Background New treatment options for ovarian cancer are urgently required. Tumor-associated macrophages (TAMs) are an attractive target for therapy; repolarizing TAMs from M2 (pro-tumor) to M1 (anti-tumor) phenotypes represents an important therapeutic goal. We have previously shown that upregulated NF-kappaB (NF-κB) signaling in macrophages promotes M1 polarization, but effects in the context of ovarian cancer are unknown. Therefore, we aimed to investigate the therapeutic potential of increasing macrophage NF-κB activity in immunocompetent mouse models of ovarian cancer. Methods We have generated a transgenic mouse model, termed IKFM, which allows doxycycline-inducible overexpression of a constitutively active form of IKK2 (cIKK2) specifically within macrophages. The IKFM model was used to evaluate effects of increasing macrophage NF-κB activity in syngeneic murine TBR5 and ID8-Luc models of ovarian cancer in two temporal windows: 1) in established tumors, and 2) during tumor implantation and early tumor growth. Tumor weight, ascites volume, ascites supernatant and cells, and solid tumor were collected at sacrifice. Populations of macrophages and T cells within solid tumor and/or ascites were analyzed by immunofluorescent staining and qPCR, and soluble factors in ascitic fluid were analyzed by ELISA. Comparisons of control versus IKFM groups were performed by 2-tailed Mann-Whitney test, and a P-value < 0.05 was considered statistically significant. Results Increased expression of the cIKK2 transgene in TAMs from IKFM mice was confirmed at the mRNA and protein levels. Tumors from IKFM mice, regardless of the timing of doxycycline (dox) administration, demonstrated greater necrosis and immune infiltration than control tumors. Analysis of IKFM ascites and tumors showed sustained shifts in macrophage populations away from the M2 and towards the anti-tumor M1 phenotype. There were also increased tumor-infiltrating CD3+/CD8+ T cells in IKFM mice, accompanied by higher levels of CXCL9, a T cell activating factor secreted by macrophages, in IKFM ascitic fluid. Conclusions In syngeneic ovarian cancer models, increased canonical NF-κB signaling in macrophages promoted anti-tumor TAM phenotypes and increased cytotoxic T cell infiltration, which was sufficient to limit tumor progression. This may present a novel translational approach for ovarian cancer treatment, with the potential to increase responses to T cell-directed therapy in future studies.

Background Current cancer immunotherapies have made tremendous impacts but generally lack high response rates, especially in ovarian cancer. New therapies are needed to provide increased benefits. One understudied approach is to target the large population of immunosuppressive tumor-associated macrophages (TAMs). Using inducible transgenic mice, we recently reported that upregulating nuclear factor-kappaB (NF-κB) signaling in TAMs promotes the M1, anti-tumor phenotype and limits ovarian cancer progression. We also developed a mannose-decorated polymeric nanoparticle system (MnNPs) to preferentially deliver siRNA payloads to M2, pro-tumor macrophages in vitro. In this study, we tested a translational strategy to repolarize ovarian TAMs via MnNPs loaded with siRNA targeting the inhibitor of NF-κB alpha (IκBα) using mouse models of ovarian cancer. Methods We evaluated treatment with MnNPs loaded with IκBα siRNA (IκBα-MnNPs) or scrambled siRNA in syngeneic ovarian cancer models. ID8 tumors in C57Bl/6 mice were used to evaluate consecutive-day treatment of late-stage disease while TBR5 tumors in FVB mice were used to evaluate repetitive treatments in a faster-developing disease model. MnNPs were evaluated for biodistribution and therapeutic efficacy in both models. Results Stimulation of NF-κB activity and repolarization to an M1 phenotype via IκBα-MnNP treatment was confirmed using cultured luciferase-reporter macrophages. Delivery of MnNPs with fluorescent payloads (Cy5-MnNPs) to macrophages in the solid tumors and ascites was confirmed in both tumor models. A three consecutive-day treatment of IκBα-MnNPs in the ID8 model validated a shift towards M1 macrophage polarization in vivo. A clear therapeutic effect was observed with biweekly treatments over 2-3 weeks in the TBR5 model where significantly reduced tumor burden was accompanied by changes in immune cell composition, indicative of reduced immunosuppressive tumor microenvironment. No evidence of toxicity associated with MnNP treatment was observed in either model. Conclusions In mouse models of ovarian cancer, MnNPs were preferentially associated with macrophages in ascites fluid and solid tumors. Evidence of macrophage repolarization, increased inflammatory cues, and reduced tumor burden in IκBα-MnNP-treated mice indicate beneficial outcomes in models of established disease. We have provided evidence of a targeted, TAM-directed approach to increase anti-tumor immunity in ovarian cancer with strong translational potential for future clinical studies.

Background: Ovarian cancer is the most lethal gynecologic malignancy. While women with BRCA-deficient tumors show sensitivity to PARP inhibitors (PARPi), new treatment options are needed for PARPi-resistant tumors. An emerging strategy to improve PARPi response is combination therapy with epigenetic drugs. A newly recognized epigenetic drug target in ovarian cancer is the bromodomain and extraterminal (BET) protein family. BET proteins such as BRD4 promote oncogenic transcription of progrowth and survival genes, including the established link between inflammation and cancer, nuclear factor-kappaB (NF-κB). A complementary strategy to targeting cancer cells with cytotoxic drugs is to activate normal immune processes in the tumor microenvironment (TME). In syngeneic mouse ovarian cancer models, we have shown that M2-like protumor macrophages are a prominent component of the TME, and that NF-κB inhibition reduces the M2 population. Thus, BET inhibitors (BETi) have the potential to induce transcriptional reprogramming in both tumors and macrophages for therapeutic benefit. Objective: To determine the cellular and molecular effects of combining BETi and PARPi in mouse ovarian cancer and peritoneal macrophage cell lines. Methods: Cultured wild-type and CRISPR-modified (TP53 and TP53/BRCA2 knockout) ID8 mouse ovarian cancer cells, and PMJ2-PC mouse peritoneal macrophages, were treated with vehicle, the PARPi olaparib, the first-in-class BETi JQ1 or the JQ1/olaparib combination for 24-72h. Sulforhodamine B (SRB) assays assessed cell growth. Immunofluorescence assays assessed adherent cell number, DNA damage (pH2AX) and cell cycle indices. Protein levels of pH2AX and the apoptosis marker cleaved PARP were assessed by Western blot. NF-κB activity was measured by luciferase assays of a transiently transfected reporter plasmid. Results: Combined JQ1 and olaparib treatment synergistically reduced cell growth in SRB assays in wild-type and TP53 knockout ID8 cells. TP53/BRCA2 knockout cells showed greater responses to PARPi alone and no synergism was observed. Consistent with these results, the JQ1/olaparib combination cooperatively reduced the number of adherent cells and cells in S phase, and increased the G0/G1 population, DNA damage and apoptosis. In contrast, the drug combination had minimal effects on DNA damage or apoptosis in PMJ2-PC macrophages, while NF-κB activity was reduced in both cancer cells and macrophages. Conclusions: BETi sensitize mouse ovarian cancer cells to the cytotoxic effects of PARPi. Combined drug treatment also has potential to inhibit NF-κB in both cancer cells and macrophages. Our novel immunomodulatory strategy will be tested in ID8 syngeneic ovarian cancer models. We believe BETi combination treatment could expand the use of PARPi in ovarian cancer patients, with the potential to benefit a substantial number of women with this devastating disease. Citation Format: Andrew J. Wilson, Alyssa Hoover, Whitney Harris, Esther Liu, Dineo Khabele, Fiona Yull. Bromodomain inhibition in ovarian cancer and the tumor microenvironment to improve PARP inhibitor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 626.

Tumor associated macrophages (TAMs) often exist in high densities in breast tumors and are associated with adverse clinical outcomes. TAM repolarization as a strategy to shift the breast cancer microenvironment from immunosuppressive to immunostimulatory is an active area of research. The canonical and non-canonical NfkappaB (NFkB) signaling pathways represent potential points of intervention to influence TAM phenotypes. We have found that upregulation of the canonical NFkB signaling pathway specifically in macrophages can be used to repolarize TAMs toward an M1, anti-tumor phenotype. Using our doxycycline inducible mouse model termed IKFM, we are exploring the effects of macrophage specific upregulation of NFkB signaling on different stages of tumor growth and metastasis. In studies using the IKFM mouse model, we found that doxycycline-induced NFkB activation in macrophages in established orthotopic mammary tumors has therapeutic effects on tumor outcomes. Compared to control mice, we found that IKFM mice have reduced tumor burden in the lung, indicating prevention of metastasis. Ongoing studies are focused on understanding how macrophage specific activation of NFkB influences the metastatic niches of lung and bone; two of the most common sites of breast cancer metastasis. To move our findings towards clinical translation, we have developed an M2-macrophage targeted nanoparticle that activates NFkB signaling by delivering an siRNA against IkappaBalpha, an inhibitor of the pathway. The M2 targeted nanoparticle is currently being tested in murine models of breast cancer metastasis to both lung and bone to translate our findings from the IKFM mouse model to a pharmacological manipulation. Citation Format: Kennady K. Bullock, Dominique Parker, Evan B. Glass, Alyssa Hoover, Alyssa Merkel, Richard Maynard, Andrew Wilson, Todd Giorgio, Julie A. Rhoades, Fiona E. Yull. Modulation of NFkappaB signaling influences tumor associated macrophage phenotypes in murine models of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2728.

It is recognized that modulation of macrophage phenotypes within the tumor microenvironment has the potential to achieve anti-tumor outcomes. However, formulating a strategy to achieve the benefits of such an approach in a cell-specific manner while minimizing off-target or uncontrolled deleterious side effects remains a challenge. We hypothesize that modulation of Nuclear Factor Kappa-B (NF-kappaB) signaling within macrophages in the proximal tumor microenvironment represents an effective approach. However, there remains controversy whether NF-kappaB signaling in macrophages induces pro- or anti-tumor outcomes. Understanding the mechanisms by which this signaling pathway defines predominant macrophage characteristics is therefore critical in order to use strategic interventions to obtain therapeutic benefits. Our goal is to gain insight into how to modulate NF-kappaB signaling in macrophages to generate anti-tumor phenotypes and to use this information to develop new treatments. We use immortalized bone marrow-derived macrophages or ex vivo macrophages in cell culture approaches to investigate impacts of modulation of NF-kappaB signaling on macrophage phenotypes. We have three methods to modulate signaling: doxycycline inducible transgenic mice, liposomal delivery of the bacterial cell mimic MTP-PE, and optimized polymeric nanoparticle-mediated delivery of siRNA targeting the NF-kappaB inhibitor. In addition to our in vitro assays, we also employ in vivo murine models of ovarian and breast tumor progression. This multi-faceted approach is providing insights into how altering NF-kappaB signaling in macrophages impacts their interactions with tumor cells and other cells in the tumor microenvironment. Our data suggests that high levels of NF-kappaB signaling in macrophages induce both direct tumor cell-killing and immune-stimulating responses. These studies are designed to better understand the mechanisms by which NF-kappaB regulates macrophage functions to inform development of a novel macrophage-based immunotherapy that will be effective across a wide spectrum of solid tumors and metastatic disease. Citation Format: Evan B. Glass, Alyssa Hoover, Whitney Harris, Zahra Mirafzali, Todd D. Giorgio, Andrew Wilson, Fiona Yull. Understanding the effects of NF-kappaB signaling on macrophage phenotype to inform therapeutic approaches [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 4138.