Standard preclinical human tumor models lack a human tumor stroma. However, as stroma contributes to therapeutic resistance, the lack of human stroma may make current models less stringent for testing new therapies. To address this, using patient-derived tumor cells, patient-derived cancer-associated mesenchymal stem/progenitor cells, and human endothelial cells, we created a human stroma-patient-derived xenograft (HS-PDX) tumor model. HS-PDX, compared to the standard PDX model, demonstrates greater resistance to targeted therapy and chemotherapy and better reflect patient response to therapy. Furthermore, HS-PDX can be grown in mice with humanized bone marrow to create humanized immune stroma patient-derived xenograft (HIS-PDX) models. The HIS-PDX model contains human connective tissues, vascular and immune cell infiltrates. RNA sequencing analysis demonstrated a 94-96% correlation with primary human tumor. Using this model, we demonstrate the impact of human tumor stroma on recruitment of TAMs and tumor immune exclusion to impact to response to immunologic therapy. We show an immunosuppressive role for human tumor stroma and that this model can be used to identify immunotherapeutic combinations to overcome stroma-mediated immunosuppression. Combined, our data confirm a critical role for human stroma in therapeutic response and indicate that HIS-PDX can be an important tool for preclinical drug testing.

EGF-like domain multiple-6 (EGFL6) is a secreted tumor growth/migration factor linked with poor outcomes in many tumor types. While EGFL6 is known to signal, in part, via its integrin-binding RGD domain, little else is known about EGFL6 receptors. We evaluated putative EGFL6 receptors and found that EGFL6 treatment of ovarian cancer cells leads to both transient phosphorylation of EGFR and prolonged phosphorylation of HER2 and HER3 and subsequent phosphorylation of ERK (pERK). We found that EGFL6 directly binds HER3. However, EGFL6-driven prolonged activation of HER3 is dependent on an intact EGFL6 integrin-binding RGD domain. Immunoprecipitation and proximity ligation assays confirmed that EGFL6 treatment of cancer cells induces HER2/3-integrin-β3 heterocomplexes. Suggesting EGFL6 could play a role in resistance to HER targeting therapies, EGFL6 is upregulated in EGFR/HER receptor inhibitor-resistant cells, and EGFL6 treatment increases resistance to EGFR/HER inhibitors in vitro. Interestingly, we found that, in EGFL6-treated ovarian cancer cells undergoing mitosis, pERK localizes to the centrosome. Both EGFL6-neutralizing antibodies and HER protein-targeted inhibitors resulted in aberrant pERK centrosomal localization with associated altered mitotic spindle alignment and mitotic catastrophe. Furthermore, combination anti-EGFL6 therapy with the pan-EGFR receptor inhibitor neratinib, compared to either therapy alone, led to an increase in aberrant pERK localization and cancer cell death in vitro and significant restricted tumor growth in vivo. Combined, our data suggests that EGFL6 is a new ligand for HER3 and that dual targeting of the EGFL6/HER signaling axis, via altered pERK localization, may be an effective therapeutic strategy in ovarian cancer. Significance: This work reveals that EGFL6 is a previously unrecognized ligand for HER3 which can increase resistance to HER family-targeted therapy. We also reveal a novel function of pERK downstream of pHER3 at the centrosome in mitosis. Importantly, we show that EGFL6 is an important therapeutic target to enhance the efficacy of EGFR/HER-targeted therapy.

Objective EGFL6, a growth factor produced by adipocytes, is upregulated in and implicated in the tumorigenesis of multiple tumor types. Given the strong link between obesity and endometrial cancer, we sought to determine the impact of EGFL6 on endometrial cancer. Methods EGFL6 expression in endometrial cancer and correlation with patient outcomes was evaluated in the human protein atlas and TCGA. EGFL6 treatment, expression upregulation, and shRNA knockdown were used to evaluate the impact of EGFL6 on the proliferation and migration of 3 endometrial cancer cell lines in vitro. Similarly, the impact of EGFL6 expression and knockdown on tumor growth was evaluated. Western blotting was used to evaluate the impact of EGFL6 on MAPK phosphorylation. Results EGFL6 is upregulated in endometrial cancer, primarily in cony-number high tumors. High tumor endometrial cancer expression of EGFL6 predicts poor patient prognosis. We find that EGFL6 acts to activate the MAPK pathway increasing cellular proliferation and migration. In xenograft models, EGFL6 overexpression increases endometrial cancer tumor growth while EGFL6 knockdown decreases endometrial cancer tumor growth. Conclusions EGFL6 is a marker of poor prognosis endometrial cancers, driving cancer cell proliferation and growth. As such EGFL6 represents a potential therapeutic target in endometrial cancer.

Abstract Epidermal growth factor EGF-like domain multiple-6 (EGFL6) is highly expressed in high grade serous ovarian cancer (HGSOC) cells and ovarian tumor vasculature. EGFL6 acts on tumor cells to drive tumor cell proliferation and migration. To better understand EGFL6 signaling and develop therapeutic strategies to target EGFL6 signaling in HGSOC, we evaluated phosphorylation of putative EGFL6 receptors and their downstream signaling. We demonstrated that EGFL6 induced phosphorylation and activation of ERBB/HER family of receptors; EGFL6 treatment of ovarian cancer cells led to rapid and transient phosphorylation of EGFR while inducing prolonged activation of both HER2 and HER3, with subsequent phosphorylation of ERK. Interestingly, we found that in EGFL6 stimulated ovarian cancer cells undergoing mitosis, pERK localized to the centrosome and the contractile ring. EGFL6 neutralizing antibodies reduce ERK phosphorylation and resulted in pERK being aberrantly localized. This resulted in altered mitotic spindle alignment and an increased number of cells undergoing mitotic catastrophe. Furthermore, combination anti-EGFL6 therapy with the pan-EGFR receptor inhibitor neratinib, compared to either therapy alone, led to an increase in aberrant pERK localization and cancer cell death in vitro. Consistent with these findings, dual anti-EGFL6 and neratinib therapy significantly restricted tumor growth in vivo, resulting in increased tumor cell death and a reduction in angiogenesis. Combined our data identify an unexplored role for pERK at the centrosome and suggest that dual targeting of the EGFL6/HER signaling axis maybe an effective therapeutic strategy in ovarian cancer. Citation Format: Shoumei Bai, Navneet Gupta, Qi Jiang, Ronald J. Buckanovich. EGFL6 induced HER2-HER3 signaling controls accurate centrosome deposition of activated ERK and mitotic spindle formation in ovarian 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 626.

Tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) play a critical role in resistance to immunotherapy. In this study, we identified epidermal growth factor-like 6 (Egfl6) as a regulator of myeloid cell functions. Our analyses indicated that Egfl6, via binding with β3 integrins and activation of p38 and SYK signaling, acts as a chemotactic factor for myeloid cell migration and promotes their differentiation toward an immunosuppressive state. In syngeneic mouse models of ovarian cancer (OvCa), tumor expression of Egfl6 increased the intratumoral accumulation of polymorphonuclear (PMN) MDSCs and TAMs and their expression of immunosuppressive factors, including CXCL2, IL-10, and PD-L1. Consistent with this, in an immune 'hot' tumor model, Egfl6 expression eliminated response to anti-PD-L1 therapy, while Egfl6 neutralizing antibody decreased the accumulation of tumor-infiltrating CD206+ TAMs and PMN-MDSCs and restored the efficacy of anti-PD-L1 therapy. Supporting a role in human tumors, in human OvCa tissue samples, areas of high EGFL6 expression colocalized with myeloid cell infiltration. scRNA-Seq analyses revealed a correlation between EGFL6 and immune cell expression of immunosuppressive factors. Our data provide mechanistic insights into the oncoimmunologic functions of EGFL6 in mediating tumor immune suppression and identified EGFL6 as a potential therapeutic target to enhance immunotherapy in patients with OvCa.

Supplementary Figure 6 from RETRACTED: Physical and Functional Interaction of DNA Methyltransferase 3A with Mbd3 and Brg1 in Mouse Lymphosarcoma Cells

Supplementary Figure 5 from RETRACTED: Physical and Functional Interaction of DNA Methyltransferase 3A with Mbd3 and Brg1 in Mouse Lymphosarcoma Cells

PDF File - 9042K, TVM antibodies do not cross-react with murine tumor vessels.

Supplementary Tables 1-3, Figures 1-3 from Aldehyde Dehydrogenase in Combination with CD133 Defines Angiogenic Ovarian Cancer Stem Cells That Portend Poor Patient Survival

PDF File - 71K, A List of Antibodies Used.

Objectives Epidermal growth factor EGF-like domain multiple-6 (EGFL6) is highly expressed in high grade serous ovarian cancer and promotes both endothelial cell proliferation/angiogenesis and cancer cell proliferation/metastasis. As such it has been implicated as a therapeutic target. As a secreted factor, EGFL6 is a candidate for antibody therapy. The objectives of this study were to create and validate humanized affinity-matured EGFL6 neutralizing antibodies for clinical development. Methods A selected murine EGFL6 antibody was humanized using CDR grafting to create 26 variant humanized antibodies. These were screened and the lead candidate was affinity matured. Seven humanized affinity-matured EGFL6 antibodies were screened for their ability to block EGFL6 activity on cancer cells in vitro, two of which were selected and tested their therapeutic activity in vivo. Results Humanized affinity matured antibodies demonstrated high affinity for EGFL6 (150 pM to 2.67 nM). We found that several humanized affinity-matured EGFL6 antibodies specifically bound to recombinant, and native human EGFL6. Two lead antibodies were able to inhibit EGFL6-mediated (i) cancer cell migration, (ii) proliferation, and (iii) increase in ERK phosphorylation in cancer cells in vitro. Both lead antibodies restricted growth of an EGFL6 expressing ovarian cancer patient derived xenograft. Analysis of treated human tumor xenografts indicated that anti-EGFL6 therapy suppressed angiogenesis, inhibited tumor cell proliferation, and promoted tumor cell apoptosis. Conclusions Our studies confirm the ability of these humanized affinity-matured antibodies to neutralize EGFL6 and acting as a therapeutic to restrict cancer growth. This work supports the development of these antibody for first-in-human clinical trials.

In ovarian cancer (OC), a desmoplastic signature is associated with poor patient prognosis and is linked to significant morbidity and mortality. In particular, OC desmoplasia is a critical driver of bowel obstruction, a common, life-limiting complication of ovarian malignancies. There are no non-surgical treatments to target complications of desmoplasia. Thus, understanding the drivers of desmoplasia can identify novel therapeutic approaches. Cancer-associated mesenchymal stem cells (CA-MSC) have been shown to differentiate into fibroblasts to facilitate tumor desmoplasia and fibrosis. Previous studies have shown that the TGF-β pathway, and the MRTF-α protein in particular, plays an important role in mesenchymal stem cell (MSC) to fibroblast differentiation and is, therefore, a viable target for this therapeutic gap. In order to explore this further, we tested the effects of a novel MRTF-α inhibitor, CCG-081, on MSCs, CA-MSCs, and ovarian tumor desmoplasia.

PDF File - 11199K, Co-localization of TVM expression with hCD31 expression.

Background Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are critical negative regulators of immunity in cancer.1 2 Understanding factors which regulate these cells could result in the identification of new approaches to enhance immunotherapy. One such factor is epidermal growth factor-like 6 (EGFL6), a secreted factor known to promote cancer stem like cell migration and regulate cancer cell differentiation.3 We found that mice which overexpress Egfl6 have an increased numbers of myeloid cells in both the bone marrow and spleen. The goal of this study is to evaluate the impact of tumor Egfl6 on myeloid cell phenotype in the ovarian cancer (OvCa) tumor microenvironment (TME). Methods Ex-vivo rEgfl6 and two syngeneic mouse models of OvCa were used to study the impact of tumor Egfl6 on the immune TME. RNA sequencing was employed to identify Egfl6-mediated gene expression in tumor infiltrating myeloid cells. Egfl6+ tumors were treated with anti-Egfl6, or anti-PD-L1 antibodies, as single agents or in combination, and tumors were harvested for immune profiling, gene and protein expression evaluation and ex vivo co-culture studies. Immunofluorescence and spatial transcriptomic were used to determine the localization of EGFL6 and myeloid cells in human OvCa tissue samples. Results In vitro and ex-vivo analysis indicated that Egfl6, via binding with beta integrins and activation of p38 and SYK signaling, (i) acts as a chemotactic factor for myeloid cells and (ii) promotes their differentiation toward a suppressive state. Suggesting an important role in promoting an immunosuppressive TME, we found that expression of Egfl6 on tumor cells increased tumor growth and shortened animal survival. This was associated with an increased accumulation of intra-tumoral MDSCs and TAMs and their expression of immunosuppressive factors, including CXCL2, IL-10 and PD-L1. Both CXCL2 and IL-10 were found to play a key role in the Egfl6-induced anti-tumor immunosuppression. In an immune ‘hot’ tumor model,4 5 Egfl6 completely inhibited response to a-PD-L-1 therapy. Finally, we found that Egfl6 antibody decreased the accumulation of tumor-infiltrating CD206+ TAMs and PMN-MDSCs, and their secreted factors, IL-10 and CXCL2, and thereby restored the efficacy of a-PD-L1 therapy. Importantly, in human OvCa tissue samples, EGFL6+ areas were highly infiltrated by myeloid cells. Conclusions Combined our data show that EGFL6 induces the recruitment of myeloid cells into the ovarian TME and subsequently promotes their immunosuppressive functions. This suggest EGFL6 is a potential novel therapeutic target to enhance response to immune therapy in OvCa patients. Acknowledgements This work was supported by the Early Investigator Award-Ovarian Cancer Research Alliance Foundation to S.C., and National Cancer Institute Grant R01CA218026 to R.B. References Hensler M, et al. M2-like macrophages dictate clinically relevant immunosuppression in metastatic ovarian cancer. J Immunother Cancer 2020;8(2). Baert T, et al. Myeloid Derived Suppressor Cells: Key Drivers of Immunosuppression in Ovarian Cancer. Front Immunol 2019;10:1273. Bai S, et al. EGFL6 Regulates the Asymmetric Division, Maintenance, and Metastasis of ALDH+ Ovarian Cancer Cells. Cancer Res 2016;76(21):6396–6409. Cascio S, et al. Cancer-associated MSC drive tumor immune exclusion and resistance to immunotherapy, which can be overcome by Hedgehog inhibition. Sci Adv 2021;7(46):eabi5790. Grabosch S, et al. Cisplatin-induced immune modulation in ovarian cancer mouse models with distinct inflammation profiles. Oncogene 2019;38(13):2380–2393. Ethics Approval Biopsies of patients with high-grade serous ovarian cancer were selected and collected at the Department of Obstetrics, Gynecology, and Reproductive Science, University of Pittsburgh.

Supplementary Data from Targeting Therapeutic Resistance and Multinucleate Giant Cells in CCNE1-Amplified HR-Proficient Ovarian Cancer

AbstractApproximately 20% of high-grade serous ovarian cancers (HGSOC) have CCNE1 amplification. CCNE1-amplified tumors are homologous recombination (HR) proficient and resistant to standard therapies. Therapy resistance is associated with increased numbers of polyploid giant cancer cells (PGCC). We sought to identify new therapeutic approaches for patients with CCNE1-amplified tumors. Using TCGA data, we find that the mTOR, HR, and DNA checkpoint pathways are enriched in CCNE1-amplified ovarian cancers. Furthermore, Interactome Mapping Analysis linked the mTOR activity with upregulation of HR and DNA checkpoint pathways. Indeed, we find that mTOR inhibitors (mTORi) downregulate HR/checkpoint genes in CCNE1-amplified tumors. As CCNE1-amplified tumors are dependent on the HR pathway for viability, mTORi proved selectively effective in CCNE1-amplified tumors. Similarly, via downregulation of HR genes, mTORi increased CCNE1-amplifed HGSOC response to PARPi. In contrast, overexpression of HR/checkpoint proteins (RAD51 or ATR), induced resistance to mTORi. In vivo, mTORi alone potently reduced CCNE1-amplified tumor growth and the combination of mTORi and PARPi increased response and tumor eradication. Tumors treated with mTORi demonstrated a significant reduction in ALDH+ PGCCs. Finally, as a proof of principle, we identified three patients with CCNE1 amplified tumors who were treated with an mTORi. All three obtained clinical benefits from the therapy. Our studies and clinical experience indicate mTORi are a potential therapeutic approach for patients with CCNE1-amplified tumors.

Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are critical negative regulators of immunity in cancer. Understanding factors which regulate these cells could result in the identification of new approaches to enhance anti-tumor immunotherapy. One such factor is Epidermal growth factor-like 6 (EGFL6). EGFL6 is a secreted factor known to promote cancer stem like cell migration and regulate cancer cell differentiation. Similarly, EGFL6 promotes endothelial cell migration and proliferation. Indicating a potential role for EGFL6 as a myeloid cell regulatory factor, we found that mice which overexpress Egfl6 have an increased numbers of granulocytes and monocytes in both the bone marrow and spleen. In vitro and ex-vivo analysis indicated that EGFL6, via binding with beta integrins and activation of Syk/ERK signaling, (i) acts as a chemotactic factor for human myeloid cells migration and (ii) promotes their differentiation toward a suppressive state. Suggesting an important role in promoting an immunosuppressive tumor microenvironment (TME), using two syngeneic mouse models of ovarian cancer, we found that expression of Egfl6 in tumor cells resulted in increased accumulation of intra-tumoral MDSCs and TAMs and fewer cytotoxic CD8+ T cells. This was associated with increased tumor growth and shortened animal survival. Gene expression profiling and flow cytometry analysis of tumor infiltrating myeloid cells indicated that Egfl6 induced the expression of immunosuppressive factors, including CXCL2, IL-10 and PD-L1. Consistent with Egfl6 driving an immune suppressive TME, EGFL6 expression in an otherwise immune ‘hot’/anti-PD-L1 responsive tumor model completely inhibited response to anti-PD-L-1 therapy. We are currently evaluating the impact of Egfl6 neutralizing antibody on the efficacy of ICI therapy and anti-tumor immunity. Combined our data show that EGFL6 acts as a chemotactic factor to both recruit myeloid cells to the ovarian TME and subsequently promotes their differentiation to an immunosuppressive phenotype. This suggests EGFL6 is a potential novel therapeutic target to ovarian tumor mediated immunosuppression and enhance response to immune therapy in ovarian cancer patients. Citation Format: Sarah Sinno, Shoumei Bai, Claudia Coronnello, Anda Vlad, Ronald J. Buckanovich, Sandra Cascio. EGFL6 induces immunosuppressive functions of tumor-associated myeloid cells and mediates resistance to anti-PDL1 therapy [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 3146.