The transcriptional repressors Snail and Slug contribute to cancer progression by mediating epithelial-mesenchymal transition (EMT), which results in tumor cell invasion and metastases. We extend this current understanding to demonstrate their involvement in the development of resistance to radiation and paclitaxel. The process is orchestrated through the acquisition of a novel subset of gene targets that is repressed under conditions of stress, effectively inactivating p53-mediated apoptosis, while another subset of targets continues to mediate EMT. Repressive activities are complemented by a concurrent derepression of specific genes resulting in the acquisition of stem celllike characteristics. Such cells are bestowed with three critical capabilities, namely EMT, resistance to p53-mediated apoptosis, and a self-renewal program, that together define the functionality and survival of metastatic cancer stem cells. EMT provides a mechanism of escape to a new, less adverse niche; resistance to apoptosis ensures cell survival in conditions of stress in the primary tumor; whereas acquisition of ''stemness'' ensures generation of the critical tumor mass required for progression of micrometastases to macrometastases. Our findings, besides achieving considerable expansion of the inventory of direct genes targets, more importantly demonstrate that such elegant cooperative modulation of gene regulation mediated by Snail and Slug is critical for a cancer cell to acquire stem cell characteristics toward resisting radiotherapy-or chemotherapy-mediated cellular stress, and this may be a determinative aspect of aggressive cancer metastases.

Chemotherapy is a double-edged sword. It is anticancer because of its cytotoxicity. Paradoxically, by increasing chemoresistance and cancer metastasis, it is also procancer. However, the underlying mechanisms for chemotherapy-induced procancer activities are not well understood. Here we describe the ability of paclitaxel (PTX), a frontline chemotherapeutic agent, to exacerbate metastasis in mouse models of breast cancer. We demonstrate that, despite the apparent benefit of reducing tumor size, PTX increased the circulating tumor cells in the blood and enhanced the metastatic burden at the lung. At the primary tumor, PTX increased the abundance of the tumor microenvironment of metastasis, a landmark microanatomical structure at the microvasculature where cancer cells enter the blood stream. At the metastatic lung, PTX improved the tissue microenvironment (the "soil") for cancer cells (the "seeds") to thrive; these changes include increased inflammatory monocytes and reduced cytotoxicity. Importantly, these changes in the primary tumor and the metastatic lung were all dependent on Atf3, a stress-inducible gene, in the noncancer host cells. Together, our data provide mechanistic insights into the procancer effect of chemotherapy, explaining its paradox in the context of the seed-and-soil theory. Analyses of public datasets suggest that our data may have relevance to human cancers. Thus, ATF3 in the host cells links a chemotherapeutic agenta stressor-to immune modulation and cancer metastasis. Dampening the effect of ATF3 may improve the efficacy of chemotherapy.chemotherapy | metastasis | stress response | immune modulation | ATF3 M odern chemotherapy can reduce tumors to an undetectable level; however, in many cases the tumors relapse, with recurrence in the original, regional, or distant sites (1-3). The mechanisms for relapse are multifaceted and complex, including intrinsic changes in cancer cells and changes in the noncancer cells in the host-the organism carrying the cancer (4-9). Although the traditional concept is that chemotherapeutic drugs provide selection pressure for drug-resistant cancer cells to thrive, recent studies showed that chemotherapeutic drugs actually induce procancer changes (reviewed in refs. 4-9 and in the references cited below). Thus, chemotherapy is a double-edged sword: It is anticancer because of its cytotoxicity on cancer cells but also can be procancer by inducing changes in cancer and/or host cells. For cancer cell-intrinsic changes, chemotherapeutic drugs have been shown to induce the migration/invasion of cancer cells (10) and to up-regulate the expression of some antiapoptotic genes (11). For noncancer cells, chemotherapy theoretically can affect all host cells, because it is administered systemically. Advances in this nascent field have benefited greatly from the extensive literature on cancerhost interaction in the recent decades (5-7, 12). Although endothelial cells have been shown to play a role in mediating the procancer effect of chemotherapy (13-15), various reports als...

Host response to cancer signals has emerged as a key factor in cancer development; however, the underlying molecular mechanism is not well understood. In this report, we demonstrate that activating transcription factor 3 (ATF3), a hub of the cellular adaptive response network, plays an important role in host cells to enhance breast cancer metastasis. Immunohistochemical analysis of patient tumor samples revealed that expression of ATF3 in stromal mononuclear cells, but not cancer epithelial cells, is correlated with worse clinical outcomes and is an independent predictor for breast cancer death. This finding was corroborated by data from mouse models showing less efficient breast cancer metastasis in Atf3-deficient mice than in WT mice. Further, mice with myeloid cell-selective KO of Atf3 showed fewer lung metastases, indicating that host ATF3 facilitates metastasis, at least in part, by its function in macrophage/myeloid cells. Gene profiling analyses of macrophages from mouse tumors identified an ATF3-regulated gene signature that could distinguish human tumor stroma from distant stroma and could predict clinical outcomes, lending credence to our mouse models. In conclusion, we identified ATF3 as a regulator in myeloid cells that enhances breast cancer metastasis and has predictive value for clinical outcomes.

Activating transcription factor 3 (ATF3) gene encodes a member of the ATF family of transcription factors and is induced by various stress signals, including many of those that induce the unfolded protein response (UPR). Emerging evidence suggests that ATF3 is a hub of the cellular adaptive–response network and studies using various mouse models indicate that ATF3 plays a role in the pathogenesis of various diseases. One way to investigate the potential relevance of ATF3 to human diseases is to determine its expression in patient samples and test whether it correlates with disease progression or clinical outcomes. Due to the scarcity and preciousness of patient samples, methods that can detect ATF3 on archival tissue sections would greatly facilitate this research. In this chapter, we briefly review the roles of ATF3 in cellular adaptive–response and UPR, and then describe the detailed steps and tips that we developed based on general immunohistochemistry (IHC) protocols to detect ATF3 on paraffin embedded sections.

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BackgroundTherapeutically targeting tumor myeloid cells has emerged as a novel and complementary strategy to existing cancer immunotherapy approaches. The interaction of tumor expressed CD47 with SIRP alpha (signal regulatory protein-alphaa, SIRPA) on macrophages, dendritic cells and neutrophils inhibits key immune effector mechanisms. Targeting SIRPa-CD47 represents a novel approach to enhance anti-tumor immunity by augmenting or reactivating critical tumor clearance mechanisms.H5F9, an antibody against CD47, has shown promising therapeutic activities in patients with MSD, AML and NHL. However, agents targeting CD47 present hematological toxicities and present a huge antigen sink leading to not achieving an optimum therapeutic window. Our approach is to target SIRP alpha, the receptor of CD47 and focus therapeutic targeting to relevant mechanisms related to phagocytosis and myeloid cell activation and at the same time avoid undesired effects of blocking CD47. SIRP gamma, a very close relative of SIRP alpha is expressed on T cells and also binds to CD47. It has been shown that blockade of SIRP gamma-CD47 interaction inhibits T cell proliferation and blocks trans-endothelial T cell migration. Hence, our aim is to generate SIRP alpha selective antibodies that do not cross-react with SIRP gamma and have minimal impact on T cell functions.MethodsUsing Apexigen’s APXiMAB™ proprietary antibody discovery platform, we have generated two novel anti-SIRP alpha antibodies (APX701 & APX702) with differentiated properties as compared to other approaches targeting the CD47/SIRP alpha axis. We have used ELISA, FACS based cell binding and blocking assays, and functional assays including in vitro phagocytosis and antibody-dependent cell phagocytosis (ADCP) in combination with tumor-opsonizing antibody to select APX701 & APX702.ResultsOur novel preclinical-stage APX701 & APX702 antibodies have demonstrated the following attributes: high binding affinity to human SIRP alpha (APX701 Kd = 0.95nM, APX702 Kd = 0.88nM), no binding to SIRP gamma, efficient blockade of SIRP alpha binding to CD47(APX701 IC50 = 1.04nM, APX702 IC50 = 0.80nM), potent macrophage mediated phagocytosis, enhancement of ADCP mediated by tumor-opsonizing antibody and favorable developability CMC profiles. In comparison with the benchmark antibody OSE-172, APX701 & APX702 showed potent phagocytosis activity and ADCP enhancement in all donors tested while OSE-172 induced phagocytosis in only 50% of the donors. This may result from the fact that APX701 and APX702 bind to all major SIRP alpha variants (V1, V2 & V8; covering ~92% population) while OSE 172 only binds to SIRPalpha V1 (~50% population).ConclusionsAPX701 and APX702 demonstrate differentiated anti-SIRPalpha activities by enhancing myeloid cell-mediated anti-tumor immunity and reactivating critical tumor clearance mechanisms within the tumor microenvironment.

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Therapeutically targeting tumor myeloid cells has emerged as a complementary strategy to existing cancer immunotherapy approaches. The interaction of tumor-expressed CD47 with SIRPα (Signal Regulatory Protein-α, SIRPA) on macrophages, dendritic cells, and neutrophils inhibit key immune effector mechanisms. Targeting the SIRPα-CD47 axis represents a novel approach to enhance anti-tumor immunity by augmenting or reactivating critical tumor clearance mechanisms. While agents that target CD47 have shown promise in clinical trials, they present hematological toxicities and antigen sink issues that narrow their therapeutic application. Our approach targets SIRPα to focus on relevant mechanisms related to myeloid cell activation and increase of tumor cell phagocytosis while simultaneously avoiding the undesired effects of targeting CD47. Furthermore, SIRPγ, a close relative of SIRPα, is expressed on T cells and potentially co-stimulates T cell proliferation and is required for trans-endothelial T cell migration. We sought to develop an anti-SIRPα therapeutic that does not cross-react with SIRPγ and avoids potentially deleterious effects on T cell functions. Here, we describe APX701, a novel anti-SIRPα antibody generated using Apexigen's APXiMAB™ proprietary antibody discovery platform, and its differentiated properties as compared to other approaches targeting the SIRPα-CD47 interaction. APX701 binds to human SIRPα at a unique epitope with high affinity (Kd = 0.95nM) and with no detectable binding to SIRPγ expressed on primary human T cells. APX701 strongly binds to the major SIRPα variants—V1 and V2—and potently blocks their interaction with CD47, thereby allowing therapeutic targeting of SIRPα in a majority of patients. APX701 reverses SIRPα-mediated suppression of macrophage phagocytosis, resulting in potent destruction of a variety of tumor cells both as a single agent and in combination with anti-TAA (tumor associated antigen) antibodies including cetuximab, rituximab, and trastuzumab. Moreover, targeting SIRPα with APX701 does not interfere with T cell function, an effect previously observed following anti-CD47 treatment. Together, these data demonstrate the unique binding profile of APX701 and its ability to potently reactivate myeloid cell-mediated anti-tumor immunity. Our data support the development of APX701, a promising myeloid checkpoint inhibitor, for the treatment of cancer. Citation Format: Ryan Alvarado, Sushma Krishnan, Minu K. Srivastava, Christine Tan, George Huang, Swati Jalgaonkar, Frances R. Bahjat, Erin L. Filbert, Xiaodong Yang. Targeting SIRPα with APX701, a Novel Myeloid Checkpoint Inhibitor [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 LB177.