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Leukemia stem cells (LSC) possess distinct self-renewal and arrested differentiation properties that are responsible for disease emergence, therapy failure and recurrence in acute myeloid leukemia (AML). Despite AML displaying extensive biological and clinical heterogeneity, LSC with high interleukin-3 receptor (IL-3R) levels are a constant yet puzzling feature as this receptor lacks tyrosine kinase activity. Here we show that the heterodimeric IL3Ra/Bc receptor assembles into hexamers and dodecamers through a unique interface in the 3D structure, where high IL3Ra/Bc ratios bias hexamer formation. Importantly, receptor stoichiometry is clinically relevant as it varies across the individual cells in the AML hierarchy, where high IL3Ra/Bc ratios in LSCs drive hexamer-mediated stemness programs and poor patient survival, whilst low ratios mediate differentiation. Our study establishes a new paradigm where alternative cytokine receptor stoichiometries differentially regulate cell fate; a signaling mechanism that may be generalizable to other transformed cellular hierarchies and of potential therapeutic significance.

Leukemia stem cells (LSC) possess distinct self-renewal and arrested differentiation properties that are responsible for disease emergence, therapy failure and recurrence in acute myeloid leukemia (AML). Pleiotropic cytokines like interleukin (IL)-3 dimerize their receptors to signal multiple functional activities of AML cells, however, the mechanistic link between cytokine receptor dimerization and LSC fate regulation is unknown. Here, we show that the Class I heterodimeric IL3Rα/βc receptors (IL-3R) assemble as hexamers and dodecamers that segregate proliferation versus differentiation signals, respectively. Receptor stoichiometry varies across the individual cells that make up the AML hierarchy, with LSCs presenting with the highest IL3Rα/βc ratio thereby driving resultant hexamer-mediated stemness programs and poor patient survival. Our study establishes a new signaling mechanism of cytokine pleiotropy and the biased instruction of cell fate that directs the AML hierarchy; a signaling mechanism that may be generalizable to other normal and transformed cellular hierarchies.

Our understanding of the biological role of the βc family of cytokines has evolved enormously since their initial identification as bone marrow colony stimulating factors in the 1960′s. It has become abundantly clear over the intervening decades that this family of cytokines has truly astonishing pleiotropic capacity, capable of regulating not only hematopoiesis but also many other normal and pathological processes such as development, inflammation, allergy and cancer. As noted in the current pandemic, βc cytokines contribute to the cytokine storm seen in acutely ill COVID-19 patients. Ongoing studies to discover how these cytokines activate their receptor are revealing insights into the fundamental mechanisms that give rise to cytokine pleiotropy and are providing tantalizing glimpses of how discrete signaling pathways may be dissected for activation with novel ligands for therapeutic benefit. Refereed/Peer-reviewed

Pathological drug withdrawal syndrome is linked to accumulation of JAK2 phosphorylation in V617F myelofibrosis., Treatment of patients with myelofibrosis with the type I JAK (Janus kinase) inhibitor ruxolitinib paradoxically induces JAK2 activation loop phosphorylation and is associated with a life-threatening cytokine-rebound syndrome if rapidly withdrawn. We developed a time-dependent assay to mimic ruxolitinib withdrawal in primary JAK2V617F and CALR mutant myelofibrosis patient samples and observed notable activation of spontaneous STAT signaling in JAK2V617F samples after drug washout. Accumulation of ruxolitinib-induced JAK2 phosphorylation was dose dependent and correlated with rebound signaling and the presence of a JAK2V617F mutation. Ruxolitinib prevented dephosphorylation of a cryptic site involving Tyr1007/1008 in JAK2 blocking ubiquitination and degradation. In contrast, a type II JAK inhibitor, CHZ868, did not induce JAK2 phosphorylation, was not associated with withdrawal signaling, and was superior in the eradication of flow-purified JAK2V617F mutant CD34+ progenitors after drug washout. Type I inhibitor–induced loop phosphorylation may act as a pathogenic signaling node released upon drug withdrawal, especially in JAK2V617F patients.

The interleukin-3 (IL-3) receptor is a cell-surface heterodimer that links the haemopoietic, vascular and immune systems and is overexpressed in acute and chronic myeloid leukaemia progenitor cells. It belongs to the type I cytokine receptor family in which the α-subunits consist of two fibronectin III-like domains that bind cytokine, and a third, evolutionarily unrelated and topologically conserved, N-terminal domain (NTD) with unknown function. Here we show by crystallography that, while the NTD of IL3Rα is highly mobile in the presence of IL-3, it becomes surprisingly rigid in the presence of IL-3 K116W. Mutagenesis, biochemical and functional studies show that the NTD of IL3Rα regulates IL-3 binding and signalling and reveal an unexpected role in preventing spontaneous receptor dimerisation. Our work identifies a dual role for the NTD in this cytokine receptor family, protecting against inappropriate signalling and dynamically regulating cytokine receptor binding and function., The N-terminal domain (NTD) of interleukin-3 receptor α-subunit (IL3Rα) is involved in IL-3 recognition but the underlying mechanism is unknown. Here, the authors present crystal structures of the IL3Rα complex and provide biochemical evidence that the NTD regulates IL-3 binding and signalling complex assembly.

The β common ([βc]/CD131) family of cytokines comprises granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5, all of which use βc as their key signaling receptor subunit. This is a prototypic signaling subunit-sharing cytokine family that has unveiled many biological paradigms and structural principles applicable to the IL-2, IL-4, and IL-6 receptor families, all of which also share one or more signaling subunits. Originally identified for their functions in the hematopoietic system, the βc cytokines are now known to be truly pleiotropic, impacting on multiple cell types, organs, and biological systems, and thereby controlling the balance between health and disease. This review will focus on the emerging biological roles for the βc cytokines, our progress toward understanding the mechanisms of receptor assembly and signaling, and the application of this knowledge to develop exciting new therapeutic approaches against human disease. Refereed/Peer-reviewed

The GM-CSF, IL-3 and IL-5 family of cytokines, also known as the βc family due to their receptors sharing the signalling subunit βc, regulates multiple biological processes such as native and adaptive immunity, inflammation, normal and malignant hemopoieis, and autoimmunity. Australian scientists played a major role in the discovery and biological characterisation of the βc cytokines and their recent work is revealing unique features of cytokine receptor assembly and signalling. Furthermore, specific antibodies have been generated to modulate their function. Characterisation of the structural and dynamic requirements for the activation of the βc receptor family and the molecular definition of downstream signalling pathways are providing new insights into cytokine receptor signalling as well as new therapeutic opportunities. Refereed/Peer-reviewed

Patients with myelofibrosis who are treated with the Type I ATP competitive JAK inhibitor ruxolitinib derive symptomatic benefit from improvement in cytokine-mediated symptoms, and show cytoreductive responses in disease manifestations, such as splenomegaly or leukocytosis. However, ruxolitinib treatment does not consistently reduce JAK2 mutant allele burden nor eradicate the disease. Secondly, when ruxolitinib is withdrawn there is rapid recrudescence of cytokine-mediated symptoms within days of treatment stopping and in rare cases this has led to a life-threatening "ruxolitinib discontinuation syndrome" characterized by acute relapse of disease symptoms, splenomegaly, worsening cytopenia, and a cytokine storm akin to septic shock. Paradoxically, Type I inhibitors can induce the accumulation of JAK activation loop phosphorylation for unknown reasons, despite blockade of kinase function and inhibition of STAT phosphorylation. Here, we developed a time-dependent assay in primary myelofibrosis samples to mimic ruxolitinib withdrawal syndrome and investigate a possible mechanism of action involving JAK2 activation loop phosphorylation. Methods: Primary peripheral blood de novo myelofibrosis samples were obtained prior to treatment with informed consent according to institutional guidelines (Stanford University Institutional Review Board No. 6453 or Board No. SACRB-APP-020). TF1.8 cells were cultured in RPMI with 10% fetal calf serum supplemented with 2 ng/ml of GM-CSF. SET-2 cells were cultured in RPMI with 10% serum. Phosphatase assays were performed with recombinant JAK2 kinase domain was mixed with recombinant PTP1B and JAK inhibitor. All antibodies were sourced from Cell Signalling Technologies. Unless otherwise stated, P values comparing two means were calculated using the two-tailed unpaired Student's t-test in Prism version 6 (GraphPad Software, Inc. La Jolla, CA). Results: We developed a time-dependent assay to mimic ruxolitinib withdrawal in primary JAK2V617F and CALR mutant myelofibrosis patient samples and observed striking activation of spontaneous STAT signaling in JAK2V617F samples after drug washout. Accumulation of ruxolitinib-induced loop phosphorylation was dose-dependent and correlated with rebound signaling and the presence of a JAK2V617F mutation. Ruxolitinib prevented dephosphorylation of a cryptic site involving Tyr1007/1008 in JAK2 blocking ubiquitination and degradation. We note that one patient with CALR mutant did not exhibit the same degree of STAT spontaneous withdrawal signalling compared to JAK2 V617F. Similarly, CALR mutant myelofibrosis cells showed an undetectable level of activated JAK2 Tyr1007/1008 phosphorylation in the presence of ruxolitinib. This is consistent with a number of emerging reports using CALR mutated models of myelofibrosis but needs to be confirmed in further patient samples. CHZ868 is a potent JAK2-biased Type II inhibitor that has efficacy in pre-clinical models of MPN at high nanomolar concentrations (250-500 nM) but its effects on withdrawal signalling have not been studied. In contrast to Type I inhibitors, CHZ868 did not prevent JAK2 activation loop dephosphorylation as shown by in vitro phosphatase assay. CHZ868 was tested in SET2 cells and primary JAK2 V617F patient samples and did not induce accumulation of JAK2 phosphorylation. Most importantly CHZ868 did not exhibit rebound JAK2 signaling upon washout and was superior in the eradication of JAK2 mutant positive progenitors purified from 14 myelofibrosis patient samples. Immunophenotypically, CD34+ progenitor cells from myelofibrosis patients most resembled CD34+CD38+CD45RA+CD123hi granulocyte-myeloid progenitors and lymphoid-restricted multipotential progenitors. Consistent with our signaling, chi-square test of 10 published cases of severe ruxolitinib-withdrawal syndrome noted a high frequency of JAK2 mutant cases (100%) compared to expected mutation frequencies (2 x 2 contingency table, P= 0.0203). In majority of cases, symptoms began within 72 hours. Conclusions: Type I inhibitor-induced loop phosphorylation acts as a pathogenic signaling node released upon drug withdrawal, especially in JAK2V617F patients. Ruxolitinib-withdrawal syndrome may be more frequent in JAK2 V617F mutant cases. Figure. Figure. Disclosures Hughes: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ross:Celgene: Research Funding; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria.

An unusual polyprenylated acylphloroglucinol derivative unsubstituted at C-2 and C-6, garcicowin A (1), together with three other new (garcicowins B-D, 2-4) and nine known analogues, was isolated and characterized from the twigs of Garcinia cowa. The structures of 1-4 were elucidated by interpretation of their spectroscopic data. The compounds isolated were evaluated for their cytotoxicity against two cancer cell lines (HT-29 and HCT116) and against normal colon cells (CCD-18Co), and the results demonstrated their selective toxicity toward the cancer cells.

Angelica sinensis is a Chinese medicinal herb for treating gynecological and gastrointestinal disorders, and also in conjunction with cancer chemotherapy. Aim of the study In the present study, the cytotoxic and anti-proliferative effects of three main Angelica sinensis phthalides, namely n -butylidenephthalide (BLP), senkyunolide A (SKA) and z -ligustilide (LGT), and their synergy on colon cancer HT-29 cells were investigated. Moreover, the results obtained in both human colon cancer HT-29 and normal colon CCD-18Co cells were compared for the investigation of selectivity. Materials and methods MTT and [ 3 H] thymidine incorporation assays were used for the evaluation of cytotoxic and anti-proliferative effects, respectively. Interactions among phthalides were determined by median-effect analysis. Results All three phthalides dose-dependently decreased cell viability more potently in HT-29 than in CCD-18Co cells. The IC 50 values for inhibition of cell proliferation for SKA, LGT and BLP were 54.17 ± 5.10, 60.63 ± 6.79 and 236.90 ± 18.22 μM, respectively, in HT-29 cells. Angelica sinensis extract demonstrated significant synergy in inhibiting cell proliferation. Conclusions The three phthalides might have anti-cancer potential, yet the phthalides, in combination with other ingredients in Angelica sinensis extract, display significant synergy leading to a stronger anti-tumor effect.

Cytokines act through their membrane-bound receptors to transmit a variety of signals including cell survival, proliferation, differentiation, and functional activity. Cytokine receptors are a conserved family of about 40 members that includes the receptors for hormones, interleukins, interferons, and colony-stimulating factors. Abnormal cytokine levels or aberrations in their signaling pathways can lead to a variety of diseases including cancers and inflammatory conditions reflecting their importance in normal hematopoiesis and immunity. Determination of the three-dimensional atomic structures of cytokines and their receptors has provided detailed insights into how cytokines transmit biological signals across cell membranes. Refereed/Peer-reviewed

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and IL-5 are members of a small family of cytokines that share a beta receptor subunit (βc). These cytokines regulate the growth, differentiation, migration and effector function activities of many hematopoietic cells in bone marrow, blood and sites of inflammation. Excessive or aberrant signaling can result in chronic inflammatory conditions and myeloid leukemias. The crystal structures of the GM-CSF ternary complex, the IL-5 binary complex and the very recent IL-3 receptor alpha subunit build upon decades of structure-function studies, giving new insights into cytokine-receptor specificity and signal transduction. Selective modulation of receptor function is now a real possibility and the structures of the βc receptor family are being used to discover novel and disease-specific therapeutics. Refereed/Peer-reviewed

Ethnopharmacological relevance Pyrrolizidine alkaloids (PAs) are a group of heterocyclic phytotoxins present in a wide range of plants. The consumption of PA-containing medicinal herbs or PA-contaminated foodstuffs has long been reported to cause human hepatotoxicity. However, the degrees of hepatotoxicity of different PAs are unknown, which makes it difficult to determine a universal threshold of toxic dose of individual PAs for safe regulation of PA-containing natural products. The aim of the present study is to develop a simple and convenient in vitro model to assess the hepatotoxicity of different PAs. Material and methods Six common cytotoxicity assays were used to evaluate the hepatotoxicity of different PAs in human hepatocellular carcinoma HepG2 cells. Results The combination of MTT and bromodeoxyuridine incorporation (BrdU) assays demonstrated to be a suitable method to evaluate the toxic potencies of various PAs in HepG2 cells, and the results indicated that otonecine-type PA (clivorine: IC 20 =0.013±0.004 mM (MTT), 0.066±0.031 mM (BrdU)) exhibited significantly higher cytotoxic and anti-proliferative effects than retronecine-type PA (retrorsine: IC 20 =0.27±0.07 mM (MTT), 0.19±0.03 mM (BrdU)). While as expected, the known less toxic platyphylline-type PA (platyphylline: IC 20 =0.85±0.11 mM (MTT), 1.01±0.40 mM (BrdU)) exhibited significantly less toxicity. The different cytotoxic and anti-proliferative potencies of various PAs in the same retronecine-type could also be discriminated by using the combined MTT and BrdU assays. In addition, the developed assays were further utilized to test alkaloid extract of Gynura segetum , a senecionine and seneciphylline-containing herb, the overall cytotoxicity of two PAs in the extract was comparable to that of these two PAs tested individually. Conclusion Using the developed in vitro model, the cytotoxicity of different PAs and the extract of a PA-containing herb were investigated in parallel in one system, and their different hepatotoxic potencies were determined and directly compared for the first time. The results suggested that the developed model has a great potential to be applied for the quick screening of the toxicity of PAs and PA-containing natural products.

Low selectivity is one of the major problems of currently used anticancer drugs, therefore, there is a high demand for novel, selective antitumor agents. In this study, the anticancer effects and mechanisms of guttiferone K (GUTK), a novel polyprenylated acylphloroglucinol derivative isolated from Garcinia cowa Roxb., were examined for its development as a novel drug targeting colon cancer. GUTK concentration- and time-dependently reduced the viability of human colon cancer HT-29 cells (IC(50) value 5.39 ± 0.22 μM) without affecting the viability of normal human colon epithelial CCD 841 CoN cells and induced G(0) /G(1) cell cycle arrest in HT-29 cells by down-regulating cyclins D1, D3 and cyclin-dependent kinases 4 and 6, while selectively restoring p21Waf1/Cip1 and p27Kip1 to levels comparable to those observed in normal colon cells, without affecting their levels in normal cells. GUTK (10.0 μM) induced cleavage of PARP, caspases-3, -8 and -9 and chromatin condensation to stimulate caspase-3-mediated apoptosis. The addition of a JNK inhibitor, SP600125, partially reversed GUTK-induced caspase-3 activity, indicating the possible involvement of JNK in GUTK-induced apoptosis. Furthermore, GUTK (10 mg/kg, i.p.) significantly decreased the tumor volume in a syngeneic colon tumor model when used alone or in combination with 5-fluorouracil without toxicity to the mice. Immunohistochemical staining of the tumor sections revealed a mechanism involving an increase in cleaved caspase-3 and a decrease in cell proliferation marker Ki-67. Our results support GUTK as a promising novel, potent and selective antitumor drug candidate for colon cancer.

Majority of traditional Chinese medicine (TCM) herbs need to undergo post-harvesting processing to convert raw material into the form readily used for prescription. In general, processing procedures are either according to China Pharmacopeia or based on traditional methods. Recently sulfur fumigation is increasingly used to replace traditional sun-drying for its pesticidal and anti-bacterial properties in a cheap and convenient manner. However, to date information on effects of sulfur fumigation on herbal safety and efficacy are limited. This article addresses potential destructive effects of sulfur fumigation on herbal efficacy and safety through reviewing currently available information. Since recently increased numbers of studies have demonstrated that sulfur fumigation-induced dramatic changes in chemical profiles of various sulfur-fumigated herbs, consequent alteration of efficacy, and/or potential incidence of toxicity are suspected. Therefore comprehensive investigations on effects of sulfur fumigation on toxicity, chemical profiles, pharmacokinetics, and bioactivities of TCM herbs are timely to provide scientific basis for standardization and regulation of this currently common but potentially harmful processing method.

The lack of selectivity and adequate potency of currently known P-glycoprotein (P-gp) inhibitors obscured their further development for clinical use to circumvent P-gp-mediated multidrug resistance (MDR), which necessitates the investigation of novel ones with higher potency and better specificity. The present study investigated the reversal effect of a new synthetic α-aminoxy lysine-peptidomimetic (Lys-P) on P-gp-mediated MDR. Effects of Lys-P on cytotoxicity of P-gp substrate doxorubicin (Dox) and intracellular accumulation of another P-gp substrate rhodamine 123 were examined in HEK293 cells. Its interaction mechanism and effect on P-gp expression were further investigated using ATPase assay and Western blot in Caco-2 cells, respectively. Lys-P restored the cytotoxicity of Dox toward the resistant MDR1-transfected HEK293 and MCF-7 TX400 cells without affecting their corresponding parental cells. It also significantly increased intracellular accumulation (21-fold) of rhodamine 123 in HEK293 MDR1 cells. Further mechanistic studies demonstrated that in the Caco-2 cell monolayer model, Lys-P abolished the P-gp-mediated efflux of Dox due to uncompetitive inhibition of P-gp ATPase without altering P-gp expression. Our findings demonstrated that Lys-P can be used as a promising lead compound for further development into selective and efficient MDR reversing agents for combination use with P-gp substrate drugs in cancer chemotherapy.

The sc family of cytokines (GM-CSF, IL-3 and IL-5) are largely products of activated T cells and regulate the survival, proliferation, differentiation and functional activation of hematopoietic cells following infection or bleeding. They are variously able to target myeloid hematopoietic stem and progenitor cells as well as mature neutrophils, eosinophils, macrophages and mast cells. Recent evidence suggests that this family of cytokines plays a role in other biological systems and importantly also in cancer, through the development and metastasis of solid tumours. Intriguingly, CD123, the IL-3 receptor alpha subunit (IL3R α ) is overexpressed by stem cells and primary blast cells of patients with acute myelogenous leukaemia (AML) and this correlates with poor prognosis leading to the development of multiple strategies that target CD123. We have now solved the structure of soluble IL3R α , in complex with a Fab fragment of CSL362, a humanised monoclonal antibody that binds IL3R α , blocks IL-3 function and is optimised for NK cell-mediated killing of leukaemic cells [1] . CSL362 is currently in a Phase 1 clinical trial for the treatment of patients with AML (Clinical Trials Gov. Identifier: NCT01632852). The three domain IL3R α structure unexpectedly revealed two alternative conformations, an open and a closed form based on the orientation of the N-terminal domain (NTD). The open conformation has not previously been reported for other Type I cytokine receptors with a similar domain structure, IL5R α , IL13R α 1 and IL13R α 2. The IL3R α structure will be presented together with data that supports a mechanism of IL-3 recognition and receptor signalling that may be applicable to other members of the Type I cytokine receptor superfamily. An unexpected dual mechanism of IL-3 antagonism utilised by CSL362 that involves direct antagonism of IL-3 binding as well as blockade of IL-3 receptor assembly, will also be presented.

Interleukin-3 (IL-3) is an activated T cell product that bridges innate and adaptive immunity and contributes to several immunopathologies. Here, we report the crystal structure of the IL-3 receptor α chain (IL3Rα) in complex with the anti-leukemia antibody CSL362 that reveals the N-terminal domain (NTD), a domain also present in the granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-5, and IL-13 receptors, adopting unique “open” and classical “closed” conformations. Although extensive mutational analyses of the NTD epitope of CSL362 show minor overlap with the IL-3 binding site, CSL362 only inhibits IL-3 binding to the closed conformation, indicating alternative mechanisms for blocking IL-3 signaling. Significantly, whereas “open-like” IL3Rα mutants can simultaneously bind IL-3 and CSL362, CSL362 still prevents the assembly of a higher-order IL-3 receptor-signaling complex. The discovery of open forms of cytokine receptors provides the framework for development of potent antibodies that can achieve a “double hit” cytokine receptor blockade. Refereed/Peer-reviewed