Purpose: Preclinical studies in HER2-amplified gastrointestinal cancer models have shown that cotargeting HER2 with a monoclonal antibody and a small molecule is superior to monotherapy with either inhibitor, but the underlying cooperative mechanisms remain unexplored. We investigated the molecular underpinnings of this synergy to identify key vulnerabilities susceptible to alternative therapeutic opportunities.Experimental Design: The phosphorylation/activation of HER2, HER3, EGFR (HER receptors), and downstream transducers was evaluated in HER2-overexpressing colorectal and gastric cancer cell lines by Western blotting and/or multiplex phosphoproteomics. The in vivo outcome of antibody-mediated HER2 blockade by trastuzumab, reversible HER2 inhibition by lapatinib, and irreversible HER2 inhibition by afatinib was assessed in patient-derived tumorgrafts and cell-line xenografts by monitoring tumor growth curves and by using antibody-based proximity assays.Results: Trastuzumab monotherapy reduced HER3 phosphorylation, with minor consequences on downstream transducers. Lapatinib alone acutely inhibited all HER receptors and effectors but led to delayed rephosphorylation of HER3 and EGFR and partial restoration of ERK and AKT activity. When combined with lapatinib, trastuzumab prevented HER3/EGFR reactivation and caused prolonged inhibition of ERK/AKT. Afatinib alone was also very effective in counteracting the reinstatement of HER3, EGFR, and downstream signaling activation. In vivo, the combination of trastuzumab and lapatinib-or, importantly, monotherapy with afatinib-resulted in overt tumor shrinkage.Conclusions: Only prolonged inhibition of HER3 and EGFR, achievable by dual blockade with trastuzumab and lapatinib or irreversible HER2 inhibition by single-agent afatinib, led to regression of HER2-amplified gastrointestinal carcinomas.

Tn5 is able to tagment compacted chromatin featuring H3K9me3. We first determined whether Tn5 is able to tagment compacted chromatin if properly redirected. To this end, we exploited a transposase-assisted chromatin multiplex immunoprecipitation (TAM-ChIP) approach, which combines the

Immunotherapy with immune checkpoint inhibitors is an approved treatment option for a subpopulation of patients with colorectal cancers that display microsatellite instability. However, not all individuals within this subgroup respond to immunotherapy, and molecular biomarkers for effective patient stratification are still lacking. In this opinion article, we provide an overview of the different biological parameters that contribute to rendering colorectal cancers with microsatellite instability potentially sensitive to immunotherapy. We critically discuss the reasons why such parameters have limited predictive value and the implications therein. We also consider that a more informed knowledge of response determinants in this tumor subtype could help understand the mechanisms of immunotherapy resistance in microsatellite stable tumors. We conclude that the dynamic nature of the interactions between cancer and immune cells complicates conventional biomarker development and argue that a new generation of adaptive metrics, borrowed from evolutionary genetics, may improve the effectiveness and reliability of clinical decision making. Immunotherapy in Colorectal Cancer (CRC): Appearances Can Be DeceivingCancer cells produce mutated antigens, neoantigens (see Glossary), that are captured by and presented at the surface of dendritic cells, in association with major histocompatibility complex (MHC) molecules. Dendritic cells deploy their cargo of tumor neoantigens to prime CD4 + T helper cells and to trigger the activation of CD8 + cytotoxic T cells, which travel to the tumor. Upon infiltration within the tumor microenvironment, activated cytotoxic T cells bind to the cancer cells and destroy them by inducing apoptosis. Cancer cells can forestall this immune attack by expressing PD-L1, a membrane-bound ligand that interacts with the PD-1 receptor exposed on effector T cells and, by doing so, prompts T cell inactivation. Antibodies against PD-L1 or PD-1 intercept this inhibitory immune checkpoint and re-engage immune-mediated cancer cell killing [1].