8-Deoxy-Rifamycin Derivatives from Amycolatopsis mediterranei S699 ΔrifT Strain

Proansamycin X, a hypothetical earliest macrocyclic precursor in the biosynthesis of rifamycin, had never been isolated and identified. According to bioinformatics analysis, it was proposed that RifT (a putative NADH-dependent dehydrogenase) may be a candidate target responsible for the dehydrogenation of proansamycin X. In this study, the mutant strain Amycolatopsis mediterranei S699 ΔrifT was constructed by deleting the rifT gene. From this strain, eleven 8-deoxy-rifamycin derivatives (1–11) and seven known analogues (12–18) were isolated. Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data and high-resolution ESI mass spectra. Compound 1 is a novel amide N-glycoside of seco-rifamycin. Compounds 2 and 3 feature conserved 11,12-seco-rifamycin W skeleton. The diverse post-modifications in the polyketide chain led to the production of 4–11. Compounds 2, 3, 5, 6, 13 and 15 exhibited antibacterial activity against Staphylococcus aureus (MIC (minimal inhibitory concentration) values of 10, 20, 20, 20, 40 and 20 μg/mL, respectively). Compounds 14, 15, 16, 17 and 18 showed potent antiproliferative activity against KG1 cells with IC50 (half maximal inhibitory concentration) values of 14.91, 44.78, 2.16, 18.67 and 8.07 μM, respectively.

Tipifarnib Modulates Interferon (IFN)-γ-Inducible Genes in Acute Myeloid Leukemia

Background Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous hematological malignancy. Chemotherapy resistance is common, and relapse is the major cause of treatment failure. Investigation of new molecularly-targeted and immunomodulating agents therefore remains a high priority. Interferon (IFN)-γ regulates inflammatory responses and tumor immunosurveillance. Prolonged IFN-γ signaling may promote immune-independent resistance to genotoxic anti-cancer therapies. Herein, we aimed to establish whether tipifarnib, a farnesyl-transferase and CXCL12/CXCR4 pathway inhibitor currently being tested in phase I/II clinical trials in individuals with myeloid malignancies, modulates IFN-γ signaling. Methods We previously defined an IFN-γ-responsiveness gene signature in AML cell lines (Vadakekolathu J, et al. Blood 2017; 130: 3945A). Herein, we employed targeted gene expression profiling for the high-dimensional analysis of canonical signaling pathways and their modulation by tipifarnib in Kasumi-1 AML cells [AML with t(8;21)] and KG1 AML cells (leukemia stem cell [LSC]-like AML). AML cells were either incubated with 100 nM or 500 nM tipifarnib for 6 hours, or left untreated, prior to in vitro challenge with 10 ng/mL IFN-γ for 24 hours. RNA (approximately 100 ng per sample) was incubated with a reporter and capture probe mix for hybridization (mRNA Pan-Cancer Pathways Panel; NanoString Technologies, Seattle, USA). Transcript counts were analyzed on the nCounter FLEX analysis system. The nSolver™ software package and nSolver Advanced Analysis module were used for quality controls. The captured transcript counts were normalized to the geometric mean of the housekeeping reference genes included in the assay and the code set's internal positive controls. Differentially expressed genes were assessed in silico for correlations with clinical-biological disease characteristics and potential prognostic value in The Cancer Genome Atlas (TCGA)-AML cases (162 sequenced AML samples with putative copy-number alterations, mutations and mRNA expression z-scores [threshold±2.0]). Results Unsupervised hierarchical clustering of mRNA expression identified a set of IFN-γ-stimulated genes (ISGs) that were up-regulated (>2.0 fold-change compared with baseline) in Kasumi-1 cells, but not in KG1 cells, in response to IFN-γ treatment. When assessed in silico for prognostic power in TCGA-AML patients treated with curative intent on a '7+3' chemotherapy backbone, ISGs in our experimentally-derived signature (SOCS1, FAS, ETV7, IL15, SOCS3, PIM1, TNFSF10, STAT1, PLAT, PRDM1, CASP7, BCL2A1) were either up-regulated, amplified, deleted or mutated in 59 (36%) of queried samples. Patients with abnormalities of ISGs experienced worse clinical outcomes, as indicated by shorter relapse-free-survival (RFS; median 12.1 versus 24.2 months in AML patients without ISG abnormalities, p=0.041) and shorter overall survival (OS; median 11.8 versus 24.8 months in AML patients without ISG abnormalities, p=0.036). Interestingly, abnormalities in ISGs significantly correlated with TP53 mutations (log ratio= 1.65; p=0.026), an established adverse prognosticator in AML. Pre-treatment with tipifarnib at either 100 nM or 500 nM concentration attenuated IFN-γ-induced changes in gene expression in Kasumi-1 AML cells (Fig. 1A), including the down-regulation of Wnt, Ras, Hedgehog and Notch signaling and the up-regulation of genes implicated in DNA damage response (Fig. 1B). In contrast, tipifarnib exerted no effects on cancer-associated canonical pathways in IFN-γ-unresponsive KG1 cells (Fig. 1A). Conclusions Our study shows that an experimentally-derived IFN-γ responsiveness gene signature correlates with poor clinical outcomes in AML and suggests that tipifarnib might attenuate IFN-γ signaling, potentially affecting AML susceptibility to DNA damage induced by chemotherapeutic agents. Grant support: Qatar National Research Fund (#NPRP8-2297-3-494) and Kura Oncology, San Diego, CA, USA. Figure 1 Disclosures Rutella: MacroGenics, Inc.: Research Funding; NanoString Technologies, Inc.: Research Funding; Kura Oncology: Research Funding.

Endoplasmic Reticulum Stress Signaling Comprises a G9a Inhibitor Tolerance Pathway and PERK Inhibition Increases Anti-Leukemia Activity of G9a Inhibitor in Leukemia Cells and Leukemia Stem-like Cells

Background: Histone methyltransferase (HMTase) G9a regulates the transcription of multiple genes by primarily catalyzing dimethylation of histone H3 lysine 9 (H3K9me2), as well as several non-histone lysine sites. Recently, pharmacological and genetic targeting of the G9a was shown to be efficient in slowing down acute myeloid leukemia (AML) cell proliferation in a mouse model and human AML cell lines thus making this HMTase potential target for epigenetic therapy of AML. Activation of adaptive mechanisms to drug plays a crucial role in drug resistance and relapse by allowing cell survival under stressful conditions. Therefore, inhibition of the adaptive response is considered as a prospective therapeutic strategy. The tolerance mechanism to HMTase regulation in leukemia cell is unclear yet. The PERK-eIF2α phosphorylation pathway is an important arm of the unfolded protein response (UPR), which is induced under conditions of endoplasmic reticulum (ER) stress. Recent previous studies showed that pro-survival ER stress is induced in cancer cells and contributes to development of drug resistance. Methods: We investigated the levels of apoptosis and ER stress by G9a inhibitor BIX-01294 in leukemia cell lines. U937, cytarabine-resistant U937 (U937/AR) and KG1 were used. U937/AR cell line was established in our laboratory by exposing parental U937 cells to stepwise increasing concentrations of cytarabine. Results: We initially examined the expression of G9a in leukemia cell lines and the primary AML cells obtained from a patient at the different time point. In U937/AR cells and primary AML cells obtained at relapse, G9a expression was increased compare to that in U937 cells and primary AML cells obtained at diagnosis, respectively. G9a expression was also increased in KG1 cells. In both of U937 and U937/AR, apoptotic cell death was induced by BIX-01294 in a dose-dependent manner. In contrast, apoptotic cell death was minimal in KG1 cells which are enriched in cells expressing a leukemia stem cell phenotype (CD34+CD38-). To address the activation of ER stress response by BIX-01294 in leukemia cells, we examined the effect of BIX-01294 treatment on PERK and eIF2α protein expression and phosphorylation levels. We found that treatment of U937, U937/AR, KG1 cells with 3μM of BIX-01294 for 24h caused an upregulation of phosphorylated PERK and eIF2α. The upregulation of PERK phosphorylation was associated with a decrease in PERK protein levels after treatment. To further address the role of the PERK-eIF2α phosphorylation in BIX-01294 sensitivity, we examined whether PERK inhibition using small interfering RNA (siRNA) or specific inhibitor could sensitize cells to BIX-01294-mediated death. The siRNA against PERK effectively inhibited BIX-01294-mediated phosphorylation of PERK and eIF2α in U937 and U937/AR cells. The addition of PERK siRNA led to a significant increase in the extent of BIX-01294-induced apoptotic cell death in U937 (P = 0.0003) and U937/AR (P < 0.0001) as compared with that of BIX-01294 treatment alone. PERK inhibitor GSK260641 significantly increased BIX-01294-induced apoptotic cell death in U937 (P < 0.0001) and U937/AR (P = 0.006) cells. To our surprise, addition of PERK siRNA or GSK260641 increased the sensitivity of KG1 cells to BIX-01294-mediated death in a dose-dependent manner (P = 0.0003 for siRNA, P = 0.0053 for GSK260641). Conclusion: These data demonstrated that PERK-eIF2α activation has a pro-survival function to G9a inhibitor in leukemia cells and mediates resistance of AML stem cells to G9a inhibitor treatment. The PERK-eIF2α phosphorylation arm may represent a suitable target for combating resistance to G9a inhibitor in AML. The mechanisms underlying the increased sensitivity of AML cells with PERK inhibition to G9a inhibitor are unclear at present and are needed to define in further studies. Disclosures No relevant conflicts of interest to declare.

Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus.

Dendrogenin_A : A Natural Liver X Receptor Modulator for the Treatment of Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is the most common type of leukemia in adults. Despite intensive research, current treatments remain unsatisfactory with only 40% of younger (<60 years) and less than 10% of older (>60 years) AML patients achieving long-term complete remission. Consequently, drugs with novel mechanism of action are urgently needed to improve the outcome of these patients. We have recently identified Dendrogenin A (DDA) as a cholesterol metabolite present in normal cells but undetectable in various cancer cell lines including AML (de Medina et al, Nat Commun, 2013). DDA, the first steroidal alkaloid identified in mammals, exhibited strong anticancer effects against different tumor models in vitro and in vivo. In this study, we investigated the antileukemic potency of DDA in AML. We demonstrated that DDA exerts potent cytotoxic effect in a large panel of AML cell lines and cytogenetically and molecularly diverse primary AML patient samples (n=50) with a median IC50 of 3.3 µM (range 1.2-10 µM). We determined that DDA triggers both apoptosis and cytotoxic autophagy on AML cells. Macroautophagy was characterized by the accumulation of autophagic vacuoles and the stimulation of autophagic flux. As opposed to conventional chemotherapies, the antileukemic effect of DDA was similarly efficient in both immature stem/progenitor CD34+CD38-CD123+ subpopulation and leukemic bulk. Interestingly, the antileukemic activity of DDA on AML patient samples was not correlated to usual prognostic factors such as adverse cytogenetic risk karyotype, clonogenic ability, white blood cells count and FLT3-ITD or NPM status. Pharmacokinetic studies revealed that both per os (PO) and intraperitoneal (IP) administration led to a good absorption with calculated bioavailability of 74% (PO) and 48% (IP), showing that these modes of administration are relevant to in vivo preclinical studies. We then examined the in vivo anti-leukemic efficacy of DDA in NOD/SCID mice injected subcutaneously with HL60 and KG1 cells. We demonstrated that daily administration of DDA (20 mg/kg IP or 40 mg/kg PO) significantly reduced KG1 and HL60 tumor growth. Immunohistochemical analysis revealed that AML xenografts from mice exposed to DDA display a 3.5 fold increase of LC3 punctated cells and a decreased P62 level highlighting that DDA induces autophagy in vivo. Furthermore, DDA significantly kills AML cells in bone marrow and brain (55±5.6% reduction of viable CD45+ cells), and strongly reduces (57±7.8%) the total cell tumor burden in bone marrow and spleen in established disease models (eg. orthotopically engraftment of HL60 cells and three primary AML patient cells via tail vein injection in NOD/SCID/IL2Rγc-deficient mice). In addition, we showed that DDA is well tolerated in mice at effective dose and spares normal hematopoietic stem/progenitor cells from healthy donor. Mechanistic studies revealed that DDA is a natural modulator of the Liver X Receptor (LXR), a nuclear receptor involved in cholesterol homeostasis, immunity and proliferation. We found that the silencing of LXRβ gene prevents the capacity of DDA to trigger both cell death and autophagy on AML cells in vitro. In addition, DDA failed to block tumor development and to trigger autophagy on LXRβ-invalidated KG1 cells xenografted on NOD/SCID mice. Moreover, DDA strongly stimulates the expression of the myeloid leukemogenesis tumor suppressors Nur77 and Nor1 through an LXRβ-dependent mechanism. Interestingly, DDA triggers the relocation of Nur77 to the mitochondria, a process associated with both apoptosis and autophagic cell death. This study provides a strong rationale to bring DDA in clinical trials for patients with AML. Disclosures de Medina: Affichem: Employment. Bize:Affichem: Employment. Paillasse:Affichem: Employment. Noguer:Affichem: Employment. Sarry:Affichem: Equity Ownership. Silvente-Poirot:Affichem: Equity Ownership. Poirot:Affichem: Equity Ownership.

Replication Factor C3 Is a Direct Target Of CREB, Promotes G1/S Transition Of Acute Myeloid Leukemia Cells, and Increases Hematopoietic Stem/Progenitor Cell Self-Renewal

CREB (cAMP Response Element Binding protein) promotes cellular transformation of hematopoietic cells and proliferation of myeloid leukemia cells. However, the underlying mechanisms of CREB function in leukemic transformation and hematopoiesis are not fully understood. To address this, we have investigated the downstream pathways of CREB activation in proliferation using a human acute myeloid leukemia (AML) cell line KG1 cells knocked-down for CREB with specific shRNAs. The CREB-knockdown KG1 cells were significantly defective in proliferative capability compared to control cells [cell number after 4d (X105), seeding (1X105), control vs. CREB-knockdown: 34.18 +/– 1.27 vs. 14.52 +/– 0.46, n=3, p< 0.01, mean +/– SEM]. In order to characterize the specific role of CREB in cell proliferation, we analyzed cell cycle progression patterns of CREB-knockdown and control KG1 cells after release from mitotic arrest. Our results indicated that G1 to S phase transition as assessed by % S phase was impeded by CREB-knockdown [S phase (%), control vs. CREB-knockdown cells, 8h after release: 53.29 +/– 0.54 vs. 23.57 +/– 1.69; 12h: 66.92 +/– 0.63 vs. 45.16 +/– 0.50, n=3, p< 0.01, mean +/– SEM]. To identify potential CREB target genes, we chose several cell cycle related genes such as CCNE1, CCNA1, CCNB1 and PCNA and compared their RNA expression levels in the CREB-knockdown with those in control KG1 cells after release from mitotic arrest. To our surprise, we failed to detect any noticeable differences in the mRNA expression levels of those genes between CREB-knockdown and control KG1 cells. In an effort to search for CREB responsive target genes, we analyzed additional CREB targets previously identified from microarray data (Pellegrini et al BMC Cancer 2008). We found that expression of replication factor C3 (RFC3), a 38kDa subunit of the RFC complex involved in DNA replication and repair processes, was significantly reduced in CREB-knockdown cells compared to control cells [38 +/– 1% of control, n=3, p<0.01]. CREB-knockdown also inhibited RFC3 mRNA expression in U937 and HL60 AML cell lines. Consistent with these results, mRNA expression levels of RFC3 appeared to be closely correlated with those of CREB when we examined bone marrow samples obtained from AML patients [n = 16, Pearson coefficient = 0.6366, p = 0.0008]. Moreover, we found that CREB directly interacted with the CRE site in the RFC3 promoter region in vivo, as assessed by chromatin immunoprecipitation assays. Exogenous overexpression of RFC3 in CREB-knockdown KG1 cells restored the defective G1/S progression [S phase (%), CREB-knockdown vs. CREB-knockdown with RFC3 overexpression, 9h after release: 38.97 +/– 0.45 vs. 62.24 +/– 1.06; 12h: 48.12 +/– 0.60 vs. 67.70 +/– 1.15, n=3, p< 0.01, mean +/– SEM]. Taken together, these results suggest that RFC3 may act as a novel downstream oncogenic target of activated CREB in AML cells. We previously reported that CREB is a critical regulator of normal myelopoiesis (Cheng et al Blood 2008). To determine whether RFC3 could exert similar effects on normal hematopoiesis, we compared human umbilical cord blood derived CD34-positive cells with and without RFC3 overexpression for the capacity to form hematopoietic colonies. Overexpression of RFC3 in the CD34-positive cells resulted in significant increases of multi-potential CFU-GEMM colony numbers [without vs. with overexpression of RFC3 (per 1000 cells): 3.2 +/– 1.3 vs. 22.3 +/– 3.3, n=3, p< 0.01, mean +/– SEM]. The RFC3 effect on stimulating colony formation was magnified in secondary colony forming assays [without vs. with overexpression of RFC3 (per 100,000 cells): 10.7 +/– 3.5 vs. 180.2 +/– 44.4, n=3, p< 0.05, mean +/– SEM]. Since the formation of secondary colonies was derived mainly from residual stem/progenitor cell populations after long-term culture, RFC3 overexpression may enhance self-renewal of stem/progenitor cells. In conclusion, our results suggest that RFC3 is able to promote G1/S transition in a human AML cell line downstream of CREB activation. In addition, we provide evidence that RFC3 is involved in normal hematopoiesis and contributes to increased self-renewal potential of hematopoietic stem/progenitor cells. Our data demonstrate that RFC3 plays multiple roles in promoting AML cells proliferation as well as normal myelopoiesis through increasing the self-renewal potential of hematopoietic stem/progenitor cells in response to CREB activation. Disclosures: No relevant conflicts of interest to declare.

Studies on the Role of TGF-β1/TGF-βR1/Smad4 In the Occurance and Development of Myelocytic Leukemia and Breast Cancer

Abstract 4714 Previous studies showed that the function of TGF-β/Smad passway is aberrant in many human cancers, the expresses of TGF-β1, TGF-βR1 and Smad4 proteins were related to the clinicopathologic characteristic of tumors and prognosis. The aim of the present work was to study the role of TGF-β1, TGF-βR1 and Smad4 in the occurance and development of myelocytic leukemia and breast cancer. Firstly, 2 myelocytic leukemia cell lines (KG1, HL60), 2 breast cancer cell lines (SKBR-3, MCF-7) and 293 cells (normal control) were selected. Secondly, we detected the expression levels of mRNA and protein in the 4 cell lines by fluorescence quantitative PCR (qRT-PCR), immunohistochemistry, and Western blotting. qRT-PCR showed that the gene copies of TGF-β1, TGF-βR1 and Smad4 in SKBR-3 cells and KG1 cells, which were poorly differentiated, were lower than that in MCF-7 cells and HL60 cells, which were well differentiated (P<0.01); and the gene copies in 293 cells were higher than all of the four cell lines (P<0.01). Western blotting showed that the protein expression levels of TGF-β1, TGF-βR1 and Smad4 in SKBR-3 and KG1 cells were also lower than in MCF-7 cells and HL60 cells (P<0.01), and higher in 293 cells (P<0.01). The same results could also be abtained by immunohistochemistry. This studies may indicate that TGF-β1, TGF-βR1 and Smad4 act possibly as the role of anti-oncogene in myelocytic leukemia and breast cancer, which also may be associated with the biological behaviors of the development of myelocytic leukemia and breast cancer. TGF-β/Smad passway might be a promising candidate target for the controling progression of malignant tumor. Disclosures: No relevant conflicts of interest to declare.

Dysregulation of Metabolic Genes May Be Normalized by Epigenetic Drugs (Vorinostat, Decitabine) and Nutritional Compounds (Genistein, Vitamin D) in Leukemic Cells.

Abstract 4423 Background Dysregulation of energy metabolism is a key feature of malignancy and known to be regulated by epigenetic mechanisms. This study explores gene expression in the KG1 cell line and a primary culture of leukemic blasts from a patient, both incubated with SAHA (suberoyl anilide hydroxamic acid, also known as Vorinostat) in comparison to other epigenetic drugs such as Decitabine (DAC) but also nutritional compounds such as genistein and vitamin D3 with a known activity on metabolism where epigenetic mechanisms play a role. Methods Following a 3 days incubation with pharmacologic concentrations of the above mentioned compounds, gene expression patterns of the KG1 cell-line were analysed on an Affymetrix Gene Expression Array and specifically evaluated by quantitative real time PCR from KG1 cells and primary leukemic cells of a patient with an AML-M2 which were cultured at the same conditions as the KG1 cells. In order to confirm epigenetic mechanisms induced by targeted drugs, methylation patterns of selected tumor suppressor genes were analyzed using specific primers for bisulfite-treated DNA. Results Our experiments show that expression of a large scale of micro RNAs which are known to be responsible for alternative splicing is stimulated by SAHA as well as DAC and to a lesser extent also Vitamin D3 and genistein, but downregulation of mitochondrial RNAs as a consequence of apoptosis was exclusively observable by SAHA. HDAC-inhibition results in clear repression of metabolic genes involved in glycolysis (5/6 genes) as well as cholesterol-biosynthesis (14/15 genes) and fatty acid synthesis (all 4 genes) which are also known to be regulated by nutrition. Classic tumour suppressor genes, transcription factors and cell-cycle regulators such as P53, P15, P16 and P21 and FOXO were (re-) activated with this treatment while MYC was down-regulated. Discussion A downregulation and thus normalization of a large scale of genes including key genes from malignancy-associated stimulation of glycolysis as well as biosynthesis of cholesterol and fatty acids was observed with SAHA and nutritional compounds such as Vitamin D and genistein. We propose that these interactions should be carefully considered in clinical application, and a combination of synergistic drugs and/or dietary strategies may provide a major advantage in future drug development. Acknowledgment Supported by Jubilaeumsfonds der Oesterreichischen Nationalbank. Disclosures: No relevant conflicts of interest to declare.