Identification of several African swine fever virus replication inhibitors by screening of a library of FDA-approved drugs.

African swine fever (ASF) caused by African swine fever virus (ASFV) is a highly infectious and lethal swine disease. Currently, there is only one novel approved vaccine and no antiviral drugs for ASFV. In the study, a high-throughput screening of an FDA-approved drug library was performed to identify several drugs against ASFV infection in primary porcine alveolar macrophages. Triapine and cytarabine hydrochloride were identified as ASFV infection inhibitors in a dose-dependent manner. The two drugs executed their antiviral activity during the replication stage of ASFV. Furthermore, molecular docking studies showed that triapine might interact with the active center Fe2+ in the small subunit of ASFV ribonucleotide reductase while cytarabine hydrochloride metabolite might interact with three residues (Arg589, Lys593, and Lys631) of ASFV DNA polymerase to block new DNA chain extension. Taken together, our results suggest that triapine and cytarabine hydrochloride displayed significant antiviral activity against ASFV in vitro.

GAS5 promotes cytarabine induced myelosuppression via inhibition of hematopoietic stem cell differentiation.

Cytarabine (Ara-C) is widely used in the induction chemotherapy for acute myeloid leukemia (AML). Association between LncRNA GAS5 genetic polymorphism and the recovery of hematopoietic function after Ara-C-based chemotherapy is observed. This study aimed to identify whether intervention of GAS5 expression and GAS5 genotype affect Ara-C-induced inhibition of hematopoietic stem cells (HSCs) differentiation. In this study, cord blood-derived CD34+ cells were cultured in vitro, and a cell model of myelosuppression was established by treatment of CD34+ cells with Ara-C. The effect of GAS5 overexpression, Ara-C treatment, and GAS5 rs55829688 genotype on the hematopoietic colony-forming ability of CD34+ cells was assessed using methylcellulose-based colony forming unit assay. GAS5 overexpression slowed down the proliferation of cord blood-derived CD34+ cells significantly (p < 0.05) and decreased their ability to form hematopoietic colonies in vitro. Ara-C significantly reduced the hematopoietic colony-forming ability of CD34+ cells in vitro (p < 0.0001), and overexpressing GAS5 further decreased the number of hematopoietic colonies. GAS5 expression was higher in CD34+ cells than in CD34- cells, and positively correlated with GATA1 mRNA expression in CD34+ cells in vitro culture. However, GAS5 genotype had no effect on the total number of hematopoietic colonies formed from cord blood-derived CD34+ cells. In conclusion, our study highlights that GAS5 inhibited the in vitro proliferation and reduced the hematopoietic colony-forming ability of cord blood-derived CD34+ cells, with the most pronounced effect observed on CFU-GEMM formation. GAS5 also enhanced the inhibitory effect of Ara-C on the in vitro hematopoietic ability of CD34+ HSCs.

Exosome-shuttled FTO from BM-MSCs contributes to cancer malignancy and chemoresistance in acute myeloid leukemia by inducing m6A-demethylation: A nano-based investigation.

Although bone marrow mesenchymal stem cells (BM-MSCs)-derived exosomes have been reported to be closely associated with acute myeloid leukemia (AML) progression and chemo-resistance, but its detailed functions and molecular mechanisms have not been fully delineated. Besides, serum RNA m6A demethylase fat mass and obesity-associated protein (FTO)-containing exosomes are deemed as important indicators for cancer progression, and this study aimed to investigate the role of BM-MSCs-derived FTO-exosomes in regulating the malignant phenotypes of AML cells. Here, we verified that BM-MSCs-derived exosomes delivered FTO to promote cancer aggressiveness, stem cell properties and Cytosine arabinoside (Ara-C)-chemoresistance in AML cells, and the underlying mechanisms were also uncovered. Our data suggested that BM-MSCs-derived FTO-exo demethylated m6A modifications in the m6A-modified LncRNA GLCC1 to facilitate its combination with the RNA-binding protein Hu antigen R (HuR), which further increased the stability and expression levels of LncRNA GLCC1. In addition, LncRNA GLCC1 was verified as an oncogene to facilitate cell proliferation and enhanced Ara-C-chemoresistance in AML cells. Further experiments confirmed that demethylated LncRNA GLCC1 served as scaffold to facilitate the formation of the IGF2 mRNA binding protein 1 (IGF2BP1)-c-Myc complex, which led to the activation of the downstream tumor-promoting c-Myc-associated signal pathways. Moreover, our rescuing experiments validated that the promoting effects of BM-MSCs-derived FTO-exo on cancer aggressiveness and drug resistance in AML cells were abrogated by silencing LncRNA GLCC1 and c-Myc. Thus, the present firstly investigated the functions and underlying mechanisms by which BM-MSCs-derived FTO-exo enhanced cancer aggressiveness and chemo-resistance in AML by modulating the LncRNA GLCC1-IGF2BP1-c-Myc signal pathway, and our work provided novel biomarkers for the diagnosis, treatment and therapy of AML in clinic.

Platelet-derived microparticles provoke chronic lymphocytic leukemia malignancy through metabolic reprogramming.

Background: It is well established that inflammation and platelets promote multiple processes of cancer malignancy. Recently, platelets have received attention for their role in carcinogenesis through the production of microvesicles or platelet-derived microparticles (PMPs), which transfer their biological content to cancer cells. We have previously characterized a new subpopulation of these microparticles (termed mito-microparticles), which package functional mitochondria. The potential of mitochondria transfer to cancer cells is particularly impactful as many aspects of mitochondrial biology (i.e., cell growth, apoptosis inhibition, and drug resistance) coincide with cancer hallmarks and disease progression. These metabolic aspects are particularly notable in chronic lymphocytic leukemia (CLL), which is characterized by a relentless accumulation of proliferating, immunologically dysfunctional, mature B-lymphocytes that fail to undergo apoptosis. The present study aimed to investigate the role of PMPs on CLL metabolic plasticity leading to cancer cell phenotypic changes. Methods: CLL cell lines were co-incubated with different concentrations of human PMPs, and their impact on cell proliferation, mitochondrial DNA copy number, OCR level, ATP production, and ROS content was evaluated. Essential genes involved in metabolic-reprogramming were identified using the bioinformatics tools, examined between patients with early and advanced CLL stages, and then validated in PMP-recipient CLLs. Finally, the impact of the induced metabolic reprogramming on CLLs' growth, survival, mobility, and invasiveness was tested against anti-cancer drugs Cytarabine, Venetoclax, and Plumbagin. Results: The data demonstrated the potency of PMPs in inducing tumoral growth and invasiveness in CLLs through mitochondrial internalization and OXPHOS stimulation which was in line with metabolic shift reported in CLL patients from early to advanced stages. This metabolic rewiring also improved CLL cells' resistance to Cytarabine, Venetoclax, and Plumbagin chemo drugs. Conclusion: Altogether, these findings depict a new platelet-mediated pathway of cancer pathogenesis. We also highlight the impact of PMPs in CLL metabolic reprogramming and disease progression.

Exploring the possible mitoprotective and neuroprotective potency of thymoquinone, betanin, and vitamin D against cytarabine-induced mitochondrial impairment and neurotoxicity in rats' brain.

It has been suggested that cytarabine (Ara-C) induces toxicity via mitochondrial dysfunction and oxidative stress. Therefore, we hypothesized that mitochondrial protective agents and antioxidants can reduce cytarabine-induced neurotoxicity. For this purpose, 48 male Wistar rats were assigned into eight equal groups include control group, Ara-C (70 mg/kg, i.p.) group, Ara-C plus betanin (25 mg/kg, i.p.) group, Ara-C plus vitamin D (500 U/kg, i.p.) group, Ara-C plus thymoquinone (0.5 mg/kg, i.p.) group, betanin group, vitamin group, and thymoquinone group. The activity of acetylcholinesterase (AChE), and butyrylcholinesterase (BChE), the concentrations of antioxidants (reduced glutathione and oxidized glutathione), oxidative stress (malondialdehyde) biomarkers, mitochondrial toxicity parameters as well as histopathological alteration in brain tissues were measured. Our results demonstrated that Ara-C exposure significantly declines the brain enzymes activity (AChE and BChE), levels of antioxidant biomarkers (GSH), and mitochondrial functions, but markedly elevate the levels of oxidative stress biomarkers (MDA) and mitochondrial toxicity. Almost all of the previously mentioned parameters (especially mitochondrial toxicity) were retrieved by betanin, vitamin D, and thymoquinone compared to Ara-C group. These findings conclusively indicate that betanin, vitamin D, and thymoquinone administration provide adequate protection against Ara-C-induced neurotoxicity through modulations of oxidative, antioxidant activities, and mitochondrial protective (mitoprotective) effects.

Atomic force microscopy measurements and model of DNA bending caused by binding of AraC protein.

Atomic force microscopy (AFM) was used to conduct single-molecule imaging of protein/DNA complexes involved in the regulation of the arabinose operon of Escherichia coli. In the presence of arabinose, the transcription regulatory protein AraC binds to a 38 bp region consisting of the araI1 and araI2 half-sites. The domain positioning of full-length AraC, when bound to DNA, was not previously known. In this study, AraC was combined with 302 and 560 bp DNA and arabinose, deposited on a mica substrate, and imaged with AFM in air. High resolution images of 560 bp DNA, where bound protein was visible, showed that AraC induces a bend in the DNA with an angle 60° ± 12° with a median of 55°. These results are consistent with earlier gel electrophoresis measurements that measured the DNA bend angle based on migration rates. By using known domain structures of AraC, geometric constraints, and contacts determined from biochemical experiments, we developed a model of the tertiary and quaternary structure of DNA-bound AraC in the presence of arabinose. The DNA bend angle predicted by the model is in agreement with the measurement values. We discuss the results in view of other regulatory proteins that cause DNA bending and formation of the open complex to initiate transcription.

The AraC/XylS Protein MxiE and Its Coregulator IpgC Control a Negative Feedback Loop in the Transcriptional Cascade That Regulates Type III Secretion in Shigella flexneri.

Members of the AraC family of transcriptional regulators (AFTRs) control the expression of many genes important to cellular processes, including virulence. In Shigella species, the type III secretion system (T3SS), a key determinant for host cell invasion, is regulated by the three-tiered VirF/VirB/MxiE transcriptional cascade. Both VirF and MxiE belong to the AFTRs and are characterized as positive transcriptional regulators. Here, we identify a novel regulatory activity for MxiE and its coregulator IpgC, which manifests as a negative feedback loop in the VirF/VirB/MxiE transcriptional cascade. Our findings show that MxiE and IpgC downregulate the virB promoter and, hence, VirB protein production, thus decreasing VirB-dependent promoter activity at ospD1, one of the nearly 50 VirB-dependent genes. At the virB promoter, regions required for negative MxiE- and IpgC-dependent regulation were mapped and found to be coincident with regions required for positive VirF-dependent regulation. In tandem, negative MxiE- and IpgC-dependent regulation of the virB promoter only occurred in the presence of VirF, suggesting that MxiE and IpgC can function to counter VirF activation of the virB promoter. Lastly, MxiE and IpgC do not downregulate another VirF-activated promoter, icsA, demonstrating that this negative feedback loop targets the virB promoter. Our study provides insight into a mechanism that may reprogram Shigella virulence gene expression following type III secretion and provides the impetus to examine if MxiE and IpgC homologs in other important bacterial pathogens, such as Burkholderia pseudomallei and Salmonella enterica serovars Typhimurium and Typhi, coordinate similar negative feedback loops. IMPORTANCE The large AraC family of transcriptional regulators (AFTRs) control virulence gene expression in many bacterial pathogens. In Shigella species, the AraC/XylS protein MxiE and its coregulator IpgC positively regulate the expression of type III secretion system genes within the three-tiered VirF/VirB/MxiE transcriptional cascade. Our findings suggest a negative feedback loop in the VirF/VirB/MxiE cascade, in which MxiE and IpgC counter VirF-dependent activation of the virB promoter, thus making this the first characterization of negative MxiE- and IpgC-dependent regulation. Our study provides insight into a mechanism that likely reprograms Shigella virulence gene expression following type III secretion, which has implications for other important bacterial pathogens with functional homologs of MxiE and IpgC.

Involvement of ORAI1/SOCE in Human AML Cell Lines and Primary Cells According to ABCB1 Activity, LSC Compartment and Potential Resistance to Ara-C Exposure.

Acute myeloid leukemia (AML) is a hematological malignancy with a high risk of relapse. This issue is associated with the development of mechanisms leading to drug resistance that are not yet fully understood. In this context, we previously showed the clinical significance of the ATP binding cassette subfamily B-member 1 (ABCB1) in AML patients, namely its association with stemness markers and an overall worth prognosis. Calcium signaling dysregulations affect numerous cellular functions and are associated with the development of the hallmarks of cancer. However, in AML, calcium-dependent signaling pathways remain poorly investigated. With this study, we show the involvement of the ORAI1 calcium channel in store-operated calcium entry (SOCE), the main calcium entry pathway in non-excitable cells, in two representative human AML cell lines (KG1 and U937) and in primary cells isolated from patients. Moreover, our data suggest that in these models, SOCE varies according to the differentiation status, ABCB1 activity level and leukemic stem cell (LSC) proportion. Finally, we present evidence that ORAI1 expression and SOCE amplitude are modulated during the establishment of an apoptosis resistance phenotype elicited by the chemotherapeutic drug Ara-C. Our results therefore suggest ORAI1/SOCE as potential markers of AML progression and drug resistance apparition.

Structural basis of transcription activation by Rob, a pleiotropic AraC/XylS family regulator.

Rob, which serves as a paradigm of the large AraC/XylS family transcription activators, regulates diverse subsets of genes involved in multidrug resistance and stress response. However, the underlying mechanism of how it engages bacterial RNA polymerase and promoter DNA to finely respond to environmental stimuli is still elusive. Here, we present two cryo-EM structures of Rob-dependent transcription activation complex (Rob-TAC) comprising of Escherichia coli RNA polymerase (RNAP), Rob-regulated promoter and Rob in alternative conformations. The structures show that a single Rob engages RNAP by interacting with RNAP αCTD and σ70R4, revealing their generally important regulatory roles. Notably, by occluding σ70R4 from binding to -35 element, Rob specifically binds to the conserved Rob binding box through its consensus HTH motifs, and retains DNA bending by aid of the accessory acidic loop. More strikingly, our ligand docking and biochemical analysis demonstrate that the large Rob C-terminal domain (Rob CTD) shares great structural similarity with the global Gyrl-like domains in effector binding and allosteric regulation, and coordinately promotes formation of competent Rob-TAC. Altogether, our structural and biochemical data highlight the detailed molecular mechanism of Rob-dependent transcription activation, and provide favorable evidences for understanding the physiological roles of the other AraC/XylS-family transcription factors.

Effect of Chemotherapy Cytarabine and Acute Myeloid Leukemia on the Development of Spermatogenesis at the Adult Age of Immature Treated Mice.

Acute myeloid leukemia (AML) accounts for around 20% of diagnosed childhood leukemia. Cytarabine (CYT) is involved in the AML treatment regimen. AML and CYT showed impairment in spermatogenesis in human and rodents in adulthood. We successfully developed an AML disease model in sexually immature mice. Monocytes and granulocytes were examined in all groups: untreated control, AML alone, CYT alone and AML+CYT (in combination). There was a significant increase in the counts of monocytes and granulocytes in the AML-treated immature mice (AML) compared to the control, and AML cells were demonstrated in the blood vessels of the testes. AML alone and CYT alone impaired the development of spermatogenesis at the adult age of the AML-treated immature mice. The damage was clear in the structure/histology of their seminiferous tubules, and an increase in the apoptotic cells of the seminiferous tubules was demonstrated. Our results demonstrated a significant decrease in the meiotic/post-meiotic cells compared to the control. However, CYT alone (but not AML) significantly increased the count of spermatogonial cells (premeiotic cells) that positively stained with SALL4 and PLZF per tubule compared to the control. Furthermore, AML significantly increased the count of proliferating spermatogonial cells that positively stained with PCNA in the seminiferous tubules compared to the control, whereas CYT significantly decreased the count compared to the control. Our result showed that AML and CYT affected the microenvironment/niche of the germ cells. AML significantly decreased the levels growth factors, such as SCF, GDNF and MCSF) compared to control, whereas CYT significantly increased the levels of MCSF and GDNF compared to control. In addition, AML significantly increased the RNA expression levels of testicular IL-6 (a proinflammatory cytokine), whereas CYT significantly decreased testicular IL-6 levels compared to the control group. Furthermore, AML alone and CYT alone significantly decreased RNA expression levels of testicular IL-10 (anti-inflammatory cytokine) compared to the control group. Our results demonstrate that pediatric AML disease with or without CYT treatment impairs spermatogenesis at adult age (the impairment was more pronounced in AML+CYT) compared to control. Thus, we suggest that special care should be considered for children with AML who are treated with a CYT regimen regarding their future fertility at adult age.

Astaxanthin decreases the growth-inhibitory dose of cytarabine and inflammatory response in the acute lymphoblastic leukemia cell line NALM-6.

BACKGROUND: In spite of the great progress in acute lymphoblastic leukemia (ALL) treatment, a large number of patients still suffer from chemotherapy drug toxicity. As a routine medication for ALL treatment, cytarabine (Ara-C) has many side effects on the patients. Astaxanthin (ASX), on the other hand, is a carotenoid with antioxidant, anti-inflammatory and anti-cancer properties. PURPOSE: The present study investigated the effects of ASX in combination with Ara-C on cell proliferation, apoptosis induction, and cell cycle arrest in NALM-6 cell line. METHODS: NALM6 cells were treated with different concentrations of ASX, Ara-C, and their co-treatment. Cytotoxic effects were evaluated using MTT assay. After treating the cells with the IC50 dose of ASX, Ara-C and their co-treatment, we studied apoptosis induction, cell cycle arrest, and expression of apoptotic, anti-apoptotic, and inflammatory genes. RESULT: MTT assay demonstrated that co-treatment of cytarabine and ASX had greater cytotoxicity effects compared with the IC50 dose of Ara-C alone. After 48 h of treatment of NALM-6 cells with the combination dose, expression levels of apoptotic genes (P53, caspase-8, 3), the anti-apoptotic gene (Bcl-xL) and inflammatory genes (IL-6, TNF-α) changed significantly compared to the untreated group (p < 0.05). CONCLUSIONS: Co-treatment of ASX and Ara-C has synergism effects on apoptosis pathways, cell proliferation inhibition, and decreased inflammation.

Whole-cell biosynthesis of cytarabine by an unnecessary protein-reduced Escherichia coli that coexpresses purine and uracil phosphorylase.

Currently, whole-cell catalysts face challenges due to the complexity of reaction systems, although they have a cost advantage over pure enzymes. In this study, cytarabine was synthesized by purified purine phosphorylase 1 (PNP1) and uracil phosphorylase (UP), and the conversion of cytarabine from adenine arabinoside reached 72.3 ± 4.3%. However, the synthesis was unsuccessful by whole-cell catalysis due to interference from unnecessary proteins (UNPs) in cells. Thus, we carried out a large-scale gene editing involving 377 genes in the genome of Escherichia coli to reduce the negative effect of UNPs on substrate conversion and cytarabine production. Finally, the PNP1 and UP activities of the obtained mutant were increased significantly compared with the parental strain, and more importantly, the conversion rate of cytarabine by whole-cell catalysis reached 67.4 ± 2.5%. The lack of 148 proteins and downregulation of 783 proteins caused by gene editing were equivalent to partial purification of the enzymes within cells, and thus, we provided inspiration to solve the problem caused by UNP interference, which is ubiquitous in the field of whole-cell catalysis.

Cytarabine-induced TNFα promotes the expansion and suppressive functions of myeloid-derived suppressor cells in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is very common haematopoietic malignancies with poor prognosis. Chemotherapy is still a mainstay therapy for AML patients. AML microenvironment plays critical roles in therapy response. However, the role of chemotherapy in AML microenvironment is poorly understood. In this study, we report that cytarabine (AraC)-triggered TNFα from AML cells expanded myeloid-derived suppressor cells (MDSCs) and enhanced MDSC functions and survival through activating IL-6/STAT3 and NFκB pathways. Blockade of TNFα in conditioned medium-derived AraC-treated AML cells (AraC_CM) impaired MDSC expansion and functions, reduced IL-6 secretion and the level of activated STAT3. Inhibiting IL6 or STAT3 abrogated AraC_CM-mediated MDSC suppressive function. Additionally, inhibiting TNFα also impaired AraC_CM-mediated NFκB activation. Blocking NFκB activation reduced MDSC viability induced by AraC_CM. Together, these results provided a role of AraC-induced TNFα in MDSC expansion and functions and suggest that targeting TNFα may benefit AML patients to current anticancer strategies by blocking MDSC-mediated immunosuppression.

Oxidative phosphorylation enhances the leukemogenic capacity and resistance to chemotherapy of B cell acute lymphoblastic leukemia.

How metabolic status controls the fates of different types of leukemia cells remains elusive. Using a SoNar-transgenic mouse line, we demonstrated that B cell acute lymphoblastic leukemia (B-ALL) cells had a preference in using oxidative phosphorylation. B-ALL cells with a low SoNar ratio (SoNar-low) had enhanced mitochondrial respiration capacity, mainly resided in the vascular niche, and were enriched with more functional leukemia-initiating cells than that of SoNar-high cells in a murine B-ALL model. The SoNar-low cells were more resistant to cytosine arabinoside (Ara-C) treatment. cyclic adenosine 3',5'-monophosphate response element-binding protein transactivated pyruvate dehydrogenase complex component X and cytidine deaminase to maintain the oxidative phosphorylation level and Ara-C-induced resistance. SoNar-low human primary B-ALL cells also had a preference for oxidative phosphorylation. Suppressing oxidative phosphorylation with several drugs sufficiently attenuated Ara-C-induced resistance. Our study provides a unique angle for understanding the potential connections between metabolism and B-ALL cell fates.

Alpha-lipoic acid ameliorates cytarabine-induced developmental anomalies in rat fetus.

Cytarabine (Ara-C) is a nucleoside analogue used in the treatment of cancers and viral infections. It has teratogenic potential and causes a variety of birth defects in fetuses. Alpha-lipoic acid (ALA) is a natural antioxidant offers protection against the developmental toxicity induced by drug- or toxicant-exposure or pathological conditions. This study was aimed at evaluating the protective effect of ALA against Ara-C induced developmental toxicity in rat fetus. Pregnant rats divided into five groups and received normal saline, ALA200 mg/kg, Ara-C12.5 mg/kg, Ara-C25 mg/kg and, Ara-C25 mg/kg plus ALA200 mg/kg respectively from gestational day (GD) 8 to GD14 and sacrificed on GD21. Ara-C treatment led to a significant and dose-dependent decrease in food intake, weight gain, placental weight, and an increase in oxidative stress in pregnant rats. Further, the in-utero exposure to Ara-C led to an increase in fetal mortality, resorptions, oxidative stress, external morphological anomalies and limb abnormalities, and impaired ossification. Co-administration of ALA resulted in amelioration of the footprints of Ara-C induced toxicity in pregnant rats as well as the fetus. These findings indicate that the ALA supplementation offers protection against developmental toxicity caused by Ara-C prenatal exposure in rats.

Downregulation of GLI3 Expression Mediates Chemotherapy Resistance in Acute Myeloid Leukemia.

Aberrant activation of the hedgehog (HH) pathway is observed in many neoplasms, including acute myeloid leukemia (AML). The glioma-associated oncogene homolog (GLI) transcription factors are the main downstream effectors of the HH signaling cascade and are responsible for the proliferation and maintenance of leukemic stem cells, which support chemotherapy resistance and leukemia relapse. Cytarabine (Ara-C)-resistant variants of AML cell lines were established through long-term cultivation with successively increasing Ara-C concentrations. Subsequently, differences in GLI expression were analyzed by RT-qPCR. GLI3 mRNA levels were detectable in parental Kasumi-1, OCI-AML3, and OCI-AML5 cells, whereas GLI3 expression was completely silenced in all resistant counterparts. Therefore, we generated GLI3-knockdown cell lines using small hairpin RNAs (shRNA) and evaluated their sensitivity to Ara-C in vitro. The knockdown of GLI3 partly abolished the effect of Ara-C on colony formation and induction of apoptosis, indicating that GLI3 downregulation results in Ara-C resistance. Moreover, we analyzed the expression of several genes involved in Ara-C metabolism and transport. Knockdown of GLI3 resulted in the upregulation of SAM and HD domain-containing protein 1 (SAMHD1), cytidine deaminase (CDA), and ATP-binding cassette C11 (ABCC11)/multidrug resistance-associated protein 8 (MRP8), each of which has been identified as a predictive marker for Ara-C response in acute myeloid leukemia. Our results demonstrate that GLI3 downregulation is a potential mechanism to induce chemotherapy resistance in AML.

Proteome activity landscapes of tumor cell lines determine drug responses.

Integrated analysis of genomes, transcriptomes, proteomes and drug responses of cancer cell lines (CCLs) is an emerging approach to uncover molecular mechanisms of drug action. We extend this paradigm to measuring proteome activity landscapes by acquiring and integrating quantitative data for 10,000 proteins and 55,000 phosphorylation sites (p-sites) from 125 CCLs. These data are used to contextualize proteins and p-sites and predict drug sensitivity. For example, we find that Progesterone Receptor (PGR) phosphorylation is associated with sensitivity to drugs modulating estrogen signaling such as Raloxifene. We also demonstrate that Adenylate kinase isoenzyme 1 (AK1) inactivates antimetabolites like Cytarabine. Consequently, high AK1 levels correlate with poor survival of Cytarabine-treated acute myeloid leukemia patients, qualifying AK1 as a patient stratification marker and possibly as a drug target. We provide an interactive web application termed ATLANTiC (http://atlantic.proteomics.wzw.tum.de), which enables the community to explore the thousands of novel functional associations generated by this work.

pH-sensitive drug delivery based on chitosan wrapped graphene quantum dots with enhanced fluorescent stability.

Graphene quantum dots (GQDs) were prepared by the pyrolysis of citric acid (CA), which were used for the loading of hydrophilic cytarabine (Cyt), an anti-cancer drug, and then wrapped with chitosan (CS) gels for the encapsulation of the loaded Cyt. The fluorescent stability of GQDs was significantly enhanced in the presence of CS, which might be attributed to the inhibited agglomeration of GQDs by the CS gels. In addition, the burst release of Cyt from the developed carrier was also effectively relieved by the CS coating. Since the incorporation of Cyt into GQDs was achieved by amidation reaction, the delivery of Cyt from the carrier was pH-sensitive due to the hydrolysis of the amido linkage between GQDs and Cyt in acidic medium.

ADORA1 Inhibition Promotes Tumor Immune Evasion by Regulating the ATF3-PD-L1 Axis.

Here, we show that tumor ADORA1 deletion suppresses cell growth in human melanoma cell lines in vitro and tumor development in vivo in immune-deficient xenografts. However, this deletion induces the upregulation of PD-L1 levels, which inactivates cocultured T cells in vitro, compromises anti-tumor immunity in vivo, and reduces anti-tumor efficacy in an immune-competent mouse model. Functionally, PD-1 mAb treatment enhances the efficacy of ADORA1-deficient or ADORA1 antagonist-treated melanoma and NSCLC immune-competent mouse models. Mechanistically, we identify ATF3 as the factor transcriptionally upregulating PD-L1 expression. Tumor ATF3 deletion improves the effect of ADORA1 antagonist treatment of melanoma and NSCLC xenografts. We observe higher ADORA1, lower ATF3, and lower PD-L1 expression levels in tumor tissues from nonresponders among PD-1 mAb-treated NSCLC patients.

18F-FAC PET Visualizes Brain-Infiltrating Leukocytes in a Mouse Model of Multiple Sclerosis.

Brain-infiltrating leukocytes contribute to multiple sclerosis (MS) and autoimmune encephalomyelitis and likely play a role in traumatic brain injury, seizure, and stroke. Brain-infiltrating leukocytes are also primary targets for MS disease-modifying therapies. However, no method exists for noninvasively visualizing these cells in a living organism. 1-(2'-deoxy-2'-18F-fluoroarabinofuranosyl) cytosine (18F-FAC) is a PET radiotracer that measures deoxyribonucleoside salvage and accumulates preferentially in immune cells. We hypothesized that 18F-FAC PET could noninvasively image brain-infiltrating leukocytes. Methods: Healthy mice were imaged with 18F-FAC PET to quantify if this radiotracer crosses the blood-brain barrier (BBB). Experimental autoimmune encephalomyelitis (EAE) is a mouse disease model with brain-infiltrating leukocytes. To determine whether 18F-FAC accumulates in brain-infiltrating leukocytes, EAE mice were analyzed with 18F-FAC PET, digital autoradiography, and immunohistochemistry, and deoxyribonucleoside salvage activity in brain-infiltrating leukocytes was analyzed ex vivo. Fingolimod-treated EAE mice were imaged with 18F-FAC PET to assess if this approach can monitor the effect of an immunomodulatory drug on brain-infiltrating leukocytes. PET scans of individuals injected with 2-chloro-2'-deoxy-2'-18F-fluoro-9-ß-d-arabinofuranosyl-adenine (18F-CFA), a PET radiotracer that measures deoxyribonucleoside salvage in humans, were analyzed to evaluate whether 18F-CFA crosses the human BBB. Results:18F-FAC accumulates in the healthy mouse brain at levels similar to 18F-FAC in the blood (2.54 ± 0.2 and 3.04 ± 0.3 percentage injected dose per gram, respectively) indicating that 18F-FAC crosses the BBB. EAE mice accumulate 18F-FAC in the brain at 180% of the levels of control mice. Brain 18F-FAC accumulation localizes to periventricular regions with significant leukocyte infiltration, and deoxyribonucleoside salvage activity is present at similar levels in brain-infiltrating T and innate immune cells. These data suggest that 18F-FAC accumulates in brain-infiltrating leukocytes in this model. Fingolimod-treated EAE mice accumulate 18F-FAC in the brain at 37% lower levels than control-treated EAE mice, demonstrating that 18F-FAC PET can monitor therapeutic interventions in this mouse model. 18F-CFA accumulates in the human brain at 15% of blood levels (0.08 ± 0.01 and 0.54 ± 0.07 SUV, respectively), indicating that 18F-CFA does not cross the BBB in humans. Conclusion:18F-FAC PET can visualize brain-infiltrating leukocytes in a mouse MS model and can monitor the response of these cells to an immunomodulatory drug. Translating this strategy into humans will require exploring additional radiotracers.