scholarly journals Small Molecule Kinase Inhibitor Sunitinib Specifically Targets Juvenile Myelomonocytic Leukemia Cells with SHP2(PTPN11) Mutation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3353-3353
Author(s):  
Chunxiao He ◽  
Yuming Zhao ◽  
Junbin Huang ◽  
Yao Guo ◽  
Hongman Xue ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Drug Screening ◽  
Leukemia Cell ◽  
Juvenile Myelomonocytic Leukemia ◽  
Oncogenic Signaling ◽  
Leukemia Cell Lines

Abstract Juvenile myelomonocytic leukemia (JMML) is a highly fatal malignant disease in early childhood. It is still unknown of the specific pathogenesis, and there is shortage of effective targeted therapeutic approaches. Gain of function SHP2 mutation encoded by PTPN11 gene is found in approximately 35% of JMML patients, which maybe contributed to its pathogenesis. JMML patients with SHP2 mutation have lower survival rate and higher recurrence rate. All of the above make development of new therapies imperative. Currently, there is no stable cell line that can accurately reflect the characteristics of JMML abnormal cells for research on JMML. In this study, we established two leukemia cell lines that depend on mutated SHP2 for survival, and discovered promising drugs that targeted mutated-SHP2-dependent oncogenic signaling pathway through drug screening method. HCD-57 cells are murine erythroleukemia cells that solely depend on exogenic erythropoietin (EPO) for survival. We constructed SHP2-D61Y and SHP2-E76K transformed HCD-57 cell lines through retroviral vectors, the survival of which dependent on mutated SHP2 mediated signaling pathway. Based on these cells, we established a drug screening platform and screened small molecule compound library containing 2862 FDA-approved drugs and 1707 kinase inhibitors. We performed cell viability, flow cytometry, Wright-Giemsa staining, and western blot to evaluate cells after drug treatment. To further assess therapeutic potential, we established in-vivo transplantation model that SHP2-D61Y transformed HCD-57 cells were implanted into immunodeficient NCG mice, and verified the effectiveness of the in-vitro screened drugs. We found that the survival and proliferation of HCD-57 cells transduced by SHP2-D61Y and SHP2-E76K no longer required EPO, but completely relied on the abnormal activation of signaling pathway mediated by mutated SHP2. Western blot results showed that the phosphorylation status of ERK1/2 and AKT of HCD-57 cells expressing SHP2 mutation were abnormally increased, consistent with SHP2-mutated JMML. Thus, we have obtained the leukemia cell lines that can represent the characteristics of activated signaling pathway in JMML with SHP2 mutation. Through drug screening, we observed that drug sunitinib (Sutent ®) selectively inhibits SHP2-mutated HCD-57 cell lines. CCK-8-based cell viability assay demonstrated a dose-dependent inhibition of SHP2-D61Y and SHP2-E76K transformed HCD-57 cell and no effects on the parental HCD-57 cells. Live cell counting with trypan blue revealed that the proliferation of SHP2-mutated HCD-57 cells was totally halted after one day upon treatment with 250 nM sunitinib, whereas the HCD-57 cells were unaffected. Wright-Giemsa staining demonstrated that SHP2-mutated HCD-57 cells showed no normal morphology change and no mitotic activity under sunitinib treatment, otherwise parental HCD-57 cells showed normal mitotic activity. Sunitinib induced apoptosis and cell cycle arrest at G1 phase in SHP2-mutated HCD-57 cells by flow cytometry, but had little effect on the parental HCD-57 cells. Sunitinib effectively downregulates the phosphorylation of ERK and AKT in SHP2-mutated cells, revealing the mechanism of sunitinib targeting SHP2-mutated cells. In addition, after transplantation of SHP2-D61Y transformed HCD-57 cells for 3 weeks, the spleen of NCG mice increased from an average of 45 mg to more than 300 mg; flow cytometry analysis showed that the implanted cells accounted for over 75% of the total nucleated cells in the bone marrow and spleen. Compared with the vehicle control, the number of monocytes in these mice was reduced to the normal range by treatment with sunitinib, and the spleen weights were reduced by about 50%. Histochemical staining showed disappearance of the myeloid infiltration in the spleen, liver and bone marrow. The above results all indicate that sunitinib has strong in-vivo anti-leukemia activity. Furthermore, western blot analysis showed that the administration of sunitinib significantly inhibited the phosphorylation expression level of AKT and ERK, indicating the effectivity of sunitinib in vivo. In conclusion, our data demonstrated that HCD-57 cell line is an effective tool for studying oncogenic signaling pathway and screening drugs that targeted JMML with SHP2 mutation. Sunitinib can be an effective drug for the targeted treatment of JMML in the future. Disclosures No relevant conflicts of interest to declare.

MDM2 Inhibitor Nutlin-3a Triggers Autophagic Cell Death In Addition to Apoptosis In Leukemia Cell Lines with Wild-Type p53

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3300-3300
Author(s):  
Seshagiri Duvvuri ◽  
Vivian Ruvolo ◽  
Duncan H. Mak ◽  
Kensuke Kojima ◽  
Marina Konopleva ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Western Blot ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Annexin V ◽  
Dependent Manner ◽  
Autophagic Vacuoles ◽  
Leukemia Cell Lines

Abstract Abstract 3300 Background: Nutlin-3a is a small molecule inhibitor of MDM2 and has been shown to induce apoptosis and cell cycle arrest in various cancer models in a p53 dependent manner. Autophagy is a programmed cell death that can occur concurrently with apoptosis or in its absence. There is significant debate whether autophagy is a protective mechanism or a bona fide mechanism of cell death. While autophagy can function as tumor cell defense mechanism against cellular stress induced death, mutation/loss of alleles of certain genes regulating autophagy have been associated with development of cancer (e.g. Beclin-1 in breast cancer [Nature, 1999, 402: 672–676]). Multiple proteins involved in autophagy are transcriptional targets of p53 but Nutlin-3a has not been evaluated for its role in inducing autophagy. Here we present data suggesting that low dose Nutlin-3a induces autophagy in addition to apoptosis in leukemia cell lines in a p53 dependent manner. Methods and results: OCI-AML-3 cells (p53-WT) treated with Nutlin-3a (2.5 and 5.0μM for 48, 72 and 96 hrs) were stained with mono-dansyl-cadaverine (MDC), a dye that accumulates in acidic autophagic vacuoles. OCI-AML-3 cells showed increasing staining with MDC in a dose and time dependent fashion by both flow cytometry (54%, 57% and 51% MDC positive after treatment with Nutlin-3a 5.0μM for 48, 72 and 96 hrs) and by confocal microscopy. Nutlin-3a treated cells also were positive for Annexin-V (flow cytometry 22%, 26% and 36% at 48, 72 and 96 hrs time points), and some of the cells were double-positive for Annexin-V and MDC (9.2%, 5% and 7% at 48, 72 and 96 hrs) suggesting that both apoptosis and autophagy can occur simultaneously. Autophagy induction was confirmed by Transmission Electron Microscopy (TEM). Large, multiple autophagic vacuoles were observed in OCI-AML-3 cells treated with Nutlin-3a. OCI-AML-3 cells with stable p53 knockdown by shRNA or HL-60 cells (p53-null) did not show increased MDC staining by flow cytometry (both cell lines) or autophagic vacuoles by TEM (HL-60) after similar treatment. Western blot analysis showed increases in LC3-II and in conjugation of Atg5/12, early and late autophagy markers respectively, in OCI-AML-3 cells after treatment with Nutlin-3a. Increased expression of the autophagy markers (LC3-II and Atg 5/12 conjugate) were also seen by Western blot analysis in the ALL cell lines REH and NALM-6 (both p53-WT) after treatment with Nutlin-3a. Western blot and/or RT-PCR analysis showed upregulation of other p53 related proteins involved in autophagy e.g. DRAM, AMPK-β, LKB1, pLKB1 in OCI-AML-3 cells treated with Nutlin-3a. As mTOR/Akt pathway inhibits autophagy, analysis of mTOR targets showed downregulation of the total and phospho-ribosomal-S6-protein levels, whereas there was no change in total or phospho-4-EBP-1 levels. Knockdown of Beclin-1 (ATG6), one of the proteins required for initiation of the formation of autophagic vacuoles, caused reduction in autophagic vacuoles (MDC staining by confocal microscopy) in OCI-AML-3 and REH cells without affecting apoptosis induction (Annexin V by flow cytometry). Pharmacologic inhibition of late autophagy by Bafilomycin (10nM for 2 hours) reduced MDC staining in OCI-AML-3 cells treated with Nutlin-3a for 48 hrs (32% without and 9% with Bafilomycin) while having limited inhibition of apoptosis (Annexin V positive 42% without and 33% with Bafilomycin). Conclusion: Nutlin-3a induces autophagy in leukemia cells by a p53 dependent manner. We also demonstrate that autophagy could go hand-in-hand with apoptosis and in a fraction of cells both processes may occur concomitantly. Inhibition of autophagy does not necessarily enhance apoptosis. Disclosures: Andreeff: Roche: Research Funding. Borthakur:ASCO: Research Funding.

scholarly journals Identification of Prostaglandin F2 Receptor Negative Regulator (PTGFRN) as an internalizable target in cancer cells for antibody-drug conjugate development

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0246197
Author(s):  
Jorge Marquez ◽  
Jianping Dong ◽  
Chun Dong ◽  
Changsheng Tian ◽  
Ginette Serrero
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Negative Regulator ◽  
Target Validation ◽  
Antibody Drug

Antibody-drug conjugates (ADC) are effective antibody-based therapeutics for hematopoietic and lymphoid tumors. However, there is need to identify new targets for ADCs, particularly for solid tumors and cancers with unmet needs. From a hybridoma library developed against cancer cells, we selected the mouse monoclonal antibody 33B7, which was able to bind to, and internalize, cancer cell lines. This antibody was used for identification of the target by immunoprecipitation and mass spectrometric analysis, followed by target validation. After target validation, 33B7 binding and target positivity were tested by flow cytometry and western blot analysis in several cancer cell lines. The ability of 33B7 conjugated to saporin to inhibit in vitro proliferation of PTFRN positive cell lines was investigated, as well as the 33B7 ADC in vivo effect on tumor growth in athymic mice. All flow cytometry and in vitro internalization assays were analyzed for statistical significance using a Welsh’s T-test. Animal studies were analyzed using Two-Way Analysis of Variance (ANOVA) utilizing post-hoc Bonferroni analysis, and/or Mixed Effects analysis. The 33B7 cell surface target was identified as Prostaglandin F2 Receptor Negative Regulator (PTGFRN), a transmembrane protein in the Tetraspanin family. This target was confirmed by showing that PTGFRN-expressing cells bound and internalized 33B7, compared to PTGFRN negative cells. Cells able to bind 33B7 were PTGFRN-positive by Western blot analysis. In vitro treatment PTGFRN-positive cancer cell lines with the 33B7-saporin ADC inhibited their proliferation in a dose-dependent fashion. 33B7 conjugated to saporin was also able to block tumor growth in vivo in mouse xenografts when compared to a control ADC. These findings show that screening antibody libraries for internalizing antibodies in cancer cell lines is a good approach to identify new cancer targets for ADC development. These results suggest PTGFRN is a possible therapeutic target via antibody-based approach for certain cancers.

Combination of Farnesyl Transferase Inhibitor (Tipifarnib) with Perifosine Induces Apoptosis through phos-PDK1 in Human Lymphoma and Leukemia Cell Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1488-1488 ◽  
Author(s):  
Ebenezer David ◽  
Rajni Sinha ◽  
Claire Torre ◽  
Jonathan L. Kaufman ◽  
Sagar Lonial
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Targeted Agents ◽  
Leukemia Cell Lines ◽  
Targeted Agent ◽  
The Impact

Abstract Introduction: Novel agents as anti-cancer therapy are used in the setting of specific molecular abnormalities that provide a survival advantage for malignant cells. One such agent, tipifarnib, is theoretically targeted at Ras mutations which are present in a number of different human cancers. Our previous experience with the FTIs (David et al, in press Blood) has demonstrated that they are ideal agents to combine with other targeted agents. We have investigated the combination of the AKT inhibitor perifosine with tipifarnib in human leukemia and lymphoma cell lines with the hypothesis that the combination of 2 targeted agents will disrupt separate survival pathways and ultimately result in synergistic tumor cell death. Methods: In this study we used the human leukemia cell lines HL-60, Jurkat, and the lymphoma cell line HT. Western blot analysis was used to assess for the effect of either single agent perifosine, tipifarnib, or the combination on AKT, p-AKT, PDK-1, and caspase cleavage. Flow cytometry was utilized to assess for Annexin V staining following combination therapy. Results:Dose escalation studies demonstrated that doses of tipifarnib up to 5μm demonstrated a significant cell death in HL-60 and HT cells. Perifosine doses of 1–5uM also induced cell death in both HL-60 and HT cells. When apoptosis was assessed using western blot analysis of caspase 3 activity and cleavage, the combination of perifosine and tipifarnib demonstrated significant apoptosis using low doses of both agents. The apoptosis was associated with downregulation of phos-PDK1, with a resultant downregulation in p-AKT. The level of phos-PDK1 was completely inhibited in less than 24 hrs in both the HL-60 and HT cell lines in combination than when either agent was given alone. Conclusion: The combination of perifosine, and AKT targeted agent, with tipifarnib, a Ras targeted agent, appear to induce significant cell death in lymphoma and leukemia cell lines with rapid downregulation of p-AKT via the PDK-1 pathway. This apoptosis occurs in vitro using concentrations well below those that have been achieved in current clinical trials using these agents. Additional studies are being carried out to further delineate the mechanism of synergy as well as to further explore the impact of sequence of administration using this combination. Further studies are also planned to xplore the impact of the combination on primary human leukemia and lymphoma cells from the blood and bone marrow.

In Vitro and In Vivo Monitoring of Valproic Acid Effects on Gene Expression Signatures in Adult Acute Myeloid Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2605-2605
Author(s):  
Lars Bullinger ◽  
Konstanze Dohner ◽  
Richard F. Schlenk ◽  
Frank G. Rucker ◽  
Jonathan R. Pollack ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Serum Levels ◽  
Leukemia Cell Lines ◽  
Acute Myeloid ◽  
Myeloid Leukemia Cell

Abstract Inhibitors of histone deacetylases (HDACIs) like valproic acid (VPA) display activity in murine leukemia models, and induce tumor-selective cytoxicity against blasts from patients with acute myeloid leukemia (AML). However, despite of the existing knowledge of the potential function of HDACIs, there remain many unsolved questions especially regarding the factors that determine whether a cancer cell undergoes cell cycle arrest, differentiation, or death in response to HDACIs. Furthermore, there is still limited data on HDACIs effects in vivo, as well as HDACIs function in combination with standard induction chemotherapy, as most studies evaluated HDACIs as single agent in vitro. Thus, our first goal was to determine a VPA response signature in different myeloid leukemia cell lines in vitro, followed by an in vivo analysis of VPA effects in blasts from adult de novo AML patients entered within two randomized multicenter treatment trials of the German-Austrian AML Study Group. To define an VPA in vitro “response signature” we profiled gene expression in myeloid leukemia cell lines (HL-60, NB-4, HEL-1, CMK and K-562) following 48 hours of VPA treatment by using DNA Microarray technology. In accordance with previous studies in vitro VPA treatment of myeloid cell lines induced the expression of the cyclin-dependent kinase inhibitors CDKN1A and CDKN2D coding for p21 and p19, respectively. Supervised analyses revealed many genes known to be associated with a G1 arrest. In all cell lines except for CMK we examined an up-regulation of TNFSF10 coding for TRAIL, as well as differential regulation of other genes involved in apoptosis. Furthermore, gene set enrichment analyses showed a significant down-regulation of genes involved in DNA metabolism and DNA repair. Next, we evaluated the VPA effects on gene expression in AML samples collected within the AMLSG 07-04 trial for younger (age<60yrs) and within the AMLSG 06-04 trial for older adults (age>60yrs), in which patients are randomized to receive standard induction chemotherapy (idarubicine, cytarabine, and etoposide = ICE) with or without concomitant VPA. We profiled gene expression in diagnostic AML blasts and following 48 hours of treatment with ICE or ICE/VPA. First results from our ongoing analysis of in vivo VPA treated samples are in accordance with our cell line experiments as e.g. we also see an induction of CDKN1A expression. However, the picture observed is less homogenous as concomitant administration of ICE, as well as other factors, like e.g. VPA serum levels, might substantially influence the in vivo VPA response. Nevertheless, our data are likely to provide new insights into the VPA effect in vivo, and this study may proof to be useful to predict AML patients likely to benefit from VPA treatment. To achieve this goal, we are currently analyzing additional samples, and we are planning to correlate gene expression findings with histone acetylation status, VPA serum levels, cytogenetic, and molecular genetic data.

TET2 Downregulation Promotes AML Cell Proliferation Via Pim-1 Expression

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5085-5085
Author(s):  
Qingxiao Chen ◽  
Jingsong He ◽  
Xing Guo ◽  
Jing Chen ◽  
Xuanru Lin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Western Blot ◽  
Cell Lines ◽  
Target Genes ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Rna Seq ◽  
Knock Down

Abstract Background: Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults which is still incurable although novel drugs and new combination of chemotherapies are used . With the development of genetic and molecular biology technologies, more and more genes are found to be related to leukemogenesis and drug resistance of AML. TET2, a member of the ten-eleven-translocation gene family which can modify DNA by catalyzing the conversion of 5-mehtyl-cytosine to 5-hydroxymethyl-cytosine , is often inactivated through mutation or deletion in myeloid malignancies. Recent research reported that TET2 knock-down can promote proliferation of hematopoietic stem cells and leukemic cells. Also, several clinical studies showed that patients with TET2 mutation or low levels of TET2 expression have more aggressive disease courses than those with normal levels of TET2. However, the mechanism of the phenomenon is unknown. Our aim is to uncover how TET2 protein level is negatively correlated with AML cell proliferation and to provide a better view of target therapy in AML. Methods: We determined the expression levels of TET2 and other target genes in acute leukemia cell lines, bone marrow AML specimens, and peripheral blood mononuclear cells from healthy donors by qRT-PCR and Western blot. We also determined the mutation status of TET2 in AML cell lines. CCK8 and flow cytometry were used to determine cell proliferation, cell apoptosis, and cell cycle profile. Methylation-specific PCR were used to examine the methylation status in gene promoter regions. Also, we developed TET2 knock-down lentivirus to transfect AML cell lines to examine the effect of TET2 depletion. Last, RNA-seq was used to compare gene expression level changes between TET2 knock-down cell lines and the control cell lines. Results: AML cells from AML cell lines (KG-1,U937, Kasumi, HL-60, THP-1, and MV4-11) and AML patients' specimens expressed lower levels of TET2 than those of PBMC from the healthy donor (P<0.05). Among AML cell lines, U937 barely expressed TET2, while KG-1 expressed TET2 at a relatively higher level than those of other AML cell lines. We constructed a TET2 shRNA to transfect KG-1,THP-1,MV-4-11,Kasumi,and HL-60, and used qRT-PCR and western blot to verify the knock-down efficiency. CCK8 confirmed that knocking down TET2 could increase leukemia cell proliferation (P<0.05). Flow cytometry showed that cell cycle profile was altered in TET2 knock-down cells compared to the negative control cells. In order to identify target genes, we performed RNA-seq on wildtype and TET2 knockdown KG-1 cells and found that the expression of cell cycle related genes, DNA replication related genes, and some oncogenes were changed. We focused on Pim-1, an oncogene related to leukemogenesis, which was significantly up-regulated in the RNA-seq profile. Western blot and qPCR verified the RNA-seq results of Pim-1 expression in the transfected cells . Also, AML patients' bone marrow samples (n=35) were tested by qPCR and 28 of them were found to express low TET2 but high Pim-1 with the other 7 being opposite. For detailed exploration in expression regulation of Pim-1 via TET2, we screened genes affecting Pim-1 expression and found SHP-1, a tumor suppress gene which is often silenced by promoter methylation in AML. Western blot band of SHP-1 was attenuated in TET2 knockdown KG-1 cells. Moreover, methylation-specific PCR showed that after knocking down TET2 in KG-1 cell line, the promoter regions were methylated much more than the control cells. These results indicated that the function of TET2 in epigenetic modulation plays an important role in regulating Pim-1 expression. Finally, using flow cytometry and CCK8 we surprisingly found that knocking down TET2 expression could lead leukemic cells (KG-1, THP-1 and MV-4-11) more sensitive to Pim-1 inhibitor (SGI-1776 free base) and decitabine (a demethylation agent treating MDS and AML) (P<0.05). Conclusion: Our study showed that knocking down TET2 promoted leukemic cell proliferation. This phenomenon may correlate to Pim-1 up-regulation. Our clinical data also showed that the expression of TET2 and Pim-1 have an inverse relationship. The mechanism of TET2 regulating Pim-1 expression may be related to the epigenetic modulation function of TET2. Finally, we found TET2 downregulation could increase leukemia vulnerability to Pim-1 inhibitor and decitbine, and provide a novel view of target therapy in AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals RUNX1/CBFβ Dosage Is Critical for MLL Leukemias Development

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2187-2187
Author(s):  
Xiaomei Yan ◽  
Yoshihiro Hayashi ◽  
Xinghui Zhao ◽  
Aili Chen ◽  
Yue Zhang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Down Regulation ◽  
Leukemia Cell Lines ◽  
Flanking Regions ◽  
The Impact

Abstract Transcription factors RUNX1/CBFβ play critical roles in hematopoiesis. Both of them are frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. We have previously shown that MLL, RUNX1, and CBFβ interact and form a regulatory complex to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity remains unknown. To determine the impact of MLL fusion protein on RUNX1/CBFβ, we introduced either MLL, MLL-BP (longer N-terminal Flag-tagged MLL construct which contains CXXC domain; 1-1406), or MLL-fusions together with RUNX1, CBFβ, or both RUNX1 and CBFβ into 293T cells. MLL-BP and MLL fusions significantly decreased RUNX1 levels compared with controls (empty vector and MLL). CBFβ protein was mildly decreased by MLL-BP and MLL-fusions when expressed alone. However, when CBFβ was co-expressed with RUNX1, it was significantly decreased compared with controls. The expression levels of RUNX1 and CBFβ proteins in LSK cells from Mll-Af9 knock-in mice were significantly lower than those from wild-type (WT) mice. To confirm these findings in human acute myeloid leukemia (AML), we measured the expression of RUNX1 and CBFβ at both mRNA and protein levels in various leukemia cell lines. The expression levels of RUNX1 and CBFβ proteins were significantly decreased in AML cells with MLL fusion and MLL partial tandem duplication (MLL-PTD) compared with those in AML cells without MLL aberrations. MLL fusions still have CXXC domain. In MLL-PTD, the CXXC domain is duplicated. Our data showed that RUNX1 protein is not only down-regulated by MLL fusion proteins, but also by MLL-BP. Thus, to determine which region is involved in the down-regulation of RUNX1, we introduced a series of MLL deletion mutants into 293T cells and measured RUNX1 protein expression. MLL deletion mutants without CXXC domain had no effect on RUNX1 stability. The construct which contains point mutations in CXXC domain also lacked the ability to reduce RUNX1 expression. Furthermore, overexpression of only CXXC domain and flanking regions could down-regulate RUNX1 protein expression. These results suggest that MLL fusion proteins and the N-terminal MLL portion of MLL fusions down-regulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. To understand the impact of RUNX1/CBFβ down-regulation on hematopoietic stem and progenitor cells (HSPCs), we generated RUNX1+/–/CBFβ+/– mice as a hypomorph model. The percentage of bone marrow (BM) LSK cells from RUNX1+/–/CBFβ+/– mice was significantly increased compared with that from WT mice. Using BM cells from these mice, we performed in vitro CFU assay and in vivo bone marrow transplantation (BMT) assay. BM cells from RUNX1+/–/CBFβ+/– mice provided more colonies in CFU assay compared with those from WT mice. To determine whether restoration of RUNX1 could repress the MLL mediated leukemogenesis, we retrovirally overexpressed WT RUNX1 in BM cells from Mll-Af9 knock-in mice. Using transduced BM cells, we performed in vitro CFU assay and in vivo BMT assay. RUNX1 overexpressed Mll-Af9 (Mll-Af9/RUNX1) cells underwent terminal differentiation after 2 times replating, while control vector transduced Mll-Af9 (Mll-Af9/Control) cells could still be replated more than 4 times. All the recipient mice transplanted with Mll-Af9/Control cells developed AML. In contrast, all the recipient mice transplanted with Mll-Af9/RUNX1 never develop AML. Furthermore, when we treated MLL leukemia cell lines with DOT1L inhibitor (EPZ-5676), RUNX1 protein levels in these MLL leukemia cell lines were significantly increased 48 hours after the treatment in comparing with controls treated with DMSO. However, there was no significant mRNA expression level change of RUNX1within 48 hours. Future studies are needed to fully understand the mechanism of whether this increasing RUNX1 protein level by DOT1L inhibitor is through blocking CXXC domain and flanking regions mediated degradation. In conclusion, MLL aberrations down-regulate RUNX1/CBFβ via their CXXC domain and flanking regions. Down-regulation of RUNX1/CBFβ plays critical role for MLL mediated leukemia development. Targeting RUNX1/CBFβ levels allows us to test novel therapies for MLL leukemias. Disclosures Mulloy: Celgene: Research Funding; Seattle Genetics: Research Funding; Amgen: Research Funding; NovImmune: Research Funding.

scholarly journals Effect of Gaillardin on Proliferation and Apoptosis of Acute Promyelocytic Leukemia Cell Lines, NB4 as Cancer Treatment

2020 ◽  
Vol 11 (1) ◽  
pp. 7445-7452
Keyword(s):  
Flow Cytometry ◽  
Cancer Treatment ◽  
Acute Promyelocytic Leukemia ◽  
Cell Lines ◽  
Mtt Assay ◽  
Apoptotic Cell ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Acute Promyelocytic Leukemia Cell ◽  
Leukemia Cell Lines

Acute promyelocytic leukemia is the most prevalent AML malignancy. The side effects caused by chemotherapeutic drugs have primarily led to the increased use of natural products for cancer treatment. Gaillardin, a medicinal herb, has been reported to have anti-proliferative effects on various cancer cells. In this study, cytotoxic and apoptotic effects of Gaillardin were investigated on acute promyelocytic leukemia cell lines, NB4. Different concentrations of Gaillardin were used to treat NB4 cells for 48 and 72 hours. Then cell viability was assessed using MTT assay. Flow cytometry was applied to the assessment of apoptosis by using Annexin V and Propidium iodide staining method. In order to determine the expression levels of Bax and Bcl-2, RT-PCR was carried out. Statistical data analysis was done by ANOVA (one-way). MTT assay results showed that Gaillardin concentrations of 7, 8, and 9 µM would significantly reduce the percentage of live cells (P< 0.001)c. Flow cytometry results showed that Gaillardin significantly increased apoptotic cell percentage in comparison with the control groups (p< 0.05). Gaillardin can be considered as a candidate for further studies on the methods for treating various cancer types.

scholarly journals Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Mediates and Sustains NF-κb Constitutive Activation in LGL Leukemia Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2762-2762
Author(s):  
Jun Yang ◽  
Francis R Leblanc ◽  
Shubha Dighe ◽  
Susan B. Nyland ◽  
David J. Feith ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Binding Activity ◽  
Leukemia Cells ◽  
Trail Expression ◽  
Constitutive Activation ◽  
Dna Binding Activity ◽  
Leukemia Cell Lines ◽  
Lgl Leukemia

Abstract Large granular lymphocyte (LGL) leukemia results from clonal expansion of CD3+ cytotoxic T-lymphocytes (CTL) or CD3-natural killer (NK) cells. Chronic antigen stimulation promotes long-term survival of LGL leukemia cells through constitutive activation of multiple survival pathways, leading to global dysregulation of apoptosis. Clinical manifestations of LGL leukemia include neutropenia, anemia and rheumatoid arthritis. Treatment for LGL leukemia patients relies on immunosuppressives such as methotrexate and is not curative. No standard therapy has been established. We reported that nuclear factor kappa B (NF-kB) is central to the leukemic LGL survival network, but the mechanisms of constitutive NF-kB activation in LGL leukemia are undefined. TNF-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis and activates NF-kB via binding to TRAIL receptor (TR) 1, 2 and decoy receptor 2 (DcR2). DcR2 is uniquely unable to transduce downstream death signals yet retains NF-kB transduction activity. The mechanisms of TRAIL expression and regulation in LGL leukemia are unknown. Thus the current study investigates these mechanisms and their potential therapeutic applications, with the use of NF-kB inhibitors ixazomib and bortezomib, in LGL leukemia. Methods: LGL leukemia cell lines TL1 (T-LGL) and NKL (NK-LGL), peripheral blood mononuclear cells (PBMC) from LGL leukemia patients, and PBMC from normal donors were studied. NF-kB DNA binding activity was determined by EMSA. Results were confirmed using probe-based NF-kB (p50/65) transcription factor assay and immunocytochemistry (ICC). Serum TRAIL levels were detected by ELISA. Cellular TRAIL expression was determined by real-time PCR, western blot and ICC. DcR2 and Mcl-1 siRNA knock-down was performed with electroporation. Flow cytometry was used to detect TR 1-3 and DcR2 expression and apoptosis. Results: The average serum levels of TRAIL in LGL leukemia patients were nearly 4-fold higher than normal (NL) control values (p ≤ 0.0001). Data from RT-PCR (p ≤ 0.04), western blot and ICC revealed that LGL leukemia cells were the major source of TRAIL overexpression. Identical expression levels of TR1, 2 and 3 were observed in PBMC from LGL leukemia patients and from NL controls. Like normal PBMC, LGL leukemia cells were resistant to TRAIL-induced apoptosis. In contrast, the expression frequency of DcR2 was at least 4-fold greater in LGL leukemia PBMC compared to NL control, and it correlated to the percentage of circulating LGL leukemia cells. We found that TRAIL activates NF-kB and NF-kB downstream target genes, including TRAIL and McL-1, in LGL leukemia samples. To confirm that TRAIL is responsible for constitutive NF-kB activation in LGL leukemia, T-LGL leukemia PBMC were treated with pooled sera from 3 each of either NL controls or T-LGL leukemia patients. Leukemia sera increased NF-kB activity on EMSA, and this effect was completely blocked by TRAIL neutralizing antibody. DcR2 siRNA knockdown specifically decreased RelA and NF-kB1 (p105/p50) levels in TL1 and NKL cells. Mcl-1 siRNA mediated increased apoptosis in the same cell lines. Likewise, ixazomib and bortezomib facilitated leukemia-selective apoptosis in LGL leukemia cell lines and in patient PBMC, via inhibition of NF-kB activity and of downstream targets (ixazomib, p≤0.0001; bortezomib, p≤0.03). Additionally, caspase-3 and PARP cleavage were observed in LGL leukemia cells treated with ixazomib or bortezomib. Serum TRAIL levels in LGL leukemia patients were significantly lower in methotrexate responders versus non-responders, corresponding with reduced NF-kB DNA binding activity and increased absolute neutrophil counts, indicative of treatment response. Conclusion: These data indicate that expression of DcR2 and constitutive activation of NF-kB are responsible for TRAIL resistance in leukemic LGLs. TRAIL triggers prolonged NF-kB activation via interaction with DcR2, and activated NF-kB in turn promotes further TRAIL production in leukemic LGLs, creating a TRAIL autocrine regulatory loop. Inhibition of NF-kB activity with ixazomib and bortezomib interrupts this loop, impairs expression of Mcl-1 and induces apoptosis of leukemia cells. Our preclinical findings provide a solid framework for clinical evaluations of ixazomib and bortezomib in the treatment of LGL leukemia. Disclosures No relevant conflicts of interest to declare.

Antitumor activity of pazopanib (P) and trametinib (T) in preclinical models of osteosarcoma (OS).

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e22509-e22509
Author(s):  
Giulia Chiabotto ◽  
Maria Laura Centomo ◽  
Alessandra Merlini ◽  
Lorenzo D'Ambrosio ◽  
Dario Sangiolo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Antitumor Activity ◽  
Western Blot ◽  
Cell Lines ◽  
Fold Change ◽  
Janus Kinase ◽  
Preclinical Models ◽  
Log2 Fold Change ◽  
In Vivo Antitumor Activity

e22509 Background: Receptor tyrosine kinases (RTKs) and their signal transducers are suitable targets for the treatment of advanced OS. We evaluated the antitumor activity of the RTK inhibitor P and the MEK inhibitor T and deeply investigated molecular mechanisms behind their activity and potential escape. Methods: Flow cytometry and western blot analyses were carried out in 7 OS cell lines to study the expression of RTK P targets and the activation of their pathways, respectively. Cell viability and colony growth were evaluated after 72h and 7-day treatment respectively, with scalar doses of both single agents and their constant combination. Cell cycle distribution and apoptosis were evaluated by flow cytometry after 72h. In vivo antitumor activity was studied in NOD/SCID mice bearing MNNG-HOS xenografts after 3 weeks of treatment. Cell migration was studied by scratch assays. The involvement of MAPK-PI3K pathway key transducers was explored by Vantage 3D RNA Panel and Nanostring technology, validated by western blot and confirmed by silencing experiments. Results: P targets are expressed on OS cell lines and their pathways are activated. P+T have synergistic antitumor activity (combination index < 1) in OS cell lines by inducing apoptosis (6/7) and inhibiting both ERK1/2(7/7) and AKT (7/7). Furthermore, in vivo antitumor activity was shown in OS bearing mice (tumor volume: P+T/untreated = 0.036, p = 0.002). P+T significantly down-modulated RTK EphaA2 (mean log2 fold change RNA P+T/untreated = -2.02±0.50) and induced Janus kinase MEK6 (mean log2 fold change RNA P+T/untreated = 2.9±0.51). EphA2 silencing reduced cellular proliferation and migration of OS cells. Impeding MEK6-up-regulation in P+T treated cells significantly increased the antitumor effect (51.5±14.3%) of the studied drugs. Conclusions: P+T exert antitumor activity in OS preclinical models through ERK and AKT inhibition and EphA2 downmodulation. MEK6-upregulation after P+T is likely implied in escape mechanism.

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