Search

Introduction: There is strong experimental and clinical data to indicate the critical involvement of immune evasion in relapsed leukemia in children. A well-defined characteristic of refractory leukemia is the accumulation of genetic aberrations and mutations that may act as drivers or passengers in the process of tumor recurrence. Many of these mutations get translated into proteins that contain tumor-specific immune-stimulatory epitopes (neoantigens) that can elicit host antitumor immune responses. Although, in general, the mutation rate is lower in pediatric tumors, recent studies have shown that almost 90% of pediatric leukemias carry potentially actionable neoepitopes. In this study, we describe the results from a comprehensive experimental approach of neoantigen prediction coupled with antigen processing and HLA-binding prediction algorithms with in vitro validation assays for the generation of neoantigen vaccines against high-risk leukemias in children. Methods: DNA and RNA from leukemia cells and matched fibroblasts were obtained. Raw reads were aligned to human reference genome and somatic variants (SNVs) were called using Strelka v1.0.1441. RNA-seq data from leukemic cells were used to predict neoantigen expression levels resulting from SNVs using STAR (2.4.1)12 and Cufflinks v2.2.1. Normalized expression data were then cross-referenced with the list of SNVs to identify leukemia-specific mutant proteins. HLA typing for each sample was carried out from RNA-seq data using seq2HLA v2.2. Using the patient's HLA phenotype, we then used NetMHCons v1.1 to predict short peptides derived from leukemia-specific mutant proteins that will bind to autologous HLA Class I molecules. These 8/9-mers were filtered to predict a high likelihood of proteasomal or immune-proteasomal processing and transporter associated with antigen processing (TAP) using NetChop v3.1 and the immune epitope database (IEDB), respectively. The peptides identified were rank-ordered based on the composite immunogenicity score derived from MHC class I binding affinities, proteasomal processing and TCR binding predictions and synthesized accordingly. Peripheral blood derived dendritic cells (DCs) and CD8+ T-cells were isolated and expanded in culture with relevant cytokines. The DCs were pulsed with peptides and then co-cultured with CD8+ T-cells. After five days, the primed CD8+ T-Cells were separated, washed and exposed to the patient's leukemic cells at varying ratios and the leukemia specific CD8+ T-cell activation was quantified by IFN gamma secretion using ELISpot assays. Results: In the leukemia specimen studied, approximately 5% of all on-target germline mutations were found only in leukemic cells. Tumor mutational burden was, on average, 0.34 mut/Mb. Analysis of the highest ranking synthetic peptides (approximately 10 per leukemia sample) showed leukemia-specific activation of patient's T-cells as measured by the mean number of spots observed in ELISpot assays. For example, in patient one (15 year old male, high-risk ALL, one year off therapy), 14 individual short sequences were identified and corresponding peptides were synthesized. Among these, three peptides were not soluble and three peptides showed significant activity above controls. Maximum leukemia specific T-cell activation was noted with peptide #7 QQSALVLL (mean 135 ELISpots compared to 72 in controls, p Discussion: Completed data, including the vaccine peptide sequences and corresponding activities showed the feasibility of identifying pediatric leukemia neoantigen sequences in personalized mutational landscapes of these patients. In addition, we have provided an in vitro experimental approach to validate the potential of such vaccines in future clinical studies and this methodology can also be used to identify agents for effective combinations such as immune checkpoint inhibitors. A clinical trial using these strategies is in development for the treatment of high-risk leukemia in children. Disclosures No relevant conflicts of interest to declare.

Introduction: In children diagnosed with leukemia, relapse and its associated morbidity and mortality remain the most dreaded consequences of the disease. Therefore, the discovery and implementation of novel and broadly applicable therapeutic strategies for these patients are urgently needed. Currently, a number of precision therapeutic approaches have been formulated where molecular analyses of the malignant cells have been used to inform, often multiple, probable targets and potential therapeutic agents. However, a common drawback of this approach has been the uncertainty involved in selecting the drug with the most and clinically relevant cytotoxic potential. In vitro xenograft approaches, although can provide key information on drug activity and side effects, are time consuming and impractical and cumbersome in most cases. We have recently demonstrated the ability of a bone marrow stromal derived cell line to sustain the growth and survival of patient leukemic cells in culture that has allowed in vitro evaluations of drug response.This methodology was combined with a previously validated molecular pathway analysis program to identify effective agents or combinations for a subsequent informed precision clinical trial. Methods: Gene expression profiles from refractory pediatric leukemic cells were analyzed against similar data from more than 12, 000 tumors and outlier analyses were carried out to generate a list of overexpressed genes. This information was computed to identify hypothetically activated pathways, druggable targets and potential agents from a panel of FDA approved drugs. A bone marrow stromal cell line was established and characterized that has been shown to support leukemia cell proliferation in vitro. Briefly, stromal cells were co-cultured with leukemic cells at pre-determined ratios with and without the drugs identified in the genomic analysis. After four days in culture, leukemic cells were re-suspended and analyzed for proliferation. Target modulation and activated cell death pathways were queried by Western blot analyses. Results: Multiple targets and potential agents for effective therapeutics were identified against an initial set of relapsed leukemia specimens. For example, in patient # P700491 (pre B-ALL) gene expression data sets revealed clustering within the area of ALL and AML in the reference cancer genomics data. Comparative tumor RNA seq outlier analysis showed molecular abnormalities in BTK, JAK3 and PIK3CD, corresponding to molecular categories of RTK, JAK-SAT and PI3K-AKT-mTOR pathways targetable by the drugs Ibrutinib, WHI-P131 and Idelalisib, respectively. However, in vitro studies showed significant cell killing with Idelalisib and not with the other two agents. Target modulation assays showed effective induction of apoptosis including PARP cleavage in Idelalisib treated leukemia cells compared to controls, indicating the feasibility of this approach to effectively identify potentially applicable agents for this individual patient. Conclusions: We demonstrate the ability of a newly cloned bone marrow stromal derived cell line to sustain the growth and survival of patient leukemic cells in culture that has allowed in vitro target modulation and target validation analyses for cytotoxicity. This methodology was combined with a previously demonstrated molecular pathway interrogation program to ascertain effective agents or combinations for an experimentally informed precision clinical trial. Importantly, our data showed that genomically identified actionable targets are not universally predictive of tumor response and an in vitro cytotoxicity analysis step may enhance the accuracy of this approach. We describe the practical advantages and versatility of this work-flow to inform the selection of agents in future umbrella trials. It is anticipated that the information obtained will lead to an applicable clinical trial the near future. Disclosures Narendran: Bayer: Honoraria, Other: CANTRK Advisory Board .

INhibitor of Growth 1 (ING1) expression is repressed in breast carcinomas, but its role in breast cancer development and metastasis is unknown. ING1 levels were quantified in >500 patient samples using automated quantitative fluorescence immunohistochemistry, and data were analysed for correlations to patient outcome. Effects of altering ING levels were examined in microarrays and metastasis assays in vitro, and in a mouse metastasis model in vivo. ING1 levels were lower in tumors compared to adjacent normal breast tissue and correlated with tumor size (p=0.019) and distant recurrence (p=0.001) in ER- or Her2+ patients. In these patients ING1 predicted disease-specific and distant metastasis-free survival. Transcriptome analysis showed that the pathway most affected by ING1 was breast cancer (p = 0.0008). Decreasing levels of ING1 increased, and increasing levels decreased, migration and invasion of MDA-MB231 cells in vitro. ING1 overexpression also blocked cancer cell metastasis in vivo and eliminated tumor-induced mortality in mouse models. Our data show that ING1 protein levels are downregulated in breast cancer and for the first time, we show that altering their levels regulates metastasis in vitro and in vivo, which indicates that ING1 may have a therapeutic role for inhibiting metastasis of breast cancer.

The ING1 epigenetic regulator and tumor suppressor plays a central role in apoptosis. The Ing1 gene is functionally inactivated in many cancer types but is rarely mutated. Although most studies have implicated the major ING1 isoform, p33ING1b, in nuclear apoptotic signalling, we recently discovered a novel and potent apoptosis-inducing effect of p33ING1b translocation to the mitochondria in response to DNA damage. In the present study, we examined the impact of cytoplasmic/mitochondrial localization of p33ING1b in oral squamous cell carcinoma (OSCC) patient samples and explored the therapeutic potential of adenovirally-overexpressed p33ING1b in OSCC cell lines in combination with ionizing radiation (IR) treatment. In contrast with previous reports, we found that p33ING1b protein and mRNA levels are higher in OSCC compared to normal epithelial cells. In OSCC patient samples, higher levels of intra-tumoral cytoplasmic p33ING1b correlated with increased apoptotic markers and significantly better patient survival. This association was strongest in patients who received post-operative radiotherapy. IR treatment induced p33ING1b translocation to the mitochondria and adenoviral-p33ING1b synergized with IR to kill OSCC cells. Our results identify a novel functional relationship between cytoplasmic p33ING1b and patient survival and highlight the potential for the use of p33ING1b as a therapeutic agent in combination with adjuvant radiotherapy in OSCC.

The INhibitor of Growth 1 (ING1) is stoichiometric member of histone deacetylase (HDAC) complexes and functions as an epigenetic regulator and a type II tumor suppressor. It impacts cell growth, aging, apoptosis, and DNA repair, by affecting chromatin conformation and gene expression. Down regulation and mislocalization of ING1 have been reported in diverse tumor types and Ser/Thr phosphorylation has been implicated in both of these processes. Here we demonstrate that both in vitro and in vivo, the tyrosine kinase Src is able to physically associate with, and phosphorylate ING1, which results in a nuclear to cytoplasmic relocalization of ING1 in cells and a decrease of ING1 stability. Functionally, Src antagonizes the ability of ING1 to induce apoptosis, most likely through relocalization of ING1 and down regulation of ING1 levels. These effects were due to both kinase-dependent and kinase-independent properties of Src, and were most apparent at elevated levels of Src expression. These findings suggest that Src may play a major role in regulating ING1 levels during tumorigenesis in those cancers in which high levels of Src expression or activity are present. These data represent the first report of tyrosine kinase-mediated regulation of ING1 levels and suggest that kinase activation can impact chromatin structure through the ING1 epigenetic regulator.