Despite the identification of MYCN amplification as an adverse prognostic marker in neuroblastoma, MYCN inhibitors have yet to be developed. Here, by integrating evidence from a whole-genome shRNA library screen and the computational inference of master regulator proteins, we identify transcription factor activating protein 4 (TFAP4) as a critical effector of MYCN amplification in neuroblastoma, providing a novel synthetic lethal target. We demonstrate that TFAP4 is a direct target of MYCN in neuroblastoma cells, and that its expression and activity strongly negatively correlate with neuroblastoma patient survival. Silencing TFAP4 selectively inhibits MYCN-amplified neuroblastoma cell growth both in vitro and in vivo, in xenograft mouse models. Mechanistically, silencing TFAP4 induces neuroblastoma differentiation, as evidenced by increased neurite outgrowth and upregulation of neuronal markers. Taken together, our results demonstrate that TFAP4 is a key regulator of MYCN-amplified neuroblastoma and may represent a valuable novel therapeutic target.

Background: The PI3K/Akt/mTOR pathway in part impacts tumorigenesis through modulation of host immune activity. To assess the effects of Akt inhibition on the tumor micro-environment (TME), we analyzed tumor tissue from patients with operable hormone receptor positive, HER2 negative breast cancer (BC) treated on a presurgical trial with the Akt inhibitor MK-2206. Methods: Quantitative multiplex immunofluorescence (qmIF) was performed using CD3, CD8, CD4, FOXP3, CD68, and pancytokeratin on biopsy and surgical specimens of MK-2206 and untreated, control patients. nanoString was performed on surgical specimens to assess mRNA expression from MK-2206-treated vs. control patients. Results: Increased CD3+CD8+ density was observed in post vs. pre-treatment tissue in the MK-2206-treated vs. control patients (87 vs. 0.2%, p < 0.05). MK-2206 was associated with greater expression of interferon signaling genes (e.g., IFI6, p < 0.05) and lower expression of myeloid genes (CD163, p < 0.05) on differential expression and gene set enrichment analyses. Greater expression of pro-apoptotic genes (e.g., BAD) were associated with MK-2206 treatment (p < 0.05). Conclusion: Akt inhibition in operable BC was associated with a favorable immune profile in the TME, including increased CD3+CD8+ density and greater expression of interferon genes. Additional studies are warranted, as this may provide rationale for combining Akt inhibition with immunotherapy.

High-dose radiation (HDRT) not only has a direct cytotoxic effect on tumor cells but can also effect the tumor vasculature. Our study examined HDRT and Notch signaling in a xenograft model of neuroblastoma. We observed an increased Notch signaling in endothelial cells after HDRT. Notch1 inhibition augments the effect of HDRT on endothelial cell loss and reduces radiationinduced endothelial-tomesenchymal transition. Purpose: The aim of this study is to characterize the effects of high-dose radiation therapy (HDRT) on Notch signaling components of the tumor vasculature. Methods and Materials: Human umbilical vein endothelial cells monolayers were exposed to different single fraction doses of irradiation; ribonucleic acid RNA was isolated and polymerase chain reaction was performed for Notch signaling components. The vascular response to radiation therapy was examined in a xenograft model of neuroblastoma. Tumors were treated with 0 Gy, 2 Gy, and 12 Gy single fraction doses and analyzed by double immunofluorescence staining for Notch1, Notch ligands Jagged1 and Dll4, and the endothelial cell (EC) marker endomucin. To assess the role of Notch in vivo, NGP xenograft tumors expressing Fc or Notch1-1-24-decoy (a novel Notch inhibitor) were treated with 0 Gy and 12 Gy. Immunofluorescence staining for endomucin and endomucin/aSMA was performed to analyze the effect of combination treatment on tumor EC and endothelial-to-mesenchymal-transition (EndMT), respectively. Results: In human umbilical vein endothelial cells monolayers doses !8 Gy increased expression of NOTCH1, JAG1, and Notch target genes HEY1 and HEY2 as early as 6 hours after irradiation. In vivo, 12 Gy significantly increased Notch1 and Jagged1 in tumor ECs compared with 0 Gy or 2 Gy after 72 hours. Combining HDRT with Notch

Purpose: Activating transcription factor 5 (ATF5), a member of the ATF/CREB family transcription factor, has been implicated in the pathogenesis of glioblastoma and other adult tumors. Recently, a novel cell penetrating (CP-d/n-ATF5) peptide has been developed to antagonize ATF5 function. The goal of the current study is to test the efficacy of CP-d/n-ATF5 in several children tumors including neuroblastoma, hepatoblastoma, Ewing sarcoma, and rhabdoid tumor, in vitro and in vivo. Methods: A panel of neuroblastoma cell lines: SK-N-Be(2)C, SK-N-DZ, NGP, IMR-32, NGP, SHEP-21N, KELLY, CHP-212, CHLA-20, CHLA-15 and SK-N-SH; hepatoblastoma cell lines HUH 6 and Hep-G2; Ewing sarcoma cell lines A673, SKNMC, SKNEP1, and TC32; and the rhabdoid cell G401, were treated with vehicle or 50, 100 and 200 μM of CP-d/n-ATF5. Cell viability and apoptosis were assessed after 72 hr by WST-8 and TUNEL assays, respectively. To test in vivo efficacy, SK-N-Be(2)C kidney xenograft tumors were treated with the peptide at dose 50mg/kg, IP injection once per day for first three days and then twice per week. Tumor growth was monitored by bioluminescence imaging and mice were sacked when flux reached a threshold value. Organ metastases were determined by ex vivo imaging. A patient-derived xenograft (PDX) model of rhabdoid tumors was employed where PDX tumors, at 150-200 mm3 size, were enrolled in the penetratin (control) and CP-d/n-ATF5 treatment (same dose as above). Tumors were measured biweekly with a calipers and mice were sacrificed when tumor volume reached a threshold (1500 mm3). Results: CP-d/n-ATF5 exerted cytotoxicity or apoptosis, in a dose dependent manner, across a wide panel of pediatric tumor cell lines. In vitro, tumor cell viabilities were reduced 40-70% (P&lt;0.05) and apoptosis was increased 50-80% at 200 μM of CP-d/n-ATF5. In vivo, CP-d/n-ATF5 significantly inhibited SK-N-Be(2)C xenograft growth in nude mice, with a median survival of 35 days for control against 21 days for CP-d/n-ATF5, P=0.0013. CP-d/n-ATF5 also reduced SK-N-Be(2)C metastatic burden in the liver (P&lt;0.05) and bone marrow (P&lt;0.01). In the rhabdoid PDX model, there was a significant inhibition of tumors treated with CP-d/n-ATF5 as compared to Penetratin treatment, with a mean tumor volume of control 1283 ± 266.6 mm3 (n=5) vs CP-d/n-ATF5 234.2 ± 50.66 mm3 (n=6), at day 10 post treatment. Penetratin treated tumors showed a median post-treatment time of 13 days to reach threshold volume. None of the CP-d/n-ATF5 treated tumors reached the threshold after 28 days of treatment with some tumors demonstrating regression, indicating a profound anti-tumor effect of the peptide. Conclusion: Our study shows that a novel ATF5-targeting peptide CP-d/n-ATF5 has broad and profound cytotoxic and apoptotic effects in several pediatric tumors in vitro and in vivo. Our study also indicates that CP-d/n-ATF5 has the potential to act as an anti-metastatic agent. Citation Format: Debarshi Banerjee, Shuobo Boboila, Cherease Street, Shunpei Okochi, Filemon S. Dela Cruz, Eileen Connolly, Angela Kadenhe-Chiweshe, Darrell Yamashiro. A novel cell-penetrating ATF5 antagonist peptide CP-d/n-ATF5 exerts in vitro and in vivo anti-tumor effects in a broad spectrum of pediatric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 702. doi:10.1158/1538-7445.AM2017-702

Background: In patients (pts) with early stage HER2-positive breast cancer (BC) who are treated with combination neoadjuvant chemotherapy (NAC) and HER2-targeted therapy, recent studies report that increased tumor infiltrating lymphocyte (TIL) density is associated with higher rates of pathologic complete response (pCR) and improved overall survival. However, in the subset of HER2-positive pts who are hormone receptor (HR)-positive, less is known about the relationship between the composition of the tumor immune infiltrate and clinical outcomes. In this study, we sought to characterize the TME of pts with early stage HR+ HER2+ BC using H&E-based TIL scoring and multiplex immunohistochemistry to assess for characteristics associated with achievement of pCR after neoadjuvant therapy. Patients & Methods: We identified 25 pts with HR+ HER2+ early stage BC who were treated with NAC at our institution between 2005 and 2016 for whom pretreatment tissue was available for analysis. Twenty-two pts (88%) received neoadjuvant HER2 antibody therapy along with NAC. Twelve pts (48%) received neoadjuvant pertuzumab in addition to trastuzumab. At the time of surgery, 11 pts achieved pCR and 14 had residual carcinoma (RC). H&E slides from pretreatment biopsy samples were scored for both stromal and intratumoral TILs by a BC pathologist using established guidelines. Additionally, quantitative immunofluorescence (qmIF) was performed on pretreatment biopsy samples for 20 pts (9 with pCR, 11 with RC) using an OPAL antibody panel targeting CD3, CD8, CD68, HLADR, FOXP3, and pancytokeratin. Images were analyzed with the Vectra/inForm software platform (Perkin Elmer) to allow for multiparameter phenotyping. Nearest neighbor analysis to assess median distance between immune cell subtypes and tumor cells was performed using phenoptr software. Results: Per TIL scoring, mean stromal TIL (sTIL) density was 28% in the pCR group and 17% in the RC group (p=0.05). Using a cutoff of ≥10% sTIL density, high sTILs were seen in pretreatment samples in 100% (11/11) of pCR pts and in 57% (8/14) of RC pts (p=0.05). Intraepithelial TIL density was not significantly associated with pCR (p=0.12). Per qmIF analysis, there was a numerical but not statistically significant increase in mean stromal CD3+CD8+ cell density (18% vs 11%) and decrease in stromal CD68+ cell density (10% vs 14%) in the pCR group compared to the RC group. Nearest neighbor analysis revealed that the median distance from tumor cells to CD68+ cells was significantly higher in pts with pCR vs. RC (p=0.02). No significant differences in median distance between CD3+CD8+ cells and tumor or CD3+CD8+ cells and CD68+ cells were detected among the two groups. Conclusions: In a population of early stage HR+ HER2+ breast cancer pts who received neoadjuvant therapy, sTIL density by pathology assessment was associated with increased rates of pCR, supporting the hypothesis that the nature of the TME is biologically significant in the subset of HER2+ pts who are also HR+. qmIF is a useful tool to further characterize the density of specific immune cell subtypes and spatial relationships between cell types in the TME. Further studies in larger cohorts are needed to determine more specific immune biomarkers that predict response to combined chemotherapy and HER2-targeted therapy in pts with HR+ HER2+ BC. Citation Format: Kathleen M. Fenn, Douglas K Marks, Rami Vanguri, Shuobo Boboila, Hua Guo, Hanina Hibshoosh, Kevin Kalinsky, Eileen Connolly. Characterization of the tumor immune microenvironment (TME) in early stage HR-positive HER2-positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-25.