General control nonderepressible 2 (GCN2) plays a major role in the cellular response to amino acid limitation. Although maintenance of amino acid homeostasis is critical for tumor growth, the contribution of GCN2 to cancer cell survival and proliferation is poorly understood. In this study, we generated GCN2 inhibitors and demonstrated that inhibition of GCN2 sensitizes cancer cells with low basal-level expression of asparagine synthetase (ASNS) to the antileukemic agent l-asparaginase (ASNase) in vitro and in vivo. We first tested acute lymphoblastic leukemia (ALL) cells and showed that treatment with GCN2 inhibitors rendered ALL cells sensitive to ASNase by preventing the induction of ASNS, resulting in reduced levels of de novo protein synthesis. Comprehensive gene-expression profiling revealed that combined treatment with ASNase and GCN2 inhibitors induced the stress-activated MAPK pathway, thereby triggering apoptosis. By using cell-panel analyses, we also showed that acute myelogenous leukemia and pancreatic cancer cells were highly sensitive to the combined treatment. Notably, basal ASNS expression at protein levels was significantly correlated with sensitivity to combined treatment. These results provide mechanistic insights into the role of GCN2 in the amino acid response and a rationale for further investigation of GCN2 inhibitors for the treatment of cancer.
Lymphocyte migration is mediated by G protein–coupled receptors (GPCRs) that respond to chemoattractive molecules. After their activation, GPCRs are phosphorylated by different GPCR kinases (GRKs), which produces distinct functional outcomes through β-arrestins. However, the molecular machinery that targets individual GRKs to activated GPCRs remains elusive. Here, we identified a protein complex consisting of copper metabolism MURR1 domain–containing (COMMD) 3 and COMMD8 (COMMD3/8 complex) as an adaptor that selectively recruits a specific GRK to chemoattractant receptors and promotes lymphocyte chemotaxis. COMMD8, whose stability depended on COMMD3, was recruited to multiple chemoattractant receptors. Deficiency of COMMD8 or COMMD3 impaired B cell migration and humoral immune responses. Using CXC-chemokine receptor 4 (CXCR4) as a model, we demonstrated that the COMMD3/8 complex selectively recruited GRK6 and induced GRK6-mediated phosphorylation of the receptor and activation of β-arrestin–mediated signaling. Thus, the COMMD3/8 complex is a specificity determinant of GRK targeting to GPCRs and represents a point of regulation for immune responses.
General
control nonderepressible 2 (GCN2) is a master regulator
kinase of amino acid homeostasis and important for cancer survival
in the tumor microenvironment under amino acid depletion. We initiated
studies aiming at the discovery of novel GCN2 inhibitors as first-in-class
antitumor agents and conducted modification of the substructure of
sulfonamide derivatives with expected type I half binding on GCN2.
Our synthetic strategy mainly corresponding to the αC-helix
allosteric pocket of GCN2 led to significant enhancement in potency
and a good pharmacokinetic profile in mice. In addition, compound 6d, which showed slow dissociation in binding on GCN2, demonstrated
antiproliferative activity in combination with the asparagine-depleting
agent asparaginase in an acute lymphoblastic leukemia (ALL) cell line,
and it also displayed suppression of GCN2 pathway activation with
asparaginase treatment in the ALL cell line and mouse xenograft model.
To investigate the binding of 5′–CpG–3′ sequences by small molecules, two pyrrole (Py)–imidazole (Im) hairpin polyamides, PyImPyIm–γ–PyImPyIm–β–Dp (1) and PyIm–β–Im–γ–PyIm–β–Im–β–Dp (2), which recognize the sequence 5′–CGCG–3′, were synthesized. The binding affinities of the 5′–CGCG–3′ sequence to the Py–Im hairpin polyamides were measured by surface plasmon resonance (SPR) analysis. SPR data revealed that dissociation equilibrium constants (Kd) of polyamides 1 and 2 were 1.1 (± 0.3) × 10–6 M and 1.7 (± 0.4) × 10–8 M, respectively. Polyamide 2 possesses great binding affinity for this sequence, 65-fold higher than polyamide 1. Moreover, when all cytosines in 5′–CpGpCpG–3′ were replaced with 5-methylcytosines (mCs), the Kd value of polyamide 2 increased to 5.8 (± 0.7) × 10–9 (M), which indicated about 3-fold higher binding than the unmethylated 5′–CGCG–3′ sequence. These results suggest that polyamide 2 would be suitable to target CpG-rich sequences in the genome.
The sequence-specific DNA alkylation by conjugates 4 and 5, which consist of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides and 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) linked with an indole linker, was investigated in the absence or presence of partner Py-Im polyamide 6. High-resolution denaturing polyacrylamide gel electrophoresis revealed that conjugate 4 alkylates DNA at the sequences 5'-(A/T)GCCTA-3' through hairpin formation, and alkylates 5'-GGAAAGAAAA-3' through an extended binding mode. However, in the presence of partner Py-Im polyamide 6, conjugate 4 alkylates DNA at a completely different sequence, 5'-AGGTTGTCCA-3'. Alkylation of 4 in the presence of 6 was effectively inhibited by a competitor 7. Surface plasmon resonance (SPR) results indicated that conjugate 4 does not bind to 5'-AGGTTGTCCA-3', whereas 6 binds tightly to this sequence. The results suggest that alkylation proceeds through heterodimer formation, indicating that this is a general way to expand the recognition sequence for DNA alkylation by Py-Im seco-CBI conjugates.
Consecutive mRNA vaccinations against SARS-CoV-2 reinforced both innate and adaptive immune responses. However, it remains unclear whether the enhanced innate immune responses are mediated by epigenetic regulation and, if so, whether these effects persist. Using mass cytometry, RNA-seq, and ATAC-seq, we show that BNT162b2 mRNA vaccination upregulated antiviral and IFN-stimulated gene expression in monocytes with greater effects after the second vaccination than those after the first vaccination. Transcription factor-binding motif analysis also revealed enriched IFN regulatory factors and PU.1 motifs in accessible chromatin regions. Importantly, although consecutive BNT162b2 mRNA vaccinations boosted innate immune responses and caused epigenetic changes in isolated monocytes, we showed that these effects occur only transiently and disappear 4 weeks after the second vaccination. Furthermore, single-cell RNA sequencing analysis revealed that a similar gene signature was impaired in the monocytes of unvaccinated COVID-19 patients with acute respiratory distress syndrome. These results reinforce the importance of the innate immune response in the determination of COVID-19 severity but indicate that, unlike adaptive immunity, innate immunity is not unexpectedly sustained even after consecutive vaccination. This study, which focuses on innate immmune memory, may provide novel insights into the vaccine development against infectious diseases.
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