Palmitoyltransferase ZDHHC3 is essential for the oncogenic activity of PML/RARα in acute promyelocytic leukemia.

The oncogenic fusion protein promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) is critical for acute promyelocytic leukemia (APL). PML/RARα initiates APL by blocking the differentiation and increasing the self-renewal of leukemic cells. The standard clinical therapies all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), which induce PML/RARα proteolysis, have dramatically improved the prognosis of APL patients. However, the emergence of mutations conferring resistance to ATRA and ATO has created challenges in the treatment of APL patients. Exploring pathways that modulate the oncogenic activity of PML/RARα could help develop novel therapeutic strategies for APL, particularly for drug-resistant APL. Herein, we demonstrated for the first time that palmitoylation of PML/RARα was a critical determinant of its oncogenic activity. PML/RARα palmitoylation was found to be catalyzed mainly by the palmitoyltransferase ZDHHC3. Mechanistically, ZDHHC3-mediated palmitoylation regulated the oncogenic transcriptional activity of PML/RARα and APL pathogenesis. The knockdown or overexpression of ZDHHC3 had respective effects on the expression of proliferation- and differentiation-related genes. Consistently, the depletion or inhibition of ZDHHC3 could significantly arrest the malignant progression of APL, particularly drug-resistant APL, whereas ZDHHC3 overexpression appeared to have a promoting effect on the malignant progression of APL. Thus, our study not only reveals palmitoylation as a novel regulatory mechanism that modulates PML/RARα oncogenic activity but also identifies ZDHHC3 as a potential therapeutic target for APL, including drug-resistant APL.

Enhanced Reactivities of Iron(IV)-Oxo Porphyrin Species in Oxidation Reactions Promoted by Intramolecular Hydrogen-Bonding.

High-valent iron-oxo species are one of the common intermediates in both biological and biomimetic catalytic oxidation reactions. Recently, hydrogen-bonding (H-bonding) has been proved to be critical in determining the selectivity and reactivity. However, few examples have been established for mechanistic insights into the H-bonding effect. Moreover, intramolecular H-bonding effect on both C-H activation and oxygen atom transfer (OAT) reactions in synthetic porphyrin model system has not been investigated yet. In this study, a series of heme-containing iron(IV)-oxo porphyrin species with or without intramolecular H-bonding are synthesized and characterized. Kinetic studies revealed that intramolecular H-bonding can significantly enhance the reactivity of iron(IV)-oxo species in OAT, C-H activation, and electron-transfer reactions. This unprecedented unified H-bonding effect is elucidated by theoretical calculations, which showed that intramolecular H-bonding interactions lower the energy of the anti-bonding orbital of iron(IV)-oxo porphyrin species, resulting in the enhanced reactivities in oxidation reactions irrespective of the reaction type. To the best of the knowledge, this is the first extensive investigation on the intramolecular H-bonding effect in heme system. The results show that H-bonding interactions have a unified effect with iron(IV)-oxo porphyrin species in all three investigated reactions.

Saikosaponin a contributed to CCIN treatment by promoting neutrophil bactericidal activity via activation CBL-dependent ERK pathway.

BACKGROUND: Despite granulocyte colony-stimulating factor (GCSF) is widely used in clinical, cancer chemotherapy induced neutropenia (CCIN) infection and infection-related mortality is high for lack of functionally mature neutrophils. Generating functional neutrophils is new therapeutic approaches to reduce CCIN-associated infection and mortality. Saikosaponin a (SSA) is one of the major bioactive components of Radix Bupleuri (RB) and exerts immunoregulatory effects. PURPOSE: The present study aims to investigate the efficacy and mechanism of SSA in CCIN therapy. METHODS: SSA was applied both in vitro and in vivo to assess the efficacy of CCIN therapy. The differentiation of neutrophils was measured by Nitroblue tetrazolium (NBT) reduction assay and Giemsa staining assay. The neutrophil differentiation related real-time transcription factors were detected by quantitative PCR (RT-qPCR) and Western Blot. Bacteria killing assay was used to assess the ability of fighting infection. Network pharmacology was employed to explore the mechanism network, and the predicted pathways were validated by Western Blot. RESULTS: We found that SSA contributed to generate functional mature neutrophils which capable of fighting infection both in vitro and in vivo. Network pharmacology prediction showed 55 pathways were predicted involved in SSA against CCIN. Further validation showed that CBL-ERK1/2 pathway was activated by SSA, which could upregulate PU.1 and CEBPß expression leading to neutrophil differentiation. CONCLUSIONS: Our findings suggest a natural regimen SSA regenerates microbicidal neutrophils to effectively reduce CCIN-associated infection via activating CBL-ERK1/2 pathway, providing a rationale for future therapeutic approaches.

Saikosaponin d contributed to cancer chemotherapy induced neutropenia therapy by promoting neutrophil differentiation via activation CBL-dependent ERK pathway.

Cancer chemotherapy induced neutropenia (CCIN) is one of the most common toxicity caused by cytotoxic anticancer agents. Despite granulocyte colony-stimulating factor (GCSF) is widely used in clinical practice, the infection and infection-related mortality rate is still high for lack of functionally mature neutrophils. Saikosaponin d (SSD) is one of the major bioactive constituents of Radix Bupleuri (RB), which exerts immune-modulatory properties. We explored the function of SSD in CCIN therapy, we found that SSD contributed to generate functional mature neutrophils which capable of fighting infection both in vitro and in vivo. Network pharmacology was employed to explore the mechanism, 61 signal pathways might play an important role in CCIN treatment. Western Blot was employed to further confirm the potential pathway involved. We found CBL-ERK1/2 pathway was activated by SSD, followed by upregulating PU.1 and CEBPß expression and leading to neutrophil differentiation. Our findings suggest a natural regimen SSD which could regenerate microbicidal neutrophils to effectively reduce CCIN-associated infection via activating CBL-ERK1/2, providing a rationale for future therapeutic approaches.

2-Sulfonylpyridines as Tunable, Cysteine-Reactive Electrophiles.

The emerging use of covalent ligands as chemical probes and drugs would benefit from an expanded repertoire of cysteine-reactive electrophiles for efficient and diverse targeting of the proteome. Here we use the endogenous electrophile sensor of mammalian cells, the KEAP1-NRF2 pathway, to discover cysteine-reactive electrophilic fragments from a reporter-based screen for NRF2 activation. This strategy identified a series of 2-sulfonylpyridines that selectively react with biological thiols via nucleophilic aromatic substitution (SNAr). By tuning the electrophilicity and appended recognition elements, we demonstrate the potential of the 2-sulfonylpyridine reactive group with the discovery of a selective covalent modifier of adenosine deaminase (ADA). Targeting a cysteine distal to the active site, this molecule attenuates the enzymatic activity of ADA and inhibits proliferation of lymphocytic cells. This study introduces a modular and tunable SNAr-based reactive group for targeting reactive cysteines in the human proteome and illustrates the pharmacological utility of this electrophilic series.

CDK2 suppression synergizes with all-trans-retinoic acid to overcome the myeloid differentiation blockade of AML cells.

A characteristic feature of leukemia cells is a blockade of differentiation in cellular maturation. All-trans-retinoic acid (ATRA) has been successfully applied for the treatment of M3-type AML (APL, 10 %), but it fails to demonstrate a significant efficacy on the remaining patients with non-APL AML (90 %). Therefore, the research for strategies to extend the efficacy of ATRA-based therapy to non-APL AML is a key avenue of investigation. Here, we evaluate the synergetic effect of CDK2 inhibition and ATRA in AML both in vitro and in vivo. We have determined that both the CDK2 depletion and pharmacological inhibitor of CDK2 significantly sensitize three subtypes of AML cells (including two non-APL cells) to ATRA-induced cell differentiation. RNA-sequence results indicate that transcription activation of differentiation and maturation pathways plays an important role in this synergetic effect. Furthermore, the down-regulation of CDK2 sensitized AML cells to ATRA-induced engraftment prevention of leukemia cells in NOD-SCID mice and promotes the primary AML blasts differentiation when combined with ATRA. Thus, our work not only provides relevant experimental evidence for further validating CDK2 as a target for differentiation therapy, but also uncovers the future clinical application of CDK2 inhibitors in ATRA-based differentiation therapeutics for AML.

2-Bromopalmitate sensitizes osteosarcoma cells to adriamycin-induced apoptosis via the modulation of CHOP.

Osteosarcoma is the most common primary malignant bone tumour, but the survival rate of patients has plateaued since the mid-1980s. Adriamycin is an integral component of the current first-line chemotherapies used for osteosarcoma, but dose-dependent severe side effects often limit its clinical application. Here, we propose a potential combination regimen in which adriamycin plus 2-bromopalmitate, a palmitoylation inhibitor, exhibited powerful therapeutic effects on osteosarcoma. First, 2-bromopalmitate strongly increased the proliferation inhibition of adriamycin in both human osteosarcoma cell lines and primary osteosarcoma cells. Adriamycin-induced apoptosis in osteosarcoma cells was enhanced when synergized with 2-bromopalmitate. Our study indicated that the reactive oxygen species scavenger NAC and GSH could largely reverse the apoptosis induced by adriamycin combined with 2-bromopalmitate, demonstrating that reactive oxygen species played an essential role in this combination therapy. Moreover, CHOP was remarkably elevated in the combination group, and silencing of CHOP almost completely blocked the apoptosis induced by the combination of 2-bromopalmitate and adriamycin. Taken together, our study provides a prospective therapeutic strategy to eliminate osteosarcoma, which is propitious to clinical combination therapy development.

KLF4 functions as an oncogene in promoting cancer stem cell-like characteristics in osteosarcoma cells.

Despite more effective chemotherapy combined with limb-salvage surgery for the osteosarcoma treatment, survival rates for osteosarcoma patients have stagnated over the past three decades due to the poor prognosis. Osteosarcoma cancer stem cells (OSCs) are responsible for the growth and metastasis of osteosarcoma. The existence of OSCs offers a theoretical explanation for therapeutic failures including tumor recurrence, metastasis, and drug resistance. Understanding the pathways that regulate properties of OSCs may shed light on mechanisms that lead to osteosarcoma and suggest better modes of treatment. In this study, we showed that the expression level of Kruppel-like factor 4 (KLF4) is highly associated with human osteosarcoma cancer stemness. KLF4-overexpressed osteosarcoma cells displayed characteristics of OSCs: increased sphere-forming potential, enhanced levels of stemness-associated genes, great chemoresistance to adriamycin and CDDP, as well as more metastasis potential. Inversely, KLF4 knockdown could reduce colony formation in vitro and inhibit tumorigenesis in vivo, supporting an oncogenic role for KLF4 in osteosarcoma pathogenesis. Furthermore, KLF4 was shown to activate the p38 MAPK signaling pathway to promote cancer stemness. Altogether, our studies uncover an essential role for KLF4 in regulation of OSCs and identify KLF4-p38 MAPK axis as a potential therapeutic target for osteosarcoma treatment.

Ubiquitin-dependent degradation of CDK2 drives the therapeutic differentiation of AML by targeting PRDX2.

A distinct hallmark of acute myeloid leukemia (AML) is the arrest of leukemic myeloblasts at an immature stage of development. Therapies that overcome differentiation arrest have emerged as a powerful strategy for treating AML, but targeting leukemia differentiation remains challenging, mainly because of an incomplete mechanistic understanding of the process. Here, we unveil a new role for cyclin-dependent kinase 2 (CDK2) in blocking myeloid differentiation in AML. We show that among several interphase CDK, only CDK2 undergoes ubiquitin-dependent proteasome degradation, which is accompanied by AML cell differentiation. By using the yeast 2-hybrid system and functional analyses, KLHL6 was identified as a specific E3 ubiquitin ligase regulating the degradation of CDK2. Importantly, inhibiting CDK2, but not other cyclin-dependent kinases CDK1/4/6, effectively induced granulocytic differentiation in AML cell lines and 5 major subtypes of primary patient-derived AML samples. Mechanistically, CDK2 depletion led to the reactivation of differentiation pathway translation, and the differentiation blockade function of CDK2 may be achieved directly by maintaining the activity of PRDX2. Finally, CDK2 depletion arrested tumor growth of AML cells in nude mice and extended survival in both AML cell line and PDX-AML cells derived xenograft mouse models. Thus, our work not only provides experimental evidence for validating CDK2 as a potential therapeutic target for differentiation, but also uncovers the biological function of the CDK2-PRDX2 axis in blocking AML differentiation.

Am80-GCSF synergizes myeloid expansion and differentiation to generate functional neutrophils that reduce neutropenia-associated infection and mortality.

Neutrophils generated by granulocyte colony-stimulating factor (GCSF) are functionally immature and, consequently, cannot effectively reduce infection and infection-related mortality in cancer chemotherapy-induced neutropenia (CCIN). Am80, a retinoic acid (RA) agonist that enhances granulocytic differentiation by selectively activating transcription factor RA receptor alpha (RARα), alternatively promotes RA-target gene expression. We found that in normal and malignant primary human hematopoietic specimens, Am80-GCSF combination coordinated proliferation with differentiation to develop complement receptor-3 (CR3)-dependent neutrophil innate immunity, through altering transcription of RA-target genes RARß2, C/EBPε, CD66, CD11b, and CD18 This led to generation of functional neutrophils capable of fighting infection, whereas neutralizing neutrophil innate immunity with anti-CD18 antibody abolished neutrophil bactericidal activities induced by Am80-GCSF Further, Am80-GCSF synergy was evaluated using six different dose-schedule-infection mouse CCIN models. The data demonstrated that during "emergency" granulopoiesis in CCIN mice undergoing transient systemic intravenous bacterial infection, Am80 effect on differentiating granulocytic precursors synergized with GCSF-dependent myeloid expansion, resulting in large amounts of functional neutrophils that reduced infection. Importantly, extensive survival tests covering a full cycle of mouse CCIN with perpetual systemic intravenous bacterial infection proved that without causing myeloid overexpansion, Am80-GCSF generated sufficient numbers of functional neutrophils that significantly reduced infection-related mortality in CCIN mice. These findings reveal a differential mechanism for generating functional neutrophils to reduce CCIN-associated infection and mortality, providing a rationale for future therapeutic approaches.

Iron-Catalyzed Decarboxylative (4+1) Cycloadditions: Exploiting the Reactivity of Ambident Iron-Stabilized Intermediates.

The first example of iron-catalyzed decarboxylative (4+1) cycloaddition reactions is described in this publication. By using this method, a wide range of functionalized indoline products were prepared from easily available vinyl benzoxazinanones and sulfur ylides in high yields and selectivities. A possible reaction pathway involving an allylic iron intermediate is discussed based on a series of control experiments and density-functional theory calculations.

Nickel-catalyzed selective oxidative radical cross-coupling: an effective strategy for inert Csp3-H functionalization.

An effective strategy for inert Csp(3)-H functionalization through nickel-catalyzed selective radical cross-couplings was demonstrated. Density functional theory calculations were conducted and strongly supported the radical cross-coupling pathway assisted by nickel catalyst, which was further confirmed by radical-trapping experiments. Different arylborates including arylboronic acids, arylboronic acid esters and 2,4,6-triarylboroxin were all good coupling partners, generating the corresponding Csp(3)-H arylation products in good yields.

Sequential treatment with aurora B inhibitors enhances cisplatin-mediated apoptosis via c-Myc.

UNLABELLED: Platinum compound such as cisplatin is the first-line chemotherapy of choice in most patients with ovarian carcinoma. However, patients with inherent or acquired cisplatin resistance often experience relapse. Therefore, novel therapies are urgently required to treat drug-resistant ovarian carcinoma. Here, we showed that compared to the non-functional traditional simultaneous treatment, sequential combination of Aurora B inhibitors followed by cisplatin synergistically enhanced apoptotic response in cisplatin-resistant OVCAR-8 cells. This effect was accompanied by the induction of polyploidy in a c-Myc-dependent manner, as c-Myc knockdown reduced the efficacy of the combination by suppressing the expression of Aurora B and impairing cellular response to Aurora B inhibitor, as indicated by the decreased polyploidy and hyperphosphorylation of histone H1. In c-Myc-deficient SKOV3 cells, c-Myc overexpression restored Aurora B expression, induced polyploidy after inhibition of Aurora B, and sensitized cells to this combination therapy. Thus, our report reveals for the first time that sequential treatment of Aurora B inhibitors and cisplatin is essential to inhibit ovarian carcinoma by inducing polyploidy and downregulating c-Myc and that c-Myc is identified as a predictive biomarker to select cells responsive to chemotherapeutical combinations targeting Aurora B. Collectively, these studies provide novel approaches to overcoming cisplatin chemotherapy resistance in ovarian cancer. KEY MESSAGE: Pretreatment of Aurora B inhibitors augment apoptotic effects of cisplatin. The synergy of Aurora B inhibitor with cisplatin is dependent on c-Myc expression. c-Myc-dependent induction of polyploidy sensitizes cells to cisplatin.

Autophagy contributes to dasatinib-induced myeloid differentiation of human acute myeloid leukemia cells.

A breakthrough in clinical oncology was achieved as All-trans-retinoic acid (ATRA) sparked intensive differentiation therapy research. However, differentiation therapy is limited because ATRA is the sole efficient agent. Dasatinib is reported to induce myeloid differentiation of acute myeloid leukemia (AML) cells in vitro, but its mechanism remains unclear. Furthermore, the ability of dasatinib to cause differentiation of AML cells has not yet been proven. We assessed the contribution of autophagy to dasatinib-induced differentiation of AML cells. We found that dasatinib induces myeloid differentiation of AML cells accompanied with autophagy induction. Pharmacological inhibition of autophagy by 3-MA, Wortmannin, LY294002 and chloroquine block dasatinib-induced AML cell differentiation, whereas the induction of autophagy by rapamycin enhances AML cell differentiation. Our results suggest that retinoic acid receptors alpha (RARα) may not be involved in dasatinib-induced differentiation. In addition, we further illustrated that even low concentration of dasatinib can enhance ATRA-induced differentiation capability through initiation of autophagy. Taken together, we conclude that autophagy enhances the dasatinib-induced differentiation, which may provide theoretical support for developing dasatinib as a promising strategy for future differentiation therapy in AML patients.

Epidermal stem cells manipulated by pDNA-VEGF165/CYD-PEI nanoparticles loaded gelatin/ß-TCP matrix as a therapeutic agent and gene delivery vehicle for wound healing.

The success of gene therapy largely relies on a safe and effective gene delivery system. The objective of this study is to design a highly efficient system for the transfection of epidermal stem cells (ESCs) and investigate the transfected ESCs (TESCs) as a therapeutic agent and gene delivery reservoir for wound treatment. As a nonviral vector, ß-cyclodextrin-linked polyethylenimines (CYD-PEI) was synthesized by linking ß-cyclodextrin with polyethylenimines (600 Da). Gelatin scaffold incorporating ß-tricalcium phosphate (ß-TCP) was utilized as a substrate for the culture and transfection of ESCs. With the CYD-PEI/pDNA-VEGF165 polyplexes incorporated gelatin/ß-TCP scaffold based 3D transfection system, prolonged VEGF expression with a higher level was obtained at day 7 in ESCs than those in two-dimensional plates. Topical application of the TESCs significantly accelerated the skin re-epithelization, dermal collagen synthesis, and hair follicle regeneration. It also exhibited a potential in scar inhibition by regulating the distribution of different types of collagen. In contrast to ESCs, an additive capacity in stimulating angiogenesis at the wound site was observed in the TESCs. The present study provides a basis for the TESCs as a promising therapeutic agent and gene delivery reservoir for wound therapy.

Transplantation of bone-marrow-derived mesenchymal and epidermal stem cells contribute to wound healing with different regenerative features.

A new concept for wound therapy is the initiation of the regeneration of epidermal and dermal layers with appendages for skin function recovery. Bone-marrow-derived mesenchymal and epidermal stem cells (BMSCs and SSCs) are hypothesized to be able to home toward or to be transplanted to wound sites for skin repair and regeneration, but this awaits confirmation by further experimental and clinical evidence. In this study, the influence of the transplantation of BMSCs and SSCs with porous gelatin-ß-tricalcium phosphate sponge as scaffolds on wound re-epithelization, collagen synthesis, skin tensile strength recovery, and skin appendage regeneration has been investigated. The transplantation of BMSCs or SSCs significantly accelerates wound re-epithelization, stimulates dermal collagen synthesis, and exhibits the trend to enhance the tensile strength recovery of skin. Furthermore, regenerative features of BMSCs and SSCs have been identified in activating blood vessel and hair follicle formation, respectively. These results not only provide experimental evidence for the application of BMSCs and SSCs as promising therapeutics for clinical wound treatment, but also display their characteristics in activating distinct skin appendage regeneration, which might have novel applications in skin tissue engineering.