Growth Of HL-60 Research Articles

Abstract 3297: Discovery and evaluation of GT19870, a GSPT1/Myc molecular glue drug (MGD) with oral bioavailability for targeting c-Myc and n-Myc-addictive blood cancer and solid tumors

Abstract c-Myc and n-Myc are oncogenic transcriptional factors driving tumor initiation, progression and poor prognosis in a variety of blood cancers and solid tumors, such as acute myeloid leukemia (AML), B-cell malignancies, castration resistance prostate cancer (mCRPC), small cell lung cancer and (SCLC) and neuroblastoma. Both c-Myc and n-Myc dysregulations have been directly linked to the poor clinical outcome in some of these cancers which are addictive to deregulated Myc proteins. Therefore, it is highly warranted to discover and develop novel therapeutical agents for targeting Myc-addictive cancers. GT19870 was discovered through a SAR effort for Myc degradation with improved bioavailability (F % 92.52) from GT19630, a GSPT1/Myc degrader series 1-4by using phenotypic screenings with c-Myc-addictive HL-60 AML cells and growth factor-dependent hematopoiesis progenitor TF-1 cells, followed by c-Myc ELISA and Western blot assays. GT19870 selectively inhibited the proliferation of HL60 cells with an IC50 of 2.6 nM as compared to an IC50 of 70.03 nM (26.5-fold selectivity) and effectively degraded c-Myc protein with an IC50 of 2.54 nM and IC90 <10 nM in HL-60 cells, as compared to an IC90>30 nM in CD3/CD28-stimulated PBMC assays. GT19870, as a MGD, was confirmed to selectively degrade GSPT1, a neo-substrate, by proteomic and Western Blot assays. Further, GT19870 was tested in SCLC, triple negative breast cancer (TNBC) and brain cancer cell panels, in which GT1987 was revealed potent anti-proliferation activities with an IC50<50 nM in 9 of 19 SCLC, 8 of 13 TNBC, and 7 of 24 brain cancer cell lines tested. Importantly GT19870 was also shown potent antitumor activities with IC50 of ≦5.4 nM in 8 of 8 SCLC patient derived organoids. Furthermore, GT19870 (salt form-GT19884) was demonstrated to induce target-engaged (c-Myc and n-Myc) tumor regression in AML, SCLC, mCRPC and neuroblastoma xenograft and PDX models with in a dose ranges of 3-12 mpk/qd/po. In brief, GT19870 is an oral MGD degrading GSPT1/c-Myc/n-Myc. This novel MGD has been demonstrated target-engaged antitumor activities in vitro and in vivo with selectivity of c-Myc degradation in c-Myc-addictive cancer cells as compared to growth factor-stimulated TF-1 cells and CD3/CD28 activated PBMC. GT19870 has been progressing IND enabling stage.

Menin Inhibitor DS-1594b Drives Differentiation and Induces Synergistic Lethality in Combination with Venetoclax in AML Cells with MLL-Rearranged and NPM1 Mutation

Introduction: Mixed-lineage-leukemia (MLL) is one of the most frequently translocated genes in hematologic malignancies, which produces aggressive leukemias that are refractory to conventional treatment resulting in a 5-year survival rate of only 35%. MLL translocations lead to fusion of the N-terminal fragment of MLL with one of over sixty protein partners, producing MLL fusion proteins that enhance proliferation, block hematopoietic differentiation, and drive leukemia development. Acute myeloid Leukemia (AML) is a heterogeneous and rapidly progressing blood cancer with a poor prognosis, especially in elderly patients where it is more common. Mutations in the nucleophosmin (NPM1) gene are one of the most common causes of AML. Interestingly, the protein-protein interaction between Menin, MLL and MLL fusion proteins plays a critical role in the pathogenesis of acute myeloid leukemias, both with MLLr as well as in leukemias carrying mutations in NPM1. Inhibition of the MLL-Menin interaction with novel Menin inhibitors has shown efficacy in both AML subsets, in pre- clinical and clinical studies. However, the responses are short-lived and hence it is imperative to identify therapeutic approaches that may synergize with Menin inhibition. Our recent work showed that MLLr t(4;11) Acute Lymphoblastic leukemia () patient cells express high levels of the anti-apoptotic BCL-2 protein and are highly sensitive to treatment with the BCL-2 selective antagonist, venetoclax (Benito, J.M., et al , Cell Reports, 2015). Here, we report experiments to establish the efficacy of the novel Menin-MLL inhibitor DS-1594b which was developed by Daiichi Sankyo co., jp, in combination with the BCL-2 antagonist venetoclax (VEN) in MLLr and NPM1-mutated acute leukemias. Methods: The efficacy of DS-1594b/VEN combination in MLLr and NPM1mut AML cell lines and primary leukemia samples was evaluated by examining the cell viability in response to serial dilutions of tested compounds as single agents or in combination using Cell Titer Glo (CTG) assay at 3, 5 and 7 days of treatment. Induction of apoptosis was assessed by Annexin V/ staining. Since down-regulation of MLL fusion target genes leads to differentiation in MLL leukemic cells, we monitored cell differentiation by flow cytometry in AML cell lines (MLLr: OCI-AML2, MOLM13, MOLM14, MV411. NPM1 Mut: OCI-AML3 . Leukemia cell lines with no MLLr or NPM1 mutations: HL60, U937) and primary leukemia samples ( blood from relapsed MLLr and NPM1mut AML) using differentiation markers CD11b, CD14, CD15. MLLr cell line MOLM13 resistant to venetoclax was also included in these studies. Results: 7 days treatment with DS-1594b reduced viability in MLLr and NPM1mut cell lines at low nM concentrations but did not affect growth of negative control U937 and HL60 . DS-1594b induced differentiation in OCI-AML3 and Ven resistant cell after 5 days and in other lines after 10 days (figure 1A). DS-1594b did not induce apoptosis when used as a single agent in any AML cell line tested indicating that differentiation is a key mechanism of Menin inhibition. Five days of treatment with single agent showed a downregulation of Menin in all cell lines and downregulation of BCL2 in OCI-AML3 and OCI-AML2 in Western blot analysis. Remarkably, combination with Venetoclax induced synergistic lethality and growth inhibition in all cell lines. Most importantly, was observed in primary leukemia samples (Figure 1B), with differentiation effects after DS-1594b single agent (bottom). Conclusions: Here we show in vitro efficacy of DS-1594b, a selective small molecule menin inhibitor, as single agent or in combination with Venetoclax in MLLr and NPM1 mutated cell lines and primary leukemia samples. Preliminary data demonstrate differentiation effects of DS-1594b as single agent in both cell lines and primary AML leukemia samples. Combination effects show synthetic lethality in almost all cell lines except OCI-AML3. The combination elicited profound apoptosis in primary leukemia samples. In vivo studies using MLLr and NPM1 PDXs models are ongoing and will be presented. Overall, the preliminary findings presented here pave the way for further studies aimed at understanding mechanisms of and further development and testing of this combination in AML with MLLr and/or NMP1 mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mitochondrial Carrier Homolog 2 (MTCH2) Regulates the Differentiation of AML Cells By Controlling Pyruvate Entry into the Mitochondria, Nuclear Localization of Pyruvate Dehydrogenase Complex and H3 and H4 Histone Acetylation

Mitochondrial carrier homolog 2 (MTCH2) is a mitochondrial outer membrane protein that functions as a receptor for pro-apoptotic BID. MTCH2 also regulates metabolism through undefined mechanisms.We identified MTCH2 as essential for AML growth and viability in a CRISPR screen. OCI-AML2 leukemia cells overexpressing CAS9 were transduced with a library of 91,320 sgRNAs in barcoded lentiviral vectors. We focused on the gRNA that targeted the 1050 mitochondrial proteins and identified MTCH2 within the top 1% of hits. We validated this finding using two individual sgRNAs, where knockout of MTCH2 reduced the growth and viability of CAS9-OCI-AML2 cells by >75%. Additionally, knockdown of MTCH2 with shRNA reduced growth and viability of OCI-AML2 (>90%), TEX (>80%), U937 (>65%), and HL60 (>75%) leukemia cells. Knockdown of MTCH2 targeted the leukemia initiating cells as it abrogated engraftment of TEX cells into immune deficient mice. We also infected hematopoietic cells from wild type and MTCH2 flox/flox Vav1-cre mice with the MLL-AF9 oncogene. Knockout of MTCH2 decreased the leukemogenic potential of these cells and increased survival of the mice.In contrast, knockout of MTCH2 has more subtle effects on normal murine hematopoiesis. flox/flox Vav1-cre MTCH2 knockout mice had normal peripheral blood counts, but a ~15% reduction in the abundance of hematopoietic stem cells with a slight increase in the restricted progenitor populations (Maryanovich et al, Nat Comm 2015).Murine hematopoietic cells from MTCH2 -/- mice have increased mitochondrial mass and increased oxidative phosphorylation. Yet, no changes in basal oxygen consumption, mitochondrial ROS, or respiratory chain activity were seen in AML cells after MTCH2 knockdown.We examined changes in gene expression by RNA-sequencing in OCI-AML-2 cells after MTCH2 knockdown. MTCH2 knockdown increased expression of genes involved in cellular differentiation and decreased genes that maintain stemness. Therefore, we analyzed differentiation of AML cells after MTCH2 knockdown and demonstrated increased non-specific esterase staining and increased expression of CD cell surface markers associated with differentiation.In lung cancer mitochondrial stress induces the translocation of pyruvate dehydrogenase (PDH) from the mitochondria to the nucleus, where it increases conversion of pyruvate to Acetyl CoA which, in turn, acetylates histones. Therefore, we examined changes in PDH localization and histone acetylation after MTCH2 knockdown in OCI-AML-2 and TEX cells. MTCH2 knockdown decreased mitochondrial pyruvate, increased nuclear localization of pyruvate dehydrogenase complex (PDH) and increased acetylation of H3 and H4 histones. Over-expression of the PDH E2 subunit with a nuclear localizing signal mimicked MTCH2 knockdown and decreased cell growth, induced differentiation, and increased H3 and H4 histone acetylation in. Likewise, the acetyltransferase inhibitor, C646, abrogated the effects of MTCH2 knockdown on histone acetylation, growth and differentiation. We also demonstrated that knockdown of MTCH2 decreased levels of mitochondrial pyruvate in OCI-AML-2 cells.The entry of pyruvate into the mitochondria is regulated by the Mitochondrial Pyruvate Carrier (MPC). Therefore, we treated AML cells (OCI-AML2, TEX, HL60, and 8227) with the selective MPC inhibitor UK5099. UK5099 mimicked the effects of MTCH2 knockdown and decreased mitochondrial levels of pyruvate, reduced cell growth and viability, increased levels of acetylated histones, increased nuclear levels of PDH and promoted differentiation of AML cells. Treatment of mice with UK5099 (40mg/kg) decreased growth of OCI-AML2 xenografts. Finally, UK5099 induced cell death by Annexin V/PI staining in undifferentiated primary AML cells, but differentiated primary AML cells (by morphology and CD marker staining) and normal hematopoietic cells were more resistant.In summary, inhibiting entry of pyruvate into the mitochondria is a new mechanism to regulate differentiation of AML cells and stem cells by increasing PDH localization to the nucleus and histone acetylation. Thus, MTCH2 or MPC inhibitors may be novel therapeutics agents for AML. DisclosuresSchimmer:Medivir: Research Funding; Takeda Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria.

Effect of Competitive Antagonist of Transmembrane Glutamine Flux V-9302 on Apoptosis of Acute Myeloid Leukemia Cell Lines HL-60 and KG-1

To investigate the effect of V-9302 (an antagonist of transmembrane glutamine flux) on the proliferation and apoptosis of acute myeloid leukemia cells HL-60 and KG-1. HL-60 and KG-1 cells at logarithmic phase were treated by different concentrations of V-9302. CCK-8 assay was used to detect the proliferation of the cells. Annexin V-FITC / PI double staining flow cytometry was used to detect the apoptosis of HL-60 and KG-1 cells. The expressions of BAX, BCL-2 and Caspase3 were detected by RT-qPCR and Western blot. V-9302 could significantly inhibit the growth of HL-60 and KG-1 cells. The concentration of V-9302 at 10, 20 μmol/L could significantly promote the apoptosis of HL-60 and KG-1 cells(P<0.05). The results of apoptosis related gene detection showed that when V-9302 was applied to HL-60 and KG-1 cell lines at 10 and 20 μmol/L, the expression levels of Pro-apoptotic protein genes BAX and Caspase3 in HL-60 and KG-1 were significantly higher than those in control group (P<0.05), while the expression level of anti-apoptotic protein gene BCL-2 was significantly lower than that in the control group (P<0.05). The results of Western blot were basically consistent with that of RT-qPCR. Competitive antagonist of transmembrane glutamine flux V-9302 can significantly promote the apoptosis of acute myeloid leukemia cell lines HL-60 and KG-1.

Bioactive Potential of Several Actinobacteria Isolated from Microbiologically Barely Explored Desert Habitat, Saudi Arabia.

Simple SummaryBioactive natural products have been regarded as promising tools for treatment of various ailments. Among natural sources, actinomycetes have been widely explored for their potential bioactivity. In this regard, the present study has focused on the phytochemical content and biological activities of several actinobacteria isolates, which were investigated for their phenolic and flavonoid content, as well as their antioxidant, antibacterial and antiprotozoal activities. The most active isolates were further investigated for their antileukemic activity, where such isolates were shown to exert cytotoxic activity against the tested cell lines, following a mechanism that might be due to the ability of the active isolate extracts to reduce cyclooxygenase and lipoxygenase activities. Overall, isolation and characterization of the active molecule from the potential actinomycetes strains will pave the way for the development of drugs against human diseases such as blood cancer.Biomolecules from natural sources, including microbes, have been the basis of treatment of human diseases since the ancient times. Therefore, this study aimed to investigate the potential bioactivity of several actinobacteria isolates form Al-Jouf Desert, Saudi Arabia. Twenty-one actinobacterial isolates were tested for their antioxidant (flavonoids, phenolics, tocopherols and carotenoids) content, and biological activities, namely FRAP, DPPH, ABTS, SOS and XO inhibition, anti-hemolytic and anti-lipid peroxidation as well as their antibacterial and antiprotozoal activities. Accordingly, five isolates (i.e., Act 2, 12, 15, 19 and 21) were selected and their 90% ethanolic extracts were used. The phylogenetic analysis of the 16S rRNA sequences indicated that the most active isolates belong to genus Streptomyces. The genus Streptomyces has been documented as a prolific producer of biologically active secondary metabolites against different cancer types. Thus, the anti-blood cancer activity and the possible molecular mechanisms by which several Streptomyces species extracts inhibited the growth of different leukemia cells, i.e., HL-60, K562 and THP-1, were investigated. In general, the five active isolates showed cytotoxic activity against the tested cell lines in a dose dependent manner. Among the potent isolates, isolate Act 12 significantly decreased the cell viability and showed maximum cytotoxic activities against both HL-60 and K562 cells, while isolate Act 15 exhibited maximum cytotoxic activity against THP-1 cells. Moreover, Act 2 and Act 12 reduced cyclooxygenase (COX-2) and lipoxygenase (LOX) activity, which is involved in the proliferation and differentiation of cancer cells and may represent a possible molecular mechanism underlying leukemia growth inhibition. The bioactive antioxidant extracts of the selected Streptomyces species inhibited leukemia cell growth by reducing the COX-2 and LOX activity. Overall, our study not only introduced a promising natural alternative source for anticancer agents, but it also sheds light on the mechanism underlying the anticancer activity of isolated actinomycetes.

Combined treatment with dissolved hydrogen molecule and platinum nanocolloid exerts carcinostatic/carcinocidal effects by increasing hydrogen peroxide generation and cell death in the human gastric cancer cell line NUGC-4

Many studies have shown that redox regulation is an effective therapeutic strategy for different types of cancer. We have previously demonstrated that combined treatment with dissolved hydrogen molecule (H2) and platinum nanocolloid (Pt-nc) has carcinostatic effects and that increased intracellular reactive oxygen species (ROS) levels were closely associated with carcinostatic effects in Ehrlich mouse ascites tumor cells. However, it is unknown whether combined treatment-induced ROS generation can occur in human cancer cells. Therefore, this study aimed to examine the carcinostatic effect of the combined treatment in human cells and investigate the relationship between treatment efficacy and ROS generation. H2 and Pt-nc treatment could exert cytostatic action by inhibiting the growth of human promyelocytic leukemia HL60 and human gastric adenocarcinoma-derived NUGC-4 cells; however, no effect was observed in normal human embryo fibroblast OUMS-36 cells by the temporary exposure. These findings indicate that combined treatment with H2 and Pt-nc may act selectively in tumor cells compared with normal cells. Additionally, combined treatment with H2 and Pt-nc resulted in an approximately 200-fold increase in intracellular ROS levels compared with the control, whereas the suppressive effect of tumor cell growth was abrogated entirely by catalase treatment in NUGC-4 cells. Furthermore, combined treatment with H2 and Pt-nc induced hydrogen peroxide generation, cellular morphological changes, cell death, and a decline in DNA synthesis-positive cells. In conclusion, combined treatment with H2 and Pt-nc can induce carcinostatic/carcinocidal effects through intracellular ROS increase, morphological changes, cell death, and DNA synthesis suppression in the human tumor cell line.

Autocrine motility factor secreted by HeLa cells inhibits the growth of many cancer cells by regulating AKT/ERK signaling

In cell competition, a secreted death signal can determine cell fate. However, the nature of such a signal remains unclear. In this study, conditioned medium from HeLa cells (HeLa CM) inhibited growth of A549 and MCF-7 cells. Through HeLa CM fractionation, glucose 6-phosphate isomerase/autocrine motility factor (GPI/AMF) was identified as the main growth inhibitor. Previously, AMF was known for its mitogenic, motogenic, and differentiation functions and was implicated in tumor progression and metastasis. HeLa CM lost its growth inhibitory property after treatment with erythrose-4-phosphate (E4P) or anti-GPI antibody. Purified HeLa recombinant AMF (rAMF) proteins inhibited the growth of A549, MDA-MB-232, MCF-7, AsPC-1, DU145, Hep-2, Hep G2, and HT-29 cells. However, growth of HL-60, SKOV3, U-87 MG, SNU-484, U-87 MG, and 3T3-L1 cells was little affected. In a Transwell assay, HeLa rAMF effectively reduced A549 cell migration and invasion. HeLa rAMF effectively induced apoptosis in A549 cells, apparently by reducing the levels of Bcl-2, GPI, and poly(ADP-ribose) polymerase (PARP)14 and activating caspase-3 and p53. HeLa rAMF antagonized HER2 and the AMF receptor (AMFR or GP78) in relation to the AKT/EKT signaling pathway. These results suggest that HeLa AMF could act as a diffusible death signal that could induce cancer cell–selective growth inhibition and apoptosis.

Characterization and anti-tumor activity of saponin-rich fractions of South Korean sea cucumbers (Apostichopus japonicus).

In this study, the saponin-rich fractions of five individual (two Red and three Black) sea cucumbers (Apostichopus japonicus) in South Korea were investigated for their antiproliferative effect against HL-60, B16F10, MCF-7, and Hep3B tumor cell lines. The red sea cucumber saponin-rich fraction (SSC) from Jeju Island (JRe) decreased the growth of HL-60 with an IC50 value of 23.55 ± 3.40μg/mL, which represented the strongest anticancer activity among the extracts. Further, SSC downregulated B-cell lymphoma extra-large (Bcl-xL), while upregulating, to different degrees, Bcl-2-associated X protein (Bax), caspase-9, caspase-3, PARP cleavage, and apoptotic bodies in cancer cells. Evidence for SSC inducing apoptosis via the mitochondria-mediated pathway was found. The contents of SSCs were determined using ultra high-performance liquid chromatography coupled with a quadrupole orbitrap mass spectrometry to comparatively evaluate the regional influence. In West Sea, the total SSC content of A. japonicus was 15.5mg/g, representing the highest content, while A. japonicus in the South Sea yielded the lowest content at 8mg/g. The major saponin constituent in SSC was identified as Holotoxin A1, which may the anti-tumor compound in A. japonicus.

Assessment of The Lnc-CCAT1/miR-155a Regulatory Network in Acute Myeloid Leukemia

Colon cancer-associated transcript_1 (CCAT1) is a long noncoding RNA that maps to chromosome 8q24.21, it was first discovered to be upregulated in colorectal cancer. Recent studies have observed the CCAT1 overexpression in primary human solid cancers and cell lines as well as in AML, moreover, it repressed monocytic differentiation and promoted cell growth of HL-60 by sequestering tumor suppressive miR-155. However, the prognostic value of CCAT1/miR-155a pathway in acute myeloid Leukemia (AML) has not been investigated on clinical samples. In this study, the expression levels of CCAT1 and miR_155a was measured in 150 AML patients with standard and high-risk factors; CCAT1 and miR_155a were increased by 2.7 and 5.7 folds; respectively in AML compared to healthy controls. Furtherly, upregulation of both biomarkers was significantly associated with high risk AML. Collectively, these results suggest that CCAT1 and miR_155a can be considered as a diagnostic and prognostic biomarker in AML.

Antiproliferative and apoptosis-inducing potential of 3β-hydroxy-Δ5-steroidal congeners purified from the soft coral Dendronephthya putteri

The exploration and identification of antiproliferative phytochemicals have received increased attention in medicinal chemistry. In particular, research focused on the toxicology of marine natural products has increased in recent years. Terpenoids, among many secondary metabolites, have been demonstrated to act as effective anticancer agents. Soft corals, a group of marine invertebrates, produce a variety of terpenoids with biofunctional properties. The current study presents the extraction, purification, and identification of sterol congeners from the soft coral Dendronephthya putteri. The method involves 50% chloroform-methanol extraction, polar column fractionation, and analysis through GC-MSn. Dose-dependent antiproliferative activity was observed within the sterol-rich fraction (DPCMH 2-4), which consisted of 3β-hydroxy-Δ5-steroidal congeners. This fraction inhibited the growth of HL-60 and MCF-7 cells with IC50 values of 25.27±1.43 and 22.81±0.15 μg/mL, respectively. Apoptotic body formation, DNA damage, cell cycle arrest, and apoptotic cell signaling pathway activation were also observed, reinforcing the dose-dependent antiproliferative and apoptosis-inducing activity of 3β-hydroxy-Δ5-steroidal congeners. To our knowledge, this is the first report of anticancer agent identification from the soft coral D. putteri. Based on the observations, these steroidal congeners are promising candidates for the development of anticancer drugs.

Two new coumarins and a new xanthone from the leaves of Rhizophora mucronata

Two new coumarins (1, 2) and a new xanthone (3), together with 14 known compounds—eight coumarins (4, 5, 9, 10, 12–15), three xanthones (11, 16, 17), a benzoic acid (6) and two flavonones (7, 8)—were isolated from the leaves of Rhizophora mucronata. The structures of the compounds were elucidated by spectroscopic (IR, MS, and NMR) analyses. The isolated compounds were tested for cytotoxicity against human cancer cell lines HL-60 and HeLa. Among these compounds, only compound 16 inhibited the growth of both HeLa (IC50 = 4.8 μM) and HL-60 (IC50 = 1.0 μM) cells. Compounds 4, 7, 10, and 12 exhibited moderate activity against HeLa cells (IC50 = 3.8–8.3 μM). Compounds 5, 9, 11, and 17 showed moderate activity against HL-60 cells (IC50 = 2.2–6.3 μM). Higher selectivity against HL-60 cell lines was observed for compounds 5, 9, 11, and 16 with SI values (NIH 3T3/HL-60) of 8.6, 19.2, 9.4, and 10.2, respectively.

Apoptotic and antiproliferative properties of 3β-hydroxy-Δ5-steroidal congeners from a partially purified column fraction of Dendronephthya gigantea against HL-60 and MCF-7 cancer cells.

Organisms belonging to the genus Dendronephthya are among a group of marine invertebrates that produce a variety of terpenoids with biofunctional properties. Many of these terpenoids have been proven effective as anticancer drugs. Here, we report the antiproliferative effect of 3β-hydroxy-Δ5-steroidal congeners against the proliferation of HL-60 human leukemia cells and MCF-7 human breast cancer cells. The sterol-rich fraction (DGEHF2-1) inhibited the growth of HL-60 and MCF-7 cells with IC50 values of 13.59±1.40 and 29.41±0.87μg ml-1 respectively. Treatment with DGEHF2-1 caused a dose-dependent increase in apoptotic body formation, DNA damage and the sub-G1 apoptotic cell population. Moreover, DGEHF2-1 downregulated the expression of Bcl-xL while upregulating Bax, caspase-9, and PARP cleavage in both HL-60 and MCF-7 cells. The steroid fraction was found to act via the mitochondria-mediated apoptosis pathway. Identification of the sterols was performed via gas chromatography-tandem mass spectrometry analysis. Studying the mechanism of the anticancer effect caused by these sterol derivatives could lead to the identification of other natural products with anticancer properties.

Beta-mangostin from Cratoxylum arborescens activates the intrinsic apoptosis pathway through reactive oxygen species with downregulation of the HSP70 gene in the HL60 cells associated with a G0/G1 cell-cycle arrest.

Xanthones are phytochemical compounds found in a number of fruits and vegetables. Characteristically, they are noted to be made of diverse properties based on their biological, biochemical, and pharmacological actions. Accordingly, the apoptosis mechanisms induced by beta-mangostin, a xanthone compound isolated from Cratoxylum arborescens in the human promyelocytic leukemia cell line (HL60) in vitro, were examined in this study. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was done to estimate the cytotoxicity effect of β-mangostin on the HL60 cell line. Acridine orange/propidium iodide and Hoechst 33342 dyes and Annexin V tests were conducted to detect the apoptosis features. Caspase-3 and caspase-9 activities; reactive oxygen species; real-time polymerase chain reaction for Bcl-2, Bax, caspase-3, and caspase-9 Hsp70 genes; and western blot for p53, cytochrome c, and pro- and cleavage-caspase-3 and caspase-9 were assessed to examine the apoptosis mechanism. Cell-cycle analysis conducted revealed that β-mangostin inhibited the growth of HL60 at 58 µM in 24 h. The administration of β-mangostin with HL60 caused cell morphological changes related to apoptosis which increased the number of early and late apoptotic cells. The β-mangostin-catalyzed apoptosis action through caspase-3, caspase-7, and caspase-9 activation overproduced reactive oxygen species which downregulated the expression of antiapoptotic genes Bcl-2 and HSP70. Conversely, the expression of the apoptotic genes Bax, caspase-3, and caspase-9 were upregulated. Meanwhile, at the protein level, β-mangostin activated the formation of cleaved caspase-3 and caspase-9 and also upregulated the p53. β-mangostin arrested the cell cycle at the G0/G1 phase. Overall, the results for β-mangostin showed an antiproliferative effect in HL60 via stopping the cell cycle at the G0/G1 phase and prompted the intrinsic apoptosis pathway.

New eremophilane and dichlororesorcinol derivatives produced by endophytes isolated from Ficus ampelas.

The novel compound, 11-O-methylpetasitol (1), was isolated from Penicillium sp. N-175-1, and two new compounds, cosmochlorins D (5) and E (6), were isolated from Phomopsis sp. N-125. In addition, three known eremophilane sesquiterpenes, sporogen-AO1 (2), petasol (3) and 6-dehydropetasol (4), were isolated from Penicillium sp. N-175-1. The structures of 1, 5 and 6 were elucidated by a combination of extensive spectroscopic analyses, including 2D NMR, high-resolution electrospray ionization time-of-flight mass spectrometry (HRESITOFMS) and chemical reactions. Compounds 2, 3, 5 and 6 exhibited cytotoxicity to HL60 and 2 and 3 to HeLa cells. Furthermore, 2 and 3 showed robust growth-restoring activity of a Saccharomyces cerevisiae (cdc2-1 rad9Δ) mutant strain, whereas 5 and 6 exhibited minor growth-restoring activity in this strain. Thus, these compounds may inhibit the growth of HL60 and HeLa cells by blocking the cell cycle, and they may be utilized as new lead compounds that act as inhibitors of survival signal transduction pathways.

GLS Inhibitor CB-839 Modulates Cellular Metabolism in AML and Potently Suppresses AML Cell Growth when Combined with 5-Azacitidine

Abstract Glutamine (Gln) is required for growth and proliferation of several tumor types including AML. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes conversion of Gln to glutamate (Glu), which provides carbons for the TCA cycle and regulates redox homeostasis through production of glutathione and NADH. CB-839 is a highly selective, reversible, allosteric inhibitor of GLS. In this study we studied metabolic and cellular consequences of GLS inhibition in AML cells cultured in normoxic or hypoxic conditions. First, we performed metabolomic analysis of HL-60 cells co-cultured with bone marrow (BM)-derived mesenchymal stem cells (MSCs). Consistent with the known mechanism of GLS inhibition, CB-839 caused a rapid and extensive decrease in intracellular Glu in both HL60 and MSC and a corresponding increase in intracellular Gln in both cell types. Unexpectedly, CB-839-treated cells exhibited a rapid increase in intracellular and extracellular concentrations of multiple amino acids (Phe, kynurenine, Trp, Leu, Ile, Met, Tyr, Val, Thr, Ala, Gln, Asn, and His), possibly reflecting inhibition of global protein synthesis. CB-839 suppressed cysteine consumption from the extracellular compartment and caused rapid increase in intracellular taurine in HL-60 cells, suggesting altered redox homeostasis (Fig. 1A). CB-839 inhibited cellular growth of HL-60 and MV4;11 AML cells cultured alone or co-cultured with MSC, under conditions mimicking the BM microenvironment (Fig. 1B). Stable isotope-resolved metabolomics (SIRM) analysis with 13C5, 15N2-Gln in HL-60 cells indicated that treatment with CB-839 severely hindered Gln anaplerosis to similar extent under normoxic or hypoxic conditions. Moreover, Gln is predominantly used to carry out oxidative metabolism. The enriched fraction of aspartate in treated cells dropped dramatically (to approximately 20% or less of the pool), suggesting that leukemia cells require Krebs cycle-derived oxaloacetate transamination for the generation of aspartate (Fig. 1C). Limiting Gln supply using CB-839 caused reduction in the concentration of alpha-ketoglutarate (α-KG) and the oncometabolite 2-hydroxyglutarate (2-HG), known to play a role in the pathogenesis of AML. We have previously shown that the leukemic BM microenvironment is highly hypoxic (Benito PLoS One 2011), andhypoxia has been reported to induce production of the L-enantiomer of 2-HG (L-2HG) (Intlekofer Cell Metabolism 2015). In AML cells, hypoxia selectively induced the production of L-2HG measured by LC-MS/MS in HL-60 (6.2 fold) and OCI-AML3 cells (2.9 fold) with wt-IDH. This increase in L-2HG was potently inhibited by CB-839, implicating Gln as a source for L-2HG production by AML cells under hypoxia. HL-60 and OCI-AML3 AML cells produced very limited amounts of the D-enantiomer of 2HG (D-2HG), and neither hypoxia nor CB-839 significantly affected D-2HG levels. We recently reported that CB-839 increased hydroxymethylation (hmc) levels using a HELP-GT assay (Velez ASH 2015), and the implications of those observations are the subject of ongoing studies. Prompted by the observation of increased hmc in response to CB-839 treatment, we next examined the efficacy of CB-839 in combination with the DNMT3A inhibitor 5-azacitidine (5-AZA). Treatment with 1µM CB-839 and escalating doses of 5-AZA caused additive or synergistic inhibition of cellular growth after 5 days of culture, both under normoxia and hypoxia, in AML cell lines (OCI-AML3, HL-60, MV4;11) and in primary AML cells (n=3) (Fig. 1D). To test the efficacy of both compounds in vivo, we injected NSG-S mice with genetically engineered MV4;11/Luc cells. Bioluminescent imaging (BLI) demonstrated significantly reduced leukemia burden in treated groups compared to controls, more prominently in the CB-839 plus 5-AZA co-treated mice. CB-839 and 5-AZA co-treatment resulted in significant extension of survival compared with 5-AZA single agent, p In summary, GLS inhibition causes AML growth arrest by multiple mechanisms, including inhibition of protein synthesis and disruption of redox homeostasis. Gln contributes to hypoxia-induced production of L-2HG and possibly epigenome regulation in AML, and concomitant blockade of GLS by CB-839 and DNMT3A with 5-AZA potently suppresses AML cell growth in vitro and in vivo. The clinical trial examining the efficacy of this combination is ongoing (Calithera, NCT02071927). Download : Download high-res image (328KB) Download : Download full-size image Disclosures Lorenzi: Erytech Pharma: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties: NIH-held patent related to L-asparaginase. DiNardo: Novartis: Research Funding; Abbvie: Research Funding; Agios: Research Funding; Celgene: Research Funding; Daiichi Sankyo: Research Funding. Konopleva: Calithera: Research Funding; Cellectis: Research Funding.

Evaluation of anticancer properties of a new α-methylene-δ-lactone DL-249 on two cancer cell lines

AbstractBackgroundThe anticancer activity of a new synthetic α-methylene-δ-lactone DL-249 was reported in leukemia HL-60 and breast cancer MCF-7 cells and compared with the activity of a natural α-methylene-γ-lactone from Tanacetum parthenium, parthenolide.MethodologyThe chemical synthesis of DL-249 was performed using Horner-Wadsworth-Emmons methodology. The cytotoxic activity of the tested compounds was assessed by an MTT test. The ability to induce apoptosis was studied by flow cytometry. The expression levels of genes were determined by quantitative real-time PCR.Principal Findings/ResultsDL-249 and parthenolide inhibited the growth of HL-60 and MCF-7 cells with IC50 values below 10 μM. DL-249 was a stronger apoptosis inducer than parthenolide in both cell lines and both compounds produced a more pronounced effect on HL-60 than on MCF-7 cells. DL-249 and PTL significantly up-regulated expression of Bax, caspase-9, caspase-3 and p53 genes and decreased the level of Bcl-2 and Bcl-xl genes in both cancer cell lines. Additionally DL-249 caused cell cycle arrest in the subG0/G1phase.Conclusion/SignificanceA new synthetic α-methylene-δ-lactone, DL-249 showed the anticancer activity on two cancer cell lines and seemed to be a better apoptosis inducer than parthenolide which makes this compound an attractive lead for further studies.

Raman spectrum reveals Mesenchymal stem cells inhibiting HL60 cells growth

Though some research results reveals that Mesenchymal stem cells (MSCs) have the ability of inhibiting tumor cells proliferation, it remains controversial about the precise interaction mechanism during MSCs and tumor cells co-culture. In this study, combing Raman spectroscopic data and principle component analysis (PCA), the biochemical changes of MSCs or Human promyelocytic leukemia (HL60) cells during their co-culture were presented. The obtained results showed that some main Raman peaks of HL60 assigned to nucleic acids or proteins were greatly higher in intensity in the late stage of co-culture than those in the early stage of co-culture while they were still lower relative to the control group, implicating that the effect of MSCs inhibiting HL60 proliferation appeared in the early stage but gradually lost the inhibiting ability in the late stage of co-culture. Moreover, some other peaks of HL60 assigned to proteins were decreased in intensity in the early stage of co-culture relative to the control group but rebounded to the level similar to the control group in the late stage, showing that the content and structure changes of these proteins might be generated in the early stage but returned to the original state in the late stage of co-culture. As a result, in the early stage of MSCs-HL60 co-culture, along with the level of Akt phosphorylation of HL60 was lowered relative to its control group, the proliferation rate of HL60 cells was decreased. And in the late stage of co-culture, along with the level of Akt phosphorylation was rebounded, the reverse transfer of Raman peaks within 875–880cm−1 appeared, thus MSCs lost the ability to inhibit HL60 growth and HL60 proliferation was increased. In addition, it was observed that the peak at 811cm−1, which is a marker of RNA, was higher in intensity in the late stage than that in the control group, indicating that MSCs might be differentiated into myofibroblast-like MSCs. In addition, PCA results also exhibited that the physiological state of MSCs can be separated by the first two main components of PC1 or PC2 easily, and the effect of MSCs inhibiting HL60 growth was greatly associated with the time of co-culture.

Targeting Glycolysis and MAPK Pathway: A Combined Pre-Clinical Approach on Acute Myeloid Leukemia

We have demonstrated that selective MEK inhibitors (MEK-I) significantly inhibit in vitro the growth of AML cell lines and primary AML blasts (Ricciardi et al., JMM 2012). However, these effects were mostly related to the inhibition of cell cycle progression, while apoptosis induction requires higher concentrations of the inhibitor and longer times of exposure. Among the many downstream effectors of MAPK pathway, metabolism is one of the key aspects that can be targeted at several levels for therapeutic perspectives (Ricciardi et al., Blood 2015). In this study, we have explored on AML cells the functional effects of combining the MEK1/2 inhibitor, PD0325901 (PD), with a metabolic modulator, dichloroacetate (DCA), which acts as pyruvate dehydrogenase kinase (PDHK) inhibitor. AML cell lines (OCI-AML3, U937, MOLM13, HL60) and AML primary samples were exposed to PD (2.5-1000nM) and DCA (0.5-2mM), alone or in combination.PD dose-dependently inhibited cell growth of OCI-AML3, HL60 and MOLM13, showing a constitutive activation of RAS/RAF/MEK/ERK pathway. This activation was not seen in U937, thus exhibiting resistance to PD. DCA shows a pro-apoptotic activity on AML cells, only at the highest concentration. The combination between PD and DCA synergistically enhanced the antiproliferative effect, with a combination index (CI) ranging between 0.37 and 0.41 in OCI-AML3, as measured by isobologram analysis from MTT data. Conversely, in U937 the combination resulted antagonistic. Co-administration of non- or sub-toxic concentrations of DCA (2mM) and of PD (10nM) resulted in a strong increase of cell death in OCI-AML3 (Annexin V). The PD/DCA combination significantly increased Annexin V+ cells, at 72 hours, (65.7±25.0%), compared to single compounds: 16.4±8.7% with 10nM PD (p=0.03) and 28.0±12.4% with 2mM DCA (p=0.04). Similar results were obtained in HL60 and MOLM13 (data not shown). In the U937 cell line, resistant to PD, the levels of apoptosis observed with DCA alone were not further enhanced by the combination: 6.19±1.7% (1000nM PD), 23.63±6.7% (2mM DCA), 31.28±9.9% (PD/DCA). At a protein level, densitometric analysis of Western blot carried out on OCI-AML3 demonstrated that PD/DCA was able to further increase PD-induced ERK inhibition (from 40% to 54%), while DCA alone, as expected, had no effect. Real time metabolic data, obtained by a Seahorse XF24 on OCI-AML3, showed that the combination of PD and DCA affected mitochondrial metabolism: basal respiration and ATP production were impaired by 37.34±10.4% (p=0.012) and by 56.31±10.0% (p=0.003), respectively, compared to control. On the other hand, both maximal respiration and spare respiratory capacity were increased by 28.80±9.5% (p=0.025) and 68.78±14.2% (p<0.001), respectively. The effects of the PD/DCA combination were then examined on AML primary samples (n=3). Preliminary results indicate that PD/DCA combination effectively killed AML cells with a net apoptosis induction at 72 hours (Annexin V+ cells: 31.79±5.9% with PD/DCA, 9.95±5.6% with 10nM PD and 8.48±7.3% with 2mM DCA) in 2 out of 3 patients. In the third sample, which proved hypersensitive to PD (58.73% with 10nM PD at 72h), the combination was ineffective (52.48%).These preliminary results suggest that the combination of a signal transduction and a metabolic inhibitor spares mitochondrial machinery while impairing at the same time its activity, thus resulting in an enhanced antiproliferative and pro-apoptotic effect on AML cells. DisclosuresNo relevant conflicts of interest to declare.

Gls Inhibitor CB-839 Modulates Cellular Metabolism in AML and Potently Suppresses AML Cell Growth When Combined with 5-Azacitidine

Glutamine (Gln) is required for growth and proliferation of several tumor types including AML. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes conversion of Gln to glutamate (Glu), which provides carbons for the TCA cycle and regulates redox homeostasis through production of glutathione and NADH. CB-839 is a highly selective, reversible, allosteric inhibitor of GLS. In this study we studied metabolic and cellular consequences of GLS inhibition in AML cells cultured in normoxic or hypoxic conditions.First, we performed metabolomic analysis of HL-60 cells co-cultured with bone marrow (BM)-derived mesenchymal stem cells (MSCs). Consistent with the known mechanism of GLS inhibition, CB-839 caused a rapid and extensive decrease in intracellular Glu in both HL60 and MSC and a corresponding increase in intracellular Gln in both cell types. Unexpectedly, CB-839-treated cells exhibited a rapid increase in intracellular and extracellular concentrations of multiple amino acids (Phe, kynurenine, Trp, Leu, Ile, Met, Tyr, Val, Thr, Ala, Gln, Asn, and His), possibly reflecting inhibition of global protein synthesis. CB-839 suppressed cysteine consumption from the extracellular compartment and caused rapid increase in intracellular taurine in HL-60 cells, suggesting altered redox homeostasis (Fig. 1A). CB-839 inhibited cellular growth of HL-60 and MV4;11 AML cells cultured alone or co-cultured with MSC, under conditions mimicking the BM microenvironment (Fig. 1B).Stable isotope-resolved metabolomics (SIRM) analysis with 13C5, 15N2-Gln in HL-60 cells indicated that treatment with CB-839 severely hindered Gln anaplerosis to similar extent under normoxic or hypoxic conditions. Moreover, Gln is predominantly used to carry out oxidative metabolism. The enriched fraction of aspartate in treated cells dropped dramatically (to approximately 20% or less of the pool), suggesting that leukemia cells require Krebs cycle-derived oxaloacetate transamination for the generation of aspartate (Fig. 1C). Limiting Gln supply using CB-839 caused reduction in the concentration of alpha-ketoglutarate (α-KG) and the oncometabolite 2-hydroxyglutarate (2-HG), known to play a role in the pathogenesis of AML.We have previously shown that the leukemic BM microenvironment is highly hypoxic (Benito PLoS One 2011), andhypoxia has been reported to induce production of the L-enantiomer of 2-HG (L-2HG) (Intlekofer Cell Metabolism 2015). In AML cells, hypoxia selectively induced the production of L-2HG measured by LC-MS/MS in HL-60 (6.2 fold) and OCI-AML3 cells (2.9 fold) with wt-IDH. This increase in L-2HG was potently inhibited by CB-839, implicating Gln as a source for L-2HG production by AML cells under hypoxia. HL-60 and OCI-AML3 AML cells produced very limited amounts of the D-enantiomer of 2HG (D-2HG), and neither hypoxia nor CB-839 significantly affected D-2HG levels. We recently reported that CB-839 increased hydroxymethylation (hmc) levels using a HELP-GT assay (Velez ASH 2015), and the implications of those observations are the subject of ongoing studies.Prompted by the observation of increased hmc in response to CB-839 treatment, we next examined the efficacy of CB-839 in combination with the DNMT3A inhibitor 5-azacitidine (5-AZA). Treatment with 1µM CB-839 and escalating doses of 5-AZA caused additive or synergistic inhibition of cellular growth after 5 days of culture, both under normoxia and hypoxia, in AML cell lines (OCI-AML3, HL-60, MV4;11) and in primary AML cells (n=3) (Fig. 1D).To test the efficacy of both compounds in vivo, we injected NSG-S mice with genetically engineered MV4;11/Luc cells. Bioluminescent imaging (BLI) demonstrated significantly reduced leukemia burden in treated groups compared to controls, more prominently in the CB-839 plus 5-AZA co-treated mice. CB-839 and 5-AZA co-treatment resulted in significant extension of survival compared with 5-AZA single agent, p<0.001 (Fig. 1E).In summary, GLS inhibition causes AML growth arrest by multiple mechanisms, including inhibition of protein synthesis and disruption of redox homeostasis. Gln contributes to hypoxia-induced production of L-2HG and possibly epigenome regulation in AML, and concomitant blockade of GLS by CB-839 and DNMT3A with 5-AZA potently suppresses AML cell growth in vitro and in vivo. The clinical trial examining the efficacy of this combination is ongoing (Calithera, NCT02071927). [Display omitted] DisclosuresLorenzi:Erytech Pharma: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties: NIH-held patent related to L-asparaginase. DiNardo:Novartis: Research Funding; Abbvie: Research Funding; Agios: Research Funding; Celgene: Research Funding; Daiichi Sankyo: Research Funding. Konopleva:Calithera: Research Funding; Cellectis: Research Funding.

Abstract 1004: GLS inhibitor CB-839 modulates cellular metabolism in AML and potently suppresses AML cell growth when combined with 5-azacitidine

Abstract Glutamine (Gln) is required for growth and proliferation of several tumor types including AML. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes conversion of Gln to glutamate (Glu), which provides carbons for the TCA cycle and regulates redox homeostasis through production of glutathione and NADH. CB-839 is a highly selective, reversible, allosteric inhibitor of GLS. In this study we studied metabolic and cellular consequences of GLS inhibition in AML cells cultured in normoxic or hypoxic conditions. First, we performed metabolic analysis of HL-60 cells co-cultured with bone marrow-derived mesenchymal stem cells (MSCs). Consistent with the known mechanism of GLS inhibition, CB-839 caused a rapid and extensive decrease in intracellular Glu in both HL60 and MSC and a corresponding increase in intracellular Gln in both cell types. Unexpectedly, CB-839-treated cells exhibited a rapid increase in intracellular and extracellular concentrations of multiple amino acids, possibly reflecting inhibition of global protein synthesis. CB-839 suppressed Cys consumption from the extracellular compartment and caused rapid increase in intracellular taurine in HL-60 cells, suggesting altered redox homeostasis. CB-839 inhibited cellular growth of HL-60 and MV4;11 AML cells cultured alone or co-cultured with MSC, demonstrating activity under conditions mimicking BM microenvironment. We have previously shown that the leukemic bone marrow microenvironment is highly hypoxic (Benito PLoS One 2011), and hypoxia has been reported to induce production of the L-enantiomer of 2-HG (L-2HG) (Intlekofer Cell Metabolism 2015). In AML cells, hypoxia selectively induced the production of L-2HG (measured by LC-MS/MS) in HL-60 (6.2+/- fold) and OCI-AML3 cells (2.9+/- fold) with wt-IDH. That increase in L-2HG was potently inhibited by CB-839. AML cells produced very little D-2HG, and neither hypoxia nor CB-839 significantly affected D-2HG levels. We recently reported that CB-839 increased hydroxymethylation (hmc) levels using a HELP-GT assay (Velez ASH 2015), and the implications of those observations are the subject of ongoing studies. Prompted by the observation of increased hmc in response to CB-839 treatment, we next examined the efficacy of CB-839 in combination with the DNMT3A inhibitor 5-azacitidine (5-AZA). Treatment with 1μM CB-839 and escalating doses of 5-AZA caused additive or synergistic inhibition of cellular growth after 5 days of culture, both under normoxia and hypoxia, in AML cell lines (OCI-AML3, HL-60, MV4;11) and primary AML cells (n = 6). In summary, GLS inhibition causes AML growth arrest by multiple mechanisms, including inhibition of protein synthesis and disruption of redox homeostasis. Gln contributes to hypoxia-induced production of L-2HG and possibly epigenome regulation in AML, and concomitant blockade of GLS by CB-839 and DNMT3A with 5-AZA potently suppresses AML cell growth. Citation Format: Tianyu Cai, Philip L. Lorenzi, Dinesh Rakheja, Michael Pontikos, Lina Han, Qi Zhang, Helen Ma, Thomas D. Horvath, Amit K. Verma, Marina Konopleva. GLS inhibitor CB-839 modulates cellular metabolism in AML and potently suppresses AML cell growth when combined with 5-azacitidine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1004.