CDC-like kinase phosphorylation of serine/arginine-rich proteins is central to RNA splicing reactions. Yet, the genomic network of CDC-like kinase-dependent RNA processing events remains poorly defined. Here, we explore the connectivity of genomic CDC-like kinase splicing functions by applying graduated, short-exposure, pharmacological CDC-like kinase inhibition using a novel small molecule (T3) with very high potency, selectivity, and cell-based stability. Using RNA-Seq, we define CDC-like kinase-responsive alternative splicing events, the large majority of which monotonically increase or decrease with increasing CDC-like kinase inhibition. We show that distinct RNA-binding motifs are associated with T3 response in skipped exons. Unexpectedly, we observe dose-dependent conjoined gene transcription, which is associated with motif enrichment in the last and second exons of upstream and downstream partners, respectively. siRNA knockdown of CLK2-associated genes significantly increases conjoined gene formation. Collectively, our results reveal an unexpected role for CDC-like kinase in conjoined gene formation, via regulation of 3′-end processing and associated splicing factors.

Neoadjuvant therapy for locally advanced rectal cancer is becoming increasingly common. However, biomarkers predicting the response to neoadjuvant therapy have not been established. Tumor‐infiltrating lymphocytes (TILs) have a crucial effect on tumor progression and survival outcome as the primary host immune response, and an antitumor immune effect has been reported to contribute to the response to radiotherapy and chemotherapy. We investigated the significance of TILs before and after neoadjuvant treatment and the change in the density of those TILs. Sixty‐four patients who underwent radical resection after neoadjuvant treatment for locally advanced rectal cancer were enrolled. The number of TIL subsets was examined using immunohistochemical staining of pretreatment biopsy samples and post‐treatment resected specimens. In both the neoadjuvant chemotherapy cohort and the neoadjuvant chemoradiotherapy cohort, a low density of CD8+ TILs in pretreatment biopsy samples was associated with a poor response, and a low density of CD8+ TILs in post‐treatment resected specimens was similarly associated with a poor response. In the neoadjuvant chemoradiotherapy cohort, the density of CD8+ TILs in post‐treatment resected specimens was significantly increased compared with that in pretreatment biopsy samples. We concluded that T lymphocyte‐mediated immune reactions play an important role in tumor response to neoadjuvant treatment for rectal cancer, and the evaluation of TILs in pretreatment biopsy samples might be a predictor of the clinical effectiveness of neoadjuvant treatment. Furthermore, neoadjuvant therapy, especially chemoradiotherapy, could induce the activation of the local immune status.

BackgroundInflammation is widely recognized to play an important role in cancer progression, and the peripheral monocyte count has been reported to correlate with the prognosis in patients with colorectal cancer. This is based on the hypothesis that the peripheral monocyte level and the density of tumor-associated macrophages (TAMs) in the cancer microenvironment correlate with each other. However, the influence of TAMs on the prognosis and the correlation between the peripheral monocyte count and the density of TAMs have not yet been elucidated.MethodsA total of 168 patients with stage II/III colorectal cancer were enrolled in this study. Preoperative blood samples were obtained at the time of the diagnosis before surgery. The expression of TAMs in the cancer microenvironment was assessed by immunohistochemistry.ResultsThe progression-free and overall survival rate were significantly worse in the high-TAMs group than in the low-TAMs group (p = 0.0012 and p = 0.0207, respectively). The peripheral monocyte count was significantly associated with the number of TAMs (correlation coefficients: 0.202, p = 0.047).ConclusionsThe peripheral monocyte count was associated with the density of the TAMs, which created a microenvironment favorable for cancer development and were correlated with a poor prognosis. Therefore, the peripheral monocyte count is a useful prognostic marker reflecting the status of the tumor microenvironment.

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The RNA helicase EIF4A3 regulates the exon junction complex and nonsense-mediated mRNA decay functions in RNA transcript processing. However, a transcriptome-wide network definition of these functions has been lacking, in part due to the lack of suitable pharmacological inhibitors. Here we employ short-duration graded EIF4A3 inhibition using small molecule allosteric inhibitors to define the transcriptome-wide dependencies of EIF4A3. We thus define conserved cellular functions, such as cell cycle control, that are EIF4A3 dependent. We show that EIF4A3-dependent splicing reactions have a distinct genome-wide pattern of associated RNA-binding protein motifs. We also uncover an unanticipated role of EIF4A3 in the biology of RNA stress granules, which sequester and silence the translation of most mRNAs under stress conditions and are implicated in cell survival and tumour progression. We show that stress granule induction and maintenance is suppressed on the inhibition of EIF4A3, in part through EIF4A3-associated regulation of G3BP1 and TIA1 scaffold protein expression.