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3D scaffolds mimicking the environment in the primary tumor or metastatic organs can deconstruct complex niche signals and facilitate the study of cancer progression and metastasis. Here, we reported that a subcutaneous 3D scaffold implant acted as a lung-mimicking dormant metastatic niche in mouse models of metastatic breast cancer, recruiting lung-tropic circulating tumor cells yet suppressing their growth through potent in situ antitumor immunity. We compared it with the immunosuppressive lungs developing lethal metastases and the dormant lungs suppressing tumor growth derived from breast cancer models with varying tumor aggressiveness and host immunity. Our data suggested that breast cancer-induced Gr1+CD11b+Ly6G+ granulocytic myeloid cells (neutrophils) infiltrated the scaffold implants and lungs, secreting the same signal to facilitate the metastatic seeding of lung-tropic cancer cells in these two types of niches. However, circulating neutrophils with opposing phenotypes and functions (N1 and N2) were selectively recruited and enriched in the dormant scaffolds/lungs and immunosuppressive lungs, respectively, responding to two distinct groups of chemoattractants. N1 or N2 neutrophils established activated or suppressive immune environments in the metastatic niches, directing different fates of cancer cells. The clinical relevance of these scientific findings was validated by the strong positive correlation of a high N1-to-N2 neutrophil chemoattractant ratio with a low-grade primary tumor, a low metastases incidence, and a better prognosis in breast cancer patients. Overall, our study revealed the multifaceted roles of neutrophils in regulating lung metastasis and underscored the importance of N1 neutrophils in driving breast cancer metastatic dormancy in the lungs, inspiring next-generation immunotherapy. Citation Format: Jing Wang, Ramon Ocadiz-Ruiz, Matthew Hall, Grace Bushnell, Sophia Orbach, Joseph Decker, Ravi Raghani, Yining Zhang, Aaron Morris, Jacqueline Jeruss, Lonnie Shea. A lung-mimicking synthetic metastatic niche reveals N1 neutrophils drive breast cancer metastatic dormancy in the lungs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB347.

s: AACR Special Conference on the Advances in Pediatric Cancer Research; September 17-20, 2019; Montreal, QC, Canada Ewing sarcoma (ES) is an aggressive bone and soft-tissue tumor, most commonly driven by the EWS/FLI1 fusion oncogene. Previous work has revealed that the scaffolding protein menin functions to promote ES tumorgenicity. Additionally, our recent studies revealed a previously undescribed role for menin in the activation of the serine biosynthetic pathway (SSP), a critical metabolic pathway that is aberrantly activated in many human cancers. The transcription factor ATF4 has been identified in other tumor types as a master transcriptional regulator of the SSP. Furthermore, regulation of the SSP by EWS/FLI1 has been described, although the mechanism of this regulation remains unknown. The biologic functions of menin are largely determined by its protein-binding partners, the best characterized of which is MLL in epigenetic trithorax complexes. In the current study, we are investigating the mechanistic link between menin, EWS/FLI1, and SSP hyperactivation to test the hypothesis that, in the context of ES, these transcriptional regulators cooperate to promote metabolic reprogramming via ATF4. Through gain- and loss-of-function studies, our results show that ATF4 regulates the SSP in ES cells, and that loss of ATF4 impairs ES cell proliferation. Additionally, both pharmacologic inhibition with the menin:MLL interaction inhibitor, MI-503, and doxycycline-inducible shRNA knockdown of menin lead to loss of ATF4, coincident with loss of SSP expression. Knockdown of EWS/FLI1 similarly results in loss of both ATF4 and SSP expression. Interrogation of publicly available ChIP-seq data further shows enrichment for EWS/FLI1 binding at the ATF4 gene promoter that is lost with EWS/FLI1 knockdown, and ChIP-qPCR shows menin binding at the ATF4 gene promoter in association with H3K4me3 enrichment. ChIP-qPCR also shows enrichment of ATF4 binding at SSP gene promoters, which is diminished upon menin inhibition with MI-503. Preliminary studies suggest that ATF4 overexpression may rescue the effects of menin loss of function on the SSP. Together these findings support the hypothesis that menin hijacks the SSP via aberrant activation of ATF4, and that this depends on cooperation with EWS/FLI1. Ongoing studies are assessing whether this is mediated by trithorax-dependent or -independent functions of menin. Citation Format: Jennifer A. Jimenez, Ramon Ocadiz Ruiz, April Apfelbaum, Abhijay Kumar, Samuel Kerk, Jolanta Grembecka, Costas A. Lyssiotis, Elizabeth R. Lawlor. Menin and EWS/FLI1 activate serine biosynthesis via ATF4 in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B37.

In colorectal cancer, APC-mediated induction of unregulated cell growth involves posttranslational mechanisms that prevent proteasomal degradation of proto-oncogene β-catenin (CTNNB1) and its eventual translocation to the nucleus. However, about 10% of colorectal tumors also exhibit increased CTNNB1 mRNA. Here, we show in colorectal cancer that increased expression of ZNF148, the gene coding for transcription factor ZBP-89, correlated with reduced patient survival. Tissue arrays showed that ZBP-89 protein was overexpressed in the early stages of colorectal cancer. Conditional deletion of Zfp148 in a mouse model of Apc-mediated intestinal polyps demonstrated that ZBP-89 was required for polyp formation due to induction of Ctnnb1 gene expression. Chromatin immunoprecipitation (ChIP) and EMSA identified a ZBP-89–binding site in the proximal promoter of CTNNB1. Reciprocally, siRNA-mediated reduction of CTNNB1 expression also decreased ZBP-89 protein. ChIP identified TCF DNA binding sites in the ZNF148 promoter through which Wnt signaling regulates ZNF148 gene expression. Suppression of either ZNF148 or CTNNB1 reduced colony formation in WNT-dependent, but not WNT-independent cell lines. Therefore, the increase in intracellular β-catenin protein initiated by APC mutations is sustained by ZBP-89–mediated feedforward induction of CTNNB1 mRNA. Cancer Res; 76(23); 6877–87. ©2016 AACR.