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Background: Aryl hydrocarbon receptor (AhR) is a transcription factor that is involved in the regulation of immunity. AhR inhibits T cell activation in tumors, which induces immune suppression in the blood and solid tumors. We identified effective small-molecule AhR antagonists for cancer immunotherapy. Methods: A new series of pyrazolopyrimidine derivatives was synthesized and evaluated for AhR antagonistic activity. Results: Compound 7k exhibited significant antagonistic activity against AhR in a transgenic zebrafish model. In addition, 7k exhibited good AhR antagonist activity, with a half-maximal inhibitory concentration (IC50) of 13.72 nM. Compound 7k showed a good pharmacokinetic profile with an oral bioavailability of 71.0% and a reasonable half-life of 3.77 h. Compound 7k selectively exerted anti-proliferative effects on colorectal cancer cells without affecting normal cells, concurrently suppressing the expression of AhR-related genes and the PD-1/PD-L1 signaling pathway. Compound 7k exhibited potent antitumor activity in syngeneic colorectal cancer models. Importantly, the combination of anti-PD1 and compound 7k enhanced antitumor immunity by augmenting cytotoxic T lymphocyte (CTL)-mediated activity. Conclusions: Collectively, a new pyrazolopyrimidine derivative, 7k, shows promise as a potential therapeutic agent for treating colorectal cancer.

AbstractTo improve ex vivo gene therapy strategies involving hematopoietic stem and progenitor cells (HSPCs), we propose a novel knock-in strategy (named KI-Ep) aiming to achieve transgene regulation of the inserted cassette through the acquisition of naturally occurring epigenetic marks. Based on this hypothesis, we selected CX3CR1 (a myeloid-specific gene presenting a poised histone signature on primitive HSPCs) as safe harbor to generate KI-Ep HSPCs. We demonstrated that, unlike the expression pattern achieved with lentiviral vectors (LVs), the insertion of a constitutive expression cassette into the intron 1 of the CX3CR1 locus (CX3CR1-I) in HSPCs resulted in very low expression levels in the more primitive HSPCs but, crucially, strong expression in HSPC-differentiated populations (especially myeloid cells), both in vitro and in vivo. Furthermore, we showed that the promoter of the expression cassette inserted into CX3CR1-I acquired epigenetic marks associated with poised genes during the HSPC stage. These marks transitioned to activated histone states upon KI-Ep HSPCs differentiation. In summary, here, we introduce the KI-Ep concept which enables the epigenetic modulation of the inserted transgene during the HSPCs stem cell stages and its subsequent activation upon differentiation. Graphical Abstract Graphical Abstract

AML is characterized by mutations in genes associated with growth regulation such as internal tandem duplications (ITD) in the receptor kinase FLT3. Inhibitors targeting FLT3 (FLT3i) are being used to treat patients with FLT3-ITD+ but most relapse and become resistant. To elucidate the resistance mechanism, we compared the gene regulatory networks (GRNs) of leukemic cells from patients before and after relapse, which revealed that the GRNs of drug-responsive patients were altered by rewiring their AP-1-RUNX1 axis. Moreover, FLT3i induces the upregulation of signaling genes, and we show that multiple cytokines, including interleukin-3 (IL-3), can overcome FLT3 inhibition and send cells back into cycle. FLT3i leads to loss of AP-1 and RUNX1 chromatin binding, which is counteracted by IL-3. However, cytokine-mediated drug resistance can be overcome by a pan-RAS inhibitor. We show that cytokines instruct AML growth via the transcriptional regulators AP-1 and RUNX1 and that pan-RAS drugs bypass this barrier. Subject area: Pharmacy, Molecular biology