BCKDK modification enhances the anticancer efficacy of CAR-T cells by reprogramming branched chain amino acid metabolism.

Altered branched chain amino acids (BCAAs), including leucine, isoleucine, and valine, are frequently observed in patients with advanced cancer. We evaluated the efficacy of chimeric antigen receptor (CAR) T cell-mediated cancer cell lysis potential in the immune microenvironment of BCAA supplementation and deletion. BCAA supplementation increased cancer cell killing percentage, while accelerating BCAA catabolism and decreasing BCAA transporter decreased cancer cell lysis efficacy. We thus designed BCKDK engineering CAR T cells for the reprogramming of BCAA metabolism in the tumor microenvironment based on the genotype and phenotype modification. BCKDK overexpression (OE) in CAR-T cells significantly improved cancer cell lysis, while BCKDK knockout (KO) resulted in inferior lysis potential. In an in vivo experiment, BCKDK-OE CAR-T cell treatment significantly prolonged the survival of mice bearing NALM6-GL cancer cells, with the differentiation of central memory cells and an increasing proportion of CAR-T cells in the peripheral circulation. BCKDK-KO CAR-T cell treatment resulted in shorter survival and a decreasing percentage of CAR-T cells in the peripheral circulation. In conclusion, BCKDK-engineered CAR-T cells exert a distinct phenotype for superior anticancer efficiency.

Identification of putative allosteric inhibitors of BCKDK via virtual screening and biological evaluation.

Abnormal accumulation of branched-chain amino acids (BCAAs) can lead to metabolic diseases and cancers. Branched-chain α-keto acid dehydrogenase kinase (BCKDK) is a key negative regulator of BCAA catabolism, and targeting BCKDK provides a promising therapeutic approach for diseases caused by BCAA accumulation. Here, we screened PPHN and POAB as novel putative allosteric inhibitors by integrating allosteric binding site prediction, large-scale ligand database virtual screening, and bioactivity evaluation assays. Both of them showed a high binding affinity to BCKDK, with Kd values of 3.9 µM and 1.86 µM, respectively. In vivo experiments, the inhibitors demonstrated superior kinase inhibitory activity and notable antiproliferative and proapoptotic effects on diverse cancer cells. Finally, bulk RNA-seq analysis revealed that PPHN and POAB suppressed cell growth through a range of signalling pathways. Taken together, our findings highlight two novel BCKDK inhibitors as potent therapeutic candidates for metabolic diseases and cancers associated with BCAA dysfunctional metabolism.

DeepS: a web server for image optical sectioning and super resolution microscopy based on a deep learning framework.

MOTIVATION: Microscopy technology plays important roles in many biological research fields. Solvent-cleared brain high-resolution (HR) 3D image reconstruction is an important microscopy application. However, 3D microscopy image generation is time-consuming and expensive. Therefore, we have developed a deep learning framework (DeepS) for both image optical sectioning and super resolution microscopy. RESULTS: Using DeepS to perform super resolution solvent-cleared mouse brain microscopy 3D image yields improved performance in comparison with the standard image processing workflow. We have also developed a web server to allow online usage of DeepS. Users can train their own models with only one pair of training images using the transfer learning function of the web server. AVAILABILITYAND IMPLEMENTATION: http://deeps.cibr.ac.cn. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Recent Advances in the Discovery of CK2 Allosteric Inhibitors: From Traditional Screening to Structure-Based Design.

Protein kinase (CK2) has emerged as an attractive cancer therapeutic target and recent efforts have been made to develop its inhibitors. However, the development of selective inhibitors remains challenging because of the highly conserved ATP-binding pocket (orthosteric site) of kinase family. As an alternative strategy, allosteric inhibitors, by targeting the much more diversified allosteric site relative to the conserved ATP-binding site, achieve better pharmacological advantages than orthosteric inhibitors. Traditional serendipitous screening and structure-based design are robust tools for the discovery of CK2 allosteric inhibitors. In this review, we summarize the recent advances in the identification of CK2 allosteric inhibitors. Firstly, we briefly present the CK2 allosteric sites. Then, the allosteric inhibitors targeting the well-elucidated allosteric sites (α/ß interface, αD pocket and interface between the Glycine-rich loop and αC-helix) are highlighted in the discovery process and possible binding modes with the allosteric sites are described. This study is expected to provide valuable clues for the design of CK2 allosteric inhibitors.

Identification and Biological Evaluation of CK2 Allosteric Fragments through Structure-Based Virtual Screening.

Casein kinase II (CK2) is considered as an attractive cancer therapeutic target, and recent efforts have been made to develop its ATP-competitive inhibitors. However, achieving selectivity with respect to related kinases remains challenging due to the highly conserved ATP-binding pocket of kinases. Allosteric inhibitors, by targeting the much more diversified allosteric site relative to the highly conserved ATP-binding pocket, might be a promising strategy with the enhanced selectivity and reduced toxicity than ATP-competitive inhibitors. The previous studies have highlighted the traditional serendipitousity of discovering allosteric inhibitors owing to the complicate allosteric modulation. In this current study, we identified the novel allosteric inhibitors of CK2α by combing structure-based virtual screening and biological evaluation methods. The structure-based pharmacophore model was built based on the crystal structure of CK2α-compound 15 complex. The ChemBridge fragment library was searched by evaluating the fit values of these molecules with the optimized pharmacophore model, as well as the binding affinity of the CK2α-ligand complexes predicted by Alloscore web server. Six hits forming the holistic interaction mechanism with the αD pocket were retained after pharmacophore- and Alloscore-based screening for biological test. Compound 3 was found to be the most potent non-ATP competitive CK2α inhibitor (IC50 = 13.0 µM) with the anti-proliferative activity on A549 cancer cells (IC50 = 23.1 µM). Our results provide new clues for further development of CK2 allosteric inhibitors as anti-cancer hits.

Adaptive functions of structural variants in human brain development.

Quantifying the structural variants (SVs) in nonhuman primates could provide a niche to clarify the genetic backgrounds underlying human-specific traits, but such resource is largely lacking. Here, we report an accurate SV map in a population of 562 rhesus macaques, verified by in-house benchmarks of eight macaque genomes with long-read sequencing and another one with genome assembly. This map indicates stronger selective constrains on inversions at regulatory regions, suggesting a strategy for prioritizing them with the most important functions. Accordingly, we identified 75 human-specific inversions and prioritized them. The top-ranked inversions have substantially shaped the human transcriptome, through their dual effects of reconfiguring the ancestral genomic architecture and introducing regional mutation hotspots at the inverted regions. As a proof of concept, we linked APCDD1, located on one of these inversions and down-regulated specifically in humans, to neuronal maturation and cognitive ability. We thus highlight inversions in shaping the human uniqueness in brain development.

De novo genes with an lncRNA origin encode unique human brain developmental functionality.

Human de novo genes can originate from neutral long non-coding RNA (lncRNA) loci and are evolutionarily significant in general, yet how and why this all-or-nothing transition to functionality happens remains unclear. Here, in 74 human/hominoid-specific de novo genes, we identified distinctive U1 elements and RNA splice-related sequences accounting for RNA nuclear export, differentiating mRNAs from lncRNAs, and driving the origin of de novo genes from lncRNA loci. The polymorphic sites facilitating the lncRNA-mRNA conversion through regulating nuclear export are selectively constrained, maintaining a boundary that differentiates mRNAs from lncRNAs. The functional new genes actively passing through it thus showed a mode of pre-adaptive origin, in that they acquire functions along with the achievement of their coding potential. As a proof of concept, we verified the regulations of splicing and U1 recognition on the nuclear export efficiency of one of these genes, the ENSG00000205704, in human neural progenitor cells. Notably, knock-out or over-expression of this gene in human embryonic stem cells accelerates or delays the neuronal maturation of cortical organoids, respectively. The transgenic mice with ectopically expressed ENSG00000205704 showed enlarged brains with cortical expansion. We thus demonstrate the key roles of nuclear export in de novo gene origin. These newly originated genes should reflect the novel uniqueness of human brain development.