Abstract 3178: A stromal integrated stress response activates perivascular cancer-associated fibroblasts to drive angiogenesis and tumor progression
Authors: Verginadis Ioannis I. , Avgousti Harris , Kim Kyle , Skoufos Giorgos , Chinga Frank , Leli Nektaria Maria , Karagounis Ilias V. , Bell Brett I. , Velalopoulou Anastasia , Wu Victoria S. , Li Yang , Ye Jiangbin , Scott David A. , Osterman Andrei L. , Sengupta Arjun , Weljie Aalim , Hatzigeorgiou Artemis G. , Ryeom Sandra , Diehl Alan J. , Fuchs Serge Y. , Puré Ellen , Koumenis Constantinos 
Published: June 2022

Abstract
Despite recent advances in prevention and treatment, including immune checkpoint inhibitors, malignant melanoma remains a particularly aggressive and deadly malignancy, which is partly attributed to its highly heterogeneous TME. However, malignant cells exhibit altered signaling pathways, which enables them to adapt to both cell-intrinsic and extrinsic stressors within TME. The activating transcription factor 4 (ATF4) is a master transcriptional effector of the Integrated Stress Response (ISR), a homeostatic mechanism coupling cell growth and survival to bioenergetic demands. We and others have established a critical tumor cell-intrinsic role of ATF4 which culminates in the promotion of primary tumor growth and in the establishment of micro- and macro-metastases in xenograft, allograft and transgenic models. However, the potential roles of the ISR and particularly of ATF4-mediated responses in host-dependent, tumor-related processes, have not been yet extensively investigated. Using novel conditional knockout ATF4 mouse models, we show that global loss of host ATF4 results in deficient tumor vascularization and a pronounced tumor growth delay in syngeneic melanoma and pancreatic tumor models. Immunofluorescence analysis revealed a severely impaired angiogenic phenotype in tumors grown in ATF4 KO mice which was accompanied by deficiencies in markers of CAF activation. Single-cell transcriptomic analysis of B16F10 melanoma tumors further localized this defect to a distinct CAF population, previously identified as vascular CAFs (vCAFs), and revealed a significant reduction in the expression of extracellular matrix components, primarily type I collagen, in tumors grown in ATF4 KO mice. Intriguingly, we identified a multifaceted impairment of the collagen biosynthetic pathway with the ATF4 to directly regulate the expression of the Col1a1 gene as well as the intracellular levels of glycine and proline, the major amino acids comprising collagen fibers. Moreover, we showed that the ATF4-deficient vCAFs secrete significantly lower levels of angiogenic factors (i.e., VEGF, SDF-1 etc.) in the perivascular area leading to an abnormal angiogenesis and significant attenuation of tumor growth. Specific deletion of ATF4 in the fibroblast compartment (Col1a1 promoter) produced a similar tumor growth delay as in the global ATF4 KO mice, and notably, co-injection of fibroblasts from ATF4-proficient mice led to significant recovery of tumor growth rates in ATF4-deficient mice. Finally, analysis of human melanoma and pancreatic tumor samples revealed a strong correlation between ATF4 and collagen levels and between an ISR gene signature and expression of collagen and CAF activation genes. Our findings uncover a novel role of stromal ATF4 in shaping CAF functionality, a key driver of disease progression, metastasis, and therapy resistance.
Citation Format: Ioannis I. Verginadis, Harris Avgousti, Kyle Kim, Giorgos Skoufos, Frank Chinga, Nektaria Maria Leli, Ilias V. Karagounis, Brett I. Bell, Anastasia Velalopoulou, Victoria S. Wu, Yang Li, Jiangbin Ye, David A. Scott, Andrei L. Osterman, Arjun Sengupta, Aalim Weljie, Artemis G. Hatzigeorgiou, Sandra Ryeom, Alan J. Diehl, Serge Y. Fuchs, Ellen Puré, Constantinos Koumenis. A stromal integrated stress response activates perivascular cancer-associated fibroblasts to drive angiogenesis and tumor progression [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 3178.