Recent clinical evidence has demonstrated that microsatellite instability (MSI) or defective mismatch repair (MMR) and high tumor mutational load can predict response to the programmed cell death 1 (PD-1) receptor inhibitor pembrolizumab in metastatic colorectal cancer (mCRC). Mutations in polymerase ε (), a DNA polymerase involved in DNA replication and repair, contribute to an ultramutated but microsatellite stable (MSS) phenotype in colorectal tumors that is uniquely distinct from MSI tumors. This report presents the first case in the literature describing a clinical response to pembrolizumab in an 81-year-old man with treatment-refractory mCRC characterized by an MSS phenotype and mutation identified on genomic profiling by next-generation sequencing. On tumor immunostaining, a large amount of CD8-positive tumor infiltrating lymphocytes (TILs) were present, with>90% of these expressing PD-1. More than 99% of PD-L1 expression was identified on nontumor cells in the tumor microenvironment that were close to the PD-1-positive CD8 TILs. mCRC tumors harboring mutations represent a hypermutated phenotype that may predict response to anti-PD-1 therapy.

Background Metastatic colorectal cancers (MCRCs) with microsatellite stability (MSS) are resistant to immunotherapy with programmed cell death protein 1 (PD‐1) and programmed death‐ligand 1 inhibitors. However, the addition of regorafenib to nivolumab was recently associated with a high response rate and a protracted progression‐free survival in a small cohort of MSS Japanese patients with metastatic colorectal cancer. Materials and Methods We evaluated the outcome of patients with MSS metastatic colorectal cancer who were treated on a compassionate basis with PD‐1 inhibitors in combination with regorafenib in a single U.S. center. Results A total of 18 patients were treated with a combination of regorafenib and PD‐1 inhibitors. No treatment‐related grade 3 or above toxicities were noted. Thirteen patients (69%) had progressive disease, and five patients (31%) experienced stable disease as best response. Four out of five stable diseases occurred in patients without liver metastases, whereas only 1 of 14 patients with history of liver metastases had a short disease stabilization. A rise in circulating tumor DNA (ctDNA) at the 4‐week time pointuniversally predicted tumor progression at 2 months, whereas a decline was associated with radiographic disease stabilization. Conclusions Regorafenib and nivolumab combination was associated with modest clinical activity in patients with MSS chemotherapy‐resistant metastatic colorectal cancer. Selection for patients without history of liver metastases may identify a cohort of patients with MSS colorectal cancer with a higher likelihood of benefit from this combination. ctDNA may represent a powerful tool for predicting early therapeutic efficacy of immunotherapy in the MSS colorectal cancer population. Implications for Practice This study showed that the combination of regorafenib and nivolumab was associated with a modest clinical activity in patients with advanced microsatellite stability (MSS) metastatic colorectal cancer. This combination should be avoided in clinical practice, especially in patients with MSS colorectal cancer with liver metastases. Further investigation of regorafenib plus PD‐1 inhibitors should be considered in MSS colorectal cancer without liver metastases.

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Key Points Question Is immunotherapy targeting programmed cell death receptor 1/programmed cell death ligand 1 (PD-1/PD-L1) associated with the presence of liver metastasis at the time of therapy and outcomes among patients with treatment-resistant microsatellite stable (MSS) metastatic colorectal cancer? Findings This cohort study included 95 patients with MSS metastatic colorectal cancer. Patients without liver metastases had a significantly superior objective response rate (19.5% vs 0) and median progression-free survival (4.0 vs 1.5 months) compared with patients with liver metastases; multivariate analysis revealed that the presence of liver metastases was an independent prognostic factor associated with poor outcome of PD-1/PD-L1 therapy. Meaning This cohort study suggests that PD-1/PD-L1 inhibitors should be reinvestigated in prospective trials in patients with MSS metastatic colorectal cancer without liver involvement.

Lgr5 þ stem cells are crucial to gut epithelium homeostasis, and therapies targeting these cells hold promise for treatment of gastrointestinal diseases. Here we report that the non-muscle-myosin-II (NMII) heavy chain Myh9 accumulates at epithelial injury sites in mice distal colon treated with dextran sulphate sodium (DSS). Gut-epithelium-specific Myh9 monoallelic deletion alleviates DSS-induced colonic crypt damage and acute colitis. Consistently, the NMII inhibitor blebbistatin can improve the survival of Lgr5 þ stem cells and the growth of Lgr5 organoids. Mechanistically, inhibition of NMII by blebbistatin or Myh9 monoallelic deletion activates Akt through Rac1 and PAK1, which is essential for the survival and pluripotency of Lgr5 þ cells. These results establish a critical role of the Myh9-Rac1-PAK1-Akt pathway in the maintenance of Lgr5 þ stem cells. As blebbistatin can mitigate DSS-induced colitis and preserve Lgr5 þ colonic stem cells in vivo, our findings provide a potential therapeutic intervention of gastrointestinal epithelium injury and degenerative diseases.

Higher densities of CD8+ TILs, PD-1-expressing CD8+ TILs, and tumor-infiltrating immune cells with a Th1 phenotype in the TME may predict response to checkpoint inhibitors in MSI-H and -mutated mCRC.

The Wnt/β-catenin signaling pathway plays important roles in the progression of colon cancer. DACT1 has been identified as a modulator of Wnt signaling through its interaction with Dishevelled (Dvl), a central mediator of both the canonical and noncanonical Wnt pathways. However, the functions of DACT1 in the WNT/β-catenin signaling pathway remain unclear. Here, we present evidence that DACT1 is an important positive regulator in colon cancer through regulating the stability and sublocation of β-catenin. We have shown that DACT1 promotes cancer cell proliferation in vitro and tumor growth in vivo and enhances the migratory and invasive potential of colon cancer cells. Furthermore, the higher expression of DACT1 not only increases the nuclear and cytoplasmic fractions of β-catenin, but also increases its membrane-associated fraction. The overexpression of DACT1 leads to the increased accumulation of nonphosphorylated β-catenin in the cytoplasm and particularly in the nuclei. We have demonstrated that DACT1 interacts with GSK-3β and β-catenin. DACT1 stabilizes β-catenin via DACT1 -induced effects on GSK-3β and directly interacts with β-catenin proteins. The level of phosphorylated GSK-3β at Ser9 is significantly increased following the elevated expression of DACT1 . DACT1 mediates the subcellular localization of β-catenin via increasing the level of phosphorylated GSK-3β at Ser9 to inhibit the activity of GSK-3β. Taken together, our study identifies DACT1 as an important positive regulator in colon cancer and suggests a potential strategy for the therapeutic control of the β-catenin-dependent pathway.

Conflict of interest statement: M.K. and D.M. are inventors on the patent application submitted by COH that covers the design of oligonucleotides presented in this report. M.K. is a co-founder of iSTAT Therapeutics Inc. and a scientific advisor to Scopus Biopharma Inc., companies focused on oligonucleotidebased cancer immunotherapies. All other authors declare no potential conflict of interest. Total word count (whole manuscript): 7872 Total number of figures and tables: 6 References: 50 Summary: The combination of STAT3 targeting and TLR9 stimulation within head and neck cancerassociated myeloid cells enhances T cell-mediated antitumor immune responses after local radiation therapy.