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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The sonic hedgehog factor GLI1 imparts drug resistance through inducible glucuronidation.

Drug resistance is a major hurdle in oncology. Responses of acute myeloid leukaemia (AML) patients to cytarabine (Ara-C)-based therapies are often short lived with a median overall survival of months. Therapies are under development to improve outcomes and include targeting the eukaryotic translation initiation factor (eIF4E) with its inhibitor ribavirin. In a Phase II clinical trial in poor prognosis AML, ribavirin monotherapy yielded promising responses including remissions; however, all patients relapsed. Here we identify a novel form of drug resistance to ribavirin and Ara-C. We observe that the sonic hedgehog transcription factor glioma-associated protein 1 (GLI1) and the UDP glucuronosyltransferase (UGT1A) family of enzymes are elevated in resistant cells. UGT1As add glucuronic acid to many drugs, modifying their activity in diverse tissues. GLI1 alone is sufficient to drive UGT1A-dependent glucuronidation of ribavirin and Ara-C, and thus drug resistance. Resistance is overcome by genetic or pharmacological inhibition of GLI1, revealing a potential strategy to overcome drug resistance in some patients.

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.

Regulation of drug metabolizing enzymes in the leukaemic bone marrow microenvironment.

The bone marrow (BM) microenvironment contributes to drug resistance in acute myeloid leukaemia (AML) and multiple myeloma (MM). We have shown that the critical drug metabolizing enzymes cytochrome P450 (CYP) 3A4 and cytidine deaminase (CDA) are highly expressed by BM stroma, and play an important role in this resistance to chemotherapy. However, what factors influence the chemoprotective capacity of the BM microenvironment, specifically related to CYP3A4 and CDA expression, are unknown. In this study, we found that the presence of AML cells decreases BM stromal expression of CYP3A4 and CDA, and this effect appears to be at least partially the result of cytokines secreted by AML cells. We also observed that stromal CYP3A4 expression is up-regulated by drugs commonly used in AML induction therapy, cytarabine, etoposide and daunorubicin, resulting in cross-resistance. Cytarabine also up-regulated CDA expression. The up-regulation of CYP3A4 associated with disease control was reversed by clarithromycin, a potent inhibitor of CYP3A4. Our data suggest that minimal residual disease states are characterized by high levels of stromal drug metabolizing enzymes and thus, strong microenvironment-mediated drug resistance. These results further suggest a potential role for clinically targeting drug metabolizing enzymes in the microenvironment.

Enzymatic rhamnosylation of anticancer drugs by an α-L-rhamnosidase from Alternaria sp. L1 for cancer-targeting and enzyme-activated prodrug therapy.

The synthesis of rhamnosylated compounds has gained great importance since these compounds have potential therapeutic applications. The enzymatic approaches for glycosylation of bioactive molecules have been well developed; however, the enzymatic rhamnosylation has been largely hindered by lacking of the glycosyl donor for rhamnosyltransferases. Here, we employed an α-L-rhamnosidase from Alternaria sp. L1 (RhaL1) to perform one-step rhamnosylation of anticancer drugs, including 2'-deoxy-5-fluorouridine (FUDR), cytosine arabinoside (Ara C), and hydroxyurea (Hydrea). The key synthesis conditions including substrate concentrations and reaction time were carefully optimized, and the maximum yields of each rhamnosylated drugs were 57.7 mmol for rhamnosylated Ara C, 68.6 mmol for rhamnosylated Hydrea, and 42.2 mmol for rhamnosylated FUDR. It is worth pointing out that these rhamnosylated drugs exhibit little cytotoxic effects on cancer cells, but could efficiently restore cytotoxic activity when incubated with exogenous α-L-rhamnosidase, suggesting their potential applications in the enzyme-activated prodrug system. To evaluate the cancer-targeting ability of rhamnose moiety, the rhamnose-conjugated fluorescence dye rhodamine B (Rha-RhB) was constructed. The fluorescence probe Rha-RhB displayed much higher cell affinity and cellular internalization rate of oral cancer cell KB and breast cancer cell MDA-MB-231 than that of the normal epithelial cells MCF 10A, suggesting that the rhamnose moiety could mediate the specific internalization of rhamnosylated compounds into cancer cells, which greatly facilitated their applications for cancer-targeting drug delivery.

Scavenger receptor class BI (SR-BI) mediates uptake of CPX-351 into K562 leukemia cells.

PURPOSE: CPX-351 is a liposomal formulation of cytarabine and daunorubicin encapsulated at a 5:1 molar ratio, for the treatment of acute myeloid leukemia. The Scavenger Receptor class B type I (SR-BI) plays an important role in mediating the uptake of high-density lipoproteins. The purpose of this study is to assess the role of the cell surface lipoprotein receptor SR-BI in the uptake of CPX-351 liposomes (Jazz Pharmaceuticals) into K562 leukemia cells. METHODS: K562 cells were pre-treated with 10 nM siRNA for 48 h and then treated with varying amount of CPX-351 for 24, 48 and 72 h. Cells were then collected and analyzed at 480/590 nm on a CytoFLEX Multicolour flow instrument to determine cellular uptake of daunorubicin. Experimental data were analyzed using two-way ANOVA with Bonferroni multiple comparisons. Significance was set at p < .05. RESULTS: K562 cells pre-treated with SR-BI siRNA for 48 h had a reduced SRB1 cell surface concentration (74-85%). Addition of CPX-351 at 10-50 nM followed by measurement of cellular daunorubicin at 48, 48 or 72 h showed a significantly lower percentage of daunorubicin positive population compared with control K562 cells (p < .05). There was significantly less daunorubicin taken up in the SR-BI knock-down cells across all drug concentrations and at all three time points, although there were no concentration-related trends. CONCLUSIONS: These preliminary studies suggest that SR-BI may be one potential mechanism by which CPX-351 is taken up into K562 cells.

Association of genetic polymorphisms in genes involved in Ara-C and dNTP metabolism pathway with chemosensitivity and prognosis of adult acute myeloid leukemia (AML).

BACKGROUND: Cytarabine arabinoside (Ara-C) has been the core of chemotherapy for adult acute myeloid leukemia (AML). Ara-C undergoes a three-step phosphorylation into the active metabolite Ara-C triphosphosphate (ara-CTP). Several enzymes are involved directly or indirectly in either the formation or detoxification of ara-CTP. METHODS: A total of 12 eQTL (expression Quantitative Trait Loci) single nucleotide polymorphisms (SNPs) or tag SNPs in 7 genes including CMPK1, NME1, NME2, RRM1, RRM2, SAMHD1 and E2F1 were genotyped in 361 Chinese non-M3 AML patients by using the Sequenom Massarray system. Association of the SNPs with complete remission (CR) rate after Ara-C based induction therapy, relapse-free survival (RFS) and overall survival (OS) were analyzed. RESULTS: Three SNPs were observed to be associated increased risk of chemoresistance indicated by CR rate (NME2 rs3744660, E2F1 rs3213150, and RRM2 rs1130609), among which two (rs3744660 and rs1130609) were eQTL. Combined genotypes based on E2F1 rs3213150 and RRM2 rs1130609 polymorphisms further increased the risk of non-CR. The SAMHD1 eQTL polymorphism rs6102991 showed decreased risk of non-CR marginally (P = 0.055). Three SNPs (NME1 rs3760468 and rs2302254, and NME2 rs3744660) were associated with worse RFS, and the RRM2 rs1130609 polymorphism was marginally associated with worse RFS (P = 0.085) and OS (P = 0.080). Three SNPs (NME1 rs3760468, NME2 rs3744660, and RRM1 rs183484) were associated with worse OS in AML patients. CONCLUSION: Data from our study demonstrated that SNPs in Ara-C and dNTP metabolic pathway predict chemosensitivity and prognosis of AML patients in China.

Low-dose CT protocols for guiding radiofrequency ablation for the treatment of small renal cell carcinomas.

OBJECTIVE: Computed tomography (CT)-guided radiofrequency ablation (RFA) results in a high radiation dose. This study aimed to assess low-dose CT protocols for guiding RFA and oncologic outcomes for the treatment of small renal cell carcinoma (RCC). MATERIALS AND METHODS: Between December 2011 and December 2014, CT-guided RFA was performed in 31 patients with 31 biopsy-proven RCCs (median, 2.1 cm). RFA included planning, targeting, monitoring and survey phases. The dose length product (DLP), CT dose index volume (CTDIvol), effective dose, number of scans, scan range, tube current and exposure time of RFA phases were compared. The 3-year recurrence-free survival rate was recorded. Nonparametric or parametric repeated-measures ANOVA with Dunn's or Tukey-Kramer multiple comparisons and Kaplan-Meier analysis were used for statistical analysis. RESULTS: The median total DLP, CTDIvol and effective dose of CT-guided RFA procedures per session were 1238.8 mGy (range 517.4-3391.7 mGy), 259.7 mGy (10.7-67.9 mGy) and 18.6 mSv (7.8-50.9 mSv), respectively. The median DLP, CTDIvol, effective dose, number of scans, tube current and exposure time during the targeting phase were higher than those during the other phases (p < 0.001). The scan range in the targeting phase was the same as that in the monitoring phase (p > 0.05) but smaller than those in the planning and survey phases (p < 0.001). The 3-year recurrence-free survival rate was 96.7%. CONCLUSIONS: Low-dose CT protocols for guiding RFA may reduce radiation dose without compromising oncologic outcomes. Reducing the number of scans during the targeting phase contributes to dose reduction.

DNA Methyltransferases Demonstrate Reduced Activity against Arabinosylcytosine: Implications for Epigenetic Instability in Acute Myeloid Leukemia.

Arabinosylcytosine (araC) is a mainstay in the initial treatment of acute myeloid leukemia (AML), although relapses are common. Given the recent recognition of altered DNA methylation patterns in relapsed AML, we considered whether araC, which acts by incorporation into DNA, could itself perturb methylation dynamics. To explore this possibility, we examined several DNA methyltransferases and find that araC embedded in DNA is consistently methylated with an efficiency diminished relative to that of deoxycytidine. Importantly, with the human maintenance methyltransferase DNMT1, the extent of araC methylation is reduced by more than ∼200-fold. These observations support a model whereby araC treatment may itself contribute to locus-specific, passive DNA demethylation in relapsed AML.

Polymorphisms at microRNA binding sites of Ara-C and anthracyclines-metabolic pathway genes are associated with outcome of acute myeloid leukemia patients.

BACKGROUND: Gene polymorphisms at microRNA-binding sites (poly-miRTS) may affect gene transcription and expression through miRNA regulation, which is associated with cancer susceptibility, sensitivity to chemotherapy and prognosis. This study investigated the association between poly-miRTS of Ara-C/anthracycline metabolic pathways genes and the outcome of acute myeloid leukemia (AML) in Chinese patients after Ara-C-based chemotherapy. METHODS: A total of 17 poly-miRTS were selected from the SNPinfo Web Server and genotyped in 206 Chinese Han non-FAB-M3 AML patients using the SEQUENOM Mass-ARRAY system. RESULTS: Among these 17 poly-miRTS, five Ara-C metabolic gene single nucleotide polymorphisms (SNPs, NT5C2 rs10786736 and rs8139, SLC29A1 rs3734703, DCTD rs7278, and RRM1 rs1042919) were identified to significantly associate with complete AML remission and/or overall and relapse-free survival (OS and RFS, respectively), and four anthracycline-metabolic gene SNPs (ABCC1 rs3743527, rs212091, and rs212090 and CBR1 rs9024) were significantly associated with chemotherapy-related toxicities. Moreover, SLC29A1 rs3734703 was shown to associate with both chemotherapy response and survival (adjusted OR 2.561 in the overdominant model; adjusted HR 2.876 for OS and 2.357 for RFS in the dominant model). CONCLUSIONS: The data from the current study demonstrated that the poly-miRTS of Ara-C/anthracyclines metabolic genes predicted the sensitivity and side effects of AML to Ara-C-based chemotherapy and patient survival. Further study will confirm them as biomarkers for AML patients after Ara-C-based chemotherapy.