Tumor Burden In Mice Research Articles

Evidence for 2-Methoxyestradiol-Mediated Inhibition of Receptor Tyrosine Kinase RON in the Management of Prostate Cancer

2-Methoxyestradiol (2-ME2) possesses anti-tumorigenic activities in multiple tumor models with acceptable tolerability profile in humans. Incomplete understanding of the mechanism has hindered its development as an anti-tumorigenic compound. We have identified for the first-time macrophage stimulatory protein 1 receptor (MST1R) as a potential target of 2-ME2 in prostate cancer cells. Human tissue validation studies show that MST1R (a.k.a RON) protein levels are significantly elevated in prostate cancer tissues compared to adjacent normal/benign glands. Serum levels of macrophage stimulatory protein (MSP), a ligand for RON, is not only associated with the risk of disease recurrence, but also significantly elevated in samples from African American patients. 2-ME2 treatment inhibited mechanical properties such as adhesion and elasticity that are associated with epithelial mesenchymal transition by downregulating mRNA expression and protein levels of MST1R in prostate cancer cell lines. Intervention with 2-ME2 significantly reduced tumor burden in mice. Notably, global metabolomic profiling studies identified significantly higher circulating levels of bile acids in castrated animals that were decreased with 2-ME2 intervention. In summary, findings presented in this manuscript identified MSP as a potential marker for predicting biochemical recurrence and suggest repurposing 2-ME2 to target RON signaling may be a potential therapeutic modality for prostate cancer.

Type 2 Innate Lymphoid Cells Protect against Colorectal Cancer Progression and Predict Improved Patient Survival.

Simple SummaryColorectal cancer is the second leading cause of cancer-related death worldwide. The immune system plays a key role in controlling tumour onset and development. However, our immune system is complex and includes many different cell types which differently impact colorectal cancer outcomes. In this study, we investigated the function of the specialised type 2 innate lymphoid cells (ILC2) in colorectal cancer development and progression. We found that ILC2 infiltrate colorectal tumours and their presence was associated with reduced tumour burden in mice. In patients, this infiltration correlated with improved overall survival. Collectively, our work reveals that ILC2s are beneficial to colorectal cancer outcomes.Chronic inflammation of the gastrointestinal (GI) tract contributes to colorectal cancer (CRC) progression. While the role of adaptive T cells in CRC is now well established, the role of innate immune cells, specifically innate lymphoid cells (ILCs), is not well understood. To define the role of ILCs in CRC we employed complementary heterotopic and chemically-induced CRC mouse models. We discovered that ILCs were abundant in CRC tumours and contributed to anti-tumour immunity. We focused on ILC2 and showed that ILC2-deficient mice developed a higher tumour burden compared with littermate wild-type controls. We generated an ILC2 gene signature and using machine learning models revealed that CRC patients with a high intratumor ILC2 gene signature had a favourable clinical prognosis. Collectively, our results highlight a critical role for ILC2 in CRC, suggesting a potential new avenue to improve clinical outcomes through ILC2-agonist based therapeutic approaches.

Bifidobacterium bifidum strains synergize with immune checkpoint inhibitors to reduce tumour burden in mice.

The gut microbiome can influence the development of tumours and the efficacy of cancer therapeutics1-5; however, the multi-omics characteristics of antitumour bacterial strains have not been fully elucidated. In this study, we integrated metagenomics, genomics and transcriptomics of bacteria, and analyses of mouse intestinal transcriptome and serum metabolome data to reveal an additional mechanism by which bacteria determine the efficacy of cancer therapeutics. In gut microbiome analyses of 96 samples from patients with non-small-cell lung cancer, Bifidobacterium bifidum was abundant in patients responsive to therapy. However, when we treated syngeneic mouse tumours with commercial strains of B. bifidum to establish relevance for potential therapeutic uses, only specific B. bifidum strains reduced tumour burden synergistically with PD-1 blockade or oxaliplatin treatment by eliciting an antitumour host immune response. In mice, these strains induced tuning of the immunological background by potentiating the production of interferon-γ, probably through the enhanced biosynthesis of immune-stimulating molecules and metabolites.

Synergistic killing effects of PD-L1-CAR T cells and colorectal cancer stem cell-dendritic cell vaccine-sensitized T cells in ALDH1-positive colorectal cancer stem cells.

Cancer stem cells (CSCs) are characterized by self-renewal and unlimited proliferation, providing a basis for tumor occurrence, metastasis, and recurrence. Because CSCs are highly resistant to conventional chemotherapy and radiotherapy, various immunotherapies, particularly chimeric antigen receptor T cell (CAR-T) therapy and dendritic cell (DC)-based vaccine therapy, are currently being developed. Accordingly, in this study, we evaluated programmed cell death ligand-1 (PD-L1) expression in colorectal CSCs (CCSCs) and non-CCSCs and designed a combination immunotherapy synchronously utilizing PD-L1-CAR-T cells together with CCSC-DC vaccine-sensitized T cells for the treatment of colorectal cancer. PD-L1-CAR-T cells specifically recognized the PD-L1 molecule on CCSCs by binding to the extracellular domain of programmed cell death-1. The CCSC-DC vaccine was prepared using CCSC lysates. We found that aldehyde dehydrogenase 1 (ALDH1)-positive CCSCs were abundant in samples from patient tumor tissues and cancer cell lines. Moreover, PD-L1 was highly expressed in ALDH1-positive CCSCs compared with that in non-CCSCs. Monotherapy with PD-L1-CAR-T cells or CCSC-DC vaccine only elicited moderate tumor remission both in vitro and in vivo. However, combination therapy markedly killed cancer cells and relieved the tumor burden in mice. Our findings may provide a novel strategy for the clinical treatment of colorectal malignancy.

Folate mediated targeted delivery of cinnamaldehyde loaded and FITC functionalized magnetic nanoparticles in breast cancer:in vitro,in vivoand pharmacokinetic studies

FiCF NPs induced apoptosis in breast cancer cells, exhibited safety, reduced tumor burden in mice due to increased pharmacological efficacy.

IFNgamma-inducible CXCL10/CXCR3 axis alters the sensitivity of HEp-2 cells to ionizing radiation

Radiotherapy is a conventional approach for anti-cancer treatment, killing tumor cells through damaging cellular DNA. While increasing studies have demonstrated that tumors generated the tolerance to radiation and tumor immune system was found to be correlated to radiotherapy resistance. Therefore, it is critical to identify potential immune factors associated with the efficacy of radiotherapy. Here in this study, we evaluated the sensitivities of different tumor cells to radiation and determined HEp-2 cells as the radio-resistant tumor cells for further investigation. IFNgamma as a key regulator of host immune response showed the potential to sensitize tumors to ionizing radiation (IR). Besides, IFNgamma-induced CXC chemokine ligand 10 (CXCL10) was found to be necessary for effective IR-induced killing of cultured HEp-2 cells. Increased clonogenic survival was observed in CXCL10-depleted HEp-2 cells and CXCL10-KO cells. Additionally, the loss of CXCL10 in HEp-2 cells showed less progression of the G0/G1 phase to G2/M when exposed to IR (8 Gy). Local IR (20 Gy) to nude mice bearing HEp-2 tumors significantly reduced tumor burden, while fewer effects on tumor burden in mice carrying CXCL10-KO tumors were observed. We furtherly evaluated the possible roles the chemokine receptor CXCR3 plays in mediating the sensitivity of cultured HEp-2 cells to IR. Altered expression of CXCR3 in HEp-2 cells affected IR-induced killing of HEp-2 cells. Our data suggest the IFNgamma-activated CXCL10/CXCR3 axis may contribute to the effective radiation-induced killing of HEp-2 cells in vitro.

Targeting 14-3-3ζ Overcomes Resistance to Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors in Lung Adenocarcinoma via BMP2/Smad/ID1 Signaling.

Background: The 14-3-3ζ protein, which acts as a putative oncoprotein, has been found to promote the proliferation, metastasis, and chemoresistance of cancer cells in several cancers including lung adenocarcinoma (LUAD); however, its significance in epidermal growth factor receptor–tyrosine kinase inhibitor (EGFR-TKI) resistance remains unknown.Methods: The Cancer Genome Atlas (TCGA) database was used to determine 14-3-3ζ expression in pancancer and LUAD. 14-3-3ζ and ID1 expression was then examined in clinical LUAD samples by immunohistochemistry (IHC). Lentiviral transfection with 14-3-3ζ-specific small hairpin RNA (shRNA) was used to establish stable 14-3-3ζ knockdown gefitinib-resistant PC9 (PC9/GR) and H1975 cell lines. The effect of 14-3-3ζ knockdown on reversing EGFR-TKI resistance was determined in vitro by Cell Counting Kit-8 (CCK-8), wound healing, Transwell assays, and flow cytometry. A xenograft tumor model was established to evaluate the role of 14-3-3ζ in EGFR-TKI resistance. Microarray analysis results showed multiple pathways regulated by 14-3-3ζ-shRNA.Results: In the present study, we demonstrated that based on the TCGA, pancancer and LUAD 14-3-3ζ expression was elevated and predicted unfavorable prognosis. In addition, high 14-3-3ζ expression was associated with advanced T stage, TNM stage, presence of lymph node metastasis and, importantly, poor treatment response to EGFR-TKIs in LUAD patients with EGFR-activating mutations. 14-3-3ζ shRNA sensitized EGFR-TKI-resistant human LUAD cells to gefitinib and reversed epithelial-to-mesenchymal transition (EMT). After 14-3-3ζ depletion, bone morphogenetic protein (BMP) signaling activation was decreased in EGFR-TKI-resistant cells in microarray analysis, which was further validated by Western blot analysis. Furthermore, the expression of 14-3-3ζ positively correlates with ID1 expression in human EGFR-mutant LUAD patient samples. In vivo, there was a reduction in the tumor burden in mice treated with 14-3-3ζ shRNA and gefitinib compared to mice treated with gefitinib alone.Conclusion: Our work uncovers a hitherto unappreciated role of 14-3-3ζ in EGFR-TKI resistance. This study might provide a potential therapeutic approach for treating LUAD patients harboring EGFR mutations.

A hemi-spleen injection model of liver metastasis for prostate cancer.

Liver metastasis is not uncommon in men with metastatic castration-resistant prostate cancer (mCRPC), estimated at ~20% to 60% of advanced late-stage patients. Liver and other visceral metastases are associated with worse overall survival. Recent evidence suggests the frequency of visceral metastases may be increasing for reasons that are unclear but may be related to selective pressures induced by modern therapies, including second-generation antiandrogen receptor signaling inhibitors such as enzalutamide and abiraterone. Consequently, robust models to study the pathobiology of prostate cancer liver metastases and their response to therapy are urgently needed. Hemi-spleen injection of human (LN95, PC3, VCaP, and MDA-PCa-2b) or syngeneic (Myc-CaP) prostate cancer cells (1 × 106 ) was performed to seed liver metastases via the splenic vessels. Plasma levels of prostate-specific antigen (PSA) were monitored longitudinally in human androgen receptor-positive (AR+) models. Immunohistochemical staining of AR and HoxB13 was performed to document the prostatic origin of hepatic lesions. LN95, PC3, and Myc-CaP produced distinct liver micrometastases that progressed to macrometastases by ~2 to 4 weeks postinoculation, while inoculation of MDA-PCa-2b and VCaP only produced occasional micrometastases and seeding of individual cells adjacent to blood vessels, respectively, at the time points analyzed. All lesions are characterized by positive staining for nuclear AR and/or the prostate-specific differentiation marker HoxB13 depending on the model. Circulating PSA levels are strongly correlated with overall tumor burden in mice seeded with LN95. Histologic micrometastases and low levels of circulating PSA are detected in mice seeded with MDA-PCa-2b at ~60 days postinoculation, but no circulating PSA was detected in animals inoculated with VCaP up to ~75 days despite the presence of rare AR+ cells in the liver. The studies reported herein establish intrasplenic injection as a robust model of mCRPC liver metastasis. In addition, circulating PSA was validated as a noninvasive biomarker to longitudinally monitor overall tumor burden when using PSA+ models. Therefore, this model can be used to interrogate the pathophysiology of prostate cancer liver metastases, the microenvironmental factors permissive to such growth, immunologic variables, and the response of hepatic lesions to therapy.

Adipocyte-Induced FABP4 Expression in Ovarian Cancer Cells Promotes Metastasis and Mediates Carboplatin Resistance.

Adipocytes are critical for ovarian cancer cells to home to the omentum, but the metabolic changes initiated by this interaction are unknown. To this end, we carried out unbiased mass spectrometry-based metabolomic and proteomic profiling of cancer cells cocultured with primary human omental adipocytes. Cancer cells underwent significant proteo-metabolomic alteration(s), typified by changes in the lipidome with corresponding upregulation of lipid metabolism proteins. FABP4, a lipid chaperone protein, was identified as the critical regulator of lipid responses in ovarian cancer cells cocultured with adipocytes. Subsequently, knockdown of FABP4 resulted in increased 5-hydroxymethylcytosine levels in the DNA, downregulation of gene signatures associated with ovarian cancer metastasis, and reduced clonogenic cancer cell survival. In addition, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated knockout of FABP4 in high-grade serous ovarian cancer cells reduced metastatic tumor burden in mice. Consequently, a small-molecule inhibitor of FABP4 (BMS309403) not only significantly reduced tumor burden in a syngeneic orthotopic mouse model but also increased the sensitivity of cancer cells toward carboplatin both in vitro and in vivo. Taken together, these results show that targeting FABP4 in ovarian cancer cells can inhibit their ability to adapt and colonize lipid-rich tumor microenvironments, providing an opportunity for specific metabolic targeting of ovarian cancer metastasis. SIGNIFICANCE: Ovarian cancer metastatic progression can be restricted by targeting a critical regulator of lipid responses, FABP4.

JZL184, A Monoacylglycerol Lipase Inhibitor, Induces Bone Loss in a Multiple Myeloma Model of Immunocompetent Mice

Multiple myeloma (MM) patients develop osteolysis characterised by excessive osteoclastic bone destruction and lack of osteoblast bone formation. Pharmacological manipulation of monoacylglycerol lipase (MAGL), an enzyme responsible for the degradation of the endocannabinoid 2-arachidonoyl glycerol (2-AG), reduced skeletal tumour burden and osteolysis associated with osteosarcoma and advanced breast and prostate cancers. MM and hematopoietic, immune and bone marrow cells express high levels of type 2 cannabinoid receptor and osteoblasts secrete 2-AG. However, the effects of MAGL manipulation on MM have not been investigated. Here, we report that treatment of pre-osteoclasts with non-cytotoxic concentrations of JZL184, a verified MAGL inhibitor, enhanced MM- and RANKL-induced osteoclast formation and size in vitro. Exposure of osteoblasts to JZL184 in the presence of MM cell-derived factors reduced osteoblast growth but had no effect on the ability of these cells to mature or form bone nodules. In vivo, administration of JZL184 induced a modest, yet significant, bone loss at both trabecular and cortical compartments of long bones of immunocompetent mice inoculated with the syngeneic 5TGM1-GFP MM cells. Notably, JZL184 failed to inhibit the in vitro growth of a panel of mouse and human MM cell lines, or reduce tumour burden in mice. Thus, MAGL inhibitors such as JZL184 can exacerbate MM-induced bone loss.

Abstract PR1: Development of new immune therapy combinations for ovarian cancer using genetically defined organoid platform

Abstract High-grade serous ovarian cancer (HGSOC) is the most common, deadly subtype of ovarian epithelial cancer. HGSOC typically presents at an advanced stage, with widespread peritoneal metastasis. Surgical debulking and platinum/taxane-based chemotherapy can result in complete responses, but disease almost always recurs, eventually in drug-resistant form. The recent addition of Avastin and PARP inhibitors benefits a subset of patients, yet survival has improved marginally in the past 30 years. Responses to immune checkpoint inhibitors have been disappointing. Genomic studies (e.g., TCGA) show that HGSOC is a complex, diverse disease with widespread copy number abnormalities affecting multiple pathways. Nevertheless, most studies aimed at developing new therapies treat HGSOC as a single entity. The absence of genetically relevant, immune-competent HGSOC models poses a major barrier to developing new therapies. By exploiting a mouse fallopian tube organoid system that we developed, along with lentiviral gene transduction and/or CRISPR/Cas9 technology, we generated multiple new HGSOC models containing combinations of mutations seen in the human disease, including homologous recombination (HR)-proficient (Tp53-/-;Ccne1OE;Akt2OE and Tp53-/-;Ccne1OE;KrasOE) -deficient (Tp53-/-;Brca1-/-;Pten-/-and Tp53-/-; Brca1-/-;MycOE and unclassified (Tp53-/-;Pten-/-;Nf1-/-) models. These cells differ in proliferation, differentiation, and polarity/organoid structure in vitro, as well as tumorigenicity and metastatic spread upon orthotopic injection into syngeneic mice. Organoids with different mutations show differential sensitivity to current HGSOC drugs and evoke distinctly different immune microenvironments in vivo. Brca1-/- and CcneOE tumors show significant T-cell infiltration/Treg cells; the latter also have increased myeloid-derived suppressor cells (MDSCs). Pten-/- tumors have lower T-cell infiltration but high levels of MDSCs and macrophages. To test the utility of this platform for new therapeutic development, we focused on CcneOE tumors, because human CCNE-amplified HGSOC responds poorly to current therapy. CcneOE organoids were sensitive to gemcitabine (GEM), probably due to CCNE-evoked replication stress. GEM treatment reduced pro-MDSC cytokines in organoid-conditioned media, and decreased tumor burden in mice. Consistent with these in vitro data, residual tumors had decreased MDSCs (CD11b+Ly6CloLy6Ghi), but persistent Treg (CD24+CD25+FoxP3+) and “exhausted” CD8 T cells (CD8+PD1+). Intriguingly, a combination regimen aimed at attacking tumor cells (gemcitabine) and the residual microenvironment (CTLA4 and PDL1 antibodies) produced complete responses in mice with widespread metastatic disease. Our results argue that genotype-informed combination therapies will be necessary for better outcomes for this devastating disease. This abstract is also being presented as Poster A42. Citation Format: Shuang Zhang, Sonia Iyer, Hao Ran, Wei Wei, Robert A. Weinberg, Benjamin G. Neel. Development of new immune therapy combinations for ovarian cancer using genetically defined organoid platform [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr PR1.

FKBP10 Regulates Protein Translation to Sustain Lung Cancer Growth

Cancer therapy is limited, in part, by lack of specificity. Thus, identifying molecules that are selectively expressed by, and relevant for, cancer cells is of paramount medical importance. Here, we show that peptidyl-prolyl-cis-trans-isomerase (PPIase) FK506-binding protein 10 (FKBP10)-positive cells are present in cancer lesions but absent in the healthy parenchyma of human lung. FKBP10 expression negatively correlates with survival of lung cancer patients, and its downregulation causes a dramatic diminution of lung tumor burden in mice. Mechanistically, our results from gain- and loss-of-function assays show that FKBP10 boosts cancer growth and stemness via its PPIase activity. Also, FKBP10 interacts with ribosomes, and its downregulation leads to reduction of translation elongation at the beginning of open reading frames (ORFs), particularly upon insertion of proline residues. Thus, our data unveil FKBP10 as a cancer-selective molecule with a key role in translational reprogramming, stem-like traits, and growth of lung cancer.

Abstract P1-12-01: Prevention of BRCA1-associated mammary cancers by selective progesterone receptor modulators (SPRM) in mice

Abstract Background: It was previously demonstrated that mifepristone (MFP), an SPRM with anti-glucocorticoid receptor (GR) activity, blocks mammary tumor formation in a BRCA1 deficient mouse model (deletion of exon 11). We sought to compare tumor prevention efficacy of MFP with other SPRMs having less GR binding, using a different BRCA1 deficient mouse model (deletion of exon 22-24). We also compared the efficacy of multiple SPRMs on suppression of cell proliferation (Ki67) in benign mammary glands, to evaluate early effects in unaffected glands. Methods: At 4-months-old, the female mice (Blg_Cre Brca1 f22-24/f22-24; p53+/-) were randomized to four treatment groups (n= 14-21 per group): no treatment control, MFP, telapristone acetate (TPA), and ulipristal acetate (UPA) and treated subcutaneously with a pellet containing 30mg of drug. Pellets were replaced every 90 days. The mice were monitored for tumor formation and growth until 15- months of age. When the tumor burden of mice became 1 cm2 or at age 15-months, the mice were euthanized to collect tumors and mammary glands for histopathologic evaluation. Ki67 and PR expression in mammary tissues were measured by immunohistochemistry and images were analyzed by ImageJ/Fiji software. Total RNA extracted from benign and tumor tissue was sequenced to discover SPRM-modulated genes and molecular pathways, in comparison with no-treatment controls. Results: The occurrence of invasive cancers (adenocarcinoma) was similar in MFP-treated and control mice in a time-adjusted analysis (72% of MFP group vs. 74% of controls, p= 0.55). TPA-treated mice displayed a non-significant lower tumor incidence (56 %) compared to control group (p=0.11). However, UPA-treated mice showed significantly less tumor incidence (43%) than controls (p=0.04). These results were supported by suppression of %Ki67 labeling Index (LI) of benign glands. Median %Ki67 LI of benign glands of tumor-affected mice were: controls (18%) = MFP (18%) > TPA (16%) > UPA groups (10 %). Median Ki67% LI of benign glands of tumor-unaffected mice were: MFP (16%) > TPA (13%) > controls (12%) > UPA group (6%). Reduction of cell proliferation by UPA was significant in benign glands of tumor-affected and un-affected mice compared to controls (p=0.007 for both) while reductions in the MFP and TPA groups were not significant. All of the invasive cancers were PR negative; PR is expressed in abnormal and benign lesions adjacent to invasive adenocarcinoma. To determine whether PR expression is related to tumor progression, we are evaluating PR expression in both benign and abnormal glands from all mice. Furthermore, RNA sequencing is underway to determine genes differentially expressed by UPA and MFP treatment compared to no treatment. Conclusions: Our mouse model (BRCA1 Δexon22-24) displayed a spectrum of sensitivity to the three SPRMs we have tested: UPA>TPA>MFP, UPA showing higher potency than TPA. The reason for the lack of efficacy of MFP may relate to its strong affinity to GR, resulting in mixed effects on both PR and GR as a reason for its failure in this model. Understanding mechanisms underlying the different anti-tumor efficacy of UPA and MFP in our model will provide important insights that will aid the clinical development of PR-directed strategies for breast cancer prevention in both BRCA1 mutation carriers. Citation Format: Oukseub Lee, Limin Sun, Maarten C. Bosland, Minhua Wang, Ali Shidfar, Omid Hosseini, Irene Helenowski, Susan E. Clare, Seema A. Khan. Prevention of BRCA1-associated mammary cancers by selective progesterone receptor modulators (SPRM) in mice [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-12-01.

Engineering Circulating Tumor Cells as Novel Cancer Theranostics.

New ways to target and treat metastatic disease are urgently needed. Tumor “self-homing” describes the recruitment of circulating tumor cells (CTCs) back to a previously excised primary tumor location, contributing to tumor recurrence, as well as their migration to established metastatic lesions. Recently, self-homing CTCs have been exploited as delivery vehicles for anti-cancer therapeutics in preclinical primary tumor models. However, the ability of CTCs to self-home and treat metastatic disease is largely unknown.Methods: Here, we used bioluminescence imaging (BLI) to explore whether systemically administered CTCs home to metastatic lesions and if CTCs armed with both a reporter gene and a cytotoxic prodrug gene therapy can be used to visualize and treat metastatic disease.Results: BLI performed over time revealed a remarkable ability of CTCs to home to and treat tumors throughout the body. Excitingly, metastatic tumor burden in mice that received therapeutic CTCs was lower compared to mice receiving control CTCs.Conclusion: This study demonstrates the noteworthy ability of experimental CTCs to home to disseminated breast cancer lesions. Moreover, by incorporating a prodrug gene therapy system into our self-homing CTCs, we show exciting progress towards effective and targeted delivery of gene-based therapeutics to treat both primary and metastatic lesions.

Abstract PR05: Development of new immune therapy combinations for ovarian cancer using genetically defined organoid platform

Abstract High-grade serous ovarian cancer (HGSOC) is the most common and deadly subtype of ovarian epithelial cancer. HGSOC typically presents at an advanced stage, with widespread peritoneal metastasis. Although surgical debulking and platinum/taxane-based chemotherapy can result in complete responses, disease almost always recurs, eventually in drug-resistant forms. Although the recent addition of Avastin and PARP inhibitors benefits a subset of patients, survival has improved only marginally in the past 30 years. Responses to immune checkpoint inhibitors have also been disappointing. Genomic studies (e.g., by TCGA) have shown that HGSOC is a complex, diverse disease with widespread copy number abnormalities affecting multiple pathways. Nevertheless, most studies aimed at developing new therapies treat HGSOC as a single entity. The absence of genomically relevant, immune-competent HGSOC models poses a major barrier to developing new therapies. Capitalizing on a mouse fallopian tube organoid system that we developed, along with lentiviral gene transduction and/or CRISPR/Cas9 technology, we generated multiple new HGSOC models containing combinations of mutations seen in human HGSOC, including homologous recombination (HR)-proficient (Tp53-/-;Ccne1OE;Akt2OE and Tp53-/-;Ccne1OE;KrasOE) and -deficient (Tp53-/-;Brca1-/-;Pten-/-and Tp53-/-;Brca1-/-;MycOE), and poorly characterized (Tp53-/-;Pten-/-;Nf1-/-) models. These models differ in proliferation, differentiation, and polarity/organoid structure in vitro, as well as tumorigenic capacity and behavior upon orthotopic injection into syngeneic mice. Organoids bearing different mutational spectra show differential sensitivity to conventional HGSOC chemotherapies and PARP inhibitors, and evoke distinctly different immune microenvironment in vivo. The immune microenvironment induced by Brca1-/- and CcneOE tumors show significant T cell infiltration/Treg cells, the latter also has increased myeloid-derived suppressor cells (MDSCs), and Pten-/-lines show lower T cell infiltration but higher levels of MDSCs and macrophages. To test the utility of this platform for new therapeutic development, we focused on CcneOE tumors, because human CCNE-amplified HGSOC responds poorly to current therapy. CcneOE organoids were sensitive to gemcitabine (GEM), probably due to CCNE-evoked replication stress. GEM treatment reduced pro-MDSC cytokines in organoid conditioned media, and decreased tumor burden in mice. Consistent with the in vitro data, residual tumors had decreased MDSCs (CD11b+Ly6CloLy6Ghi), but persistent Treg (CD24+CD25+FoxP3+) and “exhausted” CD8 T cells (CD8+PD1+) in their microenvironment. Intriguingly, a combination regimen aimed at attacking tumor cells (gemcitabine) and the residual microenvironment (CTLA4 and PDL1 antibodies) produced complete responses in mice with widepresad metastatic disease, Our results argue that genotype-informed combination therapies will be necessary for better outcomes for this devastating disease. Citation Format: Shuang Zhang, Sonia Iyer, Hao Ran, Wei wei, Robert A Weinberg, Benjamin G Neel. Development of new immune therapy combinations for ovarian cancer using genetically defined organoid platform [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PR05. doi:10.1158/1535-7163.TARG-19-PR05

First-in-Human Trial of Bcma-CD19 Compound CAR with Remarkable Donor-Specific Antibody Reduction

Ma: iCAR Bio Therapeutics Ltd: Employment. Wada:iCell Gene Therapeutics LLC: Employment. Ma:iCell Gene Therapeutics LLC: Consultancy, Equity Ownership, Research Funding; iCAR Bio Therapeutics Ltd: Consultancy, Equity Ownership, Research Funding.

Engineering High Affinity and Cleavage Resistant CD16 to Augment ADCC of Placental Hematopoietic Stem Cells-Derived Natural Killer Cells

Background: Celularity, Inc. is developing human placental hematopoietic stem cells-derived, cryopreserved, off-the-shelf, ex-vivo expanded and allogenic natural killer (PNK) cells for various hematological malignancies and solid tumors. NK cells play a central role in antibody dependent cell mediated cytotoxicity (ADCC) through Fc receptor CD16 in monoclonal antibody mediated anti-tumor therapies. Two allelic forms of CD16 have been identified. The 158Val/Val form has shown to have higher IgG binding affinity compared to the 158Phe/Phe form.1 The high IgG binding allele are found in about 10-20% of the normal population.2,3 In addition, activation of NK cells induces CD16 shedding by matrix metalloprotease ADAM17 at 197Ser, thus limiting ADCC responses. A single mutation (Ser197Pro) prevents CD16 shedding and increases ADCC activity in NK cells.4 Since the antibody binding affinity and CD16 expression of PNK could vary with different donors, we hypothesize that expressing a high affinity (158Val) and proteinase cleavage resistant (197Pro) CD16 variant (CD16VP) augments anti-tumor ADCC activity. Methods: Lentivirus expressing CD16VP was used to transduce human placental CD34+ cells. After transduction, the cells were cultured in the presence of cytokines including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2, for 35 days to generate PNK-CD16VP cells. Non-transduced PNK cells (NT) served as a control. Expression of CD16VP was evaluated by activating cells with PMA/ionomycin to induce CD16 cleavage (CD16 shedding assay) followed by immunostaining with CD16 antibody and analyzed using flowcytometry. ADCC of PNK-CD16VP cells was assessed against Daratumumab (anti-CD38) or Rituximab (anti-CD20) opsonized lymphoma cell lines at various effector to target (E:T) ratios. IgG was used as ADCC control. In vivo anti-tumor activity was assessed in a Daudi disseminated Xenograft model in NSG mice. Luciferase-expressing Daudi cells (3x106) were intravenously (IV) administered at day 0, followed by PNK-CD16VP cells (10x106) IV at day 1 and day 3, and Daratumumab at day 3. Tumor burden in mice was monitored by Bioluminescence Imaging (BLI). Statistical differences between the groups were calculated using paired t-test using Prism. Results: Lentiviral transduction of CD16VP achieved high expression efficiency in multiple placental CD34+ donors. These cells expanded [7095 ± 2998 folds (n=8)] and differentiated into PNK cells (>90% CD56+CD3-) at day 35. PNK-CD16VP expressed 64.6 ± 10.3% (n=8) of CD16, while the NT expressed 12.1 ± 3.3% (n=8) CD16. PMA/ionomycin induced >89% shedding of CD16 in NT cells, while significantly less (<11%) CD16 shedding was observed in PNK-CD16VP cells. These results indicated that CD16VP was expressed and maintained throughout the culture process. In vitro ADCC assay demonstrated improved anti-tumor activity of PNK-CD16VP cells over NT cells against Daratumumab or Rituximab opsonized lymphoma cell lines. At 10:1 E:T ratio PNK-CD16VP cells elicited higher cytotoxicity compared to NT: 47 ± 13% against Daratumumab opsonized Daudi cells versus 25 ± 5% (n=5; p<0.05); 30 ± 13% against Daratumumab opsonized HS-Sultan cells versus 21 ± 14% (n=3; p<0.05); 30 ± 7% against Daratumumab opsonized Sudhl6 cells versus 16 ± 10% (n=3; p<0.05). Improved ADCC activities in PNK-CD16VP were also observed in other cell lines including Raji and Sudhl4 with Daratumumab and Rituximab antibodies. PNK-CD16VP were used to test anti-tumor ADCC in vivo using a disseminated Daudi Xenograft model. The preliminary data demonstrated that PNK-CD16VP combined with Daratumumab reduced BLI signal (>50%) compared to vehicle or Daratumumab alone at day 10 after treatment. This observation suggested that PNK-CD16VP demonstrated in vivo ADCC anti-tumor activity. Conclusions: In this study, we genetically modified PNK to express high affinity and cleavage resistant CD16 variant using lentivirus. The PNK-CD16VP cells demonstrated enhanced ADCC function against lymphoma cell lines in vitro and in vivo. Further development of PNK-CD16VP for immune-oncology therapeutics is warranted.

Targeting bone marrow adipose tissue and the FABP family increases efficacy of dexamethasone in MM

It is well known that multiple myeloma (MM) cells have a dependency on the bone marrow (BM) microenvironment for survival and proliferation. Targeting stromal cells of the BM, rather than just the tumor cells themselves, can be useful for reducing tumor burden in mice and humans. Our lab has recently explored targeting the bone marrow adipocyte (BMA) due to correlations between obesity to increased risk of MM and increased BM adiposity. To test the effects of BMAs on MM1S in vivo, 5-week old SCID-beige female mice were treated with saline or an anti-sclerostin antibody (Scl-Ab, 100 mg/kg once per week IP) for 5 weeks (a treatment shown to increase bone volume and decrease BMAs). Then, 5x10ˆ6 human GFP+luc+MM1S cells were injected i.v. followed by 5 additional weeks of Scl-Ab or saline treatments. Mice were treated with or without dexamethasone (dex) (9mg/kg IP) during weeks 4-6 after MM1S injection. Mice were assessed with biweekly whole body bioluminescent imaging (BLI) and survival. Dex treatments led to a significantly lower total flux BLI signal (p<0.05 vs MM1S alone), and Scl-Ab+dex combination treatments had the least total flux BLI signal (p<0.01 vs MM1S alone) at day 33. Interestingly, Scl-Ab+dex combination treatments increased mouse survival significantly versus dex alone, Scl-Ab treatment alone, or no treatment. Thus, our in vivo data suggested that decreasing bone marrow adiposity enhanced anti-myeloma effects of dex. Next, to specifically examine the effects of BMAs on myeloma cells, we generated human BMAs from BM mesenchymal stem cells with adipogenic media. MM1S cells were cultured in conditioned media from BMAs (BMA-CM); BMA-CM from many, but not all donors, supported MM1S proliferation, migration, or drug resistance. Specifically, some BMAs protected MM1S cells against dex-induced apoptosis, assessed by Annexin V/DAPI flow cytometry and bioluminescence cell counting. Gene expression and protein data from MM1S cells treated with BMA-CM revealed a significant increase in fatty acid binding protein 4 (FABP4), a fatty acid shuttling molecule with broad signaling effects (pcr: p<.01, ELISA: p<0.0001). While unknown in MM, the FABP family has demonstrated pro-tumorigenic effects in other cancers. We used inhibitors of FABP4 (iFABP4), and its compensatory FABP5 family member, to study their effects on basal and dex-induced apoptosis, cell number, cell cycle and proliferation in MM1S cells. After 72 hours, FABP4i and FABP5i alone, and in combination, significantly decreased cell number and proliferation. In combination, FABP4i/FABP5i improved efficacy of dex. Decreases in MM1S cell number and proliferation, (assessed by BLI, Ki67 and cell cycle flow cytometry analyses) from FABP4i, FABP5i, or the FABP4i/FABP5i combination were attributed to increased sub G1/G0 population. Overall, targeting BMAT and FABPs may be novel approaches to inhibiting progression and decreasing the drug resistance properties of MM cells in the BM.

Abstract 4962: Repurposing bazedoxifene to suppress gastrointestinal cancer growth

Abstract The interleukin (IL)6 family of inflammatory cytokines, characterized by the shared use of the gp130 receptor, supports the progression of solid cancers. Specifically, gp130 signaling becomes rate limiting for the growth of gastrointestinal tumors arising from oncogenic driver mutations in Apc, but remains dispensable for the homeostatic renewal of the gastrointestinal mucosa. In the present study we investigate the effects of the selective estrogen receptor modulator bazedoxifene, clinically approved for the treatment of osteoporosis, as a putative small molecule inhibitor of gp130 receptor oligomerization and activation. We assessed the effects of bazedoxifene on IL6 and IL11-dependent STAT3 activation and cell proliferation in gastric and colon cancer cell lines. In patient-derived colon cancer organoid cultures, we assessed bazedoxifene effects on IL11-dependent growth. The gp130Y757F, Lgr5CreERT2- or Cdx2CreERT2-driven Apc flox mouse models of gastrointestinal cancer, were used to examine the effects of bazedoxifene on tumour growth. In silico modeling suggests that the inhibitory activity of bazedoxifene is due to its ability to mimic the interaction between the Site III interface of gp130 receptor and the tryptophan-157 in IL6, or tryptophan-168 in IL11 cytokine. We show that bazedoxifene interferes with IL6 and IL11-dependent proliferation of BAF/03 cells. Bazedoxifene suppresses gastric tumor growth in gp130Y757F mice irrespective of their gender, in which tumors arise through excessive IL11-dependent STAT3 signaling. Strikingly, in sporadic colon cancer models based on aberrant activation of the β-catenin/canonical WNT pathway, bazedoxifene treatment also reduces tumor burden in mice following conditional ablation of the Apc suppressor gene using the Lgr5CreERT2- or Cdx2CreERT2-driver alleles. Consistent with our observation that nuclear accumulation of β-catenin remains unaffected in colonic tumors of bazedoxifene-treated mice, bazedoxifenetreatment of human SW480 colon cancer cells harboring mutant APC did not affect canonical WNT signaling, but suppressed IL11-dependent STAT3 signaling. Our findings provide compelling proof-of-concept for the repurposing of bazedoxifene for the treatment of gastrointestinal cancers that arise from bona fide oncogenic driver mutations. Note: This abstract was not presented at the meeting. Citation Format: Pathum Thilakasiri, Tracy Nero, Michael W. Parker, Matthias Ernst, Ashwini L. Chand. Repurposing bazedoxifene to suppress gastrointestinal cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4962.

PF575 OVEREXPRESSION DNP73 PROMOTES THE PROLIFERATION AND DRUG RESISTANCE OF MULTIPLE MYELOMA

Background:Genomic instability and chromosomal abnormalities induced by DNA damage are important biological characteristics of multiple myeloma (MM). DNp73 is one of the main isomers of TP73, a member of the TP53 protein family. We found that DNp73 was highly expressed in MM which could resist the apoptosis induced by DNA damage in MM cells, leading to clinical drug resistance.Aims:In order to further explore the mechanism of DNp73 mediating drug resistance to DNA damage in MM, this study verified and explored from multiple levels including cell phenotype changes in vivo and in vitro.Methods:MM cell lines OCI‐MY5 and ARP1 were used to constructed DNp73 overexpression (OE) cell lines, respectively. Cell proliferation activity of DNp73 OE and EV (empty vector) cell lines was compared by absolute cell count method and CCK8 method, and cell proliferation was determined under the action of doxorubicin (5nM &amp;50 nM) or epirubicin (5nM &amp; 10 nM) at different concentrations. The cloning formation and drug sensitivity of ARP1 DNp73 OE/EV cells were determined by soft agar cloning formation assay. Flow cytometry was used to detect the apoptosis and cell cycle arrest of cells in DNp73 OE and EV cells under UV and at different concentrations of reagents treatment. Xenografted model and 5TGM1 spontaneous mouse MM model were used to detect the effect of DNp73 on the proliferation of MM cells and the treatment of DNA damage reagents in vivo, as well as the survival period of tumor‐bearing mice were analyzed. RNA‐seq and gene enrichment analysis (GSEA) were performed to explore the potential signaling pathway of DNp73 against apoptosis. Further western blotting and quantity‐real time PCR were performed for validation. ChIP and CoIP were used to search for the potential downstream key target genes and target proteins of DNp73.Results:DNp73‐OE ARP1 &amp; OCI‐MY5 cell lines showed stronger proliferation activity than EV MM cell lines, and showed significant resistance to DNA damage reagents, doxorubicin and epirubicin treatment. The cloning formation experiment of soft agar confirmed that DNp73 induced the resistance of MM cells to those drugs. Flow cytometry analysis showed that DNp73 OE suppressed the MM cells apoptosis and suppressed G1 phase arrest induced by those drugs treatment (P &lt; 0.05). In vivo experiments showed that the tumor burden of mice was significantly more severe compared with the mice of control group. The tumor‐bearing mice in DNp73 OE group were resistant to the treatment of DNA damage reagents. Moreover, the survival of DNp73 OE tumor‐bearing mice was significantly shortened compared with the control group. RNA‐seq, GSEA analysis and western blot assay revealed that DNp73 overexpression induce drug resistance and proliferation through the activation of c‐myc and AKT‐related pathways and inhibition of p21/p27 signal pathway which caused cell cycle arrested in MM.Summary/Conclusion:DNp73 promote MM cell proliferation, anti‐apoptosis and inhibit cell cycle arrest through activating c‐myc and Akt‐related pathways. DNp73 overexpression is related to the occurrence of drug resistance in MM.image