Abstract 2746: Combined inhibition of Wee1 and Poly(ADP Ribose)Polymerase 1/2 synergistically inhibits acute leukemia cells by enhancing DNA damage and inducing apoptosis in vitro and in vivo
Incorporating Hemoglobin Levels to Map Leukostasis Risk in Acute Leukemia Using Microvasculature-on-Chip Technologies
Hyperleukocytosis, most commonly defined as a white blood cell (WBC) count > 100,000/μL, is an oncologic emergency in acute leukemia that can lead to leukostasis, which occurs when leukemia cells obstruct the microvasculature resulting in significant morbidity and mortality from neurologic (CNS hemorrhage, thrombosis) or pulmonary (respiratory distress, hypoxia) symptoms. The underlying mechanisms are poorly understood but are thought to be related to increased blood viscosity, secondary to high WBC count, leukemia cell aggregation, and the abnormal mechanical properties, size, and cell-cell interactions of leukemia cells. Leukapheresis is a commonly used therapy for rapid cytoreduction in symptomatic patients, but the procedure is not without risks. No existing methods reliably predict leukostasis or guide treatment including the commonly used WBC count, which only loosely correlates with leukostasis and does not accurately describe the blood viscosity at the microvascular level. Importantly, while hematocrit/hemoglobin levels (Hgb) are known to be major contributors to blood viscosity, they have not been systematically assessed in leukostasis risk, and Hgb often decreases as leukemic cell counts rise, complicating the issue. Incorporating Hgb levels may better predict leukostasis and assist physicians balancing the risk of hyperleukocytosis compared to the interventions themselves. To that end, we investigated how the differing presentations of acute leukemia lead to microvessel occlusion, thereby affecting effective blood viscosity at the microvascular level using "microvasculature-on-a-chip" devices that mimic the microvascular geometry (Figure 1) developed by our laboratory. This physiologically relevant microvascular model allows for in vitro investigation as in vivo studies are nearly impossible due to difficulty in visualizing and manipulating the animal microvasculature and cell counts. The devices were microfabricated using polydimethylsiloxane (PDMS). Acute T-cell lymphoblastic (Jurkat) and acute monocytic (THP-1) cell lines were maintained via standard cell culture conditions. Red cells from healthy donors were isolated and mixed with leukemia cells to achieve target Hgb and WBC levels. Various physiologic leukemia "mixtures" were then perfused under physiologic microcirculatory flow conditions through the microvascular device and microchannels occlusion was tracked via videomicroscopy (Figure 2). With T-cell leukemia, Hgb levels affected the risk of "in vitro leukostasis." Specifically, with severe anemia and WBC count less than the hyperleukocytosis range, time to microchannel occlusion was longer, and was more dependent on Hgb rather than WBC count. However, in cases with severe anemia and WBC counts > 100k/μL, WBC count exhibited a stronger effect on occlusion with little dependence on Hgb (Figure 3). At Hgb > 8g/dL, microchannel occlusion was dependent on WBC count regardless of hyperleukocytosis or not. In contrast, our data to date shows that with myeloid leukemia, in vitro leukostasis is not associated with Hgb levels, and is consistent with how myeloid leukemias in vivo cause leukostasis symptoms at lower WBC counts than lymphoid leukemias, not only due to size but also adhesive interactions. These data suggest when determining risk for leukostasis, WBC count should not be the sole determinant. Here we show Hgb levels affect microvascular blood viscosity and propensity for microvascular occlusion, but it appears to have a greater impact with T-cell leukemias versus myeloid leukemias (Figure 4). These studies indicate Hgb is an important clinical parameter for leukostasis risk in acute leukemia and will help inform guidelines for leukapheresis and even phlebotomy, a much simpler and safer procedure, to mitigate hyperviscosity in acute leukemia. These results can also impact decisions regarding the need for red blood cell transfusions, which iatrogenically increase blood viscosity. Studies incorporating patient myeloid and lymphoid leukemia cells and microvasculature-on-chip devices integrating live endothelium to assess leukemia cell adhesion are ongoing. Figure Disclosures Lam: Sanguina, Inc: Current equity holder in private company.
Abstract Abstract 1496 Introduction: Although progress in the treatment of ALL has been remarkable in children, in adults ALL still carries a dismal outcome. Thus, there is a need to improve therapeutic options. In the last years, selective inhibitors of Chk1 and/or Chk2 have been discovered, developed and entered in clinical trials. However, so far, they have not yet been investigated in leukemia. Chk1 and Chk2 are serine/threonine kinases that play a critical role in response to DNA damage both by halting the cell cycle through checkpoint activation and by actively repairing DNA. Here, we explored the in vitro and in vivo activity of single-agent inhibition of Chk1/2 by PF-0477736 in B- and T-progenitor ALL and we investigated potential biomarkers of functional inhibition. Methods: Human B (BCR-ABL1-positive: BV-173, SUPB-15; BCR-ABL1- negative: NALM-6, NALM-19, REH) and T (MOLT-4, RPMI-8402, CEM) leukemia cell lines were incubated with increasing concentrations of drug (5–2000 nM) for 24, 48 and 72 hours (hrs). Results: Inhibition of Chk1/2 resulted in a dose and time-dependent cytotoxicity with RPMI-8402 and BV-173 cells being the most sensitive (IC50 at 24 hrs: 57 nM and 82 nM, respectively), while NALM-6 cells the most resistant (IC50 at 24 hrs: 1426 nM)(WST-1 assay, Roche). Sensitivity did not correlate with p53 status (BV-173, SUPB-15, NALM-6 and NALM-19 cells were p53 wild-type whereas REH, MOLT-4, RPMI-8402 and CEM cells were p53 mutated) and with baseline levels of Chk1/2 and ATR/ATM phosphorylation, indicative of intrinsic genetic stress. Consistent with the viability results, Annexin V/Propidium Iodide (PI) staining analysis showed a significant increase of apoptosis at 24 and 48 hrs in a dose and time dependent manner coupled to increased proteolytic cleavage of PARP-1. In all sensitive cell lines in addition to the induction of apoptosis, Chk1/Chk2 inhibition induced DNA damage as demonstrated by the increased number of γH2AX foci (western blot and immunofluorescence analysis) and by a marked phosphorylation of Chk1 (ser317 and ser345). Moreover, PF-0477736 efficiently triggered the Chk1-Cdc25-Cdk1 pathway as soon as 24 hrs of treatment with a decrease of the inhibitory phosphorylation of Cdc25c (ser216) and Cdk1 (tyr15), leading to the abrogation of cell cycle arrest as confirmed by PI staining analysis at 6 and 24 hrs. The efficacy of PF-0477736 was thereafter demonstrated in primary leukemic blasts separated from 14 ALL patients. Based on the viability results at 24 hrs, 3 groups of patients were identified: very good responders, 5/14, 36% (IC50: 100–500 nM); good responders, 6/14, 43% (IC50: 600–1000 nM); poor responders, 3/14, 21% (IC50 > 1000 nM). By contrast, PF-0477736 did not show efficacy in primary cultures of normal bone marrow mononuclear cells, demonstrating its specificity for leukemia cells. We extended the in vitro and ex-vivo studies by assessing the efficacy of Chk inhibition in mice transplanted with T-lymphoid leukemia, demonstrating that PF-0477736 increases the survival of treated mice compared with mice treated with vehicle (p = 0.0016). Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on treated leukemia cells and their untreated counterparts (DMSO 0.1%) after 24 hrs of incubation with concentrations equal to the IC50. Treatment resulted in a differential expression (p < 0.05) of genes involved in chromatin assembly, nucleosome organization and DNA packaging (e.g. Histone H1-H2A, 2B family clusters), DNA damage (DDIT3, GADD34 and GADD45a) and apoptosis (e.g. CDKN1A, BAX, FAS, BTG1), confirming that PF-0477736 contributes to checkpoint replication abrogation, accumulation of DNA damage and subsequent apoptosis in leukemia cells. Interestingly, N-Myc and c-Myc expression strongly decreased after treatment, as also confirmed by western blot analysis, suggesting that a negative feedback loop may exist between Chk induction and Myc expression. Conclusions: Together, these results demonstrate the efficacy of PF-0477736 both in vitro and in vivo models of ALL, arguing in favor of its future clinical evaluation in leukemia. Supported by ELN, AIL, AIRC, Fondazione Del Monte di Bologna-Ravenna, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer. Disclosures: Baccarani: ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.
Background: Functional genomic screens elegantly increase our understanding of biology of leukemias. So far, CRISPR/Cas9 screens are widely performed in cell lines and in genetically engineered mouse models, in vitro and in vivo; here, we extended their use to patient-derived leukemia cells in vivo. Methods Serially transplantable patient-derived xenograft (PDX) models were generated from children and adults with acute lymphoblastic leukemia (ALL). Cas9 was stably expressed in PDX ALL cells using a split form of Cas9 assembled by inteins, facilitating lentiviral-mediated gene delivery. Customized sgRNA library was generated using golden gate cloning, at 5 sgRNAs per target gene. The sgRNA vector additionally expressed a fluorochrome marker and a tag, for sequential magnetic-activated cell sorting (MACS) and flow cytometry (FACS) enrichment of sgRNA transduced PDX cells. Highly enriched Cas9/sgRNA double transgenic cells were transplanted into NSG mice and animals sacrificed after different periods of time. Cells were re-isolated from bone marrow, purified and subjected to PCR-based amplification of sgRNA library followed by next generation sequencing. Differential sgRNA distributions were analysed using a MAGeCK pipeline. Results We aimed to establish a comprehensive CRISPR screen pipeline allowing functional genomic screens in patients' acute leukemia cells. We investigated surface molecules required for cell homing and growth in mice, using a distinct customized sgRNA library. Quality controls of the sgRNA plasmid pool as well as transgenic PDX input samples verified standard distribution of all sgRNAs. As knockout was required at the time point of transplantation, conditions for prolonged culture of PDX ALL cells in vitro were optimized. Before injection into NSG mice, transduced PDX ALL cells were successfully enriched to above 95% using MACS and FACS. Over time in vivo, deep sequencing of re-isolated PDX cells revealed unchanged distribution of control sgRNAs, but strong loss of sgRNAs targeting CXCR4 and ITGB1, suggesting that CXCR4 and ITGB1 might be required for PDX ALL cell homing and engraftment. To validate the findings of drop-out CRISPR screen, we analyzed single sgRNAs targeting CXCR4 and ITGB1 in PDX cells. Competitive in vivo assays monitored by recombinant fluorochrome markers showed that the cells with CXCR4 or ITGB1 knockout had a significant disadvantage in vivo with respect to homing and growth in mice, compared to the control population. Taken together, we established a comprehensive workflow for CRISPR screen in PDX model of ALL in vivo. Our data identify and validate that CXCR4 and ITGB1 are required for homing and growth of PDX ALL cells in mice. Conclusion We show that CRISPR/Cas9 functional genetic screens are feasible in PDX acute leukemia models in vivo and report the first such screen, as far as to our knowledge. Extending CRISPR/Cas9 screens to patients' cells will greatly facilitate our understanding of individual leukemia biology and therapeutic targets in the future. Disclosures Becker: AVA Lifescience GmbH: Consultancy.
Abstract Abstract 2419 Introduction: Acute leukemia and solid tumors result from alterations in the essential pathways of cell physiology including apoptosis, proliferation and genome instability. In solid tumors, the proapoptotic SIVA protein modulates apoptosis, proliferation, migration and promotes Stathmin inhibition through phosphorylation. Stathmin regulates microtubules dynamics and its hyperactivity confers chromosome instability in leukemia cells. Using two-hybrid system assay, we have identified SIVA as a binding partner of ANKHD1, an ankyrin-repeat-containing protein. ANKHD1 is overexpressed in acute myeloid leukemia (AML) and acute lymphoblast leukemia (ALL) and has the potential role of regulating multiple cellular functions via their repeat motifs. We thus hypothesized that ANKHD1 and SIVA could be involved in leukemogenesis. We aimed to evaluate SIVA expression in normal and leukemia hematopoietic cells, to confirm the endogenous ANKHD1/SIVA association, and to investigate the functional role of both proteins in apoptosis, proliferation, migration, and Stathmin activation. Materials and Methods: Expression of SIVA was evaluated by qPCR in total bone marrow cells from 22 healthy donors, 42 AML and 21 ALL patients at diagnosis. All normal donors and patients provided informed written consent and the study was approved by the ethics committee of the Institution. Leukemia cell lines (Jurkat, Namalwa or U937 cells) were used for functional studies. Endogenous protein interaction was verified by immunopreciptation and cofocal microscopy. We stably knocked down the endogenous expression level of ANKHD1 or SIVA with specific shRNA-expressing lentiviral vector and in vitro apoptosis was examined by AnnexinV/PI, cell growth by MTT assay and colony formation, and migration by transwell assays. In addition, we investigated in vivo tumor growth; leukemia cells were implanted in the dorsal sub cutis of NOD/SCID mice and tumors were excised, measured and weighed after 15 days. Stathmin activation proteins (Stathmin, phospho-Stathmin, alpha tubulin and acetylated-alpha tubulin) and apoptotic proteins (BCL-XL, BAX, JNK and phospho-JNK) were evaluated by Western blot. Appropriated statistical analysis was performed. Results: SIVA expression was significantly decreased in AML and ALL cells compared with normal hematopoietic cells (P<0.05), a reverse pattern of ANKHD1 expression, when compared with published data. Immunopreciptation and confocal analyses confirmed that ANKHD1 and SIVA interact and co-localize in the cytoplasm of leukemia cells. Functional studies revealed that SIVA and ANKHD1 have antagonistic effects on migration, Stathmin activation, and in vivo tumor growth. SIVA silencing resulted in a significantly increased cell migration, Stathmin activation (decreased Stathmin phosphorylation), and augmented in vivo tumor growth (P<0.05). On the other hand, ANKHD1 silencing resulted in a significantly decreased cell migration, Stathmin inactivation (increased Stathmin phosphorylation and alpha tubulin acetylation), and reduced in vivo tumor growth (P<0.05). Regarding apoptosis and proliferation, SIVA knockdown resulted in a significant decrease in apoptosis response to UV and daunorubicin induction and a downregulation of proapoptotic proteins p-JNK and BAX, an upregulation of the antiapoptotic protein BCL-XL, but no modulation was observed in proliferation and clonal growth in vitro. In contrast, ANKHD1 knockdown resulted in a significant decrease of proliferation and clonogenicity (P<0.05), but no changes were observed in apoptosis in vitro. Conclusion: Our data indicate SIVA as a tumor suppressor gene in leukemia cells, and SIVA downmodulation may contribute to the apoptosis resistance and chromosome instability. ANKHD1 may be an oncogene, and the upregulation of this protein in leukemia cells might lead to increased proliferation and generate chromosomal instability through increased Stathmin activation. The results suggest that ANKHD1 inhibits SIVA and restoration of SIVA expression or inhibition of ANKHD1 may be an attractive approach in leukemia. Disclosures: No relevant conflicts of interest to declare.
AbstractPeposertib (M3814) is a potent and selective DNA-PK inhibitor in early clinical development. It effectively blocks non-homologous end-joining repair of DNA double-strand breaks (DSB) and strongly potentiates the antitumor effect of ionizing radiation (IR) and topoisomerase II inhibitors. By suppressing DNA-PK catalytic activity in the presence of DNA DSB, M3814 potentiates ATM/p53 signaling leading to enhanced p53-dependent antitumor activity in tumor cells. Here, we investigated the therapeutic potential of M3814 in combination with DSB-inducing agents in leukemia cells and a patient-derived tumor. We show that in the presence of IR or topoisomerase II inhibitors, M3814 boosts the ATM/p53 response in acute leukemia cells leading to the elevation of p53 protein levels as well as its transcriptional activity. M3814 synergistically sensitized p53 wild-type, but not p53-deficient, AML cells to killing by DSB-inducing agents via p53-dependent apoptosis involving both intrinsic and extrinsic effector pathways. The antileukemic effect was further potentiated by enhancing daunorubicin-induced myeloid cell differentiation. Further, combined with the fixed-ratio liposomal formulation of daunorubicin and cytarabine, CPX-351, M3814 enhanced the efficacy against leukemia cells in vitro and in vivo without increasing hematopoietic toxicity, suggesting that DNA-PK inhibition could offer a novel clinical strategy for harnessing the anticancer potential of p53 in AML therapy.
With the development of mass spectrometer technology, recent studies revealed the critical roles of cancer-specific metabolism for tumor propagation in several types of cancers. In leukemia, many studies have been conducted to elucidate a leukemia-specific metabolism, and several effective treatments such as IDH1/2 inhibitors targeting acute myeloid leukemia (AML) with IDH1/2 mutation have been developed. To identify the new metabolic pathways on which acute leukemia cells depend, we purified water-soluble metabolites from CD34+ hematopoietic stem and progenitor cells (HSPCs) of healthy donors, AML and acute lymphoblastic leukemia (ALL) patients, and we comprehensively measured 116 metabolites using mass spectrometer analysis. From this experiment, we found that the cellular content of glycerol 3-phosphate (G3P) in CD34+ AML and ALL cells was lower than that of normal CD34+ HSPCs. G3P is an intermediate metabolite in the glycolysis metabolic pathway and is utilized as a substrate for phospholipids synthesis. The initial and rate-limiting step of phospholipids synthesis is the synthesis of lysophosphatidic acid (LPA) from G3P and acyl-CoA mediated by glycerol 3-phosphate acyltransferases (GPATs). Since CD34+ acute leukemia cells contained significantly lower level of G3P, we hypothesized that leukemia cells actively consumed G3P and synthesized LPA by GPATs. GPATs are classified into four isoforms based on intracellular localization and substrate preference. GPAT1 and GPAT2 are mitochondrial GPATs that are localized to the mitochondrial outer membrane, but on the other hand, GPAT3 and GPAT4 are microsomal GPATs that are localized to the endoplasmic reticulum membrane, each encoded by independent genes. GPAT1 is identified as an essential gene for the growth of leukemia cells by RNAi screen analysis in the public database (DepMap). We found that CD34+ immature AML cells exhibited higher GPAT1 expression as compared to CD34- more differentiated AML cells and normal T cells. GPAT1 knockdown inhibited the proliferation of several acute leukemia cell lines including THP-1 and Kasumi-1 in vitro and in vivo. Moreover, a mitochondrial GPATs specific inhibitor (FSG67), which was originally developed as a drug to treat obesity and diabetes, suppressed the growth of the leukemia cell lines through the induction of G1 cell cycle arrest. Growth inhibition was rescued by exogenous administration of LPA, suggesting that the synthetic activity mediated by mitochondrial GPATs should be required for acute leukemia growth. Furthermore, FSG67 induced the apoptosis of leukemia cells derived from AML and ALL patients without affecting normal CD34+ HSPCs at least in vitro. We also confirmed that the injection of FSG67 resulted in the suppression of AML and ALL propagation in vivo using patient-derived xenograft models (see figure). GPAT1 regulates the mitochondrial function by producing LPA which is an essential metabolite for maintaining mitochondrial fusion. Actually, the amount of LPA was decreased in GPAT1 knockdown acute leukemia cells. We next examined mitochondrial energy production by extracellular flux assay, and found that GPAT1 knockdown as well as FSG67 significantly suppressed oxygen consumption rate of acute leukemia cells. Consistent with the impaired mitochondrial function, FSG67 suppressed the mitochondrial membrane potential, indicating that GPAT1 should play a pivotal role in maintaining leukemia-specific mitochondrial function. These results collectively suggest that the synthesis of LPA from G3P catalyzed by GPAT1 has a critical role in propagation of acute leukemia cells irrespective of their lineage origin. Thus, GPAT1 is a novel and common therapeutic target for human acute leukemia through suppressing leukemia-specific mitochondrial function. Figure Disclosures Akashi: Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding; Sumitomo Dainippon, Kyowa Kirin: Consultancy.
Abstract Acute leukemia with 11q23 aberrations is associated with especially poor outcome to standard treatment. HMW-MAA is a membrane bound proteoglycan that has been targeted in the treatment of malignant melanoma. It is widely distributed on 11q23-positive leukemic cells and is not expressed on normal hematopoietic cells. Finally, HMW-MAA is believed to function by signaling through the focal adhesion kinase (Fak)/proline-rich tyrosine kinase 2 (Pyk2) pathways and by supporting angiogenesis via its expression on pericytes. We hypothesized that inhibition of HMW-MAA with mAb directed against specific HMW-MAA determinants would effectively limit the growth of 11q23-positive acute leukemia cells in vitro and in vivo, by affecting Fak/Pyk2 pathways. We examined an 11q23-positive acute myeloid leukemia (AML) cell line (ML-2) and leukemia cells from two patients (one AML, one acute lymphoblastic leukemia) with 11q23 aberrations. Our results demonstrate that ML-2 cells expressed only the VT68.2 and 225.28 determinants, while primary acute leukemia cells expressed VT68.2, 225.28, and 763.74 determinants. Treatment of ML-2 cells with mAb against VT68.2 and 225.28 in vitro did not affect overall proliferation but downregulated phosphorylated (p) Pyk2 expression as determined by Western blotting. Further, anti-HMW-MAA mAb treatment enhanced the anti-proliferative effect of cytarabine (y=0.7875) in vitro. Based on these results, we then evaluated the dose-response relationships and toxicities of anti-HMW-MAA mAb alone and in combination with cytarabine in subcutaneous ML-2 xenografts grown in SCID mice. A trend towards tumor inhibition was noted following high dose (1 mg/kg 2x/week) anti-225 mAb therapy but was not seen at lower (0.25 and 0.5 mg/kg) anti-225 mAb doses. Mice demonstrated no side effects or weight loss as a result of anti-225 therapy. No downregulation of pPyk2 in tumor cells was observed following anti-225 therapy at any dose. Furthermore, combination anti-225.28 HMW-MAA mAb and cytarabine treatment (at increasing cytarabine doses) did not result in improved tumor inhibition as compared to cytarabine alone (p>0.05). We then evaluated the effects of HMW-MAA anti-225.28 and anti-763.74 mAb alone or together for treatment of NOD/SCID mice engrafted with primary 11q23-positive leukemia patient cells. Combination anti-HMW-MAA mAb therapy resulted in no prolongation in survival of engrafted mice as compared to control treated animals. We were able to confirm the presence of high levels of circulating HMW-MAA mAb in the serum of animals 4 days following mAb injection. In addition, explanted ML-2 cells harvested following xenograft growth still maintained expression of the HMW-MAA determinants. We conclude that naïve anti-HMW-MAA mAb neither caused a statistically significant inhibitory effect nor enhanced the effect of cytarabine on 11q23-positive acute leukemia cells in vivo. Future studies using radiolabelled or toxin-conjugated antibodies may enhance the anti-tumor effects of targeted HMW-MAA immunotherapy for 11q23-positive acute leukemia.
Abstract Vascular endothelial growth factor receptors -1 (FLT-1) and -2 (KDR) are expressed by subsets of acute and chronic leukemias, where they signal in paracrine and/or autocrine manner to induce cell survival, proliferation and migration. We have previously shown that acute lymphocytic leukemia migration in response to VEGF via FLT-1 modulates the onset of extramedullary disease, and thus has clinical predictive value (Fragoso et al, Blood 2006). Acute leukemia cell (AML) migration, induced by PlGF/VEGF activation of FLT-1 results in the formation of actin membrane protrusions with concomitant increased ERK1/2 and P38 phosphorylation and activation of Rho-GTPases (Casalou et al, 2007). Since we have found an in vitro association of FLT-1 with caveolin-1, actin and HSp90, we hypothesised that cholesterol-rich domains might regulate FLT-1 mediated survival, proliferation or migration of acute myeloid (AML) and lymphoid (ALL) leukemias. First we found by FACS and RQ-PCR that FLT-1 expression is up-regulated by increased cholesterol/HDL levels in vitro. As shown by sucrose gradient fractionation and western blotting, PlGF/VEGF stimulation of AML cells results in re-localization of FLT-1 to cholesterol-rich domains. Accordingly, FLT-1 localization within cholesterol-rich domains is abrogated by exposing leukemia cells to b-methyl-cyclodextrin (MbCD) which removes intracellular cholesterol. Additionally, FLT-1 phosphorylation is abolished by treatment of AML cells with MbCD or Nystatin, an inhibitor of lipid raft endocytosis. Functionally, AML cells exposure to high levels of total cholesterol/HDL for 24 hours exerted a protective effect from actinomycin D-induced apoptosis and promoted PlGF/VEGF-induced AML migration in transwell migration assays. Together, these results show that on subsets of acute leukemias cholesterol/HDL cellular-content regulates FLT-1 expression and signalling, resulting in decreased apoptosis and induction of cell migration. In vivo, we show that cholesterol-rich diet significantly increases bone marrow VEGF levels in mice; inoculation of FLT-1 expressing acute leukemias into mice fed with cholesterolrich diet significantly accelerated disease progression and worsened disease outcome. Taken together, our data show the molecular basis by which cellular and systemic cholesterol regulates VEGF and VEGFR-1 signalling on subsets of acute leukemias, modulating cell migration and survival and thereby regulating disease progression.
Objective: The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms. Background: Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression. Methods: Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage. Results: Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 µM lower than irinotecan with an IC50 of 13.86±0.80 µM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 µM lower than irinotecan (IC50±SD, 5.35±0.38 µM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 µM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10µM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells. Conclusion: Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.