Search

Existing therapeutics for autoimmune diseases remain problematic due to low efficacy, severe side effects, and difficulties to reach target tissues. Herein, we design multifunctional fusion nanovesicles that can target lesions for the treatment of autoimmune skin diseases. The grapefruit‐derived exosome‐like nanovesicles (GEVs) with anti‐inflammatory and antioxidant effects are first encapsulated with CX5461, an immunosuppressant with anti‐proliferative properties to form GEV@CX5461. In order to enhance therapeutic efficiency and safety, GEV@CX5461 are then fused with CCR6+ nanovesicles derived from membranes of engineered gingiva‐derived mesenchymal stem cells (GMSCs). The resulting FV@CX5461 not only maintain the bioactivity of GEVs, CX5461, and GMSC membranes but also home to inflamed tissues rich in chemokine CCL20 through the chemotaxis function of CCR6 on FVs. Moreover, FV@CX5461 reduce the secretion of inflammatory factors, calm down Th17 cell activation, and induce Treg cell infiltration. Finally, impressive therapeutic efficiency in both psoriasis and atopic dermatitis disease models is demonstrated using FV@CX5461 to reshape the unbalanced immune microenvironment. A nanotherapeutic drug delivery strategy is developed using fusion nanovesicles derived from plant and animal cells with high clinical potential. [ABSTRACT FROM AUTHOR]

Abstract Existing therapeutics for autoimmune diseases remain problematic due to low efficacy, severe side effects, and difficulties to reach target tissues. Herein, we design multifunctional fusion nanovesicles that can target lesions for the treatment of autoimmune skin diseases. The grapefruit‐derived exosome‐like nanovesicles (GEVs) with anti‐inflammatory and antioxidant effects are first encapsulated with CX5461, an immunosuppressant with anti‐proliferative properties to form GEV@CX5461. In order to enhance therapeutic efficiency and safety, GEV@CX5461 are then fused with CCR6+ nanovesicles derived from membranes of engineered gingiva‐derived mesenchymal stem cells (GMSCs). The resulting FV@CX5461 not only maintain the bioactivity of GEVs, CX5461, and GMSC membranes but also home to inflamed tissues rich in chemokine CCL20 through the chemotaxis function of CCR6 on FVs. Moreover, FV@CX5461 reduce the secretion of inflammatory factors, calm down Th17 cell activation, and induce Treg cell infiltration. Finally, impressive therapeutic efficiency in both psoriasis and atopic dermatitis disease models is demonstrated using FV@CX5461 to reshape the unbalanced immune microenvironment. A nanotherapeutic drug delivery strategy is developed using fusion nanovesicles derived from plant and animal cells with high clinical potential.

Ribosomal biogenesis and protein synthesis are deregulated in most cancers, suggesting that interfering with translation machinery may hold significant therapeutic potential. Here, we show that loss of the tumor suppressor adenomatous polyposis coli (APC), which constitutes the initiating event in the adenoma carcinoma sequence for colorectal cancer (CRC), induces the expression of RNA polymerase I (RNAPOL1) transcription machinery, and subsequently upregulates ribosomal DNA (rDNA) transcription. Targeting RNAPOL1 with a specific inhibitor, CX5461, disrupts nucleolar integrity, and induces a disbalance of ribosomal proteins. Surprisingly, CX5461‐induced growth arrest is irreversible and exhibits features of senescence and terminal differentiation. Mechanistically, CX5461 promotes differentiation in an MYC‐interacting zinc‐finger protein 1 (MIZ1)‐ and retinoblastoma protein (Rb)‐dependent manner. In addition, the inhibition of RNAPOL1 renders CRC cells vulnerable towards senolytic agents. We validated this therapeutic effect of CX5461 in murine‐ and patient‐derived organoids, and in a xenograft mouse model. These results show that targeting ribosomal biogenesis together with targeting the consecutive, senescent phenotype using approved drugs is a new therapeutic approach, which can rapidly be transferred from bench to bedside.

Mutations in the plant homeodomain-like finger protein 6 (PHF6) gene are strongly associated with acute myeloid (AML) and T-cell acute lymphoblastic leukemia (T-ALL). In this study, we demonstrated that PHF6 can bind to H3K9me3 and H3K27me1 on the nucleolar chromatin and recruit histone methyltransferase SUV39H1 to the rDNA locus. The deletion of PHF6 caused a decrease in the recruitment of SUV39H1 to rDNA gene loci, resulting in a reduction in the level of H3K9me3 and the promotion of rDNA transcription. The knockdown of either SUV39H1 or PHF6 significantly attenuated the effects of increase in H3K9me3 and suppressed the transcription of rDNA induced by the overexpression of the other interacting partner, thereby establishing an interdependent relationship between PHF6 and SUV39H1 in their control of rRNA transcription. The PHF6 clinical mutants significantly impaired the ability to bind and recruit SUV39H1 to the rDNA loci, resulting in an increase in rDNA transcription activity, the proliferation of in vitro leukemia cells, and the growth of in vivo mouse xenografts. Importantly, significantly elevated levels of pre-rRNA were observed in clinical AML patients who possessed a mutated version of PHF6. The specific rDNA transcription inhibitor CX5461 significantly reduced the resistance of U937 AML cells deficient in PHF6 to cytarabine, the drug that is most commonly used to treat AML. Collectively, we revealed a novel molecular mechanism by which PHF6 recruits methyltransferase SUV39H1 to the nucleolar region in leukemia and provided a potential therapeutic target for PHF6-mutant leukemia.

Ribosomal biogenesis and protein synthesis are deregulated in most cancers, suggesting that interfering with translation machinery may hold significant therapeutic potential. Here, we show that loss of the tumor suppressor adenomatous polyposis coli (APC), which constitutes the initiating event in the adenoma carcinoma sequence for colorectal cancer (CRC), induces the expression of RNA polymerase I (RNAPOL1) transcription machinery, and subsequently upregulates ribosomal DNA (rDNA) transcription. Targeting RNAPOL1 with a specific inhibitor, CX5461, disrupts nucleolar integrity, and induces a disbalance of ribosomal proteins. Surprisingly, CX5461-induced growth arrest is irreversible and exhibits features of senescence and terminal differentiation. Mechanistically, CX5461 promotes differentiation in an MYC-interacting zinc-finger protein 1 (MIZ1)- and retinoblastoma protein (Rb)-dependent manner. In addition, the inhibition of RNAPOL1 renders CRC cells vulnerable towards senolytic agents. We validated this therapeutic effect of CX5461 in murine- and patient-derived organoids, and in a xenograft mouse model. These results show that targeting ribosomal biogenesis together with targeting the consecutive, senescent phenotype using approved drugs is a new therapeutic approach, which can rapidly be transferred from bench to bedside. [ABSTRACT FROM AUTHOR]

CX5461, a compound initially identified as an RNA polymerase inhibitor and more recently as a G-quadruplex binder, binds copper to form a complex. Our previous publication showed that the complexation reaction can be leveraged to formulate copper-CX5461 inside liposomes, improving the apparent solubility of CX5461 by over 500-fold and reducing the elimination of CX5461 from the plasma compartment following intravenous administration. In mouse models of acute myeloid leukemia, the resulting formulation was more effective than the free drug solution of CX5461 (pH 3.5) currently used in clinical trials. However, the gains observed with the liposomal formulation were minimal, despite significant increases in circulation half-life. Since the formulation technology used relied on liposomes and the fate of most compounds associated with liposomes is dependent on liposomal lipid composition, the studies described here were designed to evaluate how simple changes in lipid composition could affect therapeutic activity. The previously reported formulation method was simplified to ensure an easy scale-up process. In the modified method, pre-measured solid CX5461 was added to copper-containing liposomes prior to an incubation at 60 °C, which enabled copper-CX5461 complexation inside DSPC/Chol or DMPC/Chol liposomes. Efficacy was determined in BRCA-normal (BxPC3) and BRCA-deficient (Capan-1) models of pancreatic cancer. Both liposomal formulations enhanced the circulation lifetime of CX5461 compared to the free drug solution (pH 3.5). Unlike most compounds that are loaded using a transmembrane pH-gradient, the dissociation of CX5461 from liposomes prepared using the copper complexation method were comparable for DSPC/Chol and DMPC/Chol liposomes, in vitro and in vivo. Nonetheless, copper CX5461 prepared using DMPC/Chol liposomes exhibited superior efficacy. The reason for the improved activity of DMPC/Chol copper-CX5461 was not readily explained by the release data and may be due to the fact that DMPC/Chol liposomes are less stable following localization in the tumor. The results indicate that the therapeutic effects of copper-CX5461 will be dependent on liposomal lipid composition and that liposomal CX5461 should exhibit superior benefits when used to treat BRCA-deficient cancers. [Display omitted] • A CX5461 formulation has been prepared using metal chemistry and liposomes • Liposomal formulations of CuCX5461 exhibit reduced plasma elimination rates • DMPC/Chol liposomal CX5461 is more active than DSPC/Chol liposomal CX5461 • Dissociation of the metal-CX5461 complex may be rate limiting for drug release • The DMPC/Chol formulation was most active likely due to intracellular processing [ABSTRACT FROM AUTHOR]

Mutations in the plant homeodomain-like finger protein 6 (PHF6) gene are strongly associated with acute myeloid (AML) and T-cell acute lymphoblastic leukemia (T-ALL). In this study, we demonstrated that PHF6 can bind to H3K9me3 and H3K27me1 on the nucleolar chromatin and recruit histone methyltransferase SUV39H1 to the rDNA locus. The deletion of PHF6 caused a decrease in the recruitment of SUV39H1 to rDNA gene loci, resulting in a reduction in the level of H3K9me3 and the promotion of rDNA transcription. The knockdown of either SUV39H1 or PHF6 significantly attenuated the effects of increase in H3K9me3 and suppressed the transcription of rDNA induced by the overexpression of the other interacting partner, thereby establishing an interdependent relationship between PHF6 and SUV39H1 in their control of rRNA transcription. The PHF6 clinical mutants significantly impaired the ability to bind and recruit SUV39H1 to the rDNA loci, resulting in an increase in rDNA transcription activity, the proliferation of in vitro leukemia cells, and the growth of in vivo mouse xenografts. Importantly, significantly elevated levels of pre-rRNA were observed in clinical AML patients who possessed a mutated version of PHF6. The specific rDNA transcription inhibitor CX5461 significantly reduced the resistance of U937 AML cells deficient in PHF6 to cytarabine, the drug that is most commonly used to treat AML. Collectively, we revealed a novel molecular mechanism by which PHF6 recruits methyltransferase SUV39H1 to the nucleolar region in leukemia and provided a potential therapeutic target for PHF6 -mutant leukemia. Wild-type PHF6 is recognized as H3K9me3 and H3K27me1, and it recruits SUV39H1 to catalyze the methylation of H3K9 and H3K27 to inhibit the transcription of rDNA. When PHF6 is mutated, SUV39H1 cannot be recruited for rDNA methylation, resulting in uncontrolled rDNA transcription. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract Herein, we demonstrate that NFAT, a key regulator of the immune response, translocates from cytoplasm to nucleolus and interacts with NF45/NF90 complex to collaboratively promote rDNA transcription via triggering the directly binding of NF45/NF90 to the ARRE2‐like sequences in rDNA promoter upon T‐cell activation in vitro. The elevated pre‐rRNA level of T cells is also observed in both mouse heart or skin transplantation models and in kidney transplanted patients. Importantly, T‐cell activation can be significantly suppressed by inhibiting NF45/NF90‐dependent rDNA transcription. Amazingly, CX5461, a rDNA transcription‐specific inhibitor, outperformed FK506, the most commonly used immunosuppressant, both in terms of potency and off‐target activity (i.e., toxicity), as demonstrated by a series of skin and heart allograft models. Collectively, this reveals NF45/NF90‐mediated rDNA transcription as a novel signaling pathway essential for T‐cell activation and as a new target for the development of safe and effective immunosuppressants.

Herein, we demonstrate that NFAT, a key regulator of the immune response, translocates from cytoplasm to nucleolus and interacts with NF45/NF90 complex to collaboratively promote rDNA transcription via triggering the directly binding of NF45/NF90 to the ARRE2‐like sequences in rDNA promoter upon T‐cell activation in vitro. The elevated pre‐rRNA level of T cells is also observed in both mouse heart or skin transplantation models and in kidney transplanted patients. Importantly, T‐cell activation can be significantly suppressed by inhibiting NF45/NF90‐dependent rDNA transcription. Amazingly, CX5461, a rDNA transcription‐specific inhibitor, outperformed FK506, the most commonly used immunosuppressant, both in terms of potency and off‐target activity (i.e., toxicity), as demonstrated by a series of skin and heart allograft models. Collectively, this reveals NF45/NF90‐mediated rDNA transcription as a novel signaling pathway essential for T‐cell activation and as a new target for the development of safe and effective immunosuppressants. Synopsis: This study reveals NFAT‐NF45/NF90‐mediated rDNA transcription as a key regulating axis in modulating T cell activation. Targeting ribosome biogenesis could be a novel immunosuppressive therapy for organ transplantation. NF45/NF90 protein complex positively regulates rDNA transcription by directly binding to rDNA gene promoter.Upon T cell activation, NAFT translocates to nucleolus and interacts with NF45/NF90 to cooperatively promote rDNA transcription.Knockdown of NF45/NF90 or inhibiting rDNA transcription using a polymerase I inhibitor CX5461 suppresses T cell activation.CX5461 treatment prolongs the survival of mouse skin or heart allografts even more than the most commonly used suppressant FK506. [ABSTRACT FROM AUTHOR]

Ribosomal DNA (rDNA) transcription is a limiting step in ribosome biogenesis, crucial for protein synthesis and cell growth—especially at the early stages of embryonic development—and is regulated in a mammalian target of rapamycin (mTOR)‐dependent manner. Our previous report demonstrated that treatment with mTOR inhibitors during artificial embryonic activation improved the development of embryos derived from somatic cell nuclear transfer (SCNT). We hypothesize that inhibition of ribosome biogenesis in somatic cells facilitates reactivation of embryonic nucleolar establishment and ribosome biogenesis in SCNT embryos. Herein, we show that mTOR inhibitors suppressed ribosome biogenesis in somatic cells, and more importantly, improved development potential of SCNT embryos (blastocyst rate, 34% vs 24%). SCNT embryos derived from drug‐treated somatic cells exhibited higher levels of 47S, 18S, and 5S rRNAs, upstream binding factor (UBF) mRNA, ribosomal protein S6; they also improved the rebuilding of the nucleolar ultrastructure. In addition, treatment of donor cells with the RNA polymerase I (Pol I) inhibitor cx5461 caused similar effects on SCNT embryos. These results indicated that transient inhibition of rDNA transcription in donor cells facilitated the establishment of functional nucleoli and improved preimplantation development of SCNT embryos. [ABSTRACT FROM AUTHOR]