma (BC). Prophylactic mastectomy and systemic hormonal therapy are only recommended for high-risk individuals. Prophylactic mastectomy is a drastic surgery that removes the entire breast, including the epithelial cell-lined ductal trees where BC arises but also uninvolved stroma. Even with a 90% risk reduction, many women are deterred from prophylactic mastectomy due to its associated risk of infection, lymphedema, painful recovery time, and mental health toll. Hormonal therapies, tamoxifen and raloxifene, reduce BC risk by upwards of 50% but also increase the risk of uterine cancer and stroke. Thus, new interventions to reduce the risk of BC and minimize deterrents are necessary. Intraductal (ID) injection is a minimally invasive procedure that avoids systemic toxicity by cannulating a needle directly into the ductal tree opening to directly administer a solution to the epithelial cells. We previously reported that ID infusion of 70% ethanol (EtOH) as a cell-killing solution prevents tumor formation in an aggressive genetically engineered mouse model of BC with minimum collateral tissue damage. Here, we investigated the therapeutic efficacy of EtOH-based ablative solutions in a Methyl-N-Nitrosourea (MNU) chemically induced rat model of BC to assess scalability of this procedure towards clinical trials. Two weeks after tumor induction, rats received ID infusions of a solution containing 70% EtOH to ablate epithelial cells, ethyl cellulose (EC) to minimize collateral tissue damage, and/or Tantalum Oxide (TaOx) to visualize infused ductal trees via micro-CT imaging. EtOH-infused glands had the longest tumor latency (273 days vs 97 days in control group, p &amp;lt; 0.0001). No statistically significant differences were noted in epithelial ablation or collateral tissue damage between treatment groups receiving ID injections of 70% EtOH with varying concentrations of EC, but there was a trend to lower collateral damage with EC-containing solutions. Local clearance of TaOx in infused mammary glands enabled post-procedure MR imaging surveillance after 15 d of ID procedure. This preclinical study demonstrates that ID injection of a refined solution containing 70% EtOH and TaOx is an efficacious and scalable procedure for primary prevention of BC. Citation Format: Erin Zaluzec, Mohamed Ashry, Elizabeth G. Phelps, Katarzyna Kempinska, Elizabeth Kenyon, Legend Kenny, Max Volk, Katherine Powell, Jeremy ML Hix, Christiane Mallett, Matti Kiupel, Erik M. Shapiro, Lorenzo F. Sempere. Intraductal ablative procedure for local preventive intervention in rat models of breast cancer [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 3389.

Abstract 2672: GD3 synthase (ST8SIA1) is a key immunomodulator in GD2+ breast cancer cells

Abstract Background: Gangliosides are acidic glycosphingolipids involved in cell adhesion, proliferation, and modulation of signal transduction pathways. It has been reported that tumor-shed gangliosides influence the activity of immune cells, including macrophages, NK cells, and T cells. GD3 synthase (GD3S) is the key enzyme that regulates the biosynthesis of the b and c-series gangliosides, particularly GD3 and GD2, and studies have shown that GD3S is upregulated in most tumors and plays a role in tumor progression. Similarly, we have found GD3S to be significantly upregulated in GD2+ breast cancer stem cells (BCSC) compared to GD2− cells, and the knockdown of this gene prevented tumor formation in mice. Here, we hypothesize that GD3 synthase has an immunomodulatory function in breast cancer (BC) cells through the upregulation of GD2. Methods: We stably overexpressed GD3S in BT549 and MCF-7 breast cancer cell lines by lentiviral transfection and performed CRISPR-Cas9 knockout of GD3S in BT549 and SUM 159 cell lines. We examined the effect of GD3 synthase on macrophage-mediated phagocytosis, NK cell-mediated killing, and T-cell cytotoxicity of BC cells using the live cell imaging system, IncuCyte®. Naxitamab is a US FDA-approved fully humanized anti-GD2 monoclonal antibody for the treatment of neuroblastoma. We evaluated the effects of naxitamab in GD2+ BC cell lines (HCC1395, Hs578T) using the Incucyte, and also examined its effect on in vivo tumor growth using TNBC PDX models. Results: We observed a highly significant decrease in macrophage-mediated phagocytosis (p&amp;lt;0.005; p&amp;lt;0.0001) and NK-mediated killing (p&amp;lt;0.0001; p&amp;lt;0.0001) in BT549 and MCF-7 cell overexpressing GD3S compared to the control cells. Interestingly, we observed a significant increase in phagocytosis in GD3S knockout BT549 and SUM 159 cells (p&amp;lt;0.05; p&amp;lt;0.005) compared to their wild-type counterparts. Moreover, the knockout of GD3S enhanced NK cell-mediated apoptosis in BT549 cells (p&amp;lt;0.001) and T cell killing in BT549 and SUM 159 cells (p&amp;lt;0.0001; p&amp;lt;0.0001). Finally, we found that naxitamab dose-dependently enhances macrophage-mediated phagocytosis and NK cell-mediated apoptosis in HCC1395 and Hs578T BC cells, with 20µg/ml being the most effective concentration in comparison to IgG control (p&amp;lt;0.0001; p&amp;lt;0.0001). In addition, we observed a reduction in tumor volume in naxitamab-treated mice compared to IgG control (p&amp;lt;0.05) Conclusion: GD3 synthase regulates macrophage-mediated phagocytosis, NK cell-induced apoptosis, and T-cell cytotoxicity in BC cells. Naxitamab enhances immune cell killing of breast cancer cells with high GD2 expression. Furthermore, naxitamab inhibited tumor growth in GD2+ TNBC PDX models. Citation Format: Bolutyfe Oderinde, Vivek Anand, Maryam Siddiqui, Anudishi Tyagi, Borgman Jenny, Venkata Lokesh Battula. GD3 synthase (ST8SIA1) is a key immunomodulator in GD2+ breast cancer cells [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 2672.

Prevention and treatment of HPV-related cancer through a mRNA vaccine expressing APC-targeting antigen.

Persistent human papillomavirus (HPV) infection is associated with multiple malignancies. Developing therapeutic vaccines to eliminate HPV-infected and malignant cells holds significant value. In this study, we introduced a lipid nanoparticle encapsulated mRNA vaccine expressing tHA-mE7-mE6. Mutations were introduced into E6 and E7 of HPV to eliminate their tumourigenicity. A truncated influenza haemagglutinin protein (tHA), which binds to the CD209 receptor on the surface of dendritic cells (DCs), was fused with mE7-mE6 in order to allow efficient uptake of antigen by antigen presenting cells. The tHA-mE7-mE6 (mRNA) showed higher therapeutic efficacy than mE7-mE6 (mRNA) in an E6 and E7+ tumour model. The treatment resulted in complete tumour regression and prevented tumour formation. Strong CD8+ T-cell immune response was induced, contributing to preventing and curing of E6 and E7+ tumour. Antigen-specific CD8+ T were found in spleens, peripheral blood and in tumours. In addition, the tumour infiltration of DC and NK cells were increased post therapy. In conclusion, this study described a therapeutic mRNA vaccine inducing strong anti-tumour immunity in peripheral and in tumour microenvironment, holding promising potential to treat HPV-induced cancer and to prevent cancer recurrence.

Abstract PR10: Mammary epithelial architecture modulates field cancerization

Abstract The architecture of the human breast undergoes extensive remodelling throughout puberty, the menstrual cycle, pregnancy, and involution with a risk of accumulating genetic alterations and tissue changes. Cells with mutations in cancer driver genes are abundant in tissues of healthy individuals, but rarely develop into tumors. However, the underlying mechanisms that prevent tumor formation are largely unknown. We utilized tissue clearing and imaged 15 disease-free human breasts and cancer resections with subcellular resolution to unravel the 3D architecture of thousands of mammary ducts per individual. Surprisingly, our analysis revealed that disease-free breasts carry a high burden of microscopic tissue lesions indicating widespread abnormal cell behaviour. Distinct mammary trees from the same breast differed in the kind and frequency of lesions, which coevolved scattered along the ductal routes. Combining 3D imaging of FFPE archived breast cancer resections with spatial CNV-seq revealed that, in half the cases, disease-free ductal segments neighbouring early cancer lesions contain molecular alterations. To resolve the mechanisms that regulate mutation spread, we compared the clonal behavior of cells with oncogenic mutations and neutral mutations in the mouse mammary gland epithelium. From the analysis of the clonal dynamics, we find that local tissue remodeling during the estrous cycle leads to the elimination of the majority of mutant clones, while accelerating the expansion of surviving clones. Thus, oncogenic mutations can spread along entire ducts before signs of transformation become apparent. Our data provides understanding of the earliest stages of epithelial evolution preceding disease. Citation Format: Hendrik A Messal, Cindy Cleyp0ol, Catrin Lutz, Stefan Hutten, Esther Lips, Jos Jonkers, Jelle Wesseling, Ronald Bleys, Ben Simons, Colinda Scheele, Jacco van Rheenen. Mammary epithelial architecture modulates field cancerization [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr PR10.

Inherently targeted estradiol-derived carbon dots for selective killing of ER (+) breast cancer cells via oridonin-triggered p53 pathway activation.

One of the most prevalent cancers globally is breast cancer and approximately two thirds of the breast cancers are hormone receptor positive with estrogen receptors (ER) being a prominent target. Notably, p53 that controls several cellular functions and prevents tumor formation, gets suppressed in breast cancers. Reactivation of p53 can lead to cell cycle arrest as well as apoptosis. Therefore, targeting the estrogen receptor for selective delivery of anticancer drugs that can reactivate p53 in ER (+) breast cancers can be a crucial method in breast cancer therapy. Herein, we have designed and developed estradiol-derived inherently targeted specific carbon dots (E2-CA-CD) from 17β-estradiol and citric acid following a solvothermal method. The synthesized carbon dots were characterized using spectroscopic and microscopic techniques. The water soluble, intrinsically fluorescent E2-CA-CD showed excellent biocompatibility in MCF-7, MDA-MB-231 as well as NIH3T3 cells and demonstrated target specific bioimaging in ER (+) MCF-7 cells due to the overexpressed ER receptors. Furthermore, oridonin, a well-known hydrophobic anticancer drug capable of upregulating the p53 pathway, was loaded on the carbon dots to increase its bioavailability. E2-CA-CD-Ori caused ∼2.2 times higher killing in ER (+) MCF-7 cells compared to ER (-) MDA-MB-231 cells and normal cells NIH3T3. Also, E2-CA-CD-Ori showed ∼3 fold better killing in MCF-7 cells compared to native oridonin. E2-CA-CD-Ori-induced killing of MCF-7 cells took place through the early to late apoptotic pathway along with the elevation of the intracellular ROS level. Importantly, E2-CA-CD-Ori triggered the activation of the p53 pathway in MCF-7 cells, which in turn induced apoptosis involving the upregulation of Bax and downregulation of Bcl-2 leading to the selective and efficient killing of ER (+) MCF-7 cells.

Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models.

Caveolin-1 (CAV1) is a membrane-bound protein that suppresses tumor development yet also promotes metastasis. E-cadherin is important in CAV1-dependent tumor suppression and prevents CAV1-enhanced lung metastasis. Here, we used murine B16F10 and human A375 melanoma cells with low levels of endogenous CAV1 and E-cadherin to unravel how co-expression of E-cadherin modulates CAV1 function in vitro and in vivo in WT C57BL/6 or Rag-/- immunodeficient mice and how a pro-inflammatory environment generated by treating cells with prostaglandin E2 (PGE2) alters CAV1 function in the presence of E-cadherin. CAV1 expression augmented migration, invasion, and metastasis of melanoma cells, and these effects were abolished via transient co-expression of E-cadherin. Importantly, exposure of cells to PGE2 reverted the effects of E-cadherin expression and increased CAV1 phosphorylation on tyrosine-14 and metastasis. Moreover, PGE2 administration blocked the ability of the CAV1/E-cadherin complex to prevent tumor formation. Therefore, our results support the notion that PGE2 can override the tumor suppressor potential of the E-cadherin/CAV1 complex and that CAV1 released from the complex is phosphorylated on tyrosine-14 and promotes migration/invasion/metastasis. These observations provide direct evidence showing how a pro-inflammatory environment caused here via PGE2 administration can convert a potent tumor suppressor complex into a promoter of malignant cell behavior.

Novel, Engineered, Modified Fcγ Receptor I (CD64)-Expressing T Cells (SolidT) Combined with Rituximab Exhibited Profound Anti-Tumor Activity and Memory in a CD20+ Raji Xenograft Model of Lymphoma

Background: CD19 CART cell therapy has been a major advance in the treatment of lymphoma, but is limited due to profound toxicity and relapse, in part due to loss of CD19 expression on malignant cells. Following our discovery of FcγRI (CD64)-expressing T cells in tumors and inflamed tissues (PMID 31449054), we developed novel, engineered cytotoxic T cells (“SolidT”) with increased specificity, reduced exhaustion, and attenuated cytokine secretion compared to other engineered cell therapies (e.g., CAR-T, CAR-NK). SolidT are produced by transducing a retroviral vector encoding an enhanced FcγRI into T cells for use with tumor-targeting monoclonal antibodies (mAbs). SolidT activation correlates with surface tumor antigen density. Unlike CART, where tonic signaling causes premature exhaustion and limited tumor infiltration, SolidT are not exhausted prematurely. They are activated and persist only by mAb bound to tumor-specific antigen at sufficient density, and they take on a T effector memory (T EM) phenotype. We previously showed that SolidT + mAb has anti-tumor activity in vitro and in vivo in several tumor models, including immunodeficient HER2 xenografts and an immunocompetent syngeneic B16-TYRP1 model (PMID 37070661). To further assess the SolidT platform, we examined the effect of SolidT + rituximab (R) in vitro and in vivo in a CD20+ Raji-Luciferase (Luc) xenograft model. Methods: In in vitro Raji-Luc and Daudi-Luc models, SolidT + R was compared to CD19 CART, R + sham transfected T cells (Sham), Sham only, and SolidT only. Cytotoxicity was measured by flow analysis of Annexin V and/or Luc assay. IFNγ and Granzyme B were measured by ELISA. Addition of intravenous immunoglobulin (IVIG; 5mg/ml) to cytotoxicity co-cultures was used to evaluate the effect of non-specific IgGs on the efficacy of the combination treatment. In vitro safety was evaluated in a similar manner using primary cultures of kidney and lung epithelial cells. To assess the anti-tumor activity of SolidT, NSG mice were inoculated intravenously (IV) with 1x10 6 Raji-Luc cells (n=8). SolidT (1x10 7 cells) was given on Day (D) 1. R (250 mg) was given on D1 and then weekly (125 mg). Mice were monitored for weight, clinical signs, and survival until D41 when the experiment was terminated to conduct endpoint analyses including gross pathology, histopathology, and CD3 + immunohistochemistry (IHC). Results: In vitro, SolidT + R showed higher cytotoxicity and attenuated cytokine levels against Raji-Luc compared to CD19 CART cells. Similar results were obtained in a Daudi-Luc model. Addition of IVIG did not activate SolidT cells, and though IFNγ secretion was attenuated, cytotoxicity was not affected. The in vitro co-culture of SolidT cells with primary epithelial cells demonstrated no activation of the effector cells and no death of primary target cells. In vivo, mice treated with SolidT + R had increased survival and reduced tumor burden against Raji-Luc compared to mice treated with R alone (Figure 1). On pathology, 5/8 SolidT + R mice had no tumors detected and 3 had only abdominal tumors. R mice developed tumors in the lungs (2/8), liver (7/8), and abdomen (4/8). Bone marrow immunophenotyping revealed that the presence of T EM phenotype highly correlated to better survival and lower tumor burden. Gross pathology analysis revealed no treatment-related pathology. IHC showed increased inflammation, lymphocyte infiltration, and necrosis in tumors from SolidT + R-treated mice, as well as intense infiltration of CD3 + human cells in the tumors at end point D41. The in vivo Raji-Luc model was performed twice with similar results. Conclusions: Consistent with prior results in solid tumor models targeting HER2 or TYRP1, these data demonstrated that the SolidT platform could be effectively targeted against CD20+ cell lines. A single administration of SolidT cells in combination with R prevented tumor formation and prolonged survival in a CD20+ Raji-Luc model. This experiment demonstrated that marrow-resident SolidT cells developed a T EM phenotype that positively correlated with outcome. These data confirm that the SolidT platform can be targeted against different tumor antigens by using different tumor-targeting mAbs. The SolidT platform could represent a paradigm shift in the approach to engineered cellular therapy for hematological malignancies and solid tumors.

CNSC-13. NEURODEVELOPMENTAL TRANSCRIPTION FACTORS PROMOTE PROLIFERATION, REGULATE CELL TYPE, AND PLAY OPPOSING ROLES IN CELLULAR MIGRATION IN GLIOBLASTOMA

Abstract Glioblastomas (GBM) make up ~50% of primary brain tumors and prognosis has not improved over the last 30 years. Despite high inter- and intratumoral heterogeneity, basic-helix-loop-helix (bHLH) transcription factors, ASCL1 and OLIG2, are present in the majority of tumors. Previously, we showed in patient-derived GBM xenograft (PDX-GBM) that ASCL1 binds to promoter and enhancer regions of cell cycle genes, as well as neurodevelopmental transcription factors including OLIG2. Similarly, we showed that OLIG2 binding overlaps with the majority (~90%) of ASCL1 binding sites, including at promoter and/or enhancer regions of ASCL1 and OLIG1/2 loci, illustrating their potential redundant and/or feed-forward function in GBM. It has been proposed that ASCL1 and OLIG2 contribute to the neural stem cell-like properties of tumor cells, which may promote tumor growth and progression but also the treatment resistivity and high recurrence rate of GBMs in patients. Using an immune competent glioma mouse model, we are able to efficiently induce tumors from glial progenitors surrounding the lateral ventricle while altering the levels of ASCL1 and OLIG2 to assess their combinatory roles in GBM progression. Remarkably, we found that loss of both Ascl1 and Olig2 prevents tumor formation in 80% of mice, whereas the loss of only Ascl1 resulted in reduced cellular migration from the tumor bulk while the loss of Olig2 promotes a highly migratory phenotype. Conversely, elevating the levels of Ascl1increased both tumor cell proliferation and migration similar to the loss of OLIG2. Using single cell RNA-sequencing, we found that tumor cells which express high levels of Ascl1 exhibit neural stem cell/astrocytic gene signatures, which supports ASCL1’s role as a marker of glioma-stem-cells. Collectively, these findings illustrate the role of ASCL1 and OLIG2 in regulating GBM initiation, proliferation, and migration where ASCL1 may directly be responsible for the highly invasive and proliferative phenotype of GBMs.

Long Intergenic Non-Protein Coding RNA 00342 Exerts Oncogenic Effects in Prostate Cancer by Sponging microRNA-19b-3p and Thereby Increasing Forkhead Box F2 Expression

Prostate cancer is one of the most common malignancies in males with increasing morbidity and mortality. Related reports indicate that lncRNAs may be a new class of potential tumor molecular markers. Compared with the normal tissues, the expression level of LINC00342 in prostate cancer tissues was significantly increased. Compared with normal PC3 and DU145 cells, cells silenced by the LINC00342 gene significantly decreased the ability of proliferation, migration, invasion, and induced apoptosis. Further, the FISH assay showed that LINC00342 could interact with miR-19b-3p and control the expression of Forkhead Box F2 (FOXF2), the target of miR-19b-3p. LINC00342 could activate the ERK/AKT signaling pathways. Tumor xenograft formation assay demonstrated that the down-regulated LINC00342 prevented tumor formation in vivo. Taken together, these results indicated that the interference of LINC00342 expression might inhibit the invasion, migration and promote apoptosis of prostate cancer cells by inhibiting the ERK1/2/AKT signaling pathway via sponging miRNA-19b-3p and thereby increasing FOXF2 expression.

Immunotherapy of Epstein-Barr virus (EBV) infection and EBV-associated hematological diseases with gp350/CD89-targeted bispecific antibody

Acute and persistent infection of Epstein-Barr virus (EBV) is associated with several life-threatening hematological disorders, including lymphoproliferative disorders (LPD), hemophagocytic lymphohistiocytosis (HLH), and chronic active Epstein-Barr virus infection (CAEBV). Currently, there are no efficacious virus-targeted therapies for EBV-driven hematological diseases. To explore the potential of phagocytosis-based immunotherapy, we created a bispecific antibody by targeting the viral envelope protein gp350 with a novel EBV-neutralizing antibody (named R1) that was paired with a monoclonal antibody against CD89 for redirecting macrophages and neutrophils. In vitro study showed that the bispecific antibody enabled efficient phagocytosis of EBV and killing of gp350 + lymphoma cells in the presence of PBMC. In vivo studies in NSG mice inoculated with EBV showed that bispecific antibody dramatically reduced the viral load in blood, solid organs and tissues. Treatment of mice implanted with EBV-harboring Raji lymphoma cells efficiently prevented tumor formation and massive metastasis to solid organs. Treatment of mice implanted with whole blood from EBV-HLH patients was effective in reducing viral levels in blood and solid organ. The gp350/CD89 bispecific antibody was highly effective in clearing EBV and immunotherapy of EBV-driven hematological diseases such as LPD and EBV-HLH.

Abstract IA012: Mammary epithelial architecture modulates field cancerization

Abstract The architecture of the human breast undergoes extensive remodeling throughout puberty, the menstrual cycle, pregnancy and involution with a risk of accumulating genetic alterations and tissue changes. Cells that have acquired mutations in driver genes are found to be abundant in tissues of healthy individuals, but they rarely develop into tumors. Yet, the underlying protection mechanisms that prevent tumor formation are largely unknown. Here, we utilized tissue clearing and imaged 15 normal human breasts and DCIS resections with cellular resolution to unravel the 3D architecture of thousands of mammary ducts. Surprisingly, our analysis revealed that disease-free breasts carry a high burden of microscopic tissue lesions. Distinct trees from the same breast differ in the kind and frequency of lesions, which coevolve scattered along the ductal routes. Interestingly, combining tissue clearing of FFPE archival material with spatial CNV-seq reveals that in half of the cases disease-free ductal segments neighboring DCIS lesions contain molecular alterations. To resolve the mechanisms that restrain and permit mutation spread, we compare the clonal fate behavior of cells that acquire oncogenic mutations with those bearing neutral mutations in the mouse mammary gland epithelium. From the quantitative analysis of the clonal dynamics, we find that local tissue remodeling during the estrous cycle leads to the stochastic elimination of the majority of mutant clones, while massively accelerating the expansion of a minority of clones that, by chance, survive. Thereby, oncogenic mutations can be spread along entire ducts before signs of transformation become visible. Together this data provides quantitative understanding of the earliest stages of epithelial evolution preceding disease. Citation Format: Hendrik Messal, Colinda Scheele, Esther Lips, Catrin Lutz, Stefan Hutten, Petra Kristel, Carmen Vlahu, Jos Jonkers, Jelle Wesseling, Jacco van Rheenen. Mammary epithelial architecture modulates field cancerization [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr IA012.

Inactivation of Autophagy in Keratinocytes Reduces Tumor Growth in Mouse Models of Epithelial Skin Cancer

Autophagy is a ubiquitous degradation mechanism, which plays a critical role in cellular homeostasis. To test whether autophagy suppresses or supports the growth of tumors in the epidermis of the skin, we inactivated the essential autophagy gene Atg7 specifically in the epidermal keratinocytes of mice (Atg7∆ep) and subjected such mutant mice and fully autophagy-competent mice to tumorigenesis. The lack of epithelial Atg7 did not prevent tumor formation in response to 7, 12-dimethylbenz(a)anthracene (DMBA) as the initiator and 12-O tetradecanoylphorbol-13-acetate (TPA) as the promoter of tumor growth. However, the number of tumors per mouse was reduced in mice with epithelial Atg7 deficiency. In the K5-SOS&nbsp;EGFRwa2/wa2 mouse model, epithelial tumors were initiated by Son of sevenless (SOS) in response to wounding. Within 12 weeks after tumor initiation, 60% of the autophagy-competent K5-SOS&nbsp;EGFRwa2/wa2 mice had tumors of 1 cm diameter and had to be sacrificed, whereas none of the Atg7∆ep&nbsp;K5-SOS&nbsp;EGFRwa2/wa2 mice formed tumors of this size. In summary, the deletion of Atg7 reduced the growth of epithelial tumors in these two mouse models of skin cancer. Thus, our data show that the inhibition of autophagy limits the growth of epithelial skin tumors.

RF07 | PSUN384 Hepatocyte Deletion of IGF2 Prevents DNA Damage and Tumor Formation in Hepatocellular Carcinoma

Abstract Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide with a 18% 5-year survival rate. Mice lacking the splicing factor SRSF3 in hepatocytes (SKO mice) overexpress IGF2 and are predisposed to developing HCC with age. Loss of Igf2 in the SKO background prevented hepatic fibrosis and inflammation, and completely prevented tumor formation. This was associated with decreased proliferation, apoptosis and DNA damage, and restored DNA repair enzyme expression. Mechanistically, IGF2 treatment of HepG2 cells in vitro caused DNA damage and decreased DNA repair enzyme expression, and tumors from the SKO mice show mutational signatures consistent with double strand break and defective mismatch repair. Looking at human data, HCC patients with high IGF2 mRNA expression had worse survival compared to patients with normal IGF2 expression. The patients also showed a switch in IGF2 promoter usage that correlated with increased expression. HCC patients having high SRSF3 mRNA expression also showed poor survival as did patients with alterations in known SRSF3-dependent splicing events. The level of SRSF3 protein was decreased 6-fold in human HCC tissues compared with normal liver tissues. The results indicate that IGF2 overexpression in conjunction with reduced SRSF3 splicing activity could be a major cause of DNA damage and driver of liver cancer. Presentation: Saturday, June 11, 2022 1:18 p.m. - 1:23 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Assessing the Effects of Inherited Mutations on PALB2 Structure and Function

The proper functioning of the protein PALB2 is vital to preventing tumor formation within breast tissues in individuals. Upon the detection of DNA damage, PALB2 and BRCA1 bind to each other along with BRCA2 to form a DNA repair complex. This complex then repairs DNA double-strand breaks in order to prevent the accumulation of DNA damage that leads to breast cancer. While both BRCA1 and BRCA2 have been extensively studied, a lot of information about the structure and function of PALB2 remains unknown. It is thought that BRCA1 and PALB2 bind via PALB2's coiled-coil domain; however, how variants of unknown significance (VUS) affect this binding interaction is largely unknown. Further, while some of these VUS have been studied in vivo, cheaper and easier in vitro methods to measure their effect on binding affinity have yet to be formulated. Thus, we hypothesized that isothermal titration calorimetry (ITC) could be used as an in vitro testing method for assessing the effects of VUS within the coiled-coil domain of PALB2 on the binding event between PALB2 and BRCA1. Further, we hypothesized that a decrease in binding between the two proteins as measured by ITC would correlate with a decrease in DNA repair as measured in vivo. We tested the efficacy of this method by creating seven mutations within the coiled-coil domain of PALB2 and measuring the binding event of PALB2 to BRCA1 via ITC. Our results strongly suggest that the binding event is enthalpic in nature and can be adequately measured using ITC as evidenced by the correlation between our in vitro data and previous in vivo data.

Hepatocyte Deletion of IGF2 Prevents DNA Damage and Tumor Formation in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Serine‐arginine rich splicing factor 3 (SRSF3) plays a critical role in hepatocyte function and its loss in mice promotes chronic liver damage and leads to HCC. Hepatocyte‐specific SRSF3 knockout mice (SKO mice) also overexpress insulin‐like growth factor 2 (IGF2). In the present study, double deletion of Igf2 and Srsf3 (DKO mice) prevents hepatic fibrosis and inflammation, and completely prevents tumor formation, and is associated with decreased proliferation, apoptosis and DNA damage, and restored DNA repair enzyme expression. This is confirmed in vitro, where IGF2 treatment of HepG2 hepatoma cells decreases DNA repair enzyme expression and causes DNA damage. Tumors from the SKO mice also show mutational signatures consistent with homologous recombination and mismatch repair defects. Analysis of frozen human samples shows that SRSF3 protein is decreased sixfold in HCC compared to normal liver tissue but SRSF3 mRNA is increased. Looking at public TCGA data, HCC patients having high SRSF3 mRNA expression show poor survival, as do patients with alterations in known SRSF3‐dependent splicing events. The results indicate that IGF2 overexpression in conjunction with reduced SRSF3 splicing activity could be a major cause of DNA damage and driver of liver cancer.