Single-cell multi-omics analyses reveal EZH2 as a main driver of retinoic acid resistance in PLZF-RARA leukemia.

Cancer relapse is caused by a subset of malignant cells that are resistant to treatment. To characterize resistant cells and their vulnerabilities, we studied the retinoic acid (RA)-resistant PLZF-RARA acute promyelocytic leukemia (APL) using single-cell multi-omics. We uncovered transcriptional and chromatin heterogeneity in leukemia cells and identified a subset of cells resistant to RA that depend on a fine-tuned transcriptional network targeting the epigenetic regulator Enhancer of Zeste Homolog 2 (EZH2). Epigenomic and functional analyses validated EZH2 selective dependency of PLZF-RARA leukemia and its driver role in RA resistance. Targeting pan-EZH2 activities (canonical/non-canonical) was necessary to eliminate leukemia relapse initiating cells, which underlies a dependency of resistant cells on an EZH2 non-canonical activity and the necessity to degrade EZH2 to overcome resistance. Our study provides critical insights into the mechanisms of RA resistance that allow us to eliminate treatment-resistant leukemia cells by targeting EZH2, thus highlighting a potential targeted therapy approach.

Thioesterase from Cereulide Biosynthesis Is Responsible for Oligomerization and Macrocyclization of a Linear Tetradepsipeptide

Cereulide is a cyclic depsidodecapeptide produced in <i>Bacillus cereus</i> by two non-ribosomal peptide synthetases, CesA and CesB. While highly similar in structure and with a homologous biosynthetic gene cluster to valinomycin, recent work suggests that cereulide is produced via a different mechanism, which relys on a non-canonical coupling of two didepsipeptide-PCP bound intermediates. Ultimately this alternative mechanism generates a tetradepsipeptide-PCP bound intermediate that is prosed to differ from the tetradepsipeptide predicted from canonical activity of CesA and CesB. To test this hypothesis, we chemically synthetize both tetradepsipeptides as N-acetyl cysteamine thioesters and probed the ability of the purified recombinant terminal CesB thioesterase (CesB TE) to oligomerize and macrocyclize each substrate. Only the canonical substrate is converted cereulide, ruling out this alternative mechanism. We also show that CesB TE can use related tertradepsipeptide substrates, such as the valinomycin tetradespipetide and a hybride cereulide-valinomycin tetradespispetide in conjunction with its native substrate to generate chimeric natural products. This work clarifies the biosynthetic origins of cereulide and provides a powerful biocatalyst to access analogs of these ionophoric forming natural products.

Thioesterase from Cereulide Biosynthesis Is Responsible for Oligomerization and Macrocyclization of a Linear Tetradepsipeptide

Cereulide is a cyclic depsidodecapeptide produced in <i>Bacillus cereus</i> by two non-ribosomal peptide synthetases, CesA and CesB. While highly similar in structure and with a homologous biosynthetic gene cluster to valinomycin, recent work suggests that cereulide is produced via a different mechanism, which relys on a non-canonical coupling of two didepsipeptide-PCP bound intermediates. Ultimately this alternative mechanism generates a tetradepsipeptide-PCP bound intermediate that is prosed to differ from the tetradepsipeptide predicted from canonical activity of CesA and CesB. To test this hypothesis, we chemically synthetize both tetradepsipeptides as N-acetyl cysteamine thioesters and probed the ability of the purified recombinant terminal CesB thioesterase (CesB TE) to oligomerize and macrocyclize each substrate. Only the canonical substrate is converted cereulide, ruling out this alternative mechanism. We also show that CesB TE can use related tertradepsipeptide substrates, such as the valinomycin tetradespipetide and a hybride cereulide-valinomycin tetradespispetide in conjunction with its native substrate to generate chimeric natural products. This work clarifies the biosynthetic origins of cereulide and provides a powerful biocatalyst to access analogs of these ionophoric forming natural products.

NON-CANONICAL ACTIVITY OF RETINOIC ACID AS A POSSIBLE MECHANISM OF RETINOID RESISTANCE IN CANCER THERAPY

Retinoic acid (RA) is one of the most functionally active intracellular metabolites of vitamin A, regulating the key physiological processes, including the differentiation of cells, organs and tissues. RA is successfully applied in the treatment of acute promyelocytic leukemia. Drugs based on RA and other natural and synthetic retinoids are being actively developed for the treatment of other oncopathologies, including various solid tumors. However, the use of RA in the treatment of malignant tumors is restricted by the rapid acquisition of RA-resistance. The mechanisms of RA-resistance formation are still poorly understood, what could be explained apparently by the large number of genes directly or indirectly being regulated by RA at transcription level, including genes regulating the activity and metabolism of RA itself. The situation is further complicated by the relatively recently discovered non-genomic or non-canonical activity of RA, which consists in the non-transcriptional regulation of key protein kinases involved in tumor progression. The review is devoted to the analysis of published data on non-canonical activity of RA. The review provides a modern view on the main mechanisms implementing the canonical genomic activity of the RA, presents available information on the RA-dependent non-transcriptional regulation of ERK1 / 2, PI3K / AKT, p38MAPK and PKC protein kinases and possible mechanisms mediating this activity as well as potential significance of the RA-dependent activation of intracellular signaling pathways in the formation of RA-resistance and the malignant potential of transformed cells.

Non-Canonical Regulation of Phosphatidylserine Metabolism by a Phosphatidylinositol Transfer Protein and a Phosphatidylinositol 4-OH Kinase

ABSTRACTThe phosphatidylserine (PtdSer) decarboxylase Psd2 is proposed to engage in an endoplasmic reticulum (ER)-Golgi/endosome membrane contact site (MCS) that facilitates phosphatidylserine decarboxylation to phosphatidylethanomaine (PtdEtn) in Saccharomyces cerevisiae. While this MCS is envisioned to consist of Psd2, the Sec14-like phosphatidylinositol transfer protein (PITP) Sfh4, the Stt4 phosphatidylinositol (PtdIns) 4-OH kinase, the Scs2 tether, and at least one other uncharacterized protein, functional data that address key foundations of this model are sparse. We now report that Psd2, Sfh4 and Stt4 are the only components individually required for biologically sufficient Psd2-dependent PtdEtn production. Surprisingly, neither the PtdIns-transfer activity of Sfh4 nor its capacity to activate Stt4 is required to stimulate the Psd2 pathway. Instead, Sfh4 activates the Psd2 pathway via a specific Sfh4-Psd2 physical interaction. Whereas the data indicate an Sfh4-independent association of Stt4 with Psd2 as well, we find Stt4 also regulates Psd2 activity indirectly by influencing the PtdSer pool accessible to Psd2 for decarboxylation. These collective results demonstrate that the proposed ER-Golgi/endosomal MCS model fails to provide an accurate description of the Psd2 system in yeast, and provide an example where the biological function of a Sec14-like PITP is uncoupled from its ‘canonical’ activity as a PtdIns transfer protein.

Non-canonical activity of retinoic acid in relation to the activation of protein kinases in transformed cells of different origin

Background.The non-canonical activity of retinoic acid (RA) was discovered relatively recently and consists in the rapid activation of intracellular signaling pathways by the mechanisms not related to the transcriptional activity of the RA nuclear receptors. Separate data suggest that this activity can stimulate the processes of malignancy and contribute to the formation of tumor cell resistance to RA as a therapeutic agent. However, little is known about the mechanisms of this activity. It is also unclear how universal this effect is; does the RA-dependent activation of different signaling protein kinases occur in the same cells, and whether activation of these kinases is interrelated.Materials and methods: cultivation of non-small cell lung cancer cells and neuroblastoma cells under standard conditions and with incubation with all-trans retinoic acid (ATRA); immunoblotting.Results. Here we studied the effect of ATRA on the activation of Akt and Erk1/2 protein kinases depending on the incubation time. The analysis revealed RA-dependent activation of both kinases in all studied non-small cell lung cancer and neuroblastoma cell lines. Activation of Akt and Erk1/2 occurred at five minutes of incubation, which corresponds to the non-transcriptional (non-canonical) activity of the RA, however, further activation kinetics of the two kinases differed essentially.Conclusion.We found that ATRA causes rapid activation of Erk1/2 and Akt protein kinases in both non-small cell lung cancer and neuroblastoma cells. The differences in the kinetics of RA-dependent stimulation of these two kinases suggest that their activation is mediated by independent mechanisms.

Increased Activity of Cystathionine β-Lyase Suppresses 2-Aminoacrylate Stress inSalmonella enterica

ABSTRACTReactive enamine stress caused by intracellular 2-aminoacrylate accumulation leads to pleiotropic growth defects in a variety of organisms. Members of the well-conserved RidA/YER057c/UK114 protein family prevent enamine stress by enhancing the breakdown of 2-aminoacrylate to pyruvate. InSalmonella enterica, disruption of RidA allows 2-aminoacrylate to accumulate and to inactivate a variety of pyridoxal 5′-phosphate-dependent enzymes by generating covalent bonds with the enzyme and/or cofactor. This study was initiated to identify mechanisms that can overcome 2-aminoacrylate stress in the absence of RidA. Multicopy suppressor analysis revealed that overproduction of the methionine biosynthesis enzyme cystathionine β-lyase (MetC) (EC 4.4.1.8) alleviated the pleiotropic consequences of 2-aminoacrylate stress in aridAmutant strain. Degradation of cystathionine by MetC was not required for suppression ofridAphenotypes. The data support a model in which MetC acts on a noncystathionine substrate to generate a metabolite that reduces 2-aminoacrylate levels, representing a nonenzymatic mechanism of 2-aminoacrylate depletion.IMPORTANCERidA proteins are broadly conserved and have been demonstrated to deaminate 2-aminoacrylate and other enamines. 2-Aminoacrylate is generated as an obligatory intermediate in several pyridoxal 5′-phosphate-dependent reactions; if it accumulates, it damages cellular enzymes. This study identified a novel mechanism to eliminate 2-aminoacrylate stress that required the overproduction, but not the canonical activity, of cystathionine β-lyase. The data suggest that a metabolite-metabolite interaction is responsible for quenching 2-aminoacrylate, and they emphasize the need for emerging technologies to probe metabolismin vivo.