Accurate and rapid methods for the detection of DNA damage foci in eukaryotic cells are central to DNA repair studies, which identify differences in DNA repair capacity in cell lines. Such assays have been important in delineating mechanisms of DNA repair in human cells. Previously we were the first to demonstrate the use of imaging flow cytometry for the detection of γ‐H2AX foci in cells exposed to ionizing radiation causing the induction of DNA strand breaks. In this report we extend these studies and show an enhancement of foci quantitation and image resolution using next generation imaging flow cytometry with the Amnis ImagestreamX Mark II. We demonstrate using cell lines derived from normal individuals, and DNA double strand break repair defective cells that the number of foci observed is significantly increased when using 60× as compared to 40× magnification. Also, foci numbers and resolution is further increased with the application of the focus stacking (Extended Depth of Field–EDF) capacity activated. This report represents the first such demonstration of multimagnification and EDF for the enhanced quantitation of DNA damage in cells and provides a level of resolution, which near matches in situ microscopy methods for the detection of γ‐H2AX foci. © 2015 The Authors. Published by Wiley Periodicals Inc. on behalf of ISAC.

The use of polyADPribose polymerase inhibitors in cancer treatment provides a unique opportunity to target DNA repair processes in cancer cells while leaving normal tissue intact. The PARP-1 enzyme repairs DNA single strand breaks (SSB). Therefore PARP-1 inhibition in BRCA1 negative cancers results in the formation of cytotoxic DNA double strand breaks (DSB) causing synthetic lethality. The use of PARP1 inhibitors is gaining momentum in the treatment of a variety of tumours with BRCA1 involvement including breast, ovarian, pancreatic and prostate cancer.Our previous work showed that the PARP-1 inhibitor Olaparib causes both hypersensitivity of BRCA1+/- cells following exposure to gamma radiation due to the persistence of DNA strand breaks in cells, measured by the DNA damage biomarker γ-H2AX. Therefore dual treatment of cancers with radiotherapy and PARP1 inhibition may lead to cases of increased normal tissue toxicity in cancer patients.In this study we exposed two normal lymphoblastoid cell lines and three heterozygous BRCA1 lymphoblastoid cell lines to the PARP-1 inhibitor Olaparib and gamma radiation and after measured BRCA1 protein expression and apoptosis levels following treatment. BRCA1 protein foci analysis was performed on cells exposed to 2 Gy radiation in the presence or absence of 5 μM Olaparib. Using immunofluorescence and imaging flow cytometry, foci were measured in untreated cells and at 0.5, 3, 5 and 24 hours post-irradiation. Exposing normal and BRCA1+/- cells to Olaparib followed by gamma radiation results in a dramatic change in BRCA1 protein foci expression, with a significant reduction in BRCA1 protein expression observed in the heterozygote cells, together with an increase in apoptosis levels in these cells.In conclusion, combining PARP1 inhibitors with radiotherapy in treating of BRCA1-related cancers has clinical relevance, however this study and our previous publications serve to highlight the potential problems of increased side effects in these scenarios.

Significant challenges persist in developing novel, less toxic, more effective therapies for clinically challenging cancers with relatively low response rates to treatment. Moreover, additional treatment options for refractory and drug resistant cancers are urgently required. Annexin-A1 (ANXA1), a member of a protein superfamily that binds acidic phospholipids in a calcium-dependent manner, is normally localized in cell cytoplasm or plasma membrane. However, ANXA1 can be secreted in response to cell activation and can then modulate the behaviors of nearby cells by binding to the formyl peptide receptor 2 (FPR2). Current literature strongly suggests that ANXA1 can act as a tumor suppressor or an oncogene dependent on cancer indication/subtype, playing a significant role in many upstream signalling pathways that direct cancer proliferation, angiogenesis, drug resistance, invasion and metastasis. Our hypothesis was that annexin-A1 would be a novel target for anti-cancer therapy. We have developed a humanized antibody targeting ANXA1 (MDX-124) and here we present data showing its cytotoxic effect on a panel of cancer cell lines and in a mouse model of triple negative breast cancer. Results: Incubation of a selected panel of breast, colorectal, pancreatic and ovarian cancer cell lines with MDX-124 (0-10 µM) for 72h resulted in a statistically significant reduction in cell survival compared to incubation with an IgG isotype control which binds in a non-specific manner or a commercially available anti-ANXA1 polyclonal antibody. MDX-124 has been assessed for binding affinity to ANXA1 by Biacore and found to have a low nM KD, whilst the potency of MDX-124 was evaluated to be in the low nM range using MCF-7 cells in the CellTiter-Glo® luminescent cell viability assay. ANXA1 localization was also assessed across the panel of cell lines using imaging flow cytometry to quantitate ANXA1 in different cellular compartments. We found the cytotoxic effect of MDX-124 correlated with strong ANXA1 membrane staining. Further in-vivo studies utilized MDX-124 (1 mg/kg) in the syngeneic orthotopic 4T1-luc triple negative breast cancer model. Following dosing once a week we identified a statistically significant reduction in the rate of increase in tumor volume compared to the vehicle control which was not associated with any significant change in body weight. Therefore, our results demonstrate that cancer cell growth is suppressed by inhibition of ANXA1 and indicate that MDX-124 is a promising targeted antibody therapeutic against cancers overexpressing ANXA1. Further studies are under way to determine the mechanism of cell kill by MDX-124. Citation Format: Fiona C. Dempsey, Henry C. Hays, Scott J. Crichton, Hussein Al-Ali, Christopher N. Parris. Annexin-A1 as a potential therapeutic target for solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3356.

Major recent advances have increased the understanding of both the molecular mechanisms and the immune response in cancer. This has led to the discovery of new agents that regulate the tumor immune microenvironment. Annexin-A1 (ANXA1) is a phospholipid binding protein that has been shown to have immunomodulatory effects on T-cells, macrophages and dendritic cells. Although normally localized in the cytoplasm, ANXA1 is secreted in response to a number of different stimuli and can modulate cellular functions through interactions with formyl peptide receptors (FPR1/2). Overexpression of ANXA1 has been observed in several cancer types including triple-negative breast (TNBC), lung, pancreatic and ovarian, and correlates with poor prognosis and decreased overall survival. Furthermore, ANXA1 has also been shown to influence cancer cell proliferation, angiogenesis, migration and drug resistance. Hence, we explored the role of MDX-124, a novel humanized antibody targeting ANXA1, and here present data from several pre-clinical cancer models. Results: Incubation of a panel of cell lines representing several histological cancer subtypes with MDX-124 for 72h resulted in a statistically significant dose-dependent reduction in cell viability compared to control. ANXA1 expression in the subcellular compartments (nucleus, cytoplasm and membrane) was quantified in a panel of cancer cell lines via imaging flow cytometry and the anti-proliferative effect of MDX-124 was shown to correlate with membrane ANXA1 expression. Using a transwell assay, TNBC and acute myeloid leukemia cell lines exposed to MDX-124 exhibited a statistically significant reduction in migration compared to untreated controls. We also observed that MDX-124 induced antibody dependent cellular cytotoxicity (ADCC) activity in a dose-dependent manner using a reporter bioassay (EC50 = 0.38 nM). MDX-124 was further evaluated in an additional panel of in-vitro assays and shown to exert consistent anti-cancer activity. In conclusion, our data indicate that targeting ANXA1 with MDX-124 inhibits tumorigenic processes and induces an immune response in tumors overexpressing ANXA1. MDX-124 therefore provides an innovative approach to cancer therapy. Investigations are continuing to explore the efficacy of MDX-124 in syngeneic mouse models as both a single agent and in combination with standard of care agents. Citation Format: Fiona C. Dempsey, Hussein Al-Ali, Scott J. Crichton, Chris Pepper, Christopher N. Parris. MDX-124, a novel annexin-A1 antibody, induces an anti-tumor immune response and wide-ranging anti-cancer activity in multiple preclinical models [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 1874.

590 Background: Pancreatic cancer is a highly fatal disease with poor survival and response to both chemotherapy and immunotherapy. Novel approaches to treat this disease are urgently required. Annexin-A1 (ANXA1) is secreted in response to several physiological stimuli where it activates formyl peptide receptors (FPR1/2) triggering multiple oncogenic processes. High ANXA1 expression in pancreatic cancer patients is associated with poor overall survival, and influences cancer progression, drug sensitivity, migration and invasion. MDX-124 is a novel humanized antibody targeting ANXA1 and we have previously presented data demonstrating its significant antiproliferative activity. Here we present further data showing the efficacy of MDX-124 in several preclinical models of pancreatic cancer. Methods: In-vitro models utilized MIA PaCa-2, PANC-1 or BxPC-3 human pancreatic cancer cell lines. Cell cycle progression was evaluated by measuring changes in DNA content via flow cytometry. Pancreatic cancer cell viability following incubation with MDX-124 (0-10 µM) and 5FU (IC50) was assessed via MTT assay. A transwell migration assay was used to evaluate the effect of MDX-124 (0-50 µM) on pancreatic cancer cell migration. In-vivo efficacy was evaluated using an orthotopic mouse model of metastatic pancreatic cancer (FC1242luc/zsGreen; KPC-derived cell line) with bioluminescent imaging used to quantify the incidence and burden of lung metastases. Results: When compared to untreated MIA PaCa-2 pancreatic cancer cells, MDX-124 treatment decreased the proportion of cells in S-phase by 29% and G2 phase by 9.1%, with a concomitant increase in G1 of 38.1%. This occurred in a dose-dependent manner and is consistent with an MDX-124 mediated increase in cell cycle arrest. MDX-124 significantly reduced the viability of MIA PaCa-2 and PANC-1 cell lines versus an IgG control in a dose-dependent manner. Additionally in these two cell lines, combination of MDX-124 with 5FU (IC50) had a significant synergistic impact reducing cancer cell viability by 99.8% and 91.2% respectively. Furthermore, MDX-124 significantly reduced the migratory ability of MIA PaCa-2 and BxPC-3 pancreatic cancer cells. In the orthotopic model of metastatic pancreatic cancer, the murine analog of MDX-124 (MDX-001), markedly reduced both the incidence and size of lung metastases. Conclusions: MDX-124 demonstrated significant anti-tumor efficacy in several preclinical models of pancreatic cancer as a single agent, with increased potency observed when used in combination with 5FU. Medannex will initiate a First-In-Human study in Q4 2021 to evaluate MDX-124 in solid malignancies, including pancreatic cancer.