Runt-related transcription factor 1 (RUNX1) is generally considered to function as a tumor suppressor in the development of leukemia, but a growing body of evidence suggests that it has pro-oncogenic properties in acute myeloid leukemia (AML). Here we have demonstrated that the antileukemic effect mediated by RUNX1 depletion is highly dependent on a functional p53-mediated cell death pathway. Increased expression of other RUNX family members, including RUNX2 and RUNX3, compensated for the antitumor effect elicited by RUNX1 silencing, and simultaneous attenuation of all RUNX family members as a cluster led to a much stronger antitumor effect relative to suppression of individual RUNX members. Switching off the RUNX cluster using alkylating agent–conjugated pyrrole-imidazole (PI) polyamides, which were designed to specifically bind to consensus RUNX-binding sequences, was highly effective against AML cells and against several poor-prognosis solid tumors in a xenograft mouse model of AML without notable adverse events. Taken together, these results identify a crucial role for the RUNX cluster in the maintenance and progression of cancer cells and suggest that modulation of the RUNX cluster using the PI polyamide gene-switch technology is a potential strategy to control malignancies.

Recent studies have documented frequent evolution of clones carrying common cancer mutations in apparently normal tissues, which are implicated in cancer development^1–3. However, our knowledge is still missing with regard to what additional driver events take place in what order, before one or more of these clones in normal tissues ultimately evolve to cancer. Here, using phylogenetic analyses of multiple microdissected samples from both cancer and non-cancer lesions, we show unique evolutionary histories of breast cancers harbouring der(1;16), a common driver alteration found in roughly 20% of breast cancers. The approximate timing of early evolutionary events was estimated from the mutation rate measured in normal epithelial cells. In der(1;16)(+) cancers, the derivative chromosome was acquired from early puberty to late adolescence, followed by the emergence of a common ancestor by the patient’s early 30s, from which both cancer and non-cancer clones evolved. Replacing the pre-existing mammary epithelium in the following years, these clones occupied a large area within the premenopausal breast tissues by the time of cancer diagnosis. Evolution of multiple independent cancer founders from the non-cancer ancestors was common, contributing to intratumour heterogeneity. The number of driver events did not correlate with histology, suggesting the role of local microenvironments and/or epigenetic driver events. A similar evolutionary pattern was also observed in another case evolving from an AKT1-mutated founder. Taken together, our findings provide new insight into how breast cancer evolves.

Purpose: Extramammary Paget disease (EMPD) is an uncommon skin malignancy whose genetic alterations are poorly characterized. Previous reports identified mutations in chromatin remodeling genes and PIK3CA. In order to unambiguously determine driver mutations in EMPD, we analyzed 87 EMPD samples using exome sequencing in combination with targeted sequencing. Experimental Design: First, we analyzed 37 EMPD samples that were surgically resected using whole-exome sequencing. Based on several in silico analysis, we built a custom capture panel of putative driver genes and analyzed 50 additional formalin-fixed, paraffin-embedded samples using target sequencing. ERBB2 expression was evaluated by HER2 immunohisotochemistry. Select samples were further analyzed by fluorescence in situ hybridization. Results: A median of 92 mutations/sample was identified in exome analysis. A union of driver detection algorithms identified ERBB2, ERBB3, KMT2C, TP53, PIK3CA, NUP93, AFDN, and CUX1 as likely driver mutations. Copy-number alteration analysis showed regions spanning CDKN2A as recurrently deleted, and ERBB2 as recurrently amplified. ERBB2, ERBB3, and FGFR1 amplification/mutation showed tendency toward mutual exclusivity. Copy-number alteration load was associated with likelihood to recur. Mutational signatures were dominated by aging and APOBEC activation and lacked evidence of ultraviolet radiation. HER2 IHC/fluorescence in situ analysis validated ERBB2 amplification but was underpowered to detect mutations. Tumor heterogeneity in terms of ERBB2 amplification status was observed in some cases. Conclusions: Our comprehensive, unbiased analysis shows EMPD is characterized by alterations involving the PI3K–AKT pathway. EMPD is distinct from other skin cancers in both molecular pathways altered and etiology behind mutagenesis.

Trogocytosis is defined as the transfer of cell-surface membrane proteins and membrane patches from one cell to another through contact. It is reported that human epidermal growth factor receptor 2 (HER2) could be transferred from cancer cells to monocytes via trogocytosis; however, the clinical significance of this is unknown. The aim of this study is to demonstrate the presence and evaluate the clinical significance of HER2+ tumor-infiltrated immune cells (arising through HER2 trogocytosis) in HER2-overexpressing (HER2+) breast cancer patients receiving trastuzumab-based primary systemic therapy (PST). To assess the trogocytosis of HER2 from cancer cells to immune cells, and to evaluate the up- and down-regulation of HER2 on immune and cancer cells, peripheral blood mononuclear cells from healthy volunteers and breast cancer patients were co-cultured with HER2+ and HER2-negative breast cancer cell lines with and without trastuzumab, respectively. The correlation between HER2 expression on tumor-infiltrated immune cells and a pathological complete response (pCR) in HER2+ breast cancer patients treated with trastuzumab-based PST was analyzed. HER2 was transferred from HER2+ breast cancer cells to monocytes and natural killer cells by trogocytosis. Trastuzumab-mediated trogocytosed-HER2+ effector cells exhibited greater CD107a expression than non-HER2-trogocytosed effector cells. In breast cancer patients, HER2 expression on tumor-infiltrated immune cells in treatment naïve HER2+ tumors was associated with a pCR to trastuzumab-based PST. HER2-trogocytosis is visible evidence of tumor microenvironment interaction between cancer cells and immune cells. Given that effective contact between these cells is critical for immune destruction of target cancer cells, this interaction is of great significance. It is possible that HER2 trogocytosis could be used as a predictive biomarker for trastuzumab-based PST efficacy in HER2+ breast cancer patients.

Killer immunoglobulin-like receptor (KIR) 2DL4 (CD158d) was previously thought to be a human NK cell-specific protein. Mast cells are involved in allergic reactions via their KIT-mediated and FcɛRI-mediated responses. We recently detected the expression of KIR2DL4 in human cultured mast cells established from peripheral blood of healthy volunteers (PB-mast), in the human mast cell line LAD2, and in human tissue mast cells. Agonistic antibodies against KIR2DL4 negatively regulate the KIT-mediated and FcɛRI-mediated responses of PB-mast and LAD2 cells. In addition, agonistic antibodies and human leukocyte antigen (HLA)-G, a natural ligand for KIR2DL4, induce the secretion of leukemia inhibitory factor and serine proteases from human mast cells, which have been implicated in pregnancy establishment and cancer metastasis. Therefore, KIR2DL4 stimulation with agonistic antibodies and recombinant HLA-G protein may enhance both processes, in addition to suppressing mast-cell-mediated allergic reactions.

Glioblastoma is the most common adult brain tumour, representing a high degree of malignancy. Transcription factors such as RUNX1 are believed to be involved in the malignancy of glioblastoma. RUNX1 functions as an oncogene or tumour suppressor gene with diverse target genes. Details of the effects of RUNX1 on the acquisition of malignancy in glioblastoma remain unclear. Here, we show that RUNX1 downregulates p21 by enhancing expressions of BIRC5 and PIF1, conferring anti-apoptotic properties on glioblastoma. A gene switch-off therapy using alkylating agent-conjugated pyrrole-imidazole polyamides, designed to fit the RUNX1 DNA groove, decreased expression levels of BIRC5 and PIF1 and induced apoptosis and cell cycle arrest via p21. The RUNX1-BIRC5/PIF1-p21 pathway appears to reflect refractory characteristics of glioblastoma and thus holds promise as a therapeutic target. RUNX gene switch-off therapy may represent a novel treatment for glioblastoma.